Abstract BACKGROUND: Belzutifan is an FDA approved small molecule inhibitor used for the treatment of patients with advanced clear cell renal cell carcinoma (ccRCC). Belzutifan inhibits HIF2α, a transcription factor that promotes tumor angiogenesis and metastasis. Although ccRCC patients can potentially develop resistance to belzutifan there are currently no strategies to counteract this drug resistance in patients. We have identified that serine/threonine protein phosphatase-5 (PP5) expression and activity is elevated in ccRCC, contributing to its pro-survival role. We have designed and developed small molecule inhibitors of PP5. The objective of this study was to examine whether PP5 inhibition can cause apoptosis in belzutifan-resistant ccRCC cells. METHODS: Belzutifan-resistant ccRCC cells were developed by treating 786-O cells with 10μM belzutifan. Once 70% confluent, the cells were split and treated with 10μM belzutifan again. This process was repeated until the cells appeared healthy and grew at a similar rate to 786-O without belzutifan treatment. Belzutifan was removed from the cells for at least 24 hours before additional drug treatments and/or protein extraction. Compound P053 is a second-generation small molecule inhibitor of PP5. Belzutifan-resistant ccRCC cells were treated with either 1μM or 10μM of P053 for 24 hours and apoptotic markers were evaluated by immunoblotting to examine cleaved caspase-3 and cleaved-PARP. RESULTS: The second generation PP5 inhibitor P053 has the ability to bind to the catalytic domain and inhibit the phosphatase activity of PP5. Belzutifan-resistant ccRCC cells are able to maintain growth in the presence of 10μM belzutifan and do not display any hallmarks of activation of apoptosis. Treatment of belzutifan-resistant cells with