Abstract A035: KMT2D Loss Induces Enrichment of Plod2+ Fibroblasts in Pancreatic Cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma largely composed of cancer-associated fibroblasts (CAFs), which drive tumor progression and immune evasion. CAFs are highly heterogeneous and their behavior is shaped by tumor-derived signals. KMT2D (Mll4 in mice), a histone methyltransferase responsible for H3K4 monomethylation at enhancers, is frequently mutated in PDAC and correlates with aggressive tumor phenotypes. However, its function within the PDAC stroma remains poorly defined. Methods: To evaluate KMT2D alterations in PDAC, we analyzed somatic mutations, mRNA, protein expression, and clinical data from TCGA and CPTAC. Functional studies employed genetically engineered mouse models: KPC (LSL-Kras G12D+/- ; LSL-p53 R172H/- ; Ptf1a-Cre) and KPCM (additional Mll4 flox/flox), along with KMT2D-knockout KPC, PANC-1, and BxPC-3 cell lines generated via CRISPR-Cas9. Single-cell RNA sequencing (scRNA-seq) was performed on tumors from KPC and KPCM mice, and publicly available scRNA-seq data from 48 human PDAC samples (205, 721 cells) were integrated for comparative analysis. Bulk RNA-seq, proteomics, qPCR, and ELISA further validated the results. Results: Among 535 PDAC patients in TCGA, KMT2D was the seventh most frequently mutated gene (approximately 6% of cases), with mutations linked to decreased mRNA and protein expression and reduced disease-free and overall survival. In line with these findings, KPCM mice exhibited significantly shorter survival than KPC controls. Integrating human scRNA-seq data using Scissor with bulk TCGA RNA-seq and survival data identified a CAF subset marked by high Plod2 expression that is associated with poor patient prognosis. scRNA-seq of mouse tumors revealed an increased abundance of CAFs in the KPCM cohort. Subclustering and label transfer techniques mapped an expansion of the Plod2+ CAF subset in KPCM tumors, mirroring human data. Cell-cell interaction analysis (CellChat) indicated enhanced crosstalk between tumor cells and fibroblasts in KPCM mice, particularly via activin A signaling between epithelial cells and CAFs. Bulk RNA-seq and proteomic analysis of KMT2D-deficient KPC cells demonstrated upregulation and secretion of activin A; ELISA confirmed elevated activin A levels in conditioned media. Exposure of fibroblasts to this media significantly increased Plod2 expression. Conclusion: Loss of KMT2D drives the expansion of Plod2+ fibroblasts through activin A-mediated signaling, underpinning worse outcomes in PDAC. These findings highlight a previously unrecognized role for KMT2D in shaping CAF function and contributing to our understanding of PDAC stroma biology. Citation Format: Kaiyue Ding, Hong Sun. Kim, Shungang Zhang, R Ranga. Sudharshan, Jiaqi Shi. KMT2D Loss Induces Enrichment of Plod2+ Fibroblasts in Pancreatic Cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₃): Abstract nr A035.