Background: Head and neck cancer (HNC) evades immune responses by manipulating the tumor immune microenvironment (TIME). Tumor-bound Axl has been implicated in promoting an immunosuppressive TIME in HNC, though its precise role remains unclear. Understanding Axl's contribution to immune evasion in HNC could lead to the identification of new therapeutic targets; therapies directed at these targets could be combined with and thereby enhance immunotherapies. Results: Using Axl knockout (Axl KO) cell lines derived from the immunologically "cold" MOC2 mouse model, we found that Axl loss delayed tumor growth in immunocompetent mice. This was accompanied by reduced immunosuppressive cells, including MDSCs, Tregs, B cells, and neutrophils, and increased infiltration of cytotoxic CD8 T cells and NK cells. To identify the immune population(s) responsible for these changes, Axl KO tumors were implanted in immune-deficient mice. Axl KO tumor growth in athymic nude mice (which lack T cells) was unchanged, whereas tumor growth in NCG mice (which lack NK cells) was rescued, suggesting that NK cells mediate the Axl KO tumor growth delay. Further, Axl loss enhanced NK cell cytotoxicity in vitro and in vivo, and NK cell depletion reversed delayed Axl KO tumor growth. Mechanistically, Axl KO tumors showed decreased expression of CD73 and CCL2, which inhibit NK cells, and increased expression of CCL5 and CXCL10, which promote NK cell recruitment and activation. Conclusions: These novel findings suggest that tumor-bound Axl fosters an immunosuppressive TIME by inhibiting NK cell recruitment and function, thereby promoting tumor growth. Targeting Axl may enhance NK cell-mediated tumor killing and improve immunotherapy efficacy in HNC.

Preeclampsia (PE) is a significant complication of pregnancy, occurring in approximately 10% of pregnancies. However, the underlying mechanisms of this condition remain unclear. Placentation and tumorigenesis both share many characteristics, but PE is the result of insufficient placentation, in contrast to the overaggression of tumorigenesis. AXL is a biomarker and therapeutic target for multiple metastatic cancers. We hypothesized that its downregulation could play a crucial role in the development of PE. In our study, we demonstrated that pregnant Axl -/- mice exhibited typical PE symptoms, such as hypertension, proteinuria, and inadequate trophoblast invasion and spiral artery remodeling. Cross-mating and embryo transplantation experiments confirmed that these phenotypes were caused by the decidua. RNA sequencing results revealed the abnormal expression of several transcripts in the decidua, including Corin, which encodes a cardiac protease responsible for activating atrial natriuretic peptide (ANP). ANP is a cardiac hormone that regulates sodium homeostasis and blood pressure. Chromatin immunoprecipitation-qPCR analysis indicated that the decreased CORIN in Axl -/- decidua was due to reduced signal transducer and activator of transcription 3 (STAT3) binding. Treatment with ANP successfully alleviated the PE symptoms. Furthermore, we observed that in PE decidua, the level of AXL was significantly lower compared to normal pregnancies. These findings suggest that the dysregulation of decidua-derived AXL-CORIN-ANP signaling disrupts maternal-fetal crosstalk and contributes to the development of PE.

AXL, a member of the TAM receptor family, has emerged as a potential target for advanced-stage human malignancies. It is frequently overexpressed in different cancers and plays a significant role in various tumor-promoting pathways, including cancer cell proliferation, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, stemness, DNA damage response, acquired therapeutic resistance, immunosuppression, and inflammatory responses. Beyond oncology, AXL also facilitates viral infections, including SARS-CoV-2 and Zika highlighting its importance in both cancer and virology. In preclinical models, small-molecule kinase inhibitors targeting AXL have shown promising anti-tumorigenic potential. This review primarily focuses on the induction, regulation and biological functions of AXL in mediating these tumor-promoting pathways. We discuss a range of therapeutic strategies, including recently developed small-molecule tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and antibody-drug conjugates (ADCs), anti-AXL-CAR, and combination therapies. These interventions are being examined in both preclinical and clinical studies, offering the potential for improved drug sensitivity and therapeutic efficacy. We further discuss the mechanisms of acquired therapeutic resistance, particularly the crosstalk between AXL and other critical receptor tyrosine kinases (RTKs) such as c-MET, EGFR, HER2/HER3, VEGFR, PDGFR, and FLT3. Finally, we highlight key research areas that require further exploration to enhance AXL-mediated therapeutic approaches for improved clinical outcomes.

