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Meng F, He J, Zhang X, Lyu W, Wei R, Wang S, Du Z, Wang H, Bi J, Hua X, Zhang C, Guan Y, Lyu G, Tian X, Zhang L, Xie W, Tao W. Histone Lactylation Antagonizes Senescence and Skeletal Muscle Aging by Modulating Aging-Related Pathways. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2412747. [PMID: 40388671 PMCID: PMC12165025 DOI: 10.1002/advs.202412747] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Revised: 04/11/2025] [Indexed: 05/21/2025]
Abstract
Epigenetic alterations are among the prominent drivers of cellular senescence and/or aging, intricately orchestrating gene expression programs during these processes. This study shows that histone lactylation, plays a pivotal role in counteracting senescence and mitigating dysfunctions of skeletal muscle in aged mice. Mechanistically, histone lactylation and lactyl-CoA levels markedly decrease during cellular senescence but are restored under hypoxic conditions primarily due to elevated glycolytic activity. The enrichment of histone lactylation at promoters is essential for sustaining the expression of genes involved in the cell cycle and DNA repair pathways. Furthermore, the modulation of enzymes crucial for histone lactylation, leads to reduced histone lactylation and accelerated cellular senescence. Consistently, the suppression of glycolysis and the depletion of histone lactylation are also observed during skeletal muscle aging. Modulating the enzymes can also lead to the loss of histone lactylation in skeletal muscle, downregulating DNA repair and proteostasis pathways and accelerating muscle aging. Running exercise increases histone lactylation, which in turn upregulate key genes in the DNA repair and proteostasis pathways. This study highlights the significant roles of histone lactylation in modulating cellular senescence as well as muscle aging, providing a promising avenue for antiaging intervention via metabolic manipulation.
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Affiliation(s)
- Fanju Meng
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Jianuo He
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Xuebin Zhang
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Wencong Lyu
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Ran Wei
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Shiyi Wang
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Zhehao Du
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Haochen Wang
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Jinlong Bi
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Xueyang Hua
- Hefei National Laboratory for Physical Sciences at the MicroscaleSchool of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefei230026China
| | - Chao Zhang
- Kunming Institute of ZoologyChinese Academy of SciencesKunmingYunnanChina
| | - Yiting Guan
- Zhanjiang Institute of Clinical MedicineZhanjiang Central HospitalGuangdong Medical UniversityZhanjiang524045China
| | - Guoliang Lyu
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Xiao‐Li Tian
- Department of Human Population GeneticsHuman Aging Research Institute (HARI) and School of Life SciencesNanchang UniversityNanchang330031China
| | - Lijun Zhang
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Wenbing Xie
- Hefei National Laboratory for Physical Sciences at the MicroscaleSchool of Basic Medical SciencesDivision of Life Sciences and MedicineUniversity of Science and Technology of ChinaHefei230026China
| | - Wei Tao
- The State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
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Panagopoulos A, Stout M, Kilic S, Leary P, Vornberger J, Pasti V, Galarreta A, Lezaja A, Kirschenbühler K, Imhof R, Rehrauer H, Ziegler U, Altmeyer M. Multigenerational cell tracking of DNA replication and heritable DNA damage. Nature 2025:10.1038/s41586-025-08986-0. [PMID: 40399682 DOI: 10.1038/s41586-025-08986-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2024] [Accepted: 04/04/2025] [Indexed: 05/23/2025]
Abstract
Cell heterogeneity is a universal feature of life. Although biological processes affected by cell-to-cell variation are manifold, from developmental plasticity to tumour heterogeneity and differential drug responses, the sources of cell heterogeneity remain largely unclear1,2. Mutational and epigenetic signatures from cancer (epi)genomics are powerful for deducing processes that shaped cancer genome evolution3-5. However, retrospective analyses face difficulties in resolving how cellular heterogeneity emerges and is propagated to subsequent cell generations. Here, we used multigenerational single-cell tracking based on endogenously labelled proteins and custom-designed computational tools to elucidate how oncogenic perturbations induce sister cell asymmetry and phenotypic heterogeneity. Dual CRISPR-based genome editing enabled simultaneous tracking of DNA replication patterns and heritable endogenous DNA lesions. Cell lineage trees of up to four generations were tracked in asynchronously growing cells, and time-resolved lineage analyses were combined with end-point measurements of cell cycle and DNA damage markers through iterative staining. Besides revealing replication and repair dynamics, damage inheritance and emergence of sister cell heterogeneity across multiple cell generations, through combination with single-cell transcriptomics, we delineate how common oncogenic events trigger multiple routes towards polyploidization with distinct outcomes for genome integrity. Our study provides a framework to dissect phenotypic plasticity at the single-cell level and sheds light onto cellular processes that may resemble early events during cancer development.
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Affiliation(s)
- Andreas Panagopoulos
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Merula Stout
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Sinan Kilic
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
- The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Peter Leary
- Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland
| | - Julia Vornberger
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Virginia Pasti
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Antonio Galarreta
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Aleksandra Lezaja
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Kyra Kirschenbühler
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
- NEXUS Personalized Health, ETH Zurich, Schlieren, Switzerland
| | - Ralph Imhof
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Hubert Rehrauer
- Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland
| | - Urs Ziegler
- Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland
| | - Matthias Altmeyer
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.
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Shibata Y, Peycheva M, Shibata E, Malzl D, Pavri R, Dutta A. Specific origin selection and excess functional MCM2-7 loading in ORC-deficient cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.10.30.621095. [PMID: 39554186 PMCID: PMC11565923 DOI: 10.1101/2024.10.30.621095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
The six subunit Origin Recognition Complex (ORC) loads excess MCM2-7 on chromosomes to promote initiation of DNA replication and is believed to be important for origin specification. Mapping of origins in cancer cell lines engineered to delete three of the subunits, ORC1 , ORC2 or ORC5 shows that specific origins are still used and are mostly at the same sites in the genome as in wild type cells. The few thousand origins that were up-regulated in the absence of ORC suggest that GC/TA skewness and simple repeat sequences facilitate, but are not essential for, origin selection in the absence of the six-subunit ORC. Despite the lack of ORC, excess MCM2-7 is still loaded at comparable rates in G1 phase to license dormant origins and is also repeatedly loaded in the same S phase to permit re-replication. Thus, origin specification and excess MCM2-7 loading on origins do not require the six-subunit ORC in human cancer cell lines.
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Bou Malhab LJ, Schmidt S, Fagotto-Kaufmann C, Pion E, Gadea G, Roux P, Fagotto F, Debant A, Xirodimas DP. An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway. Cells 2024; 13:1625. [PMID: 39404389 PMCID: PMC11475522 DOI: 10.3390/cells13191625] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Revised: 09/23/2024] [Accepted: 09/24/2024] [Indexed: 10/19/2024] Open
Abstract
Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.
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Affiliation(s)
- Lara J. Bou Malhab
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
- Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Susanne Schmidt
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Christine Fagotto-Kaufmann
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Emmanuelle Pion
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Gilles Gadea
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Pierre Roux
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Francois Fagotto
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Anne Debant
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
| | - Dimitris P. Xirodimas
- CRBM, Cell Biology Research Centre of Montpellier, Université de Montpellier, CNRS, 34293 Montpellier, France; (S.S.); (C.F.-K.); (E.P.); (G.G.); (P.R.); (F.F.)
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Murthy GSG, Saliba AN, Szabo A, Harrington A, Abedin S, Carlson K, Michaelis L, Runaas L, Baim A, Hinman A, Maldonado-Schmidt S, Venkatachalam A, Flatten KS, Peterson KL, Schneider PA, Litzow M, Kaufmann SH, Atallah E. A phase I study of pevonedistat, azacitidine, and venetoclax in patients with relapsed/refractory acute myeloid leukemia. Haematologica 2024; 109:2864-2872. [PMID: 38572562 PMCID: PMC11367232 DOI: 10.3324/haematol.2024.285014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2024] [Accepted: 03/27/2024] [Indexed: 04/05/2024] Open
Abstract
Azacitidine/venetoclax is an active regimen in patients with newly diagnosed acute myeloid leukemia (AML). However, primary or secondary resistance to azacitidine/venetoclax is an area of unmet need and overexpression of MCL1 is suggested to be a potential resistance mechanism. Pevonedistat inhibits MCL1 through activation of NOXA, and pevonedistat/azacitidine has previously shown activity in AML. To assess the tolerability and efficacy of adding pevonedistat to azacitidine/ venetoclax in relapsed/refractory AML, we conducted a phase I, multicenter, open-label study in 16 adults with relapsed/ refractory AML. Patients were treated with azacitidine, venetoclax along with pevonedistat intravenously on days 1, 3 and 5 of each 28-day cycle at doses of 10, 15 or 20 mg/m2 in successive cohorts in the dose escalation phase. The impact of treatment on protein neddylation as well as expression of pro-apoptotic BCL2 family members was assessed. The recommended phase II dose of pevonedistat was 20 mg/m2. Grade 3 or higher adverse events included neutropenia (31%), thrombocytopenia (13%), febrile neutropenia (19%), anemia (19%), hypertension (19%) and sepsis (19%). The overall response rate was 46.7% for the whole cohort including complete remission in five of seven (71.4%) patients who had not previously been treated with the hypomethylating agent/venetoclax. No measurable residual disease was detected in 80.0% of the patients who achieved complete remission. The median time to best response was 50 (range, 23-77) days. Four patients were bridged to allogeneic stem cell transplantation. The combination of azacitidine, venetoclax and pevonedistat is safe and shows encouraging preliminary activity in patients with relapsed/refractory AML. (NCT04172844).
