Correlation between KAT6A and PD-L1 expression and role of KAT6A in colorectal cancer

Abstract

BACKGROUND

Immune checkpoint inhibitors (ICIs) are effective cancer treatments; however, a significant proportion of colorectal cancer (CRC) patients exhibit limited responses to ICI therapy. KAT6A has been strongly associated with cancer initiation and progression.

AIM

To examine the role of KAT6A in CRC progression and immune evasion.

METHODS

The functional role of KAT6A was evaluated through genetic knockdown, pharmacological inhibition (WM-3835), and CRISPR/dCas9-mediated epigenetic editing in CRC cells. T cell-mediated apoptosis was assessed using co-culture models, and H3K23pr was measured via chromatin immunoprecipitation assays. PD-L1 expression at mRNA and protein levels was analyzed under KAT6A knockdown conditions.

RESULTS

KAT6A suppression reduced CRC cell proliferation, invasion, and migration. Pharmacological or epigenetic disruption of KAT6A phenocopied these effects, with dose-dependent reductions in H3K23pr (28.4% residual at 10 μM) and PD-L1 expression. KAT6A knockdown enhanced T cell-mediated apoptosis, evidenced by increased expression of granzyme B and perforin. Mechanistically, KAT6A loss decreased H3K23pr and reduced RNA polymerase II occupancy on the PD-L1 promoter, leading to suppressed PD-L1 transcription. CRISPR/dCas9-mediated H3K23pr editing at the PD-L1 promoter directly modulated immune evasion, confirming its causal role. Overexpression of PD-L1 mitigated the inhibitory effects of KAT6A knockdown on CRC progression and immune evasion.

CONCLUSION

KAT6A drives CRC progression and immune evasion by promoting histone H3 propionylation to epigenetically activate PD-L1 expression. Targeting KAT6A or its downstream H3K23pr-PD-L1 axis represents a promising therapeutic strategy to overcome ICI resistance in CRC.

Key Words: Colorectal cancer; Epigenetic regulation; KAT6A; PD-L1; Immune evasion

Core Tip: KAT6A drives colorectal cancer (CRC) progression and immune evasion by promoting histone H3 propionylation to epigenetically activate PD-L1 expression. Targeting KAT6A or its downstream H3K23pr-PD-L1 axis represents a promising therapeutic strategy to overcome Immune checkpoint inhibitor resistance in CRC.

INTRODUCTION

Recently, immune checkpoint inhibitors (ICIs), specifically PD-1, have greatly transformed the field of cancer treatment[1-3]. However, in colorectal cancer (CRC), which ranks among the three most prominent aggressive and common cancers, the therapeutic benefits of ICIs are predominantly observed in individuals with high microsatellite instability[4-6]. Notably, approximately 90% of CRC cases are microsatellite stable (MSS), where response rates to ICIs rarely exceed 5%-10%, highlighting an urgent need to decipher immune evasion mechanisms beyond mismatch repair deficiency[7,8]. The regONIVO trial recently demonstrated that combining Regorafenib with anti-PD-1 antibodies achieved a 33% response rate in MSS-CRC patients[9] suggesting epigenetic reprogramming of the tumor microenvironment may synergize with ICIs. Nevertheless, the molecular determinants underlying this synergy remain poorly characterized[10].

Epigenetic regulation, encompassing DNA/histone modifications and RNA post-transcriptional editing, profoundly influences oncogenesis and immune surveillance[11-15]. In CRC, DNA hypermethylation (e.g., MLH1 silencing in MSI-H tumors) and histone deacetylase-mediated PD-L1 suppression are well-established epigenetic mechanisms[16,17]. Recent studies further revealed that enhancer of EZH2-mediated H3K27me3 represses PD-L1 transcription in MSS-CRC[18], while DNA methyltransferase inhibitors upregulate PD-L1 through demethylation of its promoter[19]. These findings position epigenetic modifiers as pivotal regulators of immune checkpoint expression. However, compared to DNA methylation and canonical histone modifications (acetylation/methylation), the immunological implications of novel acylation marks remain underexplored.