Background and objective: Cannabinoids are commonly used as adjuvant cancer drugs to overcome numerous adverse side effects for patients. The aim of this study was to identify the target genes that show a synergistic anti-tumor role in combination with the cannabinoid WIN55212-2 in vitro and in vivo. Methods: A human kinome RNAi library was used to screen the targeted gene that silencing plus WIN55212-2 treatment synergistically inhibited cancer cell growth in an INCELL Analyzer 2000. Cell viability, cell phase arrest and apoptosis were evaluated by MTT and flow cytometry assay. In vivo combined anti-tumor effects and regulatory mechanisms were detected in immunocompromised and immunocompetent mice. Results: Using RNAi screening, we identified the tyrosine receptor kinase AXL as a potential gene whose silencing plus WIN55212-2 treatment synergistically inhibited the proliferation of cancer cells in an INCELL Analyzer 2000. Subsequently, we demonstrated that inhibition of AXL by TP-0903 potentiated the inhibitory role of WIN55212-2 on cellular viability, colony formation and 3D tumor sphere in HCT-8 cells. Meanwhile, TP-0903 plus WIN55212-2 treatment promoted the apoptosis of HCT-8 cells. We then investigated the synergistic anti-tumor effect of TP-0903 and WIN55212-2 using colon cancer cell xenografts in immunocompromised and immunocompetent mice. The in vivo study demonstrated that combined administration of TP-0903 plus WIN55212-2 effectively reduced tumor volume and microvessel density and promoted apoptotic cells of tumor tissues in HCT-8 exogenous mice compared to either TP-0903 or WIN55212-2 treatment alone. Moreover, in addition to tumor suppression, the combination therapy of TP-0903 and WIN55212-2 induced the infiltration of cytotoxic CD8+ T cells and significantly reduced mTOR and STAT3 activation in tumor tissues of C57BL/6J mice bearing MC-38 cells. Conclusions: This study demonstrated that targeting AXL could sensitize cannabinoids to cancer therapy by interfering with tumor cells and tumor-infiltrating CD8+ T cells.

Head and neck squamous cell carcinoma (HNSCC) is diagnosed in more than 71,000 patients each year in the United States, with nearly 16,000 associated deaths. One significant hurdle in the treatment of HNSCC is acquired and intrinsic resistance to existing therapeutic agents. Over the past several decades, the University of Wisconsin has formed a multidisciplinary team to move basic scientific discovery along the translational spectrum to impact the lives of HNSCC patients. In this review, we outline key discoveries made throughout the years at the University of Wisconsin to deepen our understanding of therapeutic resistance in HNSCC and how a strong, interdisciplinary team can make significant advances toward improving the lives of these patients by combatting resistance to established therapeutic modalities. We are profoundly grateful to the many scientific teams worldwide whose groundbreaking discoveries, alongside evolving clinical paradigms in head and neck oncology, have been instrumental in making our work possible.

Cancer-associated fibroblasts (CAFs) play critical roles in the metastasis and therapeutic response of high-grade serous ovarian cancer (HGSC). Our study intended to select HGSC patients with unfavorable prognoses and therapeutic responses based on CAF-enriched prognostic genes. The bulk RNA and single-cell RNA sequencing (scRNA-seq) data of tumor tissues were collected from the TCGA and GEO databases. The infiltrated levels of immune and stromal cells were estimated by multiple immune deconvolution algorithms and verified through immunohistochemical analysis. The univariate Cox regression analyses were used to identify prognostic genes. Gene Set Enrichment Analysis (GSEA) was conducted to annotate enriched gene sets. The Genomics of Drug Sensitivity in Cancer (GDSC) database was used to explore potential alternative drugs. We found the infiltered levels of CAFs were remarkedly elevated in advanced and metastatic HGSC tissues and identified hundreds of genes specifically enriched in CAFs. Then we selected 6 CAF-enriched prognostic genes based on which HGSC patients were reclassified into 2 subclusters with discrepancy prognoses. Further analysis revealed that the HGSC patients in cluster-2 tended to undergo poor responses to traditional chemotherapy and immunotherapy. Subsequently, we selected 24 novel potential therapeutic drugs for cluster-2 HGSC patients. Moreover, we discovered a positive correlation of infiltrated levels between CAFs and monocytes/macrophages in HGSC tissues. Collectively, our study successfully reclassified HGSC patients into 2 different subgroups that have discrepancy prognoses and responses to current therapeutic methods.

