Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance
Authors: Steinbrunn Torsten , Shirasaki Ryosuke , Dashevsky Olga , Tang Huihui , Yamano Shizuka , Mizuno Rin , Sugihara Takeru , Teoh Phaik Ju , Leypoldt Lisa B , Kaji Mariko , Sorrell Jeffrey , Glassner Brian J , De Matos Simoes Ricardo , Christensen James , Mitsiades Constantine S 
Published: December 2023

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents.
We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination.
RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies.
Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.