P-371: Mutant-specific pharmacological inhibition of KRAS in multiple myeloma and functional genomics studies to identify mechanisms regulating myeloma cell response vs. resistance to KRAS inhibition.

Introduction: Oncogenic RAS mutations are frequent genomic drivers in MM. Specific inhibitors of the G12C and G12D KRAS mutants are clinically active in solid tumors with these mutations, which however are rare in MM patients. Nevertheless, we studied these inhibitors in G12C/D KRAS mutant MM cells to assess its functional consequences and potential mechanisms of escape from KRAS inhibition, and facilitate potential similar efforts when other KRAS mutations, more prevalent in MM, may become targetable in the future.

Methods: We assessed the response of G12C (KHM1B, XG7) and G12D (KARPAS620, KP6) KRAS mutant MM cell lines to G12C- (MRTX1257) and G12D- (MRTX1133) specific KRAS inhibitors (within the concentration range achievable in patients with solid tumors) both as single agents and in combination with clinically established anti-MM agents. Effects of these inhibitors on cell viability and proliferation (bioluminescence assays), signaling downstream of KRAS (Western blots) and transcriptional networks (RNA-seq) were analyzed. Induction of cell death, apoptosis and cell cycle distribution were assessed by flow cytometry. Genome-scale CRISPR activation studies identified genes whose gain-of-function (GOF) can enhance or suppress the MM cell response to KRAS inhibition.

Results: Both KRAS inhibitors exhibited potent and mutant-specific activity in their respective MM lines (IC50 in the range of 5-150 nM after 3-7d treatment), with effective abrogation of downstream phosphorylation for MAPK and AKT pathway members. MRTX1257 predominantly reduced proliferation of XG7 cells, whereas MRTX1133 led to potent cell death induction of KARPAS620 and KP6 cells. Combinations of MRTX1257 with investigational or established anti-MM drugs, including melphalan, bortezomib or pomalidomide, led to no antagonism and in some cases – including combination with MEKi trametinib – caused supra-additive effects. MRTX1257 downregulated known Ras effector genes (e.g., ETV4, ETV5), negative regulators of Ras signaling (e.g., DUSP6, SPRY4, reflecting a Ras-driven negative feedback loop controlling expression of these genes) and genes involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair; and upregulated genes involved in cell cycle arrest and stress responses. In CRISPR activation studies, GOF of KRAS itself, upstream surface receptors (e.g., EGFR), SHOC2 (which encodes for a scaffold protein serving as positive regulator of RAS-MAPK signaling) were among key “hits” promoting MM cell escape from KRAS inhibitor treatment.

Conclusions: This study documents mutant-specific activity of KRAS G12C and G12D inhibitors in MM cells and provides functional insights into the pharmacological inhibition of KRAS in MM. Ongoing in vitro and in vivo studies are examining the targeting of genes/pathways associated with escape from KRAS inhibition, as a framework for future efforts to improve the rates, depth and durability of responses to KRAS inhibition in MM.