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Affiliation(s)
| | | | - Aniko Szabo
- Department of Biostatistics, Medical College of Wisconsin, Milwaukee, Wisconsin
| | | | - Sameem Abedin
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Karen Carlson
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Laura Michaelis
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Lyndsey Runaas
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Arielle Baim
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
| | - Alex Hinman
- Clinical Trials Office, Medical College of Wisconsin, Milwaukee, Wisconsin
| | | | | | - Karen S Flatten
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; Division of Oncology Research, Mayo Clinic, Rochester, MN
| | - Kevin L Peterson
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; Division of Oncology Research, Mayo Clinic, Rochester, MN
| | - Paula A Schneider
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; Division of Oncology Research, Mayo Clinic, Rochester, MN
| | - Mark Litzow
- Division of Hematology, Mayo Clinic, Rochester, Minnesota
| | - Scott H Kaufmann
- Division of Hematology, Mayo Clinic, Rochester, Minnesota; Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN; Division of Oncology Research, Mayo Clinic, Rochester, MN
| | - Ehab Atallah
- Division of Hematology-Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin
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6
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Foster JH, Reid JM, Minard C, Woodfield S, Denic KZ, Isikwei E, Voss SD, Nelson M, Liu X, Berg SL, Fox E, Weigel BJ. Phase 1 study of NEDD8 activating enzyme inhibitor pevonedistat in combination with chemotherapy in pediatric patients with recurrent or refractory solid tumors (ADVL1615). Eur J Cancer 2024; 209:114241. [PMID: 39096851 PMCID: PMC11392690 DOI: 10.1016/j.ejca.2024.114241] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2024] [Revised: 06/24/2024] [Accepted: 07/22/2024] [Indexed: 08/05/2024]
Abstract
PURPOSE The objective of this study was to determine the recommended Phase 2 dose (RP2D) of pevonedistat, a first in class inhibitor of NEDD8 activating enzyme, in combination with irinotecan (IRN) and temozolomide (TMZ) in children with cancer. METHODS This Phase 1 study used a rolling 6 design to evaluate escalating doses of pevonedistat in combination with standard doses of IRN and TMZ in pediatric patients with recurrent/refractory solid or CNS tumors. During cycle 1, pevonedistat was administered intravenously on days 1, 8, 10, and 12, with IRN (IV, 50 mg/m2) and TMZ (orally, 100 mg/m2), on days 8-12 of a 28-day cycle. In subsequent cycles, pevonedistat was administered on days 1, 3, and 5, with IRN/TMZ on days 1-5 of a 21-day cycle. RESULTS Thirty patients enrolled; all were eligible and evaluable for toxicity. Six patients each enrolled on pevonedistat dose levels (DL) 1 (15 mg/m2), 2 (20 mg/m2), 3 (25 mg/m2) and 4 (35 mg/m2) as well as an expanded pharmacokinetic (PK) cohort at DL4. The maximum tolerated dose (MTD) was not exceeded. 2/12 (17 %) patients treated at the RP2D (35 mg/m2) experienced a cycle 1 dose limiting toxicity (DLT). IRN is unlikely to affect the pharmacokinetics of pevonedistat. Two patients had a partial response and 6 patients had prolonged stable disease (> 6 cycles). CONCLUSIONS Pevonedistat in combination with IRN/TMZ is well tolerated in children with solid or CNS tumors. The RP2D of pevonedistat is 35 mg/m2 on days 1, 3, 5 in combination with IRN/TMZ.
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Affiliation(s)
- Jennifer H Foster
- Texas Children's Hospital, Baylor College of Medicine, Dan L Duncan Comprehensive Cancer Center, Houston, TX, USA.
| | | | - Charles Minard
- Institute for Clinical and Translational Research, Baylor College of Medicine, Houston, TX, USA
| | - Sarah Woodfield
- Texas Children's Hospital, Baylor College of Medicine, Dan L Duncan Comprehensive Cancer Center, Houston, TX, USA
| | | | | | - Stephan D Voss
- Department of Radiology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Marvin Nelson
- Children's Hospital Los Angeles, Radiology, Keck USC School of Medicine, Los Angeles, CA, USA
| | - Xiaowei Liu
- Children's Oncology Group, Monrovia, CA, USA
| | - Stacey L Berg
- Texas Children's Hospital, Baylor College of Medicine, Dan L Duncan Comprehensive Cancer Center, Houston, TX, USA
| | - Elizabeth Fox
- Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Brenda J Weigel
- Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
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Benamar M, Eki R, Du KP, Abbas T. Break-induced replication drives large-scale genomic amplifications in cancer cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.27.609980. [PMID: 39253455 PMCID: PMC11383296 DOI: 10.1101/2024.08.27.609980] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/11/2024]
Abstract
DNA double-strand breaks (DSBs) are highly toxic lesions that underly the efficacy of ionizing radiation (IR) and a large number of cytotoxic chemotherapies 1-3 . Yet, abnormal repair of DSBs is associated with genomic instability and may contribute to cancer heterogeneity and tumour evolution. Here, we show that DSBs induced by IR, by DSB-inducing chemotherapeutics, or by the expression of a rare-cutting restriction endonuclease induce large-scale genomic amplification in human cancer cells. Importantly, the extent of DSB-induced genomic amplification (DIGA) in a panel of melanoma cell lines correlated with the degree of cytotoxicity elicited by IR, suggesting that DIGA contributes significantly to DSB-induced cancer cell lethality. DIGA, which is mediated through conservative DNA synthesis, does not require origin re-licensing, and is enhanced by the depletion or deletion of the methyltransferases SET8 and SUV4-20H1, which function sequentially to mono- and di-methylate histone H4 lysine 20 (H4K20) at DSBs to facilitate the recruitment of 53BP1-RIF1 and its downstream effector shieldin complex to DSBs to prevent hyper-resection 4-11 . Consistently, DIGA was enhanced in cells lacking 53BP1 or RIF1, or in cells that lacked components of the shieldin complex or of other factors that help recruit 53BP1 to DSBs. Mechanistically, DIGA requires MRE11/CtIP and EXO1, factors that promote resection and hyper-resection at DSBs, and is dependent on the catalytic activity of the RAD51 recombinase. Furthermore, deletion or depletion of POLD3, POLD4, or RAD52, proteins involved in break-induced replication (BIR), significantly inhibited DIGA, suggesting that DIGA is mediated through a RAD51-dependent BIR-like process. DIGA induction was maximal if the cells encountered DSBs in early and mid S-phase, whereas cells competent for homologous recombination (in late S and G2) exhibited less DIGA induction. We propose that unshielded, hyper-resected ends of DSBs may nucleate a replication-like intermediate that enables cytotoxic long-range genomic DNA amplification mediated through BIR.
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Saurat N, Minotti AP, Rahman MT, Sikder T, Zhang C, Cornacchia D, Jungverdorben J, Ciceri G, Betel D, Studer L. Genome-wide CRISPR screen identifies neddylation as a regulator of neuronal aging and AD neurodegeneration. Cell Stem Cell 2024; 31:1162-1174.e8. [PMID: 38917806 PMCID: PMC11405001 DOI: 10.1016/j.stem.2024.06.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 03/25/2024] [Accepted: 06/03/2024] [Indexed: 06/27/2024]
Abstract
Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APPswe/swe mutation. Aged APPswe/swe but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSENM146V/M146V cortical and LRRK2G2019S/G2019S midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.
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Affiliation(s)
- Nathalie Saurat
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA.
| | - Andrew P Minotti
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA
| | - Maliha T Rahman
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
| | - Trisha Sikder
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA
| | - Chao Zhang
- Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA; Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA; Section of Computational Biomedicine, Boston University School of Medicine, Boston, MA, USA
| | - Daniela Cornacchia
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA
| | - Johannes Jungverdorben
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
| | - Gabriele Ciceri
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA
| | - Doron Betel
- Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA; Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA.
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9
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Jones RM, Ruiz JH, Scaramuzza S, Nath S, Liu C, Henklewska M, Natsume T, Bristow RG, Romero F, Kanemaki MT, Gambus A. Characterizing replisome disassembly in human cells. iScience 2024; 27:110260. [PMID: 39055910 PMCID: PMC11269944 DOI: 10.1016/j.isci.2024.110260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 03/22/2024] [Accepted: 06/10/2024] [Indexed: 07/28/2024] Open
Abstract
To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.
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Affiliation(s)
- Rebecca M. Jones
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Joaquin Herrero Ruiz
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Shaun Scaramuzza
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Sarmi Nath
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Chaoyu Liu
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Marta Henklewska
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Toyoaki Natsume
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Robert G. Bristow
- Cancer Research UK – Manchester Institute, Manchester Cancer Research Center, Manchester, UK
| | - Francisco Romero
- Department of Microbiology, University of Seville, Seville, Spain
| | - Masato T. Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
- Department of Biological Science, The University of Tokyo, Tokyo, Japan
| | - Agnieszka Gambus
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
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10
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Chen KH, Sun JM, Lin L, Liu JW, Liu XY, Chen GD, Chen H, Chen ZY. The NEDD8 activating enzyme inhibitor MLN4924 mitigates doxorubicin-induced cardiotoxicity in mice. Free Radic Biol Med 2024; 219:127-140. [PMID: 38614228 DOI: 10.1016/j.freeradbiomed.2024.04.221] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 04/08/2024] [Accepted: 04/10/2024] [Indexed: 04/15/2024]
Abstract
Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.
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Affiliation(s)
- Kang Hui Chen
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Jian Min Sun
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Li Lin
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Jian Wen Liu
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Xin Yue Liu
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Guang Duo Chen
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China
| | - Hang Chen
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China.
| | - Zhao Yang Chen
- Department of Cardiology, Fujian Medical Center for Cardiovascular Diseases, Fujian Medical University Union Hospital, Fuzhou, Fujian, 350001, China.
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11
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Powell RT, Rinkenbaugh AL, Guo L, Cai S, Shao J, Zhou X, Zhang X, Jeter-Jones S, Fu C, Qi Y, Baameur Hancock F, White JB, Stephan C, Davies PJ, Moulder S, Symmans WF, Chang JT, Piwnica-Worms H. Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer. NPJ Breast Cancer 2024; 10:37. [PMID: 38802426 PMCID: PMC11130334 DOI: 10.1038/s41523-024-00644-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Accepted: 05/09/2024] [Indexed: 05/29/2024] Open
Abstract
Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.
