Abstract B120: Mapping and modulating epithelial-mesenchymal plasticity under RAS(ON) multi-selective inhibition in PDAC through lineage tracing and Perturb-seq

Abstract KRAS is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC), mutationally activated in approximately 90% of cases. Mutations in this oncogene have been associated with more aggressive disease, poorer outcomes, and have remained hard to drug for over three decades. Recently developed small molecule inhibitors of KRAS (KRASi) have shown promising efficacy in advanced, previously treated PDAC patients but ultimately, most patients develop acquired resistance. Approximately half of these resistance cases do not present a putative genomic driver. In preclinical studies using genetically engineered mouse models of PDAC, we and others have demonstrated that cell state identity along the classical/epithelial-mesenchymal axis is a key determinant of response and resistance to KRASi. Notably, acute KRASi treatment initially induces a strong and selective bottlenecking of the malignant population in vivo, resulting in tumors that are predominantly classical with depleted mesenchymal characteristics. While these findings highlight a potent cell state-selective effect of acute KRASi treatment on PDAC cells, the underlying mechanisms driving the drug-induced cell state selection and plasticity remain poorly characterized. These mechanisms may reveal novel therapeutic targets for developing combination therapies that improve therapeutic responses. To comprehensively map and modulate epithelial-mesenchymal (E-M) cell state plasticity in response to KRASi treatment, we performed CRISPRi Perturb-seq (single-cell gene expression readout) with lineage tracing on a patient-derived PDAC cell line treated with the RAS(ON) multi-selective inhibitor RMC-7977. We captured 734,092 high-quality single-cell transcriptomic profiles and 156,119 unique clones across 60 genetic perturbations, including E- and M-specific transcription factors (TF) inferred to influence E-M plasticity from previous single-cell lineage tracing experiments. To optimally model clonal growth and transition rates, we profiled cells across a time series at day 6, day 15 and day 22, after 1 week of DMSO or KRASi treatment. We developed a hierarchical generative probabilistic model to jointly infer E↔M transition rates and cell state-specific growth rates, while capturing the effects of perturbations and KRASi treatment on state dynamics. Analysis of longitudinally tracked clones revealed that KRASi treatment specifically reduced the M-state growth rate while increasing the probability of M→E transitions compared to DMSO. Notably, we identified several TFs, displaying KRASi-dependent changes in state transition dynamics, either promoting (ELF3, MEIS2 among others) or decreasing (ZNF281, IRF9, among others) probabilities of M→E transitions when compared to non-targeting controls. These preclinical findings suggest that perturbation of these factors may be used as a paradigm to prevent cell state plasticity upon acute KRASi treatment and could be used to homogenize tumor populations towards a treatment-sensitive state. Citation Format: Julien Dilly, Mike Bogaev, Lynn Bi, Abigail Collins, Martin Jankowiak, Aziz Al'Khafaji, Kyle E. Evans, Mehrtash Babadi, Thouis R. Jones, Elisa Donnard, David T. Ting, Nir Hacohen, Dana Pe'er, Eric S. Lander, Andrew J. Aguirre, Arnav Mehta. Mapping and modulating epithelial-mesenchymal plasticity under RAS(ON) multi-selective inhibition in PDAC through lineage tracing and Perturb-seq [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(18_Suppl_3):Abstract nr B120.