Advanced epithelial ovarian cancer is the commonest cause of gynaecological cancer deaths. First-line treatment for advanced disease includes a combination of platinum-taxane chemotherapy (post-operatively or peri-operatively) and maximal debulking surgery whenever feasible. Initial response rate to chemotherapy is high (up to 80%) but most patients will develop recurrence (approximately 70-90%) and succumb to the disease. Recently, poly-ADP-ribose polymerase (PARP) inhibition (by drugs such as Olaparib, Niraparib or Rucaparib) directed synthetic lethality approach in BRCA germline mutant or platinum sensitive disease has generated real hope for patients. PARP inhibitor (PARPi) maintenance therapy can prolong survival but therapeutic response is not sustained due to intrinsic or acquired secondary resistance to PARPi therapy. Reversion of BRCA1/2 mutation can lead to clinical PARPi resistance in BRCA-germline mutated ovarian cancer. However, in the more common platinum sensitive sporadic HGSOC, the clinical mechanisms of development of PARPi resistance remains to be defined. Here we provide a comprehensive review of the current status of PARPi and the mechanisms of resistance to therapy.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by microvascular injury, extracellular matrix deposition, autoimmunity, inflammation, and fibrosis. The clinical complexity and high heterogeneity of the disease make the discovery of potential therapeutic targets difficult. However, the recent progress in the comprehension of its pathogenesis is encouraging. Growth Arrest-Specific 6 (Gas6) and Tyro3, Axl, and MerTK (TAM) receptors are involved in multiple biological processes, including modulation of the immune response, phagocytosis, apoptosis, fibrosis, inflammation, cancer development, and autoimmune disorders. In the present manuscript, we review the current evidence regarding SSc pathogenesis and the role of the Gas6/TAM system in several human diseases, suggesting its likely contribution in SSc and highlighting areas where further research is necessary to fully comprehend the role of TAM receptors in this condition. Indeed, understanding the involvement of TAM receptors in SSc, which is currently unknown, could provide valuable insights for novel potential therapeutic targets.

AXL, a receptor tyrosine kinase (RTK), plays a pivotal role in various cellular functions. It is primarily involved in processes such as epithelial-mesenchymal transition (EMT) in tumor cells, angiogenesis, apoptosis, immune regulation, and chemotherapy resistance mechanisms. Therefore, targeting AXL is a promising therapeutic approach for the treatment of cancer. AXL inhibitors that have entered clinical trials, such as BGB324(1), have shown promising efficacy in the treatment of melanoma and non-small cell lung cancer. Additionally, novel AXL-targeted drugs, such as AXL degraders, offer a potential solution to overcome the limitations of traditional small-molecule AXL inhibitors targeting single pathways. We provide an overview of the structure and biological functions of AXL, discusses its correlation with various cancers, and critically analyzes the structure-activity relationship of AXL small-molecule inhibitors in cellular contexts. Additionally, we summarize multiple research and development strategies, offering insights for the future development of innovative AXL inhibitors.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. More than 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response and subsequent tumor recurrence is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration, and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration, and chemotherapy response, and whether targeting of AXL with small-molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDX), primary cultures and cell line models of RMS, consistent with its stem cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacologic blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong antitumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor burden compared with single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in patients with RMS.

Anexelekto (AXL) is an attractive molecular target for ovarian cancer therapy because of its important role in ovarian cancer initiation and progression. To date, several AXL inhibitors have entered clinical trials for the treatment of ovarian cancer. However, the disadvantages of low AXL affinity and severe off-target toxicity of these inhibitors limit their further clinical applications. Herein, by rational design of a nonapeptide derivative Nap-Phe-Phe-Glu-Ile-Arg-Leu-Arg-Phe-Lys (Nap-IR), a strategy of in situ nanofiber formation is proposed to suppress ovarian cancer growth. After administration, Nap-IR specifically targets overexpressed AXL on ovarian cancer cell membranes and undergoes a receptor-instructed nanoparticle-to-nanofiber transition. In vivo and in vitro experiments demonstrate that in situ formed Nap-IR nanofibers efficiently induce apoptosis of ovarian cancer cells by blocking AXL activation and disrupting subsequent downstream signaling events. Remarkably, Nap-IR can synergistically enhance the anticancer effect of cisplatin against HO8910 ovarian tumors. It is anticipated that the Nap-IR can be applied in clinical ovarian cancer therapy in the near future.

