Abstract 3874: Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues, with full pathway inhibition at 4 hours post treatment that was restored by 24 hours. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. To understand the dynamic response of malignant cells following RAS-GTP inhibition, we did single cell RNA sequencing on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms [Maroling., et al. 2006; Alvarez., et al. 2016], we performed regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of >5000 transcriptional regulatory proteins per cell. Additionally, we assessed the impact of treatment on malignant cell subtypes as previously identified by our team based on their sets of hyperactivated developmental transcription factors [Laise., et al. 2022]. As expected, RAS pathway transcription factors, such as FOS and JUN, were repressed relative to controls in malignant cells by 4 hours after treatment with RMC-7977. However, at 24 hours, responses varied by cellular subtype: well-differentiated Gastrointestinal Lineage State (GLS) cells had restored MAPK activity, while poorly differentiated Morphogenic State (MOS) cells were still inactivated. Strikingly, we found that after one week of treatment (in actively regressing tumors, representing a state of residual disease), nearly all malignant cells had assumed the GLS state. Acquisition of resistance after weeks or months of treatment was associated with a restored diversity of malignant cell states, suggesting that the GLS state serves as a regulatory “safe harbor” in which residual malignant cells can undergo evolution and adaptation. Mechanistically, we found that RAS pathway signaling directly activates transcriptional regulators that drive the MOS state, elucidating an early adaptive response to RAS-GTP inhibition that enables residual cells to survive treatment prior to development of acquired resistance. Citation Format: Lorenzo Tomassoni, Urszula N. Wasko, Alvaro C. Garcia, Tanner C. Dalton, Pasquale Laise, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3874.