Abstract A060: Targeting metabolic–DNA repair vulnerabilities in SDHB- and FH-deficient renal cell carcinoma with the novel alkylating agent KL-50
Loss of the tricarboxylic acid (TCA) cycle enzymes succinate dehydrogenase B (SDHB) and fumarate hydratase (FH) in renal cell carcinoma (RCC) leads to accumulation of the oncometabolites succinate and fumarate, which impair homologous recombination (HR) repair through inhibition of KDM4A/B and aberrant H3K9 hypermethylation at DNA break sites, respectively. These alterations disrupt epigenetic regulation and DNA damage response pathways, potentially creating therapeutic vulnerabilities to DNA-damaging agents, including alkylators. We investigated this vulnerability in SDHB- and FH-deficient RCC models by comparing the activity of KL-50, a novel imidazotetrazine alkylator, with the clinically established agent temozolomide (TMZ). TMZ mediates cytotoxicity predominantly via O6-methylguanine DNA adducts, which are directly reversed by O6-methylguanine-DNA methyltransferase (MGMT). In the absence of MGMT, O6-methylguanine mispairs during replication and triggers the mismatch repair (MMR) pathway, leading to futile repair cycling, replication stress, and cell death. However, TMZ efficacy is frequently limited by acquired MMR inactivation. Mechanistically, KL-50 transfers a 2-fluoroethyl group to O6-guanine to generate O6-(2-fluoroethyl)guanine (O6FEtG), which undergoes spontaneous chemical rearrangement in MGMT-deficient cells to form DNA interstrand crosslinks (ICLs), driving MMR-independent cell death. Using CRISPR, we generated SDHB- and FH-deficient Renca RCC models. We tested KL50 sensitivity in Renca isogenic cells by performing in vitro cell viability assays using serial dilutions of TMZ or KL50 to determine half-maximal inhibitory concentration (IC50) values. For in vivo studies, murine flank tumor models were treated with TMZ or KL-50 (25 mg/kg) or vehicle control via oral gavage on a 5-days-on/2-days-off schedule for two cycles. Tumor volumes were monitored twice weekly, with study endpoints defined as tumor burden >2000 mm3 or ≥20% body weight loss, and Kaplan–Meier survival analyses performed using the Mantel–Cox test. To assess the role of MGMT, MGMT-isogenic SDHB-deficient models were generated through stable MGMT overexpression. SDHB- and FH-deficient models demonstrated marked sensitivity to KL-50 relative to TMZ in both in vitro and in vivo settings. Both parental models exhibited baseline MGMT silencing, consistent with the hypermethylated phenotype previously reported in FH- and SDHB-mutant tumors, suggesting enrichment of MGMT-deficient tumors within these RCC subtypes. Restoration of MGMT expression conferred resistance to both KL-50 and TMZ, establishing MGMT status as a key determinant of therapeutic response. Collectively, these findings identify FH- and SDHB-mutant RCC as a molecularly defined subset characterised by epigenetic MGMT silencing, which may confer selective vulnerability to KL-50. Given that KL-50 retains activity independent of MMR status, it has the potential to overcome resistance mechanisms that have historically constrained the clinical utility of TMZ in this disease context. Suparna M. Basu, Ranjini Sundaram, Brian Shuch, Susan Gueble, Juan C. Vasquez. Targeting metabolic–DNA repair vulnerabilities in SDHB- and FH-deficient renal cell carcinoma with the novel alkylating agent KL-50 [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A060.