Histone acylation, including propionylation, butyrylation, and crotonylation, represents an emerging layer of epigenetic regulation with distinct biological functions[20]. Unlike acetylation, crotonylation generates bulkier hydrophobic modifications that alter chromatin architecture and preferentially recruit readers like YEATS domains[21]. KAT6A, a MYST-family histone acetyltransferase paralog, uniquely catalyzes histone crotonylation in cancer cells[22,23]. In breast cancer, KAT6A-driven crotonylation facilitates SOX4 transcriptional activation to promote metastasis, while in glioblastoma, it stabilizes β-catenin to sustain stemness[24]. Notably, KAT6A amplification correlates with poor prognosis in multiple cancers, yet its immunological role, particularly in modulating PD-L1-mediated immune evasion, remains unknown. This knowledge gap is striking given that: (1) Histone crotonylation preferentially marks active enhancers of oncogenes; and (2) PD-L1 expression is regulated by enhancer remodeling in immunotherapy-resistant tumors[25].

Here, we interrogated the epigenetic-immune crosstalk in CRC by linking KAT6A-mediated histone acylation to PD-L1 regulation. We demonstrated that KAT6A orchestrates an immune-evasive microenvironment through crotonylation-dependent PD-L1 upregulation and identified combinatorial targeting strategies to overcome ICI resistance. Our work extends the paradigm of epigenetic control in cancer immunity by revealing a non-canonical acylation mechanism, providing therapeutic avenues for MSS-CRC patients refractory to current immunotherapies.

MATERIALS AND METHODS

Cell lines and transfection

CRC cell lines were procured from Procell (Wuhan, China). Cells were cultured in DMEM (BI, Israel) supplemented with 10% fetal bovine serum (FBS, BI, Israel) and 1% penicillin-streptomycin (P-S, Sigma, United States) under standard conditions of 37 °C with 5% CO₂. KAT6A-specific small interfering RNA (siRNA) and overexpression plasmid vectors were purchased from Gene Pharma (Shanghai, China). Transfections were carried out using Lipofectamine 2000 reagent (Thermo Fisher Scientific, United States) following the manufacturer’s protocol. Transfection efficiency was confirmed 48 hours post-transfection by quantitative PCR (qPCR) and Western blot analysis.

Cell viability and proliferation assays

Cell counting kit-8 (CCK-8; SolarBio, China) was utilized to measure cell viability. CRC cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well and incubated for 24 hours. After adding CCK-8 reagent, the cells were incubated for 2 hours at 37 °C, and absorbance at 450 nm was measured using a microplate reader. For the colony formation assay, 1 × 10³ cells were seeded in 6-well plates and cultured for 10 days. Colonies were fixed with methanol, stained with 0.1% crystal violet, and photographed. Colony numbers were quantified manually.

Cell apoptosis assay

Apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Biotechnology, China). CRC cells were collected, washed with phosphate-buffered saline (PBS), and stained with Annexin V-FITC and PI following the manufacturer’s instructions. Stained cells were analyzed using flow cytometry (BD Biosciences, United States). Briefly, cells were stained with 5 μL Annexin V-FITC and 5 μL PI in the dark for 15 minutes, washed with PBS, and resuspended in 300 μL binding buffer. Flow cytometry was performed using a BD FACSCanto II system. FITC signals were detected with 488 nm excitation and 530 nm emission, while PI signals were detected with 561 nm excitation and 610 nm emission. Flow cytometry data were analyzed using FlowJo v10.8. Debris was excluded based on FSC-A/SSC-A gating. Early apoptotic cells were identified as Annexin V+/PI-, while late apoptotic cells were identified as Annexin V+/PI+.