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Affiliation(s)
- Reid T Powell
- Center for Translational Cancer Research, Institute of Bioscience and Technology Texas A&M Health Science Center, Houston, TX, USA
| | - Amanda L Rinkenbaugh
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Lei Guo
- Center for Translational Cancer Research, Institute of Bioscience and Technology Texas A&M Health Science Center, Houston, TX, USA
| | - Shirong Cai
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jiansu Shao
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Xinhui Zhou
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Xiaomei Zhang
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Sabrina Jeter-Jones
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Chunxiao Fu
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yuan Qi
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Faiza Baameur Hancock
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jason B White
- Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Clifford Stephan
- Center for Translational Cancer Research, Institute of Bioscience and Technology Texas A&M Health Science Center, Houston, TX, USA
| | - Peter J Davies
- Center for Translational Cancer Research, Institute of Bioscience and Technology Texas A&M Health Science Center, Houston, TX, USA
| | - Stacy Moulder
- Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Eli Lilly and Company, Indianapolis, IN, USA
| | - W Fraser Symmans
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jeffrey T Chang
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Helen Piwnica-Worms
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
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12
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Zhang S, Yu Q, Li Z, Zhao Y, Sun Y. Protein neddylation and its role in health and diseases. Signal Transduct Target Ther 2024; 9:85. [PMID: 38575611 PMCID: PMC10995212 DOI: 10.1038/s41392-024-01800-9] [Citation(s) in RCA: 34] [Impact Index Per Article: 34.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Revised: 02/22/2024] [Accepted: 03/04/2024] [Indexed: 04/06/2024] Open
Abstract
NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8) is an ubiquitin-like protein that is covalently attached to a lysine residue of a protein substrate through a process known as neddylation, catalyzed by the enzyme cascade, namely NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). The substrates of neddylation are categorized into cullins and non-cullin proteins. Neddylation of cullins activates CRLs (cullin RING ligases), the largest family of E3 ligases, whereas neddylation of non-cullin substrates alters their stability and activity, as well as subcellular localization. Significantly, the neddylation pathway and/or many neddylation substrates are abnormally activated or over-expressed in various human diseases, such as metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers, among others. Thus, targeting neddylation becomes an attractive strategy for the treatment of these diseases. In this review, we first provide a general introduction on the neddylation cascade, its biochemical process and regulation, and the crystal structures of neddylation enzymes in complex with cullin substrates; then discuss how neddylation governs various key biological processes via the modification of cullins and non-cullin substrates. We further review the literature data on dysregulated neddylation in several human diseases, particularly cancer, followed by an outline of current efforts in the discovery of small molecule inhibitors of neddylation as a promising therapeutic approach. Finally, few perspectives were proposed for extensive future investigations.
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Affiliation(s)
- Shizhen Zhang
- Department of Breast Surgery, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310029, China
- Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310029, China
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China
| | - Qing Yu
- Department of Thyroid Surgery, Zhejiang Cancer Hospital, Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, 310022, China
- Key Laboratory of Head & Neck Cancer Translational Research of Zhejiang Province, Hangzhou, 310022, China
| | - Zhijian Li
- Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310029, China
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China
| | - Yongchao Zhao
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.
- Department of Hepatobiliary and Pancreatic Surgery, Zhejiang University School of Medicine, Hangzhou, 310029, China.
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310029, China.
- Zhejiang University Cancer Center, Hangzhou, 310029, China.
| | - Yi Sun
- Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310029, China.
- Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, 310029, China.
- Zhejiang University Cancer Center, Hangzhou, 310029, China.
- Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, Hangzhou, 310024, China.
- Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, 310053, China.
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13
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Qin A, Wells L, Malhotra B, Gadgeel S, Schneider BJ, Ramnath N, Rice JD, Kalemkerian GP. A Phase II Trial of Pevonedistat and Docetaxel in Patients With Previously Treated Advanced Non-Small-Cell Lung Cancer. Clin Lung Cancer 2024; 25:128-134. [PMID: 37977950 DOI: 10.1016/j.cllc.2023.10.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2023] [Revised: 10/20/2023] [Accepted: 10/24/2023] [Indexed: 11/19/2023]
Abstract
BACKGROUND Postimmunotherapy (IO) treatment options for stage IV non-small-cell lung cancer (NSCLC) remain limited. Docetaxel alone or in combination with ramucirumab remains a standard of care, but response rates and survival benefit are suboptimal. Cullin-RING ligases (CRL) catalyze degradation of tumor suppressor proteins and are overactivated in NSCLC. Neddylation, which is catalyzed by the NEDD8 activating enzyme (NAE), is required for the activation of CRLs. Pevonedistat, a first-in-class small molecule NAE inhibitor, exerted antitumor activity when combined with docetaxel in preclinical studies. METHODS We conducted a phase II, single-arm, investigator-initiated study evaluating the efficacy of pevonedistat plus docetaxel in patients with relapsed/refractory stage IV NSCLC. Patients received docetaxel 75 mg/m2 on day 1 and pevonedistat 25 mg/m2 on days 1, 3 and 5 of a 21-day cycle. The primary endpoint was objective response rate (ORR). RESULTS From March 5, 2018 to January 26, 2021, we enrolled 31 patients. The ORR was 22% (1 CR, 5 PR), median PFS was 4.1 months, and median OS was 13.2 months. The incidence of Grade ≥3 adverse events (AE) was 53% in patients (n = 30) who received at least 1 dose of both drugs, with the most frequent being neutropenia and AST/ALT elevation. One patient was taken off study for a Grade 4 transaminase elevation. There were no Grade 5 toxicities. CONCLUSION Our data suggest that the combination of docetaxel and pevonedistat is safe and exerts activity in patients with relapsed NSCLC. These encouraging results suggest that the neddylation pathway is an antitumor pathway that should be further studied.
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Affiliation(s)
- Angel Qin
- University of Michigan Rogel Cancer Center, Ann Arbor, MI.
| | - Leah Wells
- University of Michigan Rogel Cancer Center, Ann Arbor, MI
| | | | | | | | - Nithya Ramnath
- University of Michigan Rogel Cancer Center, Ann Arbor, MI; VA Ann Arbor Healthcare System, Ann Arbor, MI
| | - John D Rice
- Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, MI
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14
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Huang D, Zhao Q, Yang K, Lei J, Jing Y, Li H, Zhang C, Ma S, Sun S, Cai Y, Wang G, Qu J, Zhang W, Wang S, Liu GH. CRL2 APPBP2-mediated TSPYL2 degradation counteracts human mesenchymal stem cell senescence. SCIENCE CHINA. LIFE SCIENCES 2024; 67:460-474. [PMID: 38170390 DOI: 10.1007/s11427-023-2451-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Accepted: 09/13/2023] [Indexed: 01/05/2024]
Abstract
Cullin-RING E3 ubiquitin ligases (CRLs), the largest family of multi-subunit E3 ubiquitin ligases in eukaryotic cells, represent core cellular machinery for executing protein degradation and maintaining proteostasis. Here, we asked what roles Cullin proteins play in human mesenchymal stem cell (hMSC) homeostasis and senescence. To this end, we conducted a comparative aging phenotype analysis by individually knocking down Cullin members in three senescence models: replicative senescent hMSCs, Hutchinson-Gilford Progeria Syndrome hMSCs, and Werner syndrome hMSCs. Among all family members, we found that CUL2 deficiency rendered hMSCs the most susceptible to senescence. To investigate CUL2-specific underlying mechanisms, we then applied CRISPR/Cas9-mediated gene editing technology to generate CUL2-deficient human embryonic stem cells (hESCs). When we differentiated these into hMSCs, we found that CUL2 deletion markedly accelerates hMSC senescence. Importantly, we identified that CUL2 targets and promotes ubiquitin proteasome-mediated degradation of TSPYL2 (a known negative regulator of proliferation) through the substrate receptor protein APPBP2, which in turn down-regulates one of the canonical aging marker-P21waf1/cip1, and thereby delays senescence. Our work provides important insights into how CRL2APPBP2-mediated TSPYL2 degradation counteracts hMSC senescence, providing a molecular basis for directing intervention strategies against aging and aging-related diseases.
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Affiliation(s)
- Daoyuan Huang
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China
| | - Qian Zhao
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China
| | - Kuan Yang
- University of Chinese Academy of Sciences, Beijing, 100049, China
- CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics and China National Center for Bioinformation, Chinese Academy of Sciences, Beijing, 100101, China
- Sino-Danish College, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Jinghui Lei
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China
| | - Ying Jing
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China
| | - Hongyu Li
- University of Chinese Academy of Sciences, Beijing, 100049, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Chen Zhang
- The Fifth People's Hospital of Chongqing, Chongqing, 400062, China
| | - Shuai Ma
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China
| | - Shuhui Sun
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China
| | - Yusheng Cai
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China
| | - Guibin Wang
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 102206, China
| | - Jing Qu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China
| | - Weiqi Zhang
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics and China National Center for Bioinformation, Chinese Academy of Sciences, Beijing, 100101, China.
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China.
- Sino-Danish College, University of Chinese Academy of Sciences, Beijing, 101408, China.
| | - Si Wang
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China.
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
- The Fifth People's Hospital of Chongqing, Chongqing, 400062, China.
| | - Guang-Hui Liu
- Advanced Innovation Center for Human Brain Protection, National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing, 100053, China.
- Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China.
- Institute for Stem Cell and Regeneration, CAS, Beijing, 100101, China.
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15
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Yadav AK, Polasek-Sedlackova H. Quantity and quality of minichromosome maintenance protein complexes couple replication licensing to genome integrity. Commun Biol 2024; 7:167. [PMID: 38336851 PMCID: PMC10858283 DOI: 10.1038/s42003-024-05855-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Accepted: 01/25/2024] [Indexed: 02/12/2024] Open
Abstract
Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.
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Affiliation(s)
- Anoop Kumar Yadav
- Department of Cell Biology and Epigenetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
| | - Hana Polasek-Sedlackova
- Department of Cell Biology and Epigenetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.
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16
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Chen YN, Chan YH, Shiau JP, Farooqi AA, Tang JY, Chen KL, Yen CY, Chang HW. The neddylation inhibitor MLN4924 inhibits proliferation and triggers apoptosis of oral cancer cells but not for normal cells. ENVIRONMENTAL TOXICOLOGY 2024; 39:299-313. [PMID: 37705323 DOI: 10.1002/tox.23951] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/04/2023] [Revised: 07/26/2023] [Accepted: 08/13/2023] [Indexed: 09/15/2023]
Abstract
Increased neddylation benefits the survival of several types of cancer cells. The inhibition of neddylation has the potential to exert anticancer effects but is rarely assessed in oral cancer cells. This study aimed to investigate the antiproliferation potential of a neddylation inhibitor MLN4924 (pevonedistat) for oral cancer cells. MLN4924 inhibited the cell viability of oral cancer cells more than that of normal oral cells (HGF-1) with 100% viability, that is, IC50 values of oral cancer cells (CAL 27, OC-2, and Ca9-22) are 1.8, 1.4, and 1.9 μM. MLN4924 caused apoptotic changes such as the subG1 accumulation, activation of annexin V, pancaspase, and caspases 3/8/9 of oral cancer cells at a greater rate than in normal oral cells. MLN4924 induced greater oxidative stress in oral cancer cells compared to normal cells by upregulating reactive oxygen species and mitochondrial superoxide and depleting the mitochondrial membrane potential and glutathione. In oral cancer cells, preferential inductions also occurred for DNA damage (γH2AX and 8-oxo-2'-deoxyguanosine). Therefore, this investigation demonstrates that MLN4924 is a potential anti-oral-cancer agent showing preferential inhibition of apoptosis and promotion of DNA damage with fewer cytotoxic effects on normal cells.
