Co-targeting cystathionine gamma-lyase and mTOR: a promising therapeutic strategy in clear cell ovarian cancer.

Clear cell ovarian carcinoma (CCOC) is a rare, aggressive ovarian cancer subtype, comprising approximately 12% of epithelial ovarian carcinoma (EOC) cases in Western countries yet associated with the worst prognoses due to its chemoresistance. Most CCOCs harbor activating PIK3CA mutations that drive PI3K/AKT/mTOR signaling and HIF1α expression. Despite their clinical success in some cancers, mTOR inhibitors have shown limited efficacy in CCOC, even though this pathway is highly active. Consequently, CCOC continues to be managed like other epithelial ovarian cancers. We recently uncovered cystathionine γ-lyase (CTH), a key enzyme in the transsulfuration pathway, as a metabolic vulnerability in CCOC, supporting HIF1α expression, survival and metastasis. This study aims to address whether simultaneously targeting CTH and mTOR could enhance therapeutic outcomes beyond those achieved by mTOR inhibition alone.CCOC lines were engineered with CTH knockout (KO) or treated with the CTH inhibitor Aviglycine hydrochloride (AVG; ABG-3168); mTOR was blocked with everolimus, a clinical stage mTOR inhibitor. Single and combined treatments were tested in two-dimensional cultures and three-dimensional spheroids under normoxia (21% O2) and hypoxia (1% O2). We measured viability, proliferation, colony formation, apoptosis, and protein signaling. Drug interaction was quantified with a synergy model.Everolimus reduced viability and increased apoptosis across CCOC cell lines, but activity was modest as a single agent. Adding CTH inhibition produced synergistic growth suppression at low doses, with the effect being stronger under hypoxia. The combination reduced colony formation over 14 days and decreased spheroid size while increasing caspase-3/7 activity. Dual treatment decreased HIF1α protein expression more than either monotherapy while maintaining strong inhibition of mTOR/S6 kinase signaling; total mTOR and S6 kinase were unchanged. In CTH-deficient models, the synergy was maintained; however, pharmacological inhibition of CTH with AVG did not produce additional effects, indicating dependence on CTH activity and supporting an on-target mechanism. Similar antiproliferative effects with combined treatment were observed in a CTH-expressing Ewing sarcoma (EwS) model.Together, these findings support biomarker-guided strategies and rational combination therapies that achieve dual targeting of mTOR and CTH, thereby disrupting protein translation and hypoxia adaptation in CCOC and other CTH-expressing cancers.