The identification of a novel orally available ferroptosis inducer for the treatment of clear cell renal carcinoma

BACKGROUND: Background: Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene resulting in constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. HIF-2α is a central driver of kidney cancer progression and is a well validated therapeutic target for ccRCC. Additionally, VHL loss and HIF activation promote lipid accumulation that increases the sensitivity of ccRCC cells to ferroptosis, a form of cell death characterized by iron dependent lipid peroxidation. However, classical inducers of ferroptosis, which induce ferroptosis in vitro, show limited activity in vivo due to possible redundancies in the extracellular space, suggesting a need for novel molecular targets for the induction of ferroptosis in vivo for therapeutic benefit. METHODS: Method: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α and performed additional validation and structure activity relationship studies to identify compounds with improved potency of HIF-2α inhibition. The molecular target of these inhibitors was determined by mass spectrometry using the Drug Affinity Responsive Target Stability (DARTS) assay, and target validation was confirmed using siRNA knockdown and NMR. In vivo anti-tumor efficacy was verified using syngeneic RENCA cells injected into Balb/C mice. Exposure to KD061 was verified using LC-MS/MS detection of KD061 in the plasma and tumors of treated mice. RESULTS: Results: We identified a novel molecule, KD061, which decreases levels of HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. Cell death induced by KD061 was decreased by re-expression of VHL, suggesting synthetic lethality in the context of VHL loss. ISCA2 inhibition also decreased HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreased HIF-1α translation through unknown mechanisms. Treatment of tumor-bearing RENCA-Balb/C mice with KD061 significantly reduced tumor growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Treatment with KD061 was well tolerated at the therapeutic dose with no detectable effects on blood chemistry and blood counts. CONCLUSIONS: Conclusions: Here, we describe the first orally available inducer of ferroptosis with anti-tumor efficacy in vivo. The targeting of ISCA2 using KD061 is a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells. CDMRP DOD Funding: yes