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl-/- mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl-/- tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment.

The TAM receptor family of TYRO3, AXL and MERTK regulates tissue and immune homeostasis. Aberrant TAM receptor signalling has been linked to a range of diseases, including cancer, fibrosis and viral infections. Specifically, the dysregulation of TAM receptors can enhance tumour growth and metastasis due to their involvement in multiple oncogenic pathways. For example, TAM receptors have been implicated in the epithelial-mesenchymal transition, maintaining the stem cell phenotype, immune modulation, proliferation, angiogenesis and resistance to conventional and targeted therapies. Therapeutically, multiple TAM receptor inhibitors are in preclinical and clinical development for cancers and other indications, with those targeting AXL being the most clinically advanced. Although there has been notable clinical advancement in recent years, challenges persist. This Review aims to provide both biological and clinical insights into the current therapeutic landscape of TAM receptor inhibitors, and evaluates their potential for the treatment of cancer and non-malignant diseases.

Brain tumors are a diverse collection of neoplasms affecting the brain with a high prevalence rate in people of all ages around the globe. In this pathological context, glioblastoma, a form of glioma that belongs to the IV-grade astrocytoma group, is the most common and most aggressive form of the primary brain tumors. Indeed, despite the best treatments available including surgery, radiotherapy or a pharmacological approach with Temozolomide, glioblastoma patients' mortality is still high, within a few months of diagnosis. Therefore, to increase the chances of these patients surviving, it is critical to keep finding novel treatment opportunities. In the past, efforts to treat glioblastoma have mostly concentrated on customized treatment plans that target specific mutations such as epidermal growth factor receptor (EGFR) mutations, Neurotrophic Tyrosine Receptor Kinase (NTRK) fusions, or multiple receptors using multi-kinase inhibitors like Sunitinib and Regorafenib, with varying degrees of success. Here, we focused on the receptor tyrosine kinase AXL that has been identified as a mediator for tumor progression and therapy resistance in various cancer types, including squamous cell tumors, small cell lung cancer, and breast cancer. Activated AXL leads to a significant increase in tumor proliferation, tumor cell migration, and angiogenesis in different in vitro and in vivo models of cancer since this receptor regulates interplay with apoptotic, angiogenic and inflammatory pathways. Based on these premises, in this review we mainly focused on the role of AXL in the course of glioblastoma, considering its primary biological mechanisms and as a possible target for the application of the most recent treatments.

The role of AXL in various oncogenic processes has made it an attractive target for cancer therapy. Currently, kinase selectivity profiles, especially circumventing MET inhibition, remain a scientific issue of great interest in the discovery of selective type II AXL inhibitors. Starting from a dual MET/AXL-targeted lead structure from our previous work, we optimized a 1,6-naphthyridinone series using molecular modeling-assisted compound design to improve AXL potency and selectivity over MET, resulting in the potent and selective type II AXL-targeted compound 25c. This showed excellent AXL inhibitory activity (IC50 = 1.1 nM) and 343-fold selectivity over the highly homologous kinase MET in biochemical assays. Moreover, compound 25c significantly inhibited AXL-driven cell proliferation, dose-dependently suppressed 4T1 cell migration and invasion, and induced apoptosis. Compound 25c also showed noticeable antitumor efficacy in a BaF3/TEL-AXL xenograft model at well-tolerated doses. Overall, this study presented a potent and selective type II AXL-targeted lead compound for further drug discovery.

Few treatment options exist for pleural mesothelioma (PM), which is a progressive malignant tumor. However, the efficacy of molecular-targeted monotherapy is limited, and further therapeutic strategies are warranted to treat PM. Recently, the cancer cell-cycle checkpoint inhibitors have attracted attention because they disrupt cell-cycle regulation. Here, we aimed to establish a novel combinational therapeutic strategy to inhibit the cell-cycle checkpoint kinase, ATR in PM cells. The siRNA screening assay showed that anexelekto (AXL) knockdown enhanced cell growth inhibition when exposed to ATR inhibitors, demonstrating the synergistic effects of the ATR and AXL combination in some PM cells. The AXL and ATR inhibitor combination increased cell apoptosis via the Bim protein and suppressed cell migration when compared with each monotherapy. The combined therapeutic targeting of AXL and ATR significantly delayed regrowth compared with monotherapy. Thus, optimal AXL and ATR inhibition may potentially improve the PM outcome.

