Abstract Background The cellular origin of lung squamous cell carcinoma (LUSC) remains poorly understood due to the absence of native squamous epithelium in the lung. While LKB1 loss can promote lung adenocarcinoma (LUAD)-to-LUSC lineage switching, this mechanism does not explain many cases. KMT2D, a histone H3K4 methyltransferase mutated in ∼20% of LUSC, is associated with poor outcome in early-stage disease. We investigated whether Kmt2d loss drives initiation of lung adenosquamous (LUAS) lesions and promotes lineage infidelity in AT2 and Club cells. Methods We evaluated whether Kmt2d loss is necessary and sufficient to induce LUSC in Pik3caH1047R/p53-/- (PP) and KrasMut/p53-/- (KP) mouse lung cancer models. A R26-LSL-Tdtomato (TdT) reporter allele was crossed into KP mice, and TdT+ tumor cells were isolated at the moribund stage for 10x single-nuclei multiomics profiling (snRNA-seq and snATAC-seq). To define mechanisms underlying Kmt2d-loss-mediated transdifferentiation, cell lines representing different tumor states and histologies were subjected to histone mark ChIP-seq (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) and CUT= 0.01). Mechanistically, KMT2D cooperates with FOXA1/FOXA2 to maintain lineage-specific enhancers and TFs networks required for endodermal differentiation. Upon KMT2D loss, FOXA1 partners with the KMT2A/B-MENIN complex, activating aberrant squamous programs that accelerate tumorigenesis. Consequently, Kmt2d-null cells become dependent on MENIN for their excessive proliferation. NFE2L2 is identified as a key aberrantly activated oncogene contributing to the growth of Kmt2d-null cells. Conclusions Our findings identify KMT2D as a critical guardian of lung lineage fidelity and reveal how its loss reconfigures core regulatory programs to drive squamous transdifferentiation and aggressive tumor progression. Citation Format: Yingda Jiang, Manrong Wu, Uttara Saran, Archit Ghosh, Kunal Rai. KMT2D is a master epigenetic regulator of lineage plasticity in lung squamous cell carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1362.
Jiang et al. (Fri,) studied this question.