Glutamine is a major nutrient involved in several aspects of cancer metabolism. Glutaminase1 (GLS1) initiates that process by converting glutamine to glutamate that is subsequently used in multiple reactions that support tumor cell survival. Accordingly, GLS1 inhibition is a promising approach to treat tumors dependent on glutamate. Here we focus on targeted inhibition of GLS1 to provide a new strategy to enhance the effect of prostate-specific membrane antigen-based radiopharmaceutical therapy (PSMA-RPT) to treat metastatic clear cell renal cell carcinoma (mRCC). mRCC is lethal, with a 5-year survival of only 12%. Although immunotherapy with checkpoint inhibitors has demonstrated improved overall survival, only a minority of patients reliably respond. PSMA displays high expression within the neovasculature of aggressive mRCC, and is a negative prognostic indicator. Because tumor neovasculature is an established target for approved therapies for mRCC, we hypothesize that PSMA-RPT can be a new and effective anti-vascular radiotherapy. Many cancer-associated mutations reprogram the metabolism of mRCC with increased glutamine utilization through GLS1. Inhibition of GLS1 reduces DNA repair in mRCC through decreased nucleotide production and reduces glutathione synthesis while independently making the tumor cells vulnerable to DNA damage. We hypothesize that inhibition of GLS1 will enhance PSMA-RPT by inducing further DNA damage to the cancer cells. Inhibition of GLS1 has not been evaluated with RPT in general, or in PSMA-RPT, specifically. The availability of clinically tested GLS1 inhibitors and PSMA-RPT renders this combined approach rapidly translatable. We discovered small-molecule PSMA targeting, particularly for imaging, and have developed the corresponding radiotherapeutic agents over many years. We recently translated an optimized 177Lu-labeled β- particle-emitting compound (PSMA-R2/L1) for treating prostate cancer. While maintaining tumor uptake, our compound showed significantly lower salivary gland uptake than existing agents in human studies, suggesting that PSMA-RPT with 225Ac-L1 will improve the therapeutic window of α-particle-based radiotherapy. We plan to evaluate 177Lu-L1 and 225Ac-L1 because of the different radiobiologic effects of the particles, including DNA damage activity in the hypoxic tumor microenvironment of mRCC. We have developed several mRCC cell lines in vitro and tumor models with variable GLS1 and PSMA levels and a patient-drived tumor model for a proof-of- concept study. Specific Aims: Aim 1. To access the efficacy of 177Lu-L1 or 225Ac-L1 in combination with a GLS1 inhibitor in vitro. Aim 2. To assess the efficacy of 177Lu-L1 or 225Ac-L1 in combination with a GLS1 inhibitor in vivo in relevant tumor models in orthotopic implantation. If successful, our research may have a near-term and significant impact on improving patient outcomes.

PROJECT NARRATIVE Glutaminase1 (GLS1) is over-expressed in metastatic clear cell renal cell carcinoma (mRCC), providing necessary glutamate for this malignancy to thrive. Here we focus on increasing the efficacy of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical therapy (RPT) by combining it with GLS1 inhibition in mRCC, which expresses abundant PSMA in its vasculature. Because both GLS1 inhibitors and PSMA-RPT have been administered to patients, we intend for rapid translation of our proposed combination therapy.