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Affiliation(s)
- Yan-Ning Chen
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Yu-Hsuan Chan
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Science, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Jun-Ping Shiau
- Division of Breast Oncology and Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
| | | | - Jen-Yang Tang
- Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- School of Post-Baccalaureate Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Kuan-Liang Chen
- Department of Oral and Maxillofacial Surgery, Chi-Mei Medical Center, Tainan, Taiwan
| | - Ching-Yu Yen
- Department of Oral and Maxillofacial Surgery, Chi-Mei Medical Center, Tainan, Taiwan
- School of Dentistry, Taipei Medical University, Taipei, Taiwan
| | - Hsueh-Wei Chang
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Science, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
- Center for Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
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17
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Taylor B, Tang N, Hao Y, Lee M, Peng S, Bybee R, Hartman L, Garcia-Mansfield K, Sharma R, Pirrotte P, Ma J, Parisian AD, Furnari F, Dhruv HD, Berens ME. Glioblastoma vulnerability to neddylation inhibition is dependent on PTEN status, and dysregulation of the cell cycle and DNA replication. Neurooncol Adv 2024; 6:vdae104. [PMID: 39119276 PMCID: PMC11306933 DOI: 10.1093/noajnl/vdae104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/10/2024] Open
Abstract
Background Neddylation (NAE) inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report the cytotoxic vulnerability to NAE inhibitors in a subset of glioblastoma (GBM) preclinical models and identify genetic alterations and biological processes underlying differential response. Methods GBM DNA sequencing and transcriptomic data were queried for genes associated with response to NAE inhibition; candidates were validated by molecular techniques. Multi-omics and functional assays revealed processes implicated in NAE inhibition response. Results Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and DNA repair pathways as significant differentiators between sensitive and resistant models. Vulnerability to MLN4924, a NAE inhibitor, is associated with elevated S-phase populations, DNA re-replication, and DNA damage. In a panel of GBM models, loss of WT PTEN is associated with resistance to different NAE inhibitors. A NAE inhibition response gene set could segregate the GBM cell lines that are most resistant to MLN4924. Conclusions Loss of WT PTEN is associated with non-sensitivity to 3 different compounds that inhibit NAE in GBM. A NAE inhibition response gene set largely consisting of DNA replication genes could segregate GBM cell lines most resistant to NAEi and may be the basis for future development of NAE inhibition signatures of vulnerability and clinical trial enrollment within a precision medicine paradigm.
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Affiliation(s)
- Brett Taylor
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Nanyun Tang
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Yue Hao
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Matthew Lee
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Sen Peng
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Rita Bybee
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Lauren Hartman
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Krystine Garcia-Mansfield
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Ritin Sharma
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Patrick Pirrotte
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Jianhui Ma
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Alison D Parisian
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Frank Furnari
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Harshil D Dhruv
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
| | - Michael E Berens
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, Arizona, USA
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18
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Kayesh MEH, Kohara M, Tsukiyama-Kohara K. Effects of neddylation on viral infection: an overview. Arch Virol 2023; 169:6. [PMID: 38081982 DOI: 10.1007/s00705-023-05930-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 10/19/2023] [Indexed: 12/18/2023]
Abstract
Neddylation is a post-translational modification that plays an important role not only in cancer development but also in regulating viral infection and replication. Upregulation of neddylation occurs in viral infections, and inhibition of neddylation can suppress viral replication. Neddylation is thought to enhance viral protein stability and replication. Neddylation has been reported to enhance the stability of the regulatory hepatitis B virus (HBV) X protein, modulate viral replication, and enhance hepatocarcinogenesis. Inhibition of neddylation using the NEDD8-activating enzyme E1 inhibitor MLN4924 inhibits viral replication, including that of HBV. Understanding of the role of neddylation in viral infections is critical for developing new therapeutic targets and potential treatment strategies. In this review, we discuss recent progress in the understanding of the effects of neddylation during viral infection, particularly in HBV infection, and strategies for curing viral infection by targeting the neddylation pathway.
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Affiliation(s)
- Mohammad Enamul Hoque Kayesh
- Department of Microbiology and Public Health, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University, Barishal, 8210, Bangladesh.
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-8506, Japan
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, 890-0065, Japan.
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19
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Wong B, Bergeron A, Maznyi G, Ng K, Jirovec A, Birdi HK, Serrano D, Spinelli M, Thomson M, Taha Z, Alwithenani A, Chen A, Lorimer I, Vanderhyden B, Arulanandam R, Diallo JS. Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, sensitizes cancer cells to VSVΔ51 oncolytic virotherapy. Mol Ther 2023; 31:3176-3192. [PMID: 37766429 PMCID: PMC10638453 DOI: 10.1016/j.ymthe.2023.09.017] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Revised: 08/23/2023] [Accepted: 09/23/2023] [Indexed: 09/29/2023] Open
Abstract
The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy.
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Affiliation(s)
- Boaz Wong
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Anabel Bergeron
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Glib Maznyi
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Kristy Ng
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Anna Jirovec
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Harsimrat K Birdi
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Daniel Serrano
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Marcus Spinelli
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Max Thomson
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Zaid Taha
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Akram Alwithenani
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Andrew Chen
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Ian Lorimer
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Barbara Vanderhyden
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada
| | - Rozanne Arulanandam
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
| | - Jean-Simon Diallo
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
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20
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He ZX, Yang WG, Zengyangzong D, Gao G, Zhang Q, Liu HM, Zhao W, Ma LY. Targeting cullin neddylation for cancer and fibrotic diseases. Theranostics 2023; 13:5017-5056. [PMID: 37771770 PMCID: PMC10526667 DOI: 10.7150/thno.78876] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Accepted: 04/12/2023] [Indexed: 09/30/2023] Open
Abstract
Protein neddylation is a post-translational modification, and its best recognized substrates are cullin family proteins, which are the core component of Cullin-RING ligases (CRLs). Given that most neddylation pathway proteins are overactivated in different cancers and fibrotic diseases, targeting neddylation becomes an emerging approach for the treatment of these diseases. To date, numerous neddylation inhibitors have been developed, of which MLN4924 has entered phase I/II/III clinical trials for cancer treatment, such as acute myeloid leukemia, melanoma, lymphoma and solid tumors. Here, we systematically describe the structures and biological functions of the critical enzymes in neddylation, highlight the medicinal chemistry advances in the development of neddylation inhibitors and propose the perspectives concerning targeting neddylation for cancer and fibrotic diseases.
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Affiliation(s)
- Zhang-Xu He
- Pharmacy College, Henan University of Chinese Medicine, 450046, Zhengzhou, China
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Wei-guang Yang
- Children's hospital affiliated of Zhengzhou university; Henan children's hospital; Zhengzhou children's hospital, Henan Zhengzhou 450000, China
| | - Dan Zengyangzong
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Ge Gao
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Qian Zhang
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Hong-Min Liu
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Wen Zhao
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
| | - Li-Ying Ma
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
- China Meheco Topfond Pharmaceutical Co., Zhumadian 463000, China
- Key Laboratory of Cardio-cerebrovascular Drug, Henan Province, Zhumadian 463000, China
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21
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Aubry A, Pearson JD, Charish J, Yu T, Sivak JM, Xirodimas DP, Avet-Loiseau H, Corre J, Monnier PP, Bremner R. Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses. Cell Rep 2023; 42:112925. [PMID: 37552601 DOI: 10.1016/j.celrep.2023.112925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 05/29/2023] [Accepted: 07/18/2023] [Indexed: 08/10/2023] Open
Abstract
The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.
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Affiliation(s)
- Arthur Aubry
- Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada; Department of Lab Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Centre Hospitalo-universitaire (CHU) de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole (IUCT-O), Université de Toulouse, UPS, Unité de Génomique du Myélome, Toulouse, France
| | - Joel D Pearson
- Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada
| | - Jason Charish
- Department of Ophthalmology and Vision Science, University of Toronto, Toronto, ON, Canada; Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, ON, Canada
| | - Tao Yu
- Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada
| | - Jeremy M Sivak
- Department of Lab Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, ON, Canada; Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, ON, Canada
| | | | - Hervé Avet-Loiseau
- Centre Hospitalo-universitaire (CHU) de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole (IUCT-O), Université de Toulouse, UPS, Unité de Génomique du Myélome, Toulouse, France; Centre de Recherches en Cancérologie de Toulouse (CRCT), INSERM, Toulouse, France
| | - Jill Corre
- Centre Hospitalo-universitaire (CHU) de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole (IUCT-O), Université de Toulouse, UPS, Unité de Génomique du Myélome, Toulouse, France; Centre de Recherches en Cancérologie de Toulouse (CRCT), INSERM, Toulouse, France
| | - Philippe P Monnier
- Department of Ophthalmology and Vision Science, University of Toronto, Toronto, ON, Canada; Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, ON, Canada
| | - Rod Bremner
- Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada; Department of Lab Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, ON, Canada.
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22
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Fu DJ, Wang T. Targeting NEDD8-activating enzyme for cancer therapy: developments, clinical trials, challenges and future research directions. J Hematol Oncol 2023; 16:87. [PMID: 37525282 PMCID: PMC10388525 DOI: 10.1186/s13045-023-01485-7] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Accepted: 07/20/2023] [Indexed: 08/02/2023] Open
Abstract
NEDDylation, a post-translational modification through three-step enzymatic cascades, plays crucial roles in the regulation of diverse biological processes. NEDD8-activating enzyme (NAE) as the only activation enzyme in the NEDDylation modification has become an attractive target to develop anticancer drugs. To date, numerous inhibitors or agonists targeting NAE have been developed. Among them, covalent NAE inhibitors such as MLN4924 and TAS4464 currently entered into clinical trials for cancer therapy, particularly for hematological tumors. This review explains the relationships between NEDDylation and cancers, structural characteristics of NAE and multistep mechanisms of NEDD8 activation by NAE. In addition, the potential approaches to discover NAE inhibitors and detailed pharmacological mechanisms of NAE inhibitors in the clinical stage are explored in depth. Importantly, we reasonably investigate the challenges of NAE inhibitors for cancer therapy and possible development directions of NAE-targeting drugs in the future.
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Affiliation(s)
- Dong-Jun Fu
- Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Ting Wang
- Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China.
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23
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Sun Y, Baechler SA, Zhang X, Kumar S, Factor VM, Arakawa Y, Chau CH, Okamoto K, Parikh A, Walker B, Su YP, Chen J, Ting T, Huang SYN, Beck E, Itkin Z, McKnight C, Xie C, Roper N, Nijhawan D, Figg WD, Meltzer PS, Yang JC, Thomas CJ, Pommier Y. Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex. Nat Commun 2023; 14:3762. [PMID: 37353483 PMCID: PMC10290057 DOI: 10.1038/s41467-023-39374-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2022] [Accepted: 06/09/2023] [Indexed: 06/25/2023] Open
Abstract
Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.
