Unraveling the Impact of KRAS Accessory Proteins on Oncogenic Signaling Pathways

The oncogene KRAS drives tumor growth by activating pathways such as MAPK and PI3K-AKT in a constitutive manner. Although direct KRAS inhibitors exist, they are often limited in clinical use due to therapeutic resistance and toxicity. Therefore, alternative combinatorial therapeutic strategies are urgently needed. This study examined the knockout of five KRAS-related proteins—galectin-3 (GAL3), phosphodiesterase delta (PDEδ), nucleophosmin (NPM1), IQ motif-containing GTPase-activating protein 1 (IQGAP1), and SHOC2—using CRISPR-Cas9 in adenocarcinoma cell lines harboring the KRAS(G12V) oncogenic mutation, as well as in the noncancerous HEK-293 cell line. These proteins act as critical modulators that regulate KRAS activity, cellular localization, and that of its downstream signaling components. We analyzed the downstream activation of ERK and AKT kinases and evaluated subsequent cancer cell proliferation. Knockout of GAL3 and PDEδ was highly effective, significantly reducing MAPK and PI3K-AKT pathway activity and substantially impairing cell proliferation. SHOC2 knockout selectively and potently disrupted MAPK activation, while NPM1 knockout resulted in the complex, reciprocal modulation of the two major pathways. Notably, knocking out IQGAP1 enhanced PI3K–AKT and mTORC2–AKT signaling without affecting the MAPK pathway. These distinct modulatory roles highlight the non-redundant functions of the accessory proteins. In conclusion, our findings establish GAL3 and PDEδ, two KRAS-associated proteins, as promising combinatorial drug targets. Targeting these modulators provides an effective alternative strategy to overcome resistance mechanisms and enhance the clinical utility of existing KRAS inhibitors.