p53 R248Q upregulation of VTCN1 via dual mechanisms as a driver of immune evasion and adaptive resistance to CDK4/6 inhibitors: Rationale for combination immunotherapy in endometrial cancer.

5618 Background: TP53-mutated endometrial cancer (EC) is a heterogeneous disease with variable responses to immunotherapy. While generic p53 mutation status is a standard biomarker, the distinct clinical impact of specific hotspot mutants remains underappreciated. Our bioinformatics analysis of TCGA data suggested that the R248Q hotspot—but not others—is specifically linked to an "immune-desert" phenotype. We aimed to mechanistically define this subtype and validate its diagnostic value as a predictor for immune exclusion and CDK4/6 inhibitor sensitivity. Methods: We performed a comprehensive bioinformatic analysis of the TCGA-UCEC cohort to correlate specific p53 hotspot mutations with immune landscapes. To elucidate the underlying mechanism, we engineered a panel of isogenic EC cell lines (p53-null, R248Q, R175H, R248W, R273H/C) and assessed VTCN1 (B7-H4) regulation via biochemical assays. To validate these findings clinically, we established an independent cohort of 254 EC patients with tissue microarrays (TMA); whole-exome sequencing (WES) is currently underway to pinpoint hotspot mutations and correlate them with VTCN1 expression and immune infiltration. Results: Discovery of Diagnostic Target: In the TCGA cohort, stratifying p53 mutants revealed that R248Q carriers exhibited the most profound "cold" tumor microenvironment compared to other hotspots, suggesting it as a potential negative predictive biomarker. Mechanistic Validation: Using our isogenic models, we observed that R248Q specifically drives high VTCN1 expression. Mechanistically, R248Q appears to promote VTCN1 via a dual pathway: transcriptional upregulation and, uniquely, protein stabilization by inhibiting proteasomal degradation. This indicates VTCN1 as a potential surrogate marker for the R248Q subtype. Therapeutic Implications: In preclinical models, abemaciclib treatment paradoxically upregulated VTCN1 in R248Q cells, suggesting adaptive resistance. However, this induced vulnerability was associated with enhanced sensitivity to combined CDK4/6 inhibition and immunotherapy (anti-VTCN1), which elicited superior tumor regression compared to monotherapy. Conclusions: Our study suggests the potential value of refining p53 diagnostics from binary (mutant/WT) to hotspot-specific resolution. Current data indicate that p53 R248Q may function as a distinct driver that contributes to an immunosuppressive "cold" TME in EC, likely through synergistic transcriptional and post-translational upregulation of VTCN1. Furthermore, abemaciclib-induced VTCN1 elevation points to a possible mechanism of monotherapy resistance. These findings provide a strong preclinical rationale to explore combining CDK4/6 inhibitors with immunotherapy, specifically for patients with the high-risk p53 R248Q subtype.