Functional Analysis of Rare RAS Variants of Unknown Significance.

Abstract

The RAS gene is frequently mutated in human cancers. Whereas the functional significance of frequent mutations is well established, the significance of rare mutations remains unknown. This study aimed to comprehensively investigate the function of rare RAS variants and provide new insights about their clinical relevance. A total of 298 K/N/HRAS variants (169, 72, and 57 variants, respectively) reported in the COSMIC database v100 were introduced into 3T3 cells. Subsequently, the drug sensitivity of KRAS variants to BI-2865, a noncovalent pan-KRAS inhibitor, was evaluated using the mixed-all-nominated-in-one method. The 3T3 focus formation assay newly identified 35 KRAS, 10 NRAS, and 21 HRAS variants as transforming competent. The oncogenicity assessed in the present study was consistent with that reported in the database. The drug sensitivity assay identified 15 KRAS variants sensitive to BI-2865. BI-2865 treatment inhibited the RAS downstream signaling pathways and induced apoptosis in cells with the sensitive variants. The present study identified 66 new oncogenic RAS variants. The sensitivity of KRAS variants to BI-2865 varies by variant. Functional analysis provides clues for the treatment of patients with rare RAS variants.

Significance: This study presents the first comprehensive functional analysis of 298 rare RAS variants, identifying 66 novel oncogenic mutations and 15 KRAS variants sensitive to the noncovalent pan-KRAS inhibitor BI-2865. The heterogeneity in drug responses among KRAS variants underscores the need for variant-specific therapeutic strategies. These findings provide a preclinical framework for guiding personalized treatment in RAS-driven cancers and a valuable resource for understanding the clinical relevance of rare RAS mutations.