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Affiliation(s)
- Yilun Sun
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
| | - Simone A Baechler
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Xiaohu Zhang
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA
| | - Suresh Kumar
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Valentina M Factor
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Yasuhiro Arakawa
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Cindy H Chau
- Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Kanako Okamoto
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Anup Parikh
- Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Bob Walker
- Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Yijun P Su
- Advanced Imaging and Microscopy Resource, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Jiji Chen
- Advanced Imaging and Microscopy Resource, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Tabitha Ting
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Shar-Yin N Huang
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Erin Beck
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA
| | - Zina Itkin
- Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Crystal McKnight
- Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Changqing Xie
- Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Nitin Roper
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Deepak Nijhawan
- Advanced Imaging and Microscopy Resource, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA
| | - William Douglas Figg
- Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Paul S Meltzer
- Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - James C Yang
- Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Craig J Thomas
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA
| | - Yves Pommier
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
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24
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Mao W, Zhang L, Rong Y, Kuang T, Wang D, Xu X, Lou W, Li J. NEDD8-Activating Enzyme Inhibitor MLN4924 Inhibits Both the Tumor Stroma and Angiogenesis in Pancreatic Cancer via Gli1 and REDD1. Dig Dis Sci 2023; 68:1351-1363. [PMID: 36098876 DOI: 10.1007/s10620-022-07671-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2022] [Accepted: 08/12/2022] [Indexed: 12/09/2022]
Abstract
PURPOSE Pancreatic cancer is characterized by a dense desmoplasia stroma, which hinders efficient drug delivery and plays a critical role in tumor progression and metastasis. MLN4924 is a first-in-class NEDD8-activating enzyme inhibitor that exhibits anti-tumor activities toward pancreatic cancer, and given the comprehensive effects that MLN4924 could have, we ask what impact MLN4924 would have on the stroma of pancreatic cancer and its underlying mechanisms. METHODS Primary pancreatic stellate cells (PSCs) and human HMEC-1 cells were treated with MLN4924 in vitro. The proliferation and extracellular matrix protein levels of PSCs were tested, and their relationship with transcription factor Gli1 in PSCs was investigated. The angiogenic phenotypes of HMEC-1 cells were evaluated using capillary-like tube formation assay, and their relationship with REDD1 in HMEC-1 cells was investigated. RESULTS In this study, we found that MLN4924 inhibited the proliferation of pancreatic stellate cells and their secretion of collagen and CXCL-1, and the collagen secretion inhibiting effect of MLN4924 was related with transcription factor Gli1. MLN4924 inhibited multiple angiogenic phenotypes of HMEC-1 cells, and mTOR agonist partially relieved the inhibition of MLN4924 on HEMCs. MLN4924 increased the expression of REDD1 and REDD1 knockdown promoted the angiogenic phenotypes of HMEC-1 cells. CONCLUSIONS Our study suggests that MLN4924 inhibits both the tumor stroma and angiogenesis in pancreatic cancer, and the inhibition effect is related with Gli1 in pancreatic stellate cells and REDD1 in vascular endothelial cells, respectively.
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Affiliation(s)
- Weilin Mao
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Lei Zhang
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Yefei Rong
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Tiantao Kuang
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Dansong Wang
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Xuefeng Xu
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China
| | - Wenhui Lou
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China.
| | - Jianang Li
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China.
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25
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Ye C, Zhang S, Zhang D, Shen Y, Wang Z, Wang H, Ren J, Jiang XD, Du J, Shang R, Wang G. Engineering J-aggregates for NIR-induced meso-CF3-BODIPY nanoparticles by activated apoptosis mechanism in photothermal therapy. CHINESE CHEM LETT 2023. [DOI: 10.1016/j.cclet.2023.108223] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
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Ratnayeke N, Baris Y, Chung M, Yeeles JTP, Meyer T. CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis. Mol Cell 2023; 83:26-42.e13. [PMID: 36608667 DOI: 10.1016/j.molcel.2022.12.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 09/16/2022] [Accepted: 12/08/2022] [Indexed: 01/07/2023]
Abstract
Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.
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Affiliation(s)
- Nalin Ratnayeke
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA
| | - Yasemin Baris
- Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK
| | - Mingyu Chung
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Joseph T P Yeeles
- Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK
| | - Tobias Meyer
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA.
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Petropoulos M, Champeris Tsaniras S, Nikou S, Maxouri S, Dionellis VS, Kalogeropoulou A, Karamichali A, Ioannidis K, Danalatos IR, Obst M, Naumann R, Delinasios GJ, Gorgoulis VG, Roukos V, Anastassiadis K, Halazonetis TD, Bravou V, Lygerou Z, Taraviras S. Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage. J Pathol 2023; 259:10-20. [PMID: 36210634 DOI: 10.1002/path.6017] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2022] [Revised: 09/20/2022] [Accepted: 10/05/2022] [Indexed: 11/11/2022]
Abstract
Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.
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Affiliation(s)
- Michalis Petropoulos
- Department of Physiology, Medical School, University of Patras, Patras, Greece.,Department of General Biology, Medical School, University of Patras, Patras, Greece
| | | | - Sofia Nikou
- Department of Anatomy-Histology-Embryology, School of Medicine, University of Patras, Patras, Greece
| | - Styliani Maxouri
- Department of General Biology, Medical School, University of Patras, Patras, Greece
| | | | | | | | | | | | - Mandy Obst
- Stem Cell Engineering, Biotechnology Center, Center for Molecular and Cellular Bioengineering, University of Technology Dresden, Dresden, Germany
| | - Ronald Naumann
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | | | - Vassilis G Gorgoulis
- Department of Histology and Embryology, School of Medicine, National Kapodistrian University of Athens, Athens, Greece.,Biomedical Research Foundation of the Academy of Athens, Athens, Greece.,Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
| | | | - Konstantinos Anastassiadis
- Stem Cell Engineering, Biotechnology Center, Center for Molecular and Cellular Bioengineering, University of Technology Dresden, Dresden, Germany
| | | | - Vasiliki Bravou
- Department of Anatomy-Histology-Embryology, School of Medicine, University of Patras, Patras, Greece
| | - Zoi Lygerou
- Department of General Biology, Medical School, University of Patras, Patras, Greece
| | - Stavros Taraviras
- Department of Physiology, Medical School, University of Patras, Patras, Greece
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Mittler F, Obeïd P, Haguet V, Allier C, Gerbaud S, Rulina AV, Gidrol X, Balakirev MY. Mechanical stress shapes the cancer cell response to neddylation inhibition. J Exp Clin Cancer Res 2022; 41:115. [PMID: 35354476 PMCID: PMC8966269 DOI: 10.1186/s13046-022-02328-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Accepted: 03/13/2022] [Indexed: 12/28/2022] Open
Abstract
Background The inhibition of neddylation by the preclinical drug MLN4924 represents a new strategy to combat cancer. However, despite being effective against hematologic malignancies, its success in solid tumors, where cell–cell and cell-ECM interactions play essential roles, remains elusive. Methods Here, we studied the effects of MLN4924 on cell growth, migration and invasion in cultured prostate cancer cells and in disease-relevant prostate tumoroids. Using focused protein profiling, drug and RNAi screening, we analyzed cellular pathways activated by neddylation inhibition. Results We show that mechanical stress induced by MLN4924 in prostate cancer cells significantly affects the therapeutic outcome. The latter depends on the cell type and involves distinct Rho isoforms. In LNCaP and VCaP cells, the stimulation of RhoA and RhoB by MLN4924 markedly upregulates the level of tight junction proteins at cell–cell contacts, which augments the mechanical strain induced by Rho signaling. This “tight junction stress response” (TJSR) causes the collapse of cell monolayers and a characteristic rupture of cancer spheroids. Notably, TJSR is a major cause of drug-induced apoptosis in these cells. On the other hand, in PC3 cells that underwent partial epithelial-to-mesenchymal transition (EMT), the stimulation of RhoC induces an adverse effect by promoting amoeboid cell scattering and invasion. We identified complementary targets and drugs that allow for the induction of TJSR without stimulating RhoC. Conclusions Our finding that MLN4924 acts as a mechanotherapeutic opens new ways to improve the efficacy of neddylation inhibition as an anticancer approach. Graphical Abstract ![]()
Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02328-y.
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Ferdosi SR, Taylor B, Lee M, Tang N, Peng S, Bybee R, Reid G, Hartman L, Garcia-Mansfield K, Sharma R, Pirrotte P, Ma J, Parisian AD, Furnari F, Dhruv HD, Berens ME. PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors. Neuro Oncol 2022; 24:1857-1868. [PMID: 35305088 PMCID: PMC9629460 DOI: 10.1093/neuonc/noac067] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. METHODS GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. RESULTS Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. CONCLUSIONS We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.
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Affiliation(s)
- Shayesteh R Ferdosi
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Brett Taylor
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Matthew Lee
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Nanyun Tang
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Sen Peng
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Rita Bybee
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - George Reid
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Lauren Hartman
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Krystine Garcia-Mansfield
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Ritin Sharma
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Patrick Pirrotte
- Collaborative Center for Translational Mass Spectrometry, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Jianhui Ma
- Ludwig Cancer Research, San Diego Branch, La Jolla, CA 92093, USA
| | | | - Frank Furnari
- Ludwig Cancer Research, San Diego Branch, La Jolla, CA 92093, USA
- Department of Pathology, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA
| | - Harshil D Dhruv
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Michael E Berens
- Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ 85004, USA
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Adaptive exchange sustains cullin-RING ubiquitin ligase networks and proper licensing of DNA replication. Proc Natl Acad Sci U S A 2022; 119:e2205608119. [PMID: 36037385 PMCID: PMC9456757 DOI: 10.1073/pnas.2205608119] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.
