Abstract 4426: Chromatin constraints underpin adeno-to-squamous cell state transitions during acquired resistance to KRAS inhibition

The advent of single-cell RNA-seq has permitted a global view of the transcriptomic variation across cancer cells in individual tumors and revealed functionally-related sets of genes - a gene module - underpin a cancer cell state. While single-cell transcriptomic atlases have provided a descriptive characterization of cancer cell states across tumor types, we lack a mechanistic framework on the constraints of tumor heterogeneity. Therefore, we propose a ‘developmental constraint’ model - where cancer cell state transitions are restricted by the organism’s developmental map, wherein from the cell-of-origin the malignant cells traverse states that are accessible by its developmental trajectory, both toward progenitor-like states and differentiated-like states of adjacent lineages. Our model posits a constraint on the emergence of states, even under evolutionary pressures such as therapeutic intervention. To test this framework, we modeled non-genetic mechanisms of acquired resistance to KRAS inhibitors - histological adeno-to-squamous transition (AST). Multi-omic analysis of 68 patients enrolled in the KRYSTAL-1 clinical trial revealed a squamous gene signature correlated with a poor response specifically in patients with co-occurring KRASG12C;STK11 (KCL) mutations. We recapitulated AST upon resistance to KRASG12C inhibition in KCL genetically engineered mouse models and tumor-derived organoids. Single-cell transcriptomic and epigenomic of tumor-derived organoid revealed that AST is mediated by a two-step transition - from adeno-like states to an intermediate highly plastic cell state expressing markers of mixed-identity to enriched for squamous-like states. These findings support the developmental constraint model where cell state transitions are mediated by progenitor states in contrast to a trans-differentiation model. Furthermore, we hypothesized that cancer cell state transitions are restricted by the cell’s chromatin context and specific chromatin modifiers are required for cell state transitions analogous to development. Therefore, we leveraged a time-course Perturb-seq approach against 191 chromatin modifiers to identify functional requirements for the adeno-to-intermediate and intermediate-to-squamous transition. Using a module-based method, we found cells with perturbations of the SWI/SNF complex (Arid1a, Smarca4 and Smarce1) did not transition to the squamous cell state but were restricted to the intermediate highly plastic cell state, suggesting that the SWI/SNF complex is required for the intermediate-to-squamous transition. Together, we have showed evidence that cell state transitions in cancer may be explained by the developmental constraint model and revealed that these constraints may act at the chromatin level where chromatin modifiers and chromatin states enable AST plasticity during adaptation to KRAS inhibitors. Citation Format: Ayushi S. Patel, Samuel Greene, Arianne Parvaresh-Rizi, Gustavo S. França, Kwok-Kin Wong, Itai Yanai. Chromatin constraints underpin adeno-to-squamous cell state transitions during acquired resistance to KRAS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4426.