The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. Inhibitor design and screening rely on tools to study the kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl, and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 (AF2) to predict peptide-kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases and observed that, remarkably, every sequence for which it predicted a putative catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide-protein interaction modeling with AF2 for predicting the likelihood of constructive catalytic interactions.

Ovarian cancer (OC) is the most common lethal gynecologic cause of death in women worldwide, with a high mortality rate and increasing incidence. Despite advancements in the treatment, most OC patients still die from their disease due to late-stage diagnosis, the lack of effective diagnostic methods, and relapses. Aptamers, synthetic, short single-stranded oligonucleotides, have emerged as promising anticancer therapeutics. Their ability to selectively bind to target molecules, including cancer-related proteins and receptors, has revolutionized drug discovery and biomarker identification. Aptamers offer unique insights into the molecular pathways involved in cancer development and progression. Moreover, they show immense potential as drug delivery systems, enabling targeted delivery of therapeutic agents to cancer cells while minimizing off-target effects and reducing systemic toxicity. In the context of OC, the integration of aptamers with non-coding RNAs (ncRNAs) presents an opportunity for precise and efficient gene targeting. Additionally, the conjugation of aptamers with nanoparticles allows for accurate and targeted delivery of ncRNAs to specific cells, tissues, or organs. In this review, we will summarize the potential use and challenges associated with the use of aptamers alone or aptamer-ncRNA conjugates, nanoparticles, and multivalent aptamer-based therapeutics for the treatment of OC.

Novel treatment approaches are needed to overcome innate and acquired mechanisms of resistance to current anticancer therapies in cancer cells and the tumour immune microenvironment. The TAM (TYRO3, AXL and MERTK) family receptor tyrosine kinases (RTKs) are potential therapeutic targets in a wide range of cancers. In cancer cells, TAM RTKs activate signalling pathways that promote cell survival, metastasis and resistance to a variety of chemotherapeutic agents and targeted therapies. TAM RTKs also function in innate immune cells, contributing to various mechanisms that suppress antitumour immunity and promote resistance to immune-checkpoint inhibitors. Therefore, TAM antagonists provide an unprecedented opportunity for both direct and immune-mediated therapeutic activity provided by inhibition of a single target, and are likely to be particularly effective when used in combination with other cancer therapies. To exploit this potential, a variety of agents have been designed to selectively target TAM RTKs, many of which have now entered clinical testing. This Review provides an essential guide to the TAM RTKs for clinicians, including an overview of the rationale for therapeutic targeting of TAM RTKs in cancer cells and the tumour immune microenvironment, a description of the current preclinical and clinical experience with TAM inhibitors, and a perspective on strategies for continued development of TAM-targeted agents for oncology applications.

Ovarian cancer is the leading cause of gynecological cancer death. Improved understanding of the biologic pathways and introduction of poly (ADP-ribose) polymerase inhibitors (PARPi) during the last decade have changed the treatment landscape. This has improved outcomes, but unfortunately half the women with ovarian cancer still succumb to the disease within 5 years of diagnosis. Pathways of resistance to PARPi and chemotherapy have been studied extensively, but there is an unmet need to overcome treatment failure and improve outcome. Major mechanisms of PARPi resistance include restoration of homologous recombination repair activity, alteration of PARP function, stabilization of the replication fork, drug efflux, and activation of alternate pathways. These resistant mechanisms can be targeted to sensitize the resistant ovarian cancer cells either by rechallenging with PARPi, overcoming resistance mechanism or bypassing resistance pathways. Augmenting the PARPi activity by combining it with other targets in the DNA damage response pathway, antiangiogenic agents and immune checkpoint inhibitors can potentially overcome the resistance mechanisms. Methods to bypass resistance include targeting non-cross-resistant pathways acting independent of homologous recombination repair (HRR), modulating tumour microenvironment, and enhancing drug delivery systems such as antibody drug conjugates. In this review, we will discuss the first-line management of ovarian cancer, resistance mechanisms and potential strategies to overcome these.

