P-248: CRISPR-based functional genomics studies identify mechanisms of multiple myeloma (MM) cell response to vs. resistance against novel pharmacological mutant-specific KRAS inhibitors.

Introduction: RAS genes are frequently mutated oncogenic drivers in MM. Several novel pharmacological inhibitors targeting specific KRAS mutations have become available. We previously reported that G12C or G12D mutation-specific KRAS inhibitors, which are clinically active in solid tumors with these mutations, are also potently active in vitro against MM lines with the respective mutations. Here, we performed 8 genome-wide CRISPR gene editing (knockout, KO) or gene activation studies to assess the mechanisms of sensitization vs. resistance against these inhibitors in KRAS-mutant MM cells.

Methods: The KRAS G12D-mutant MM cell lines KP-6 and KARPAS620 and the KRAS G12C-mutant lines XG-7 and KHM-1B were treated with the small molecule KRAS inhibitors MRTX1133 (G12D-specific) and MRTX1257 (G12C-specific), respectively, in different concentrations within the clinically achievable concentration range in patients with solid tumors. A total of 8 genome-scale CRISPR-based gene activation or editing screens were conducted to identify genomic perturbations altering response to KRAS inhibitors. The in vitro activity of both compounds as single agents or in combination with clinically established anti-MM drugs was assessed with bioluminescence assays and flow cytometry, analyzing cell viability/proliferation and cell cycle distribution/apoptosis induction, respectively. Downstream RAS signaling was assessed by Western blotting.

Results: The KRAS inhibitors showed strong and specific activity in their respective cell lines (IC50 of 5-150 nM) and downstream signaling (pERK1/2 and pAKT) was effectively abrogated. Apoptosis induction was most prominent in G12D-mutant cells. CRISPR activation studies identified KRAS itself, upstream surface receptors (e.g., EGFR), SHOC2 (a positive regulator of RAS-MAPK signaling) as key genes promoting MM cell resistance against KRAS inhibitor treatment. CRISPR gene KO studies suggest a role of the KEAP1/NF2L1 pathway (regulating cell homeostasis), GTPase-activating proteins as well as negative regulators of RAS-MAPK (e.g. LZTR1) as molecular determinants of MM cell response to KRAS inhibition.

Conclusions: Mutant-specific KRAS G12C and G12D inhibitors show strong in vitro activity in MM cells with these mutations. CRISPR KO or gene activation studies provide functional insights into the pharmacological inhibition of KRAS in MM, identifying potential targets which regulate the response vs. resistance of MM cells to KRAS inhibitors.