Abstract IA009: Differential Biology of KRAS Splice Variants

The excitement generated by the advent of direct inhibitors of KRAS (KRASi) has been dampened by the observation that they do not produce durable responses in KRAS-driven cancer. This has stimulated renewed interest in the biology of KRAS signaling that might reveal vulnerabilities that synergize with KRASi. Metabolic vulnerabilities are among those that hold the most promise. The KRAS locus produces two proteins, KRAS4A and KRAS4B, both of which are oncogenic when the locus sustains an activating mutation. The isoforms differ exclusively in their hypervariable regions (HVR), which dictate differential trafficking within the cell. We have reported that when KRAS4A becomes depalmitoylated it translocates to the outer mitochondrial membrane (OMM) where it interacts with and activates hexokinase 1 (HK1). Using a variety of biochemical and cell biological methods, including single molecule tracking, we now show that KRAS4A promotes oligomerization of HK1 on the OMM. To better characterize the differential interactomes of the two KRAS splice variants we performed a proximity labeling screen. Among the 24 proteins preferentially labeled by KRAS4A was BIRC6 and methionine synthase (MTR). BIRC6 is the largest member of the inhibitor of apoptosis protein (IAP) family and functions as an E2/E3 ubiquitin ligase. We found that BIRC6 mono- and di-ubiquitinates KRAS4A on the Golgi rendering it relatively insensitive to GAP. MTR is one of only two mammalian enzymes that utilize B12 as a cofactor and is essential to sustain the folate cycle and thereby one-carbon metabolism. One-carbon metabolism has been the target of chemotherapeutic drugs for more than 7 decades. We validated a direct, isoform-specific interaction between KRAS4A and MTR that depends on both the KRAS4A G-domain and its HVR. We employed radiometric and colorimetric assays to measure MTR activity. Strikingly, KRAS4A stimulated enzyme activity. Kinetic analysis reveals an increase in the Vmax without change in KM, suggesting that KRAS4A is an allosteric activator of MTR. These studies demonstrate differential biology among the two KRAS isoforms that must be taken into account when designing inhibitors and suggest metabolic vulnerabilities for KRAS-driven tumors that express relatively high levels of KRAS4A. Citation Format: Mercedes Fissore-O'Leary, Wenjuan Su, Cristina Nuevo Tapioles, Juan Kochen-Rossi, Mark R. Philips. Differential Biology of KRAS Splice Variants [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr IA009.