This study aimed to investigate the aggressive behavior of triple-negative breast cancer (TNBC) cells that had survived ionizing radiation and explore the potential targets of TNBC combination treatment. Consistent with the previous literature, Axl was highly expressed in TNBC and closely associated with the degree of malignancy based on immunohistochemical staining. Using a gradient irradiation method, the ionizing radiation-resistant mouse TNBC cell line 4T-1/IRR was established. It was found that Axl expression was upregulated in 4T-1/IRR cells. After irradiation by X-ray, the cell viability and colony formation ability of 4T-1/IRR cells were significantly increased when compared with the 4T-1 cells. Combined radiotherapy with Axl inhibition by treatment with R428 and small interfering RNA lentivirus targeting Axl infection significantly reduced cell viability, colony formation ability, DNA double-stranded break repair, and the invasive and migratory ability of 4T-1/IRR cells. In vivo, the small animal radiation research platform was applied to precisely administer radiotherapy of the tumor-bearing mice. R428 treatment combined with 6 Gy X-ray significantly inhibited the growth of 4T-1/IRR cells-derived xenograft tumors in the BALB/c mouse. The results of western blotting showed that the critical molecular mechanism involved in the radioresistance of TNBC cells was the PI3K/Akt/mTOR signaling pathway induced by Axl activation. Thus, it is hypothesized that targeted Axl therapy combined with radiotherapy may have significant potential for the treatment of TNBC.

The current obesity epidemic and high prevalence of metabolic diseases necessitate efficacious and safe treatments. Brown adipose tissue in this context is a promising target with the potential to increase energy expenditure, however no pharmacological treatments activating brown adipose tissue are currently available. Here, we identify AXL receptor tyrosine kinase as a regulator of adipose function. Pharmacological and genetic inhibition of AXL enhance thermogenic capacity of brown and white adipocytes, in vitro and in vivo. Mechanistically, these effects are mediated through inhibition of PI3K/AKT/PDE signaling pathway, resulting in induction of nuclear FOXO1 localization and increased intracellular cAMP levels via PDE3/4 inhibition and subsequent stimulation of the PKA-ATF2 pathway. In line with this, both constitutive Axl deletion as well as inducible adipocyte-specific Axl deletion protect animals from diet-induced obesity concomitant with increases in energy expenditure. Based on these data, we propose AXL receptor as a target for the treatment of obesity.

AXL is a receptor tyrosine kinase that is often overexpressed in cancers. It contributes to pathophysiology in cancer progression and therapeutic resistance, making it an emerging therapeutic target. The first-in-class AXL inhibitor bemcentinib (R428/BGB324) has been granted fast track designation by the U.S. Food and Drug Administration (FDA) in STK11-mutated advanced metastatic non-small cell lung cancer and was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal molecular subtype. In this study, we further explored AXL's role in mediating DNA damage responses by using OC as a disease model. AXL inhibition using R428 resulted in the increase of DNA damage with the concurrent upregulation of DNA damage response signalling molecules. Furthermore, AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress. Combinatory use of AXL and ATR inhibitors showed additive effects in OC. Through SILAC co-immunoprecipitation mass spectrometry, we identified a novel binding partner of AXL, SAM68, whose loss in OC cells harboured phenotypes in DNA damage responses similar to AXL inhibition. In addition, AXL- and SAM68-deficiency or R428 treatment induced elevated levels of cholesterol and upregulated genes in the cholesterol biosynthesis pathway. There might be a protective role of cholesterol in shielding cancer cells against DNA damage induced by AXL inhibition or SMA68 deficiency.

Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC).

pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC).

pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals.

pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

Bile duct cancer, or cholangiocarcinoma, is a rare disease with limited treatment options that include surgery and cytotoxic chemotherapy. The high recurrence rate and poor prognosis of this type of cancer highlights the need to identify new and more effective therapeutic targets. In this study, we found that AXL, a receptor tyrosine kinase, is highly expressed in biliary cancer patients and significantly correlated with poor patient outcomes, including metastasis and low survival rates. We also demonstrated that targeting AXL inhibits tumor progression. In vitro studies with bile duct cancer cells (SNU1196 and HUCCT1) showed that genetic knockdown of AXL significantly reduced both tumor cell growth and invasion. In addition, in vivo studies using subcutaneous and orthotopic intrahepatic models demonstrated that genetic inhibition of AXL resulted in tumor-growth delay. To further examine the possible clinical translation of AXL inhibition in the clinic, we tested the efficacy of AVB-500, a soluble AXL receptor, in reducing AXL activation and tumor growth. AVB-500 was effective at inhibiting AXL activation and decreasing the growth and invasion of SNU1196 and HUCCT1 tumors which possess high AXL expression. Most importantly, AVB-500 was highly effective at decreasing tumor dissemination of bile duct tumor cells in the peritoneal cavity. This study strongly supports the idea of using the AXL receptor as a new therapeutic target to treat the growth and progression of biliary cancer.

