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.

Figure 1.

RAS protein domain and distribution of RAS mutations. A, RAS proteins comprise a G-domain consisting of five α-helix structures and six β-sheet structures, followed by a hypervariable region (HVR; 166–189 aa). The G-domain contains three GTP-binding domains and an effector-binding domain. The G-domain contains the P-loop (10–14 aa), switch I (30–40 aa), and Switch II (60–76 aa). B, Frequency of each cancer type associated with all mutations, canonical mutations, and noncanonical mutations in RAS, KRAS, NRAS, and HRAS, as observed in the C-CAT database. The values in each row represent percentages and sum to 100%, indicating the relative distribution of cancer types within each RAS mutation class. C, Frequency of each cancer type associated with codon-specific canonical and noncanonical RAS mutations, as observed in the C-CAT database. The values in each row represent percentages and sum to 100%, indicating the relative distribution of cancer types within each RAS mutation variant. D, Frequency of each RAS isoform among RAS mutations, as observed in the C-CAT database.

Figure 2.

Lollipop chart and transforming activity of RAS variants. A, The results of the FFA for each RAS isoform are shown in the lollipop chart. The circle color indicates FFA scores, and the frame color of the circles indicates mutation type. The size of the circles was proportional to COSMIC count. B, The triplicate data of a representative variant in the FFA. C, Correlation of the FFA score of RAS variants with the variant count in the public databases or the OncoKB annotation. The oncogenicity evaluated as the FFA score is shown in colors and compared with the variant count number in the COSMIC, C-CAT, and GENIE databases. The distribution of FFA scores compared with OncoKB annotation is shown in the pie chart.

Figure 3.

Drug sensitivity of KRAS variants to BI-2865. A, 3T3 cells expressing KRAS variants were treated with the indicated concentrations of BI-2865 (a pan-KRAS inhibitor). The viability of cells treated with BI-2865 was measured, and the results are illustrated using the color-coded scale. The IC₅₀ values of BI-2865, the clinical significance annotated in the OncoKB and ClinVar databases, and the mutation type and domain are described at the bottom of the charts. B, 3T3 cells with KRAS variants, HRAS G12V, or GFP were incubated with the indicated concentrations of BI-2865 for 5 days. The cell viability was measured using the PrestoBlue cell viability assay and plotted relative to that of the untreated control. Data are presented as the mean ± SD (n = 6). The IC₅₀ values of inhibitors are shown in the table on the right.