Abstract A075: SHP2 inhibition is active in a RAS mutation-dependent manner and synergizes with RAS(ON) inhibition in models of fusion-negative rhabdomyosarcoma.

Abstract Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas and are characterized by the presence or absence of an oncogenic chromosomal fusion between PAX3/PAX7 and FOXO1. The subset lacking this fusion, fusion-negative RMS (FN-RMS), are frequently driven by mutations in RAS signaling pathways. Therapeutic strategies to target oncogenic RAS include inhibitors of RAS regulatory proteins, such as the nonreceptor protein tyrosine phosphatase SHP2. SHP2 activates RAS signaling by dephosphorylating key RAS tyrosine residues Y32 and Y64, and by recruiting the RAS guanine nucleotide exchange factor (GEF) SOS1. We set out to determine whether SHP2 inhibition (SHP2i) demonstrates preclinical activity in models of RAS-dependent FN-RMS. Oncogenic RAS mutations at codons 12, 13, and 61 slow RAS-GTP hydrolysis and we therefore hypothesized that they would cause variable sensitivity to SHP2i. We tested the effects of SHP2i in a panel of FN-RMS cell lines. SHP2i reduced MEK-ERK signaling and anchorage-independent cell growth in cells with WT RAS driven by aberrant RTK activation, or those with G12 or G13-mutant HRAS, but not those with Q61-mutant H/NRAS. In vivo, SHP2i demonstrated anti-tumor activity against the WT RAS/FGFR1-amplified RMS-YM cell line derived xenograft (CDX) but not the HRAS Q61K SMS-CTR CDX. To confirm that RAS mutation determines sensitivity to SHP2i, we transduced murine C2C12 myoblast cells with mutant H/N/KRAS constructs and tested their sensitivity to SHP2i. C2C12 cells expressing WT and G12/G13-mutant H/N/KRAS showed significant growth inhibition and decreased ERK signaling in response to SHP2i, but those expressing Q61-mutant H/N/KRAS showed no such changes. To determine if RAS tyrosyl phosphorylation plays a role in SHP2-mediated activation of RAS, and therefore SHP2i sensitivity, we transduced C2C12 cells with tyrosine-mutant HRAS and treated these cells with SHP2i, SOS1i, and RAS(ON)i. Our data suggest that Y64 and Y96 may be involved in RAS regulation via RAS-GEFs or RAS-GAPs in a manner that determines SHP2i sensitivity. RAS(ON)i inhibits RAS-GTP by inducing a complex between RAS and Cyclophilin A (CYPA) via RAS Y64 that confers RAS-GAP function to CYPA, leading to RAS inactivation. By restoring GTPase activity in mutant RAS protein, we predicted that RAS(ON)i would be additive with SHP2i. We treated a panel of RAS-mutant C2C12 isogenic cell lines, as well as RAS-mutant FN-RMS cell lines, with a combination of SHP2i and RAS(ON)i. The combination further reduced cell growth and ERK signaling compared to either compound alone in RAS-mutant isogenic and FN-RMS models, but not RAS WT models. Our data supports the concept that RAS mutations determine sensitivity to SHP2i and that Y64 and Y96 are critical residues for RAS signaling that modulate sensitivity to SHP2i. We have also found that SHP2i increases sensitivity to RAS(ON)i in RAS-mutant, but not WT models. Citation Format: Andrew R. Baker, Elizabeth Fenner, Marios Constantinides, Alla Lisok, Marielle E. Yohe, Angelina Vaseva, Patience I. Odeniyide, Jiawan Wang, Christine A. Pratilas. SHP2 inhibition is active in a RAS mutation-dependent manner and synergizes with RAS(ON) inhibition in models of fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A075.