Abstract B061: Epigenetic EZH2 dependency in KRAS inhibitor-resistant PDAC cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, mainly due to limited therapeutic options, late diagnosis and rapid development of drug resistance. Recently, multiple pan- and mutation-selective KRAS inhibitors have been developed, showing significant survival benefit, including the KRASG12D inhibitor MRTX1133. Unfortunately, as observed with the KRASG12C inhibitors, resistance mechanisms often limit its long-term efficacy. Epigenetic remodeling, particularly via the histone methyltransferase enhancer of zeste homolog 2 (EZH2), has been implicated in cell plasticity and therapy resistance by repressing epithelial programs while inducing more aggressive cellular states. Here, we investigated the mechanisms driving resistance to MRTX1133 in PDAC. We established three MRTX1133-resistant (MRTX1133_R) cell lines through chronic exposure to escalating drug concentrations. After, we employed bulk RNA sequencing followed by gene set enrichment analysis to reveal alterations between MRTX1133_R and parental counterparts. We then performed multiple functional assays to characterize the mechanisms driving the resistant phenotype. Additionally, we stained human samples from a tissue microarray by immunohistochemistry to assess the translation of our findings. We found that KRAS inhibitor-resistant cells exhibited profound molecular rearrangements, including loss of epithelial markers (E-cadherin, Claudin-1), and increased expression of mesenchymal players (N-cadherin, Slug), concomitantly with enhanced PI3K-mTOR pathway activity. RNA sequencing analysis revealed enhanced cellular plasticity and enrichment of neuroendocrine (NE) and EZH2-associated transcriptional programs, marked by higher expression of several NE markers, including ASCL1, CHGA, NKX2-2, and PAX6. Importantly, treatment with an EZH2 inhibitor – Tazemetostat – markedly reduced the proliferation of MRTX1133_R cells, abolished H3K27me3 protein levels and decreased expression of NE markers. Combined treatment of Tazemetostat and MRTX1133 re-sensitized resistant cells, leading to a significant reduction in cell viability alongside cell death induction. Remarkably, Tazemetostat synergized with the dual mTOR/PI3K inhibitor Dactolisib to significantly reduce cell viability, providing an alternative therapeutic option. Importantly, in human samples, high percentage of EZH2+ cells significantly associated with advanced stages, lymph node-positive cases, as well as recurrence at distant sites, particularly in the lung. Overall, our findings uncover an epigenetically-driven resistance mechanism in which EZH2 promotes NE differentiation and provides a bypass signaling under KRAS inhibition. Targeting EZH2 may therefore represent a promising therapeutic strategy to overcome KRAS inhibitor resistance and improve the clinical outcome for PDAC patients.