Vascular endothelial growth factor receptors (VEGFRs) and Axl are receptor tyrosine kinases (RTK) that are targeted in ovarian cancer therapy. Two-dimensional monolayer culture and three-dimensional spheroids are common models for RTK-targeted drug screening: monolayers are simple and economical while spheroids include several genetic and histological tumor features. RTK membrane localization dictates RTK signaling and drug response, however, it is not characterized in these models. We quantify plasma membrane RTK concentrations and show differential RTK abundance and heterogeneity in monolayers versus spheroids. We show VEGFR1 concentrations on the plasma membrane to be 10 times higher in OVCAR8 spheroids than in monolayers; OVCAR8 spheroids are more heterogeneous than monolayers, exhibiting a bimodal distribution of a low-Axl (6200/cell) and a high-Axl subpopulation (25,000/cell). In addition, plasma membrane Axl concentrations differ by 100 times between chemosensitive (OVCAR3) and chemoresistant (OVCAR8) cells and by 10 times between chemoresistant cell lines (OVCAR5 vs. OVCAR8). These systematic findings can guide ovarian cancer model selection for drug screening.

GalNAc-type O-glycosylation and its initiating GalNAc transferases (GALNTs) play crucial roles in a wide range of cellular behaviors. Among 20 GALNT members, GALNT2 is consistently associated with poor survival of patients with colorectal cancer in public databases. However, its clinicopathological significance in colorectal cancer remains unclear. In this study, immunohistochemistry showed that GALNT2 was overexpressed in colorectal tumors compared with the adjacent nontumor tissues. GALNT2 overexpression was associated with poor survival of colorectal cancer patients. Forced expression of GALNT2 promoted migration and invasion as well as peritoneal metastasis of colorectal cancer cells. In contrast, GALNT2 knockdown with siRNAs or knockout with CRISPR/Cas9 system suppressed these malignant properties. Interestingly, we found that GALNT2 modified O-glycans on AXL and determined AXL levels via the proteasome-dependent pathway. In addition, the GALNT2-promoted invasiveness was significantly reversed by AXL siRNAs. These findings suggest that GALNT2 promotes colorectal cancer invasion at least partly through AXL.

For non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations, the initial therapeutic interventions will have crucial impacts on their clinical outcomes. Drug tolerant factors reportedly have an impact on EGFR-tyrosine kinase inhibitor sensitivity. This prospective study investigated the impacts of drug tolerant-related protein expression in tumors based on the efficacy of osimertinib in the first-setting of EGFR-mutated advanced NSCLC patients. A total of 92 patients with EGFR-mutated advanced or postoperative recurrent NSCLC were analyzed and treated with osimertinib at 14 institutions in Japan. AXL, p53, and programmed death-ligand 1 (PD-L1) expression in patient tumors was determined using immunohistochemistry. The AXL signaling pathway was investigated using a cell line-based assay and AXL-related gene expression in The Cancer Genome Atlas (TCGA) database. High levels of AXL and positive-p53 expression were detected in 26.1% and 53.3% of the pretreatment EGFR-mutated NSCLC tumors, respectively. High AXL expression levels were significantly associated with a shorter progression-free survival compared with low AXL expression levels, irrespective of the EGFR activating mutation status (p = 0.026). Cell line-based assays indicated that the overexpression of AXL protein accelerated PD-L1 expression, which induced insensitivity to osimertinib. In the TCGA database, AXL RNA levels were positively correlated with PD-L1 expression in the lung adenocarcinoma cohort. The results show that high AXL expression levels in tumors impact clinical predictions when using osimertinib to treat EGFR-mutated NSCLC patients. Trial Registration: UMIN000043942.

CT053PTSA is a novel tyrosine kinase inhibitor that targets MET, AXL, VEGFR2, FLT3 and MERTK. Here, we present preclinical data about CT053PTSA, and we conducted the first-in-human (FIH) study to evaluate the use of CT053PTSA in adult patients with pretreated advanced solid tumors.

The selectivity and antitumor activity of CT053PTSA were assessed in cell lines in vitro through kinase and cellular screening panels and in cell line-derived tumor xenograft (CDX) and patient-derived xenograft (PDX) models in vivo. The FIH, phase I, single-center, single-arm, dose escalation (3 + 3 design) study was conducted, patients received at least one dose of CT053PTSA (15 mg QD, 30 mg QD, 60 mg QD, 100 mg QD, and 150 mg QD). The primary objectives were to assess safety and tolerability, to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and the recommended dose of CT053PTSA for further study. Secondary objectives included pharmacokinetics, antitumor activity.

CT053 (free-base form of CT053PTSA) inhibited MET, AXL, VEGFR2, FLT3 and MERTK phosphorylation and suppressed tumor cell angiogenesis by blocking VEGF and HGF, respectively, in vitro. Moreover, cell lines with high MET expression exhibited strong sensitivity to CT053, and CT053 blocked the MET and AXL signaling pathways. In an in vivo study, CT053 significantly inhibited tumor growth in CDX and PDX models. Twenty eligible patients were enrolled in the FIH phase I trial. The most common treatment-related adverse events were transaminase elevation (65%), leukopenia (45%) and neutropenia (35%). DLTs occurred in 3 patients, 1/6 in the 100 mg group and 2/4 in the 150 mg group, so the MTD was set to 100 mg. CT053PTSA was rapidly absorbed after the oral administration of a single dose, and the Cmax and AUC increased proportionally as the dose increased. A total of 17 patients in this trial underwent tumor imaging evaluation, and 29.4% had stable disease.

CT053PTSA has potent antitumor and antiangiogenic activity in preclinical models. In this FIH phase I trial, CT053PTSA was well tolerated and had a satisfactory safety profile. Further trials evaluating the clinical activity of CT053PTSA are ongoing.

Commonly overexpressed in many cancers and associated with tumor growth, metastasis, drug resistance, and poor overall survival, Axl has emerged as a promising target for cancer therapy. However, the availability of new chemical forms for Axl inhibition is limited. Herein, we present the development and characterization of novel Axl inhibitors, including the design, synthesis, and structure-activity relationships (SARs) of a series of diphenylpyrimidine-diamine derivatives. Most of these compounds exhibited remarkable activity against the Axl kinase. In particular, the promising compound m16 showed the highest enzymatic inhibitory potency (IC₅₀ = 5 nM) and blocked multiple tumor cells' proliferation potencies (the CC₅₀ of 4 out of 42 cancer cell lines <100 nM). Furthermore, compound m16 also possessed preferable pharmacokinetic profiles and liver microsome stability. All these favorable results make m16 a good leading therapeutic candidate for further development.

Chemotherapy is often ineffective in advanced-stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis. However, the mechanism of how inhibition of AXL improves response to chemotherapy is still largely unknown. Thus, we aimed to determine whether treatment with AVB-500, a selective inhibitor of GAS6-AXL, improves endometrial cancer cell sensitivity to chemotherapy particularly through metabolic changes. We found that both GAS6 and AXL expression were higher by immunohistochemistry in patient tumors with a poor response to chemotherapy compared with tumors with a good response to chemotherapy. We showed that chemotherapy-resistant endometrial cancer cells (ARK1, uterine serous carcinoma and PUC198, grade 3 endometrioid adenocarcinoma) had improved sensitivity and synergy with paclitaxel and carboplatin when treated in combination with AVB-500. We also found that in vivo intraperitoneal models with ARK1 and PUC198 cells had decreased tumor burden when treated with AVB-500 + paclitaxel compared with paclitaxel alone. Treatment with AVB-500 + paclitaxel decreased AKT signaling, which resulted in a decrease in basal glycolysis. Finally, multiple glycolytic metabolites were lower in the tumors treated with AVB-500 + paclitaxel than in tumors treated with paclitaxel alone. Our study provides strong preclinical rationale for combining AVB-500 with paclitaxel in aggressive endometrial cancer models.