Functional Genomics Studies Decipher the Genetic Perturbations and Unravel Mechanisms of Response/Resistance upon Mutant-Specific KRAS Inhibition in Multiple Myeloma
Authors: Steinbrunn Torsten , Shirasaki Ryosuke , Dashevsky Olga , Tang Huihui , Yamano Shizuka , Teoh Phaik Ju , Leypoldt Lisa , Glassner Brian , De Matos Simoes Ricardo , Christensen James , Mitsiades Constantine S 
Published: December 2024

INTRODUCTION
Mutated RAS, a key oncogenic driver in multiple myeloma (MM), can be present in up to 50% of primary MM. Several novel pharmacological inhibitors developed to target specific KRAS point mutations, have been successfully incorporated in the treatment of solid cancers, but their potential impact in MM has yet to be determined. Here, we performed genome-scale CRISPR studies to decipher the functional gene perturbation landscape of KRAS-mutated MM after treatment with specific KRAS inhibitors as well as the potential mechanisms of resistance to these molecules.

METHODS
We performed a total of 11 genome-scale CRISPR gene activation or CRISPR gene editing (knockout, KO) studies in 5 MM lines with distinct KRAS point mutations (KHM-1B and XG-7 [G12C]; KARPAS-620 and KP-6 [G12D]; or MM.1S [G12A]) after treatment with specific KRAS inhibitors (MRTX-1133, MRTX-1257, BI-2865, RMC-6236) in clinically achievable concentration ranges (as used in patients with solid tumors). Potency and mutant-specific in vitro activity of these inhibitors in the respective cell lines was assessed with bioluminescence assays and flow cytometry and confirmed to be similar to KRAS-mutant lines from our own in vitro studies (eg. MIA PaCa-2 [G12C]) as well as from other groups. Functional downstream abrogation of KRAS-dependent growth and survival pathways via MEK/MAPK and PI3K/AKT was determined by immunoblotting to confirm dephosphorylation of ERK and/or AKT. The level of apoptosis induction was assessed with flow cytometry after AnnexinV/PI staining. Cell cycle analyses were performed using DAPI staining. Furthermore, we performed RNA sequencing in 6 KRAS mutant cell lines to investigate their transcriptomic response to KRAS-inhibitor treatment.

RESULTS
Our genome-scale CRISPR activation and KO studies in 5 MM lines with mutant KRAS (G12A/G12C/G12D) revealed a heterogeneous and cell line-specific pattern of gene perturbations regulating response vs. resistance to KRAS inhibition. CRISPR activation screens identified positive regulators of RAS-MAPK signaling (eg. SHOC2) and upstream surface receptors (eg. EGFR) among the highest scoring genes involved in resistance to KRAS inhibitors. Interestingly, CRISPR activation of KRAS itself was identified as a promoter of resistance, possibly due to reduced target saturation. Notably, many of the top “hit” genes are not recurrently genetically altered or differentially expressed in MM nor do they represent clinical “high-risk” features. In turn, CRISPR gene KO studies pointed to a key role of regulators of cell homeostasis (eg. KEAP1, NF2L1) in conferring KRAS inhibitor sensitization vs. resistance. Moreover, genes encoding for GTPase-activating proteins (GAPs) and negative regulators of RAS/MAPK signaling (eg. LZTR1) were functionally important. Some of the genes identified here are not typically expressed in newly diagnosed MM (eg. EGFR), but their upregulation during KRAS inhibitor treatment may be a candidate biomarker for decreased response or early relapse.
We examined the RNA sequencing profile of 6 KRAS-mutant MM lines after short-term treatment with specific KRAS inhibitors. Transcriptional responses to KRAS inhibition were also heterogeneous between cell lines, and included downregulation of RAS effector genes (eg. ETV4, ETV5) and negative regulators of RAS signaling (eg. DUSP6, SPRY4), pointing to a RAS-driven negative feedback loop. Other recurrently differentially expressed genes include cell cycle regulators (e.g., D-type cyclin) and genes involved in apoptosis and DNA repair. Upregulation of gene expression was predominant in genes involved in cell cycle arrest and stress responses.

CONCLUSION
Our genome-scale CRISPR activation and knockout studies revealed a heterogeneous landscape of genomic perturbations regulating the response vs. resistance of MM cell to mutant-specific KRAS inhibitors. These compounds exhibit potent and specific activity against MM cells with the respective KRAS mutations, but our functional studies point to individual lines, even those harboring the same KRAS mutation, exhibiting their own distinct “resistome” against these inhibitors. These results underscore the complex functional genomics of MM cell sensitivity vs. resistance to KRAS inhibitors and have implications for the choice of potential combination partners of these inhibitors in future preclinical or clinical studies.