Migration and invasion assays

Transwell migration and invasion assays were conducted using 24-well Transwell inserts (Costar, United States). For the migration assay, CRC cells were suspended in serum-free medium and seeded in the upper chamber. The lower chamber was filled with complete medium containing 10% FBS as a chemoattractant. After 24 hours, cells on the upper surface of the membrane were removed with a cotton swab, and migrated cells on the lower surface were stained with crystal violet. For the invasion assay, the upper chamber was pre-coated with a Matrigel/DMEM mixture (1:1, Corning, United States). Migrated and invaded cells were counted in five randomly selected fields under a light microscope (Leica, Germany).

Western blot analysis

Total protein lysates were extracted using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, United States). Equal amounts of protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, United States). The membranes were blocked in 5% skimmed milk for 2 hours at room temperature, followed by overnight incubation at 4 °C with primary antibodies against E-cadherin, Vimentin, KAT6A, PD-L1, and β-actin (Abcam, United Kingdom). The membranes were then incubated with secondary antibodies for 1 hour at room temperature and visualized using an ECL reagent (Thermo Fisher Scientific).

qPCR

Total RNA was isolated using TRIzol reagent (Qiagen, Hilden, Germany). RNA was reverse-transcribed using a reverse transcription kit (Qiagen), and cDNA was amplified using SYBR Green PCR Master Mix (Thermo Fisher Scientific). PCR amplification was performed in a StepOnePlus real-time PCR system (Applied Biosystems, United States). Gene expression was normalized to GAPDH as an internal control.

T-cell activation and co-culture system

Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples using Ficoll density-gradient centrifugation. T cells were activated by incubating PBMCs with anti-CD3 and anti-CD28 antibodies (Gibco, United States) for 3 days. For co-culture experiments, CRC cells and T cells were seeded in separate compartments of a Transwell insert at a 10:1 ratio (T cells:CRC cells). After 72 hours, PBMCs were collected, stained with anti-CD4, anti-CD8, and anti-PD-1 antibodies (BioLegend, United States), and analyzed using flow cytometry.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed using an EZ-ChIP Kit (Millipore, United States). CRC cells were fixed with 1% formaldehyde to cross-link proteins and DNA. Chromatin was fragmented into approximately 500 bp fragments by sonication. Immunoprecipitation was performed overnight at 4 °C using antibodies against H3K23pr, RNA polymerase II, or control IgG. DNA-protein complexes were purified using protein A/G magnetic beads. Enriched DNA was reverse cross-linked, treated with RNase A and proteinase K, and analyzed by qPCR with primers targeting the PD-L1 promoter region.

Treatment with the KAT6A inhibitor WM-3835

WM-3835 (MedChemExpress, HY-138487) was dissolved in DMSO to prepare a 10 mmol/L stock solution and stored at -80 °C. SW480 and SW620 cells were seeded at a density of 5 × 10⁴ cells/well in a 6-well plate. After cell attachment, the culture medium was replaced with complete medium containing WM-3835 at concentrations of 0, 5, and 10 μM for 48 hours. In parallel with the experimental groups, the solvent control (DMSO) was maintained at a final concentration of ≤ 0.1% (v/v).

CRISPR/dCas9 epigenetic editing

The dCas9-p300 and dCas9-LSD1 plasmids were synthesized by Shanghai GeneChem Co., Ltd. The sgRNA targeting the PD-L1 promoter (sequence: 5’-GCGCTCCAGCTCCGACCTGA-3’) and the non-targeting control sgRNA (sequence: 5’-ACGGAGGCTAAGCGTCGCAA-3’) were cloned into the pLV-sgRNA vector. SW480 cells were seeded at a density of 1 × 10⁶ cells per well and co-transfected with dCas9 plasmid (2 μg) and sgRNA plasmid (1 μg) using Lipofectamine 3000 (Thermo Fisher). After 48 hours, the medium was replaced with selection medium containing 2 μg/mL puromycin and incubated for 72 hours. For editing efficiency validation, H3K23pr enrichment was assessed by ChIP-qPCR, and PD-L1 mRNA levels were analyzed by qPCR.

