Abstract B049: Overcoming treatment adaptation to RAS(ON) inhibition in <i>NRAS/HRAS</i> mutant pediatric tumors

Abstract Pediatric cancers frequently harbor activating alterations of the RAS signaling pathway. In fusion-negative RMS (FN-RMS), the predominant molecular subtype of rhabdomyosarcoma, 56% of patient tumors carry RAS pathway mutations – with a majority of these tumors harboring Q61X hyperactivating mutations in NRAS or HRAS. We evaluated 16 in vitro RMS models for 32 inhibitors targeting the RAS-RAF-MEK-ERK, PI3K-Akt, or mTOR pathways. RAS(ON) inhibitors RMS-7977 and RMC-6236 (daraxonrasib) emerged as potent, selective agents against NRAS- and HRAS-mutant FN-RMS, including models with trametinib resistance. Treatment suppressed of MEK and ERK phosphorylation and reduced DUSP6 expression. Daraxonrasib also induced expression of differentiation markers, confirmed by bulk RNA-sequencing and immunofluorescence. To define mechanisms of adaptation to RAS(ON) inhibition, we performed single-cell RNA-sequencing combined with intracellular protein staining using oligo-conjugated antibodies. Two persistent subpopulations were identified that restore Akt and MEK phosphorylation during therapy. Combination drug-screening revealed that vertical co-inhibition of RAS signaling with Akt or MEK blockade may overcome these adaptive states. Using perturbation sequencing, a method that integrates scRNA-seq with pooled CRISPR screening, we assessed the impact of 199 gene knockdowns on cellular heterogeneity in NRAS Q61H- and HRAS Q61K-mutant FN-RMS. Eight transcription factors whose knockout selectively depleted drug-tolerant cell states, were identified. We are now evaluating in vitro and in vivo efficacy of genetic and pharmacologic blockade of these eight targets in combination with RAS(ON) inhibition. Moreover, we are extending these approaches to additional RAS-driven pediatric cancers. Citation Format: Roketa Henry, Ayush Attery, Anang Shelat, Anand Patel. Overcoming treatment adaptation to RAS(ON) inhibition in NRAS/HRAS mutant pediatric tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B049.