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31
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Zhou LN, Xiong C, Cheng YJ, Song SS, Bao XB, Huan XJ, Wang TY, Zhang A, Miao ZH, He JX. SOMCL-19-133, a novel, selective, and orally available inhibitor of NEDD8-activating enzyme (NAE) for cancer therapy. Neoplasia 2022; 32:100823. [PMID: 35907292 PMCID: PMC9352467 DOI: 10.1016/j.neo.2022.100823] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 07/08/2022] [Accepted: 07/11/2022] [Indexed: 11/29/2022]
Abstract
Inhibition of the NEDD8-activating enzyme (NAE), the key E1 enzyme in the neddylation cascade, has been considered an attractive anticancer strategy with the discovery of the first-in-class NAE inhibitor, MLN4924. In this study, we identified SOMCL-19-133 as a highly potent, selective, and orally available NAE inhibitor, which is an analog to AMP. It effectively inhibited NAE with an IC50 value of 0.36 nM and exhibited more than 2855-fold selectivity over the closely related Ubiquitin-activating enzyme (UAE). It is worth noting that treatment with SOMCL-19-133 prominently inhibited Cullin neddylation and delayed the turnover of a panel of Cullin-RING ligases (CRLs) substrates (e.g., Cdt1, p21, p27, and Wee1) at lower effective concentrations than that of MLN4924, subsequently caused DNA damage and Chk1/Chk2 activation, and thus triggered cell cycle arrest and apoptosis. Moreover, SOMCL-19-133 exhibited potent antiproliferative activity against a broad range of human tumor cell lines (mean IC50 201.11 nM), which was about 5.31-fold more potent than that of MLN4924. In vivo, oral delivery treatments with SOMCL-19-133, as well as the subcutaneous injection, led to significant tumor regression in mouse xenograft models. All of the treatments were well tolerated on a continuous daily dosing schedule. Compared with MLN4924, SOMCL-19-133 had a 5-fold higher peak plasma concentration, lower plasma clearance, and a 4-fold larger area under the curve (AUClast). In conclusion, SOMCL-19-133 is a promising preclinical candidate for treating cancers owing to its profound in vitro and in vivo efficacy and favorable pharmacokinetic properties.
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Affiliation(s)
- Li-Na Zhou
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Chaodong Xiong
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Yong-Jun Cheng
- School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
| | - Shan-Shan Song
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Xu-Bin Bao
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Xia-Juan Huan
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Tong-Yan Wang
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China
| | - Ao Zhang
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; Pharm-X Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China.
| | - Ze-Hong Miao
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China.
| | - Jin-Xue He
- State Key Laboratory of Drug Research, Cancer Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
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Hussain M, Lu Y, Tariq M, Jiang H, Shu Y, Luo S, Zhu Q, Zhang J, Liu J. A small-molecule Skp1 inhibitor elicits cell death by p53-dependent mechanism. iScience 2022; 25:104591. [PMID: 35789855 PMCID: PMC9249674 DOI: 10.1016/j.isci.2022.104591] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Revised: 04/21/2022] [Accepted: 06/08/2022] [Indexed: 11/28/2022] Open
Abstract
Skp1 overexpression promotes tumor growth, whereas reduced Skp1 activity is also linked with genomic instability and neoplastic transformation. This highlights the need to gain better understanding of Skp1 biology in cancer settings. To this context, potent and cellularly active small-molecule Skp1 inhibitors may be of great value. Using a hypothesis-driven, structure-guided approach, we herein identify Z0933M as a potent Skp1 inhibitor with KD ∼0.054 μM. Z0933M occupies a hydrophobic hotspot (P1) – encompassing an aromatic cage of two phenylalanines (F101 and F139) – alongside C-terminal extension of Skp1 and, thus, hampers its ability to interact with F-box proteins, a prerequisite step to constitute intact and active SCF E3 ligase(s) complexes. In cellulo, Z0933M disrupted SCF E3 ligase(s) functioning, recapitulated previously reported effects of Skp1-reduced activity, and elicited cell death by a p53-dependent mechanism. We propose Z0933M as valuable tool for future efforts toward probing Skp1 cancer biology, with implications for cancer therapy.
Z0933M manifests strong binding with Skp1 and inhibits Skp1-F-box PPIs Z0933M interacts with a P1 hotspot alongside C-terminal extension of Skp1 Z0933M alters SCF E3 ligase functioning, leading to substrate accumulation/modulation Z0933M causes cell-cycle arrest, and elicits cell death by p53-dependent mechanism
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Affiliation(s)
- Muzammal Hussain
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Guangdong Provincial Key Laboratory of Biocomputing, Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yongzhi Lu
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China
| | - Muqddas Tariq
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China
| | - Hao Jiang
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
| | - Yahai Shu
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
| | - Shuang Luo
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China
| | - Qiang Zhu
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China
| | - Jiancun Zhang
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Guangdong Provincial Key Laboratory of Biocomputing, Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Jinsong Liu
- State Key Laboratory of Respiratory Disease, Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China
- Guangdong Provincial Key Laboratory of Biocomputing, Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- China-New Zealand Joint Laboratory on Biomedicine and Health, Guangzhou 510530, China
- Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
- Corresponding author
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Tarcan Z, Poovathumkadavil D, Skagia A, Gambus A. The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase. J Biol Chem 2022; 298:102234. [PMID: 35798141 PMCID: PMC9358472 DOI: 10.1016/j.jbc.2022.102234] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 06/22/2022] [Accepted: 06/23/2022] [Indexed: 11/20/2022] Open
Abstract
Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2Lrr1 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2Lrr1 replisome unloading pathway in unperturbed S-phase.
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Molecular relation between biological stress and carcinogenesis. Mol Biol Rep 2022; 49:9929-9945. [PMID: 35610338 DOI: 10.1007/s11033-022-07543-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Accepted: 04/29/2022] [Indexed: 10/18/2022]
Abstract
This paper aims to overview different types of stress, including DNA replication stress, oxidative stress, and psychological stress. Understanding the processes that constitute a cellular response to varied types of stress lets us find differences in how normal cells and cancer cells react to the appearance of a particular kind of stressor. The revealed dissimilarities are the key for targeting new molecules and signaling pathways in anticancer treatment. For this reason, molecular mechanisms that underlay DNA replication stress, oxidative stress, and psychological stress have been studied and briefly presented to indicate biochemical points that make stressors contribute to cancer development. What is more, the viewpoint in which cancer constitutes the outcome and the cause of stress has been taken into consideration. In a described way, this paper draws attention to the problem of cancer-related post-traumatic stress disorder and proposes a novel, multidimensional oncological approach, connecting anticancer treatment with psychiatric support.
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Karantzelis N, Petropoulos M, De Marco V, Egan DA, Fish A, Christodoulou E, Will DW, Lewis JD, Perrakis A, Lygerou Z, Taraviras S. Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells. Front Pharmacol 2022; 13:860682. [PMID: 35548337 PMCID: PMC9083542 DOI: 10.3389/fphar.2022.860682] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2022] [Accepted: 03/30/2022] [Indexed: 01/18/2023] Open
Abstract
DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.
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Affiliation(s)
| | - Michalis Petropoulos
- Department of General Biology, Medical School, University of Patras, Patras, Greece
| | - Valeria De Marco
- Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | - David A Egan
- Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | - Alexander Fish
- Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | | | - David W Will
- Chemical Biology Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Joe D Lewis
- Chemical Biology Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Anastassis Perrakis
- Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
| | - Zoi Lygerou
- Department of General Biology, Medical School, University of Patras, Patras, Greece
| | - Stavros Taraviras
- Department of Physiology, Medical School, University of Patras, Patras, Greece
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The Nuclear Proteins TP73 and CUL4A Confer Resistance to Cytarabine by Induction of Translesion DNA Synthesis via Mono-ubiquitination of PCNA. Hemasphere 2022; 6:e0708. [PMID: 35519003 PMCID: PMC9067361 DOI: 10.1097/hs9.0000000000000708] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 03/10/2022] [Indexed: 12/03/2022] Open
Abstract
Resistance to cytarabine is a key problem in the treatment of acute myeloid leukemia (AML). To understand the molecular biology of resistance to cytarabine, a viability-based chemosensitizer screen was utilized. We screened synthetic lethal targets using 437 different small interfering RNAs (siRNAs) directed against factors involved in DNA repair mechanisms and cytarabine as the chemical compound. Three hits were identified: CUL4A, TP73, and RFC2. We show here that the ubiquitin ligase CULLIN 4A (CUL4A) and the tumor-suppressive transcription factor p73 contribute to drug resistance by modulating DNA damage response. P73 confers resistance to cytarabine therapy by transactivation of REV3L, encoding the catalytic subunit of translesion DNA polymerase ζ, and CUL4A probably by influencing proliferating cell nuclear antigen (PCNA) and the polymerase switch towards error-prone translesion DNA polymerases. Abrogation of the polymerase ζ by siRNA causes identical effects as siRNAs against CUL4A or TP73 and resensitizes cells towards cytarabine therapy in vitro. As CUL4A needs to be activated by neddylation to facilitate the degradation of several proteins including PCNA, we propose a novel explanation for the synergism between cytarabine and the neddylation inhibitor pevonedistat by inhibition of translesion synthesis. In keeping with this, in AML patients treated with cytarabine, we found high expression of CUL4A and TP73 to be associated with poor prognosis.
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Jones TM, Espitia CM, Ooi A, Bauman JE, Carew JS, Nawrocki ST. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cell Death Dis 2022; 13:350. [PMID: 35428778 PMCID: PMC9012827 DOI: 10.1038/s41419-022-04798-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Revised: 03/21/2022] [Accepted: 03/30/2022] [Indexed: 12/24/2022]
Abstract
Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.
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Affiliation(s)
- Trace M Jones
- University of Arizona Cancer Center, Tucson, AZ, USA
| | | | - Aikseng Ooi
- Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA
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Zhang J, Li C, Zhang L, Heng Y, Wang S, Pan Y, Cai L, Zhang Y, Xu T, Chen X, Hoffman RM, Jia L. Andrographolide, a diterpene lactone from the Traditional Chinese Medicine Andrographis paniculate, induces senescence in human lung adenocarcinoma via p53/p21 and Skp2/p27. PHYTOMEDICINE : INTERNATIONAL JOURNAL OF PHYTOTHERAPY AND PHYTOPHARMACOLOGY 2022; 98:153933. [PMID: 35121394 DOI: 10.1016/j.phymed.2022.153933] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 01/04/2022] [Accepted: 01/07/2022] [Indexed: 06/14/2023]
Abstract
BACKGROUND Senescence leads to permanent cell-cycle arrest and is a potential target for cancer therapy. Andrographolide (AD) is a diterpene lactone isolated from Traditional Chinese Medicine (TCM) Andrographis paniculate, which has been used as an anti-inflammatory drug in clinical practice with the potential to target senescence in recalcitrant lung cancer. PURPOSE To determine whether AD can induce senescence in human lung adenocarcinoma in vitro and in vivo and to elucidate the underlying mechanisms. METHODS SA-β-Gal staining was used to detect the expression of senescence-associated β-galactosidase (SA-β-Gal) in human lung adenocarcinoma cells A549 and NCI-H1795. DNA damage was examined by the detection of γH2AX foci. Cell cycle was analyzed by flow cytometry. Cancer cell proliferation was determined by ATPlite assay and clonogenic survival assay in vitro. Tumor growth was determined in a mouse model of A549. The expression level of proteins and mRNA was estimated by Western blotting and Quantitative RT-PCR, respectively. Small interfering RNA (siRNA) was used to knock down p21, p27 and p53 to explore the potential mechanism of AD-induced senescence in human lung adenocarcinoma cells. RESULTS AD-induced A549 and NCI-H1795 cell senescence determined by increased cell size, flattened morphology, DNA damage, cell cycle arrest as well as the increased expression of β-galactosidase. AD inhibited cell proliferation in lung cells in vitro and lung cells xenograft growth in nude mice. p21 and p27, the major cell cycle regulators and mediators of senescence, were upregulated at the protein level in AD-treated A549 lung adenocarcinoma in vitro and in vivo. Further studies demonstrated that AD induced cell senescence via p53/p21 and Skp2/p27. CONCLUSION In the present study, we found that the primary anti-inflammatory drug AD could have a potential antitumor effect in lung cancer. We demonstrated that AD induced lung adenocarcinoma senescence in vitro and in vivo via p53/p21 and Skp2/p27 for the first time. AD is therefore a promising senescence-inducing therapeutic for recalcitrant human lung adenocarcinoma.
