Abstract A031: A metabolic weak spot: GJB3–SLC7A11 Synthetic lethality in colon cancer

Abstract Colon adenocarcinoma frequently harbors activating KRAS mutations (∼35–45%), which are associated with aggressive disease, poor prognosis, and resistance to anti-EGFR therapies. Oncogenic KRAS constitutively activates downstream effector signaling, particularly the RAS–RAF–MEK–ERK cascade, driving uncontrolled proliferation and survival. As direct targeting of KRAS remains clinically limited, identifying downstream effectors and context-specific vulnerabilities is essential for developing alternative therapeutic strategies. Methods Publicly available colorectal cancer gene expression datasets from GEO were analyzed to identify KRAS-regulated genes through comparisons of KRAS-mutant and wild-type samples. Dependency and expression data from DepMap were used to assess context-specific vulnerabilities. Colorectal cancer cell lines were subjected to lentiviral shRNA-mediated silencing of GJB3 and SLC7A11, individually or in combination, with knockdown confirmed by qPCR and immunoblotting. Proliferation, migration, and anchorage-independent growth were assessed using standard functional assays. RNA sequencing following GJB3 silencing was performed to identify altered stress and metabolic pathways. FRA1 binding to the GJB3 promoter was assessed by ChIP-PCR, and gap junction–mediated cystine transfer was evaluated using metabolite transport assays. AI-based language tools were used to assist with grammar and clarity during manuscript preparation. Results Analysis of GEO datasets identified GJB3 as one of the most strongly upregulated genes in KRAS-mutant colorectal cancers. GJB3 expression positively correlated with KRAS signaling, and mechanistic studies demonstrated direct transcriptional regulation by the KRAS-controlled transcription factor FRA1, establishing GJB3 as a downstream KRAS effector. Functional assays revealed that GJB3 silencing significantly impaired proliferation, migration, and spheroid formation. Transcriptomic profiling following GJB3 depletion showed activation of the integrated stress response, marked by GCN2 signaling, indicating metabolic stress. As GJB3 encodes the gap junction protein connexin-31, we demonstrated that cystine is transferred through CX31-mediated gap junctions. Dependence on this pathway was cell line–specific: GJB3-dependent cells such as SW480 exhibited low expression of the canonical cystine transporter SLC7A11, whereas SLC7A11-high cell lines (HCT116, HT29) were resistant to GJB3 loss. Importantly, dual silencing of GJB3 and SLC7A11 induced a strong synthetic lethal effect in otherwise GJB3-independent cells. Conclusion These findings identify GJB3 as a previously unrecognized KRAS effector that supports colorectal cancer growth through gap junction–mediated cystine uptake. Variable SLC7A11 expression dictates reliance on GJB3, revealing a context-dependent metabolic vulnerability. Targeting the GJB3–SLC7A11 cystine uptake axis represents a potential therapeutic strategy for KRAS-driven colorectal cancers resistant to current treatments. Citation Format: Disha Acharya, Balkrishna Chaube, Sudhanshu Shukla. A metabolic weak spot: GJB3–SLC7A11 Synthetic lethality in colon cancer [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 A031.