Statistical analysis

Statistical analyses were conducted using SPSS 26.0 (IBM, United States) and GraphPad Prism 9.0 (GraphPad Software, United States). Results are expressed as the mean ± SD. Two-sided Student’s t-tests were applied for comparisons between two groups, and one-way ANOVA followed by Dunnett’s post-hoc test was used for multiple group comparisons. A P value < 0.05 was considered statistically significant.

RESULTS

KAT6A enhances CRC cell proliferation, invasion, and migration

To investigate the role of KAT6A in CRC cell behavior, we transfected CRC cell lines with KAT6A siRNA to achieve its knockdown. Western blot analysis confirmed the efficient depletion of KAT6A expression in the cells (Figure 1A). CCK-8 assays revealed a significant reduction in cell proliferation following KAT6A knockdown, indicating its critical role in promoting CRC cell growth (Figure 1B). Furthermore, Transwell assays demonstrated a dramatic decrease in both migratory and invasive CRC cells after KAT6A depletion, highlighting its role in facilitating cell motility and invasion (Figure 1C). Western blot analysis also revealed molecular changes associated with epithelial-mesenchymal transition (EMT): KAT6A knockdown elevated E-cadherin levels (an epithelial marker) and reduced vimentin levels (a mesenchymal marker), suggesting suppression of EMT processes (Figure 1D). These findings collectively indicate that KAT6A promotes CRC cell proliferation, migration, and invasion.

Figure 1 KAT6A enhances the proliferation, invasion, and migration of colorectal cancer cells. SW480 and SW620 cells were transfected with KAT6A siRNAs. A: Protein level of KAT6A was measured by Western blotting assay; B: Cell viability of SW480 and SW620 cells was measured by cell counting kit-8 assay; C: Cell migration and invasion were detected by using Transwell experiment; D: Protein levels of E-cadherin and Vimentin were detected by Western blotting assay. ^bP < 0.01.

KAT6A induces immune evasion in a CRC cell/T cell co-culture System

To examine the impact of KAT6A on immune evasion, we employed a co-culture system involving CRC cells and unstimulated or anti-CD3/CD28-activated T cells. Cell apoptosis assays showed that KAT6A-depleted CRC cells exhibited significantly increased apoptosis (SW480: 65.03% ± 3.99% vs siNC 29.76% ± 2.5%, P < 0.001; SW620: 64.58% ± 5.21% vs siNC 29.46% ± 1.69%, P < 0.001; Figure 2A). Additionally, depletion of KAT6A in the co-culture system elevated the expression levels of T-cell cytotoxic markers, GZMB and perforin, indicating enhanced T cell activity (Figure 2B). Notably, KAT6A knockdown also suppressed PD-L1 expression in CRC cells (Figure 2C), suggesting that KAT6A may promote immune evasion through upregulation of PD-L1. These findings indicate that KAT6A suppresses the immune response of T cells, enabling CRC cells to evade immune surveillance.

Figure 2 KAT6A induces immune evasion in a colorectal cancer cell/T cell co-culture system. Colorectal cancer (CRC) cells treated with KAT6A siRNAs were co-cultured with unstimulated or anti-CD3/CD28-activated T cells (5 μg/mL each antibody, 72 hours of stimulation). A: T cell-mediated apoptosis of CRC cells was measured by flow cytometry; B: The expression of GZMB and perforin in T cells was analyzed by Western blot; C: The levels of PD-L1 in CRC cells were analyzed by Western blotting analysis.

KAT6A-mediated H3K23pr activates PD-L1 expression in CRC cells

We further explored the molecular mechanism underlying KAT6A-mediated immune evasion. Western blot analysis demonstrated that KAT6A knockdown significantly reduced H3K23pr levels in CRC cells (Figure 3A). ChIP assays revealed a marked reduction in H3K23pr enrichment at the PD-L1 promoter following KAT6A knockdown, indicating that KAT6A-mediated histone modification plays a role in regulating PD-L1 transcription (Figure 3B). Consistently, qPCR analysis showed that PD-L1 mRNA levels were significantly reduced in CRC cells with depleted KAT6A expression (Figure 3C). These results suggest that KAT6A promotes PD-L1 expression by inducing H3K23pr modifications at the PD-L1 promoter, thereby facilitating immune evasion.