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Affiliation(s)
- Junqian Zhang
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Chunjie Li
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Li Zhang
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Yongqing Heng
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Shiwen Wang
- Department of Laboratory Medicine, Huadong Hospital, Affiliated to Fudan University, Shanghai, China
| | - Yongfu Pan
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Lili Cai
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Yunjing Zhang
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Tong Xu
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Xihui Chen
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
| | - Robert M Hoffman
- Department of Surgery, University of California, San Diego, CA, USA; Anticancer, Inc., San Diego, CA, USA
| | - Lijun Jia
- Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China.
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He ZX, An Q, Wei B, Zhou WJ, Wei BF, Gong YP, Zhang X, Gao G, Dong GJ, Huo JL, Zhang XH, Yang FF, Liu HM, Ma LY, Zhao W. Discovery of Potent and Selective 2-(Benzylthio)pyrimidine-based DCN1-UBC12 Inhibitors for Anticardiac Fibrotic Effects. J Med Chem 2022; 65:163-190. [PMID: 34939411 DOI: 10.1021/acs.jmedchem.1c01207] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
DCN1, a co-E3 ligase, interacts with UBC12 and activates cullin-RING ligases (CRLs) by catalyzing cullin neddylation. Although DCN1 has been recognized as an important therapeutic target for human diseases, its role in the cardiovascular area remains unknown. Here, we first found that DCN1 was upregulated in isolated cardiac fibroblasts (CFs) treated by angiotensin (Ang) II and in mouse hearts after pressure overload. Then, structure-based optimizations for DCN1-UBC12 inhibitors were performed based on our previous work, yielding compound DN-2. DN-2 specifically targeted DCN1 at molecular and cellular levels as shown by molecular modeling studies, HTRF, cellular thermal shift and co-immunoprecipitation assays. Importantly, DN-2 effectively reversed Ang II-induced cardiac fibroblast activation, which was associated with the inhibition of cullin 3 neddylation. Our findings indicate a potentially unrecognized role of DCN1 inhibition for anticardiac fibrotic effects. DN-2 may be used as a lead compound for further development.
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Affiliation(s)
- Zhang-Xu He
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Qi An
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Bo Wei
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Wen-Juan Zhou
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Bing-Fei Wei
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Yun-Peng Gong
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Xin Zhang
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Ge Gao
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Guan-Jun Dong
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Jin-Ling Huo
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Xin-Hui Zhang
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Fei-Fei Yang
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Hong-Min Liu
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
| | - Li-Ying Ma
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
- China Meheco Topfond Pharmaceutical Co., Zhumadian 463000, China
| | - Wen Zhao
- State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
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40
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Li JA, Rong Y, Mao W, Zhang L, Kuang T, Lou W. Gene expression profiling reveals the genomic changes caused by MLN4924 and the sensitizing effects of NAPEPLD knockdown in pancreatic cancer. Cell Cycle 2022; 21:152-171. [PMID: 34874801 PMCID: PMC8837228 DOI: 10.1080/15384101.2021.2014254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 08/25/2021] [Accepted: 11/22/2021] [Indexed: 10/19/2022] Open
Abstract
MLN4924 inhibits the proteolytic degradation of Cullin-Ring E3 ligase (CRL) substrates and exhibits antitumor activity toward various malignancies, including pancreatic cancer. MLN4924 suppresses tumor growth by altering various key regulator proteins; however, its impact on gene expression in tumors remains unknown. In this study, the genomic changes caused by MLN4924 in pancreatic cancer were examined by gene chip analysis and ingenuity pathway analysis. Eleven pathways were significantly altered (5 activated and 6 inhibited), 45 functions were significantly changed (21 activated and 24 inhibited), and the most activated upstream factor was predicted to be TNF. Of 691 differentially expressed genes, NAPEPLD knockdown showed synergism with MLN4924, as determined by real-time quantitative PCR and high content screening. NAPEPLD knockdown enhanced the effect of MLN4924 on inhibiting proliferation and inducing apoptosis in vitro. In a pancreatic cancer nude mouse model, MLN4924 inhibited tumor growth more significantly in the NAPEPLD knockdown group than in the control group. NAPEPLD expression was higher in pancreatic cancer tissues than in the normal pancreas but was not associated with prognosis. These findings indicate that MLN4924 causes extensive genomic changes in pancreatic cancer cells, and targeting NAPEPLD may increase the efficacy of MLN4924.
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Affiliation(s)
- Jian-Ang Li
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Yefei Rong
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Weilin Mao
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Lei Zhang
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Tiantao Kuang
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Wenhui Lou
- Department of Pancreatic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
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41
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Li W, Li F, Zhang X, Lin HK, Xu C. Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduct Target Ther 2021; 6:422. [PMID: 34924561 PMCID: PMC8685280 DOI: 10.1038/s41392-021-00825-8] [Citation(s) in RCA: 114] [Impact Index Per Article: 28.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 11/02/2021] [Accepted: 11/05/2021] [Indexed: 12/11/2022] Open
Abstract
More and more in-depth studies have revealed that the occurrence and development of tumors depend on gene mutation and tumor heterogeneity. The most important manifestation of tumor heterogeneity is the dynamic change of tumor microenvironment (TME) heterogeneity. This depends not only on the tumor cells themselves in the microenvironment where the infiltrating immune cells and matrix together forming an antitumor and/or pro-tumor network. TME has resulted in novel therapeutic interventions as a place beyond tumor beds. The malignant cancer cells, tumor infiltrate immune cells, angiogenic vascular cells, lymphatic endothelial cells, cancer-associated fibroblastic cells, and the released factors including intracellular metabolites, hormonal signals and inflammatory mediators all contribute actively to cancer progression. Protein post-translational modification (PTM) is often regarded as a degradative mechanism in protein destruction or turnover to maintain physiological homeostasis. Advances in quantitative transcriptomics, proteomics, and nuclease-based gene editing are now paving the global ways for exploring PTMs. In this review, we focus on recent developments in the PTM area and speculate on their importance as a critical functional readout for the regulation of TME. A wealth of information has been emerging to prove useful in the search for conventional therapies and the development of global therapeutic strategies.
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Affiliation(s)
- Wen Li
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China
| | - Feifei Li
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China
- Guangxi Collaborative Innovation Center for Biomedicine (Guangxi-ASEAN Collaborative Innovation Center for Major Disease Prevention and Treatment), Guangxi Medical University, 530021, Nanning, Guangxi, China
| | - Xia Zhang
- Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), 400038, Chongqing, China
| | - Hui-Kuan Lin
- Department of Cancer Biology, Wake Forest Baptist Medical Center, Wake Forest University, Winston Salem, NC, 27101, USA
| | - Chuan Xu
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China.
- Department of Cancer Biology, Wake Forest Baptist Medical Center, Wake Forest University, Winston Salem, NC, 27101, USA.
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42
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Cheng F, Cheng Y, Zhao X, An L, Yang L, Li Z, Zhang L, He R. NEDD4 E3 ubiquitin protein ligase serves an important role in cutaneous melanoma occurrence and development. Exp Ther Med 2021; 22:1382. [PMID: 34650630 PMCID: PMC8506948 DOI: 10.3892/etm.2021.10818] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 06/25/2021] [Indexed: 11/18/2022] Open
Abstract
The present study aimed to discuss the effects and relative mechanisms of NEDD4 E3 ubiquitin protein ligase (NEDD4) in cutaneous melanoma (CMM) occurrence and development. Clinical cancer and adjacent normal tissues samples were collected to analyze pathological changes and protein expression of NEDD4. Moreover, small interfering (si)RNA was used to knockdown NEDD4 expression in SK-MEL-2 and Malme-3M cells. Cellular proliferation, apoptosis, invasiveness and migration were examined using colony formation, flow cytometric, Transwell and wound-healing assays, respectively. In addition, the relative mRNA and protein expression levels of NEDD4, notch receptor 1 (Notch1) and PTEN were evaluated via reverse transcription-quantitative (RT-q) PCR and western blotting. It was found that NEDD4 mRNA and protein expression were significantly upregulated (both P<0.01). Following NEDD4-knockdown, colony number was significantly decreased, while the apoptotic rate was significantly increased, the invasive cell number was significantly inhibited and the wound-healing capacity was significantly decreased. Following si-NEDD4 transfection, RT-qPCR and western blotting revealed that NEDD4 and Notch1 mRNA and protein expression levels were significantly downregulated, while those of PTEN were significantly upregulated in the SK-MEL-2 and Malme-3M cell lines. Collectively, the current results suggest that NEDD4-knockdown effectively suppressed CMM biological activity by regulating the Notch1/PTEN pathway in vitro.
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Affiliation(s)
- Fang Cheng
- Department of Dermatology, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
| | - Yi Cheng
- Department of Dermatology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, P.R. China
| | - Xiaoling Zhao
- Department of Dermatology, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
| | - Lihui An
- Department of Dermatology, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
| | - Linfang Yang
- Department of Dermatology, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
| | - Zihan Li
- Department of Dermatology, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
| | - Lei Zhang
- Department of Dermatology, The Second People's Hospital of Guiyang, Guiyang, Guizhou 550023, P.R. China
| | - Runzhi He
- Department of Neurosurgery, Affiliated Xingtai People's Hospital of Hebei Medical University, Xingtai, Hebei 054001, P.R. China
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43
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Wittig KA, Sansam CG, Noble TD, Goins D, Sansam CL. The CRL4DTL E3 ligase induces degradation of the DNA replication initiation factor TICRR/TRESLIN specifically during S phase. Nucleic Acids Res 2021; 49:10507-10523. [PMID: 34534348 PMCID: PMC8501952 DOI: 10.1093/nar/gkab805] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Revised: 08/26/2021] [Accepted: 09/13/2021] [Indexed: 01/02/2023] Open
Abstract
A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.
