Transcriptomic and functional characterization of KRASG12D and KRASG12V NSCLC clones reveals AXL as a potential target

Introduction. In Non-Small Cell Lung Cancer (NSCLC), KRAS (Kirsten rat sarcoma virus) mutations occur in about 30% of adenocarcinoma cases. Although the oncogene has long been recognized, it is only recently that Sotorasib (AMG510) and Adagrasib (MRTX849), drugs specifically targeting the KRAS G12C mutation, which accounts for approximately 40% of KRASmutated cases, have been approved for clinical use. Other mutations at the same codon, such as KRASG12V and KRASG12D, represent around 19% and 15% of KRAS-mutated cases, respectively, in various studies and currently lack targeted therapies. Methods. Clones from H322 and H292 NSCLC cells carrying the KRASG12D and KRASG12V mutations were generated by lentiviral particles. Gene expression was investigated by RNA sequencing, Gene Ontology, and Cytoscape analysis. Protein expression and phosphorylation were evaluated by Western blotting; the hypoxia condition was established using a H35 incubator for hypoxia. Results. We isolated two clones carrying the KRASG12D and KRASG12V mutations. Clones were selected using a Western blot with antibodies specific to the G12D and G12V mutations. The mutated lines exhibited a higher proliferation index compared to control cells. Subsequently, RNA sequencing revealed more than 600 differentially expressed genes in the H322 cell line with the G12D mutation, and over 400 differentially expressed genes in the G12V mutant, compared to the control. Among the most upregulated genes in both mutations, we identified AXL, a well-known oncogene implicated in resistance mechanisms in various tumors. Mutant cell clones from H322 and H292 showed increased AXL protein expression, which was more pronounced under hypoxic conditions and serum starvation, consistent with AXL promoter regulation by HIF1α (Hypoxiainducible factor 1-alpha). Notably, treatment with the pan-RAS inhibitor RMC-6236 induced further AXL expression, suggesting AXL as a potential target for a combination. Conclusions Our data suggest that both mutations, KRASG12D and KRASG12V, increase the transcription and protein expression of the AXL gene. AXL induction also occurred under hypoxic conditions and serum starvation. The use of RMC-6236 led to increased AXL expression. These data suggest the rationale for a combination between RMC-6236 and an AXL inhibitor in KRASG12D and KRASG12V NSCLC cells.