Abstract 5774: Small cell lung cancer subtype plasticity is regulated by KDM6A

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that accounts for ~15% of lung cancers. While nearly all SCLCs are genetically driven by near universal loss of function (LOF) mutations in RB1 and TP53; several recent studies show that there are different phenotypic SCLC molecular subtypes characterized by expression of lineage transcription factors. These include the neuroendocrine ASCL1 and NEUROD1 subtypes which together comprise ~70-80% of SCLCs. Initially subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoral subtype heterogeneity and plasticity between subtypes. A recent study found that 35-40% of human SCLCs express both ASCL1 and NEUROD1, but the mechanisms driving ASCL1 and NEUROD1 intra-tumoral heterogeneity are not well understood. My laboratory previously developed an autochthonous CRISPR-based SCLC genetically-engineered mouse model (GEMM) generated by intratracheally injecting adenoviruses encoding Cre recombinase and sgRNAs targeting Rb1, Trp53, and Rbl2. Cre turns on Cas9 expression and allows for CRISPR/Cas9 editing of Rb1, Trp53, and Rbl2 in somatic cells in the lungs. The unique advantage of this model is that it allows the inclusion of sgRNAs targeting additional genes of interest in the same adenovirus. Using this CRISPR-based autochthonous SCLC GEMM approach, we studied the consequences of inactivating the epigenetic modifier KDM6A during SCLC tumorigenesis. KDM6A functions as an H3K27 histone demethylase and also exists in the COMPASS complex with KMT2C/D to promote H3K4 mono-/di-methylation at enhancers. KDM6A along with its protein binding partner KMT2D are mutated in SCLC and KDM6A has been implicated in controlling differentiation in other lineages. Strikingly, we found that KDM6A inactivation in SCLC GEMMs induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumors that expressed both ASCL1 and NEUROD1. ATAC-sequencing showed open chromatin at the promoters of NEUROD1 and NEUROD1 target genes in KDM6A inactivated tumors. Interestingly, KDM6A inactivated tumors showed a spectrum of ASCL1 to NEUROD1 heterogeneity where some KDM6A inactivated tumors completely lost ASCL1 and solely expressed NEUROD1, some tumors expressed ASCL1 and NEUROD1 in a mutually exclusive manner, while others primarily expressed ASCL1 with very few NEUROD1 positive cells. Mechanistically, KDM6A binds and maintains ASCL1 target genes in an active chromatin state with its loss increasing H3K27me3 near both promoters and enhancers, and decreasing H3K4me1/2 at enhancers together leading to a cell state primed for ASCL1 to NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC GEMM to model ASCL1 and NEUROD1 subtype heterogeneity, which is found in 35-40% of human SCLCs. Citation Format: Leslie Duplaquet, Yixiang Li, Matthew A. Booker, Yingtian Xie, Radhika A. Patel, Deli Hong, Thomas Denize, Emily Walton, Yasmin N. Laimon, Roderick Bronson, Jackson Southard, Shuqiang Li, Sabina Signoretti, Michael Y. Tolstorukov, Paloma Cejas, Henry W. Long, Michael C. Haffner, Matthew G. Oser. Small cell lung cancer subtype plasticity is regulated by KDM6A. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5774.