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Affiliation(s)
- Kimberlie A Wittig
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.,Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Courtney G Sansam
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Tyler D Noble
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.,Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Duane Goins
- Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Christopher L Sansam
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.,Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
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44
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Wu K, Hopkins BD, Sanchez R, DeVita RJ, Pan ZQ. Targeting Cullin-RING E3 Ubiquitin Ligase 4 by Small Molecule Modulators. JOURNAL OF CELLULAR SIGNALING 2021; 2:195-205. [PMID: 34604860 PMCID: PMC8486283 DOI: 10.33696/signaling.2.051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4's core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents.
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Affiliation(s)
- Kenneth Wu
- Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA
| | - Benjamin D Hopkins
- Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA.,Genetics and Genomics, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA
| | - Roberto Sanchez
- Department of Pharmacological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA.,Drug Discovery Institute, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA
| | - Robert J DeVita
- Department of Pharmacological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA.,Drug Discovery Institute, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA
| | - Zhen-Qiang Pan
- Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029-6574, USA
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45
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Kittai AS, Danilova OV, Lam V, Liu T, Bruss N, Best S, Fan G, Danilov AV. NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells. Oncotarget 2021; 12:2068-2074. [PMID: 34611480 PMCID: PMC8487718 DOI: 10.18632/oncotarget.28063] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2021] [Accepted: 08/18/2021] [Indexed: 11/25/2022] Open
Abstract
Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.
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Affiliation(s)
- Adam S Kittai
- The Ohio State University, Columbus, OH 43210, USA.,These authors contributed equally to this work
| | - Olga V Danilova
- City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA.,These authors contributed equally to this work
| | - Vi Lam
- City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA
| | - Tingting Liu
- City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA
| | - Nur Bruss
- Oregon Health and Science University, Portland, OR 97239, USA
| | - Scott Best
- Oregon Health and Science University, Portland, OR 97239, USA
| | - Guang Fan
- Oregon Health and Science University, Portland, OR 97239, USA
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El-Far YM, El-Mesery M. Pevonedistat attenuates cisplatin-induced nephrotoxicity in mice by downregulating the release of inflammatory mediators. J Biochem Mol Toxicol 2021; 35:e22908. [PMID: 34476871 DOI: 10.1002/jbt.22908] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 07/09/2021] [Accepted: 08/20/2021] [Indexed: 01/11/2023]
Abstract
Pevonedistat (MLN4924) is a specific NEDD8-activating enzyme inhibitor that inactivates cullin-RING ligases involved in ubiquitylation and turnover of different signaling molecules. In the current study, we evaluated the effect of pevonedistat on cisplatin (CIS)-induced nephrotoxicity in mice. Serum creatinine and urea levels were analyzed in different groups. Histopathological examination of renal tissue was done using hematoxylin and eosin staining. In addition, renal IL-6 and TNF-α expressions were analyzed using the enzyme-linked immunosorbent assay technique, and IL-1β and NF-κB expressions were analyzed by immunohistochemical staining of renal tissue. Caspase-3, A20, β-catenin, and Nrf2 gene expressions in renal tissue were analyzed using the reverse-transcription polymerase chain reaction technique. Western blot analysis was adopted to assess cleaved caspase-3 and β-catenin expressions in renal tissue. Pevonedistat coadministration with CIS improved kidney functions and attenuated CIS-induced nephrotoxicity as indicated by the significant decrease in serum creatinine and urea levels. In addition, pevonedistat coadministration with CIS showed a significant decrease in caspase-3 and a significant increase in A20, β-catenin, and Nrf2 gene expressions. Also, pevonedistat decreased caspase-3 cleavage to p19 in mice treated with CIS. Moreover, pevonedistat decreased CIS-induced upregulation of IL-6, TNF-α, IL-1β, and NF-κB protein expressions in renal tissue. Thus, pevonedistat alleviated CIS-induced nephrotoxicity that might be attributed to suppression of the inflammation induced in renal tissue.
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Affiliation(s)
- Yousra M El-Far
- Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
| | - Mohamed El-Mesery
- Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
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Lee‐Law PY, Olaizola P, Caballero‐Camino FJ, Izquierdo‐Sanchez L, Rodrigues PM, Perugorria MJ, Azkargorta M, Elortza F, Martinez‐Chantar ML, Aspichueta P, Marzioni M, Bujanda L, Drenth JPH, Banales JM. Inhibition of NAE-dependent protein hyper-NEDDylation in cystic cholangiocytes halts cystogenesis in experimental models of polycystic liver disease. United European Gastroenterol J 2021; 9:848-859. [PMID: 34310849 PMCID: PMC8435261 DOI: 10.1002/ueg2.12126] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Accepted: 04/01/2021] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND Polycystic liver diseases (PLDs) are genetic inherited disorders characterized by the progressive growth of numerous intrahepatic biliary cysts, which are the main cause of morbidity. Previous studies revealed that cystic cholangiocytes are characterized by endoplasmic reticulum stress and aberrant posttranslational modification (PTM) of proteins, in particular hyper-SUMOylation, that promote PLD pathobiology. Protein NEDDylation is a newly characterized PTM that modulates a plethora of biological processes and its dysregulation is associated with the development and progression of several human diseases. However, the role of NEDDylation in PLD remains elusive. OBJECTIVE To explore the role of protein NEDDylation in PLD and its potential therapeutic regulatory value. METHODS Levels and functional effects of NEDDylation, including response to Pevonedistat (first-in-class selective inhibitor of the NEDDylation E1 enzyme NAE), were assessed in vitro, in vivo, and/or in patients with PLD. NEDDylated protein levels in normal and cystic human cholangiocytes were assessed by immunoprecipitation, and the proteomic profile was further analyzed by mass spectrometry. RESULTS AND CONCLUSION The genes involved in the NEDDylation pathway were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture, compared to controls. Elevated levels of NEDDylated proteins were further confirmed in cystic cholangiocytes in vitro, which diminished under Pevonedistat incubation. Pevonedistat promoted apoptotic cell death and reduced proliferation in cystic cholangiocytes in vitro. Comparative proteomic profiling of NEDD8-immunoprecipitated proteins between normal and cystic cholangiocytes in culture reported candidate proteins involved in cystogenesis, mostly associated with protein biogenesis and quality control. All these data indicate that cystic cholangiocytes display increased protein NEDDylation, contributing to cell survival and proliferation, ultimately supporting hepatic cystogenesis. Targeting of protein hyper-NEDDylation in cystic cholangiocytes inhibits cystogenesis in experimental models, representing a novel therapeutic opportunity in PLD.
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Affiliation(s)
- Pui Y. Lee‐Law
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- Department of Gastroenterology & HepatologyRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
| | - Paula Olaizola
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
| | - Francisco J. Caballero‐Camino
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
| | - Laura Izquierdo‐Sanchez
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
| | - Pedro M. Rodrigues
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
| | - Maria J. Perugorria
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
| | - Mikel Azkargorta
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
- Proteomics PlatformCIC bioGUNEProteoRed‐ISCIIIBizkaia Science and Technology ParkDerioSpain
| | - Felix Elortza
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
- Proteomics PlatformCIC bioGUNEProteoRed‐ISCIIIBizkaia Science and Technology ParkDerioSpain
| | - Maria L. Martinez‐Chantar
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
- Liver Disease LaboratoryCIC bioGUNE, Basque Research and Technology Alliance (BRTA)DerioSpain
| | - Patricia Aspichueta
- Department of PhysiologyFaculty of Medicine and NursingUPV/EHULeioaSpain
- Biocruces Bizkaia Health Research InstituteCruces University HospitalBarakaldoSpain
| | - Marco Marzioni
- Department of GastroenterologyUniversità Politecnica delle MarcheAnconaItaly
| | - Luis Bujanda
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
| | - Joost P. H. Drenth
- Department of Gastroenterology & HepatologyRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
| | - Jesus M. Banales
- Department of Liver and Gastrointestinal DiseasesBiodonostia Health Research Institute—Donostia University HospitalUniversity of the Basque Country (UPV/EHU)Donostia‐San SebastianSpain
- National Institute for the Study of Liver and Gastrointestinal Diseases(CIBERehd, “Instituto de Salud Carlos III”)MadridSpain
- IKERBASQUEBasque Foundation for ScienceBilbaoSpain
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Jones TM, Carew JS, Bauman JE, Nawrocki ST. Targeting NEDDylation as a Novel Approach to Improve the Treatment of Head and Neck Cancer. Cancers (Basel) 2021; 13:3250. [PMID: 34209641 PMCID: PMC8268527 DOI: 10.3390/cancers13133250] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2021] [Revised: 06/24/2021] [Accepted: 06/25/2021] [Indexed: 12/24/2022] Open
Abstract
Head and neck cancer is diagnosed in nearly 900,000 new patients worldwide each year. Despite this alarming number, patient outcomes, particularly for those diagnosed with late-stage and human papillomavirus (HPV)-negative disease, have only marginally improved in the last three decades. New therapeutics that target novel pathways are desperately needed. NEDDylation is a key cellular process by which NEDD8 proteins are conjugated to substrate proteins in order to modulate their function. NEDDylation is closely tied to appropriate protein degradation, particularly proteins involved in cell cycle regulation, DNA damage repair, and cellular stress response. Components of the NEDDylation pathway are frequently overexpressed or hyperactivated in many cancer types including head and neck cancer, which contribute to disease progression and drug resistance. Therefore, targeting NEDDylation could have a major impact for malignancies with alterations in the pathway, and this has already been demonstrated in preclinical studies and clinical trials. Here, we will survey the mechanisms by which aberrant NEDDylation contributes to disease pathogenesis and discuss the potential clinical implications of inhibiting NEDDylation as a novel approach for the treatment of head and neck cancer.
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Affiliation(s)
| | | | | | - Steffan T. Nawrocki
- Department of Medicine, The University of Arizona Cancer Center, Tucson, AZ 85724, USA; (T.M.J.); (J.S.C.); (J.E.B.)
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The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma. Int J Mol Sci 2021; 22:ijms22126565. [PMID: 34207315 PMCID: PMC8234433 DOI: 10.3390/ijms22126565] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 06/11/2021] [Accepted: 06/14/2021] [Indexed: 12/01/2022] Open
Abstract
Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.
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Abstract
Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.
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