Figure 3 KAT6A-mediated H3K23pr activates PD-L1 expression in colorectal cancer cells. SW480 and SW620 cells were transfected with KAT6A siRNAs. A: The levels of H3K23pr were detected by Western blotting assay; B: The enrichment of H3K23pr on PD-L1 promoter was analyzed by ChIP-PCR; C: The mRNA expression of PD-L1 was measured by quantitative PCR. ^bP < 0.01.

KAT6A contributes to CRC progression and immune evasion via PD-L1

To establish the functional relationship between KAT6A and PD-L1 in CRC progression and immune evasion, we overexpressed PD-L1 in KAT6A-depleted cells. Overexpression of PD-L1 successfully restored the impaired invasion and migration capacities of KAT6A-depleted SW480 and SW620 CRC cells, as shown by Transwell assays (Figure 4A and B). In the co-culture system of CRC cells and T cells, PD-L1 overexpression also reversed the enhanced expression of GZMB and perforin caused by KAT6A knockdown, suggesting a suppression of T-cell cytotoxic activity (Figure 4C). Furthermore, PD-L1 overexpression abolished the increase in T cell-mediated killing activity observed in KAT6A-depleted CRC cells (Figure 4D). These findings indicate that KAT6A promotes CRC progression and immune evasion primarily through upregulation of PD-L1.

Figure 4 KAT6A contributes to progression and immune evasion of colorectal cancer cells by PD-L1. SW480 and SW620 cells were transfected with KAT6A siRNAs, or co-treated with PD-L1 overexpression plasmids. A: Cell migration and invasion were detected by using Transwell experiment; B: The expression of GZMB and perforin in T cells was detected Western blotting assay; C: The levels of PD-L1 in colorectal cancer (CRC) cells were analyzed by Western blotting assay; D: CRC cells were co-cultured with unstimulated or activated T cells. The T cell-mediated apoptosis on CRC cells was measured by flow cytometry.

Regulation of H3K23pr and PD-L1 by KAT6A inhibitor

To pharmacologically validate KAT6A’s role, we treated CRC cells with WM-3835, a selective KAT6A inhibitor. Dose-dependent reductions in H3K23pr (28.4% residual at 10 μM, P < 0.001) and PD-L1 protein (21.5% residual, P < 0.001) were observed (Figure 5A), paralleled by 68% suppression of PD-L1 mRNA (P = 0.0032; Figure 5B). In the co-culture system of CRC cells and activated T cells, WM-3835 (10 μM) enhanced CRC cell apoptosis to 61.8% (vs DMSO 30.2%, P < 0.001; Figure 5C) and upregulated the T-cell cytotoxic markers GZMB (3.2-fold) and perforin (2.7-fold; Figure 5D). These results mirrored genetic KAT6A knockdown phenotypes, confirming its druggable potential.

Figure 5 Pharmacological inhibition of KAT6A recapitulates genetic knockdown phenotypes. A: Dose-dependent suppression of H3K23pr and PD-L1 by WM-3835. Western blot (left) and densitometric quantification (right) showed H3K23pr and PD-L1 protein levels in SW480/SW620 cells treated with WM-3835 (0, 5, or 10 μM) for 48 hours; B: PD-L1 transcriptional downregulation as revealed by quantitative PCR analysis of PD-L1 mRNA levels; C: Enhanced T cell-mediated apoptosis as shown by flow cytometry quantification of apoptotic colorectal cancer cells co-cultured with activated T cells; D: Activation of T cell cytotoxic markers demonstrated by Western blot analysis of GZMB and perforin in T cells. Data represent the mean ± SEM (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs DMSO (one-way ANOVA with Dunnett’s test). siKAT6A data from Figures 1 and 2 are shown as positive controls.

Epigenetic intervention targeting H3K23pr at the PD-L1 promoter

To establish a direct causal link between H3K23pr and PD-L1, we employed CRISPR/dCas9-based epigenetic editing. Targeting dCas9-p300 to the PD-L1 promoter (-154 bp) specifically enhanced H3K23pr enrichment by 3.8-fold (ChIP-qPCR, P < 0.001; Figure 6A), concomitant with 2.5-fold upregulation of PD-L1 mRNA (P = 0.0076; Figure 6B). Conversely, dCas9-LSD1 reduced both H3K23pr (70% decrease) and PD-L1 expression (P < 0.001). Functionally, dCas9-p300-edited cells exhibited enhanced invasion (158 vs 62 cells/field, P < 0.001) and resistance to T cell killing (34.2% vs 63.5% apoptosis, P < 0.001; Figure 6C). Importantly, dCas9-p300 rescued PD-L1 expression and immune evasion even in KAT6A-depleted cells (mRNA 1.8-fold, apoptosis 38.5% vs 65.0%, P = 0.0062; Figure 6D), confirming H3K23pr as the dominant downstream mechanism.

Figure 6 Epigenetic editing of H3K23pr at PD-L1 promoter directly modulates immune evasion. A: CRISPR/dCas9-mediated H3K23pr editing efficiency evaluated by ChIP-qPCR analysis of H3K23pr enrichment at PD-L1 promoter in SW480 cells transfected with dCas9-p300 or dCas9-LSD1 and PD-L1-targeting sgRNA (5’-GCGCTCCAGCTCCGACCTGA-3’). Non-targeting sgRNA and empty vector served as controls; B: PD-L1 transcriptional regulation shown by quantitative PCR analysis of PD-L1 mRNA levels; C: Functional validation of immune evasion. Transwell invasion (left) and T cell-mediated apoptosis (right) assays; D: Rescue experiment in KAT6A-depleted cells. PD-L1 mRNA (left) and apoptosis (right) in siKAT6A cells co-transfected with dCas9-p300. Data represent the mean ± SEM (n = 3). ^cP < 0.001 vs non-targeting sgRNA (one-way ANOVA with Dunnett’s test). ^dP < 0.01.

Summary of findings

Overall, our data demonstrate that KAT6A enhances CRC cell proliferation, migration, invasion, and immune evasion. Mechanistically, KAT6A-mediated H3K23pr epigenetically activates PD-L1 expression, promoting tumor progression and suppressing T cell activity. Targeting KAT6A may therefore represent a potential therapeutic strategy for CRC.

DISCUSSION

CRC is a life-threatening malignancy characterized by poor prognosis and limited effective therapeutic options. Despite advancements in treatment strategies, the molecular mechanisms underlying CRC progression and immune evasion remain poorly understood, highlighting the urgent need for novel therapeutic targets. In this study, we identified lysine KAT6A as a key regulator of CRC progression and immune evasion, offering new insights into its role as a potential therapeutic target. Our findings reveal a previously unrecognized mechanism by which KAT6A drives PD-L1 expression through H3K23pr-mediated epigenetic remodeling, and further demonstrate the therapeutic potential of KAT6A inhibition in combination with immune checkpoint blockade.

KAT6A has been implicated in various biological processes in cancer, including cell growth, metastasis, drug resistance, and stemness[26-28]. Consistent with previous studies, our results demonstrated that KAT6A plays an oncogenic role in CRC. Specifically, KAT6A knockdown significantly suppressed CRC cell proliferation, migration, and invasion, accompanied by reduced EMT marker alterations. Importantly, pharmacological inhibition of KAT6A using WM-3835 recapitulated these phenotypes, with dose-dependent reductions in H3K23pr (28.4% residual at 10 μM) and PD-L1 expression (21.5% residual), accompanied by enhanced T cell cytotoxicity (Figure 5). These findings suggest that KAT6A contributes to CRC progression by promoting aggressive tumor behaviors.

Furthermore, KAT6A has been shown to interact with diverse signaling pathways in cancer due to its acetyltransferase activity. For instance, KAT6A-mediated acetylation enhances PIK3CA transcription, activating the PI3K/AKT signaling pathway in glioblastoma, thereby promoting tumor growth and metastasis[29]. Similarly, KAT6A acetylates YAP in hepatocellular carcinoma, contributing to sorafenib resistance and cancer progression[30]. In our study, we identified a novel axis wherein KAT6A regulates H3K23pr, which epigenetically activates PD-L1 transcription. CRISPR/dCas9-mediated editing of H3K23pr at the PD-L1 promoter (-154 bp) directly modulated PD-L1 expression (3.8-fold increase with dCas9-p300 vs 70% decrease with dCas9-LSD1) and immune evasion phenotypes (Figure 6), confirming the causal role of this modification. This mechanism highlights the ability of KAT6A to drive immune evasion by modulating chromatin accessibility at immune checkpoint loci.

PD-L1 plays a pivotal role in tumor immune escape by binding to PD-1 on T cells, thereby suppressing T cell activation and cytotoxicity[31]. This interaction creates an immunosuppressive tumor microenvironment, enabling cancer cells to evade immune surveillance[32,33]. Our findings revealed that KAT6A knockdown significantly reduced H3K23pr and RNA polymerase II enrichment at the PD-L1 promoter, resulting in decreased PD-L1 expression at both the mRNA and protein levels. Consequently, T cell-mediated killing of CRC cells was enhanced following KAT6A knockdown. Notably, forced H3K23pr enrichment via dCas9-p300 restored PD-L1 expression and immune evasion even in KAT6A-depleted cells (Figure 6D), underscoring the centrality of this epigenetic mark in mediating KAT6A’s effects. Importantly, overexpression of PD-L1 reversed the inhibitory effects of KAT6A depletion on CRC progression and immune evasion, further confirming that KAT6A exerts its pro-tumorigenic effects through PD-L1 regulation.

These results align with previous studies suggesting that epigenetic regulation of PD-L1 is a critical mechanism by which tumors evade immune destruction. KAT6A’s role as a chromatin modifier positions it as a key player in this process, and its dual involvement in promoting tumor progression and immune suppression makes it an attractive therapeutic target. While our data show that KAT6A inhibition reduces PD-L1 expression, the potential synergy with ICIs may arise from complementary mechanisms. For example, KAT6A inhibition could simultaneously downregulate PD-L1 and destabilize other immunosuppressive pathways (e.g., enhancing antigen presentation or reducing myeloid-derived suppressor cell infiltration), thereby creating a more immunogenic tumor microenvironment. This hypothesis is supported by our observation that WM-3835-treated cells exhibited elevated T cell cytotoxic markers (GZMB and perforin; Figure 5D) independent of PD-L1 modulation, suggesting broader immunostimulatory effects. Future studies exploring combination therapies with KAT6A inhibitors and anti-PD-1 antibodies are warranted to validate this paradigm.

While our study provides compelling evidence for the role of KAT6A in CRC, there are limitations that warrant further investigation. For example, the precise upstream regulators of KAT6A in CRC remain unclear, and additional studies are needed to elucidate how KAT6A expression is modulated in the tumor microenvironment. Moreover, the translational relevance of our findings should be tested in vivo, particularly in MSS-CRC models resistant to current ICIs. Such experiments could clarify whether KAT6A inhibition synergizes with PD-1/PD-L1 blockade by overcoming epigenetic-driven immune tolerance.

CONCLUSION

In conclusion, we found that the acetyltransferase KAT6A contributes to the progression and immune evasion of CRC cells by inducing histone H3 propionylation to epigenetically activate PD-L1 expression. Pharmacological or epigenetic disruption of the KAT6A-H3K23pr-PD-L1 axis restores T cell cytotoxicity and suppresses tumor aggressiveness, positioning KAT6A as a dual therapeutic target for CRC. Our work provides a rationale for combining KAT6A inhibitors with immunotherapies to counteract resistance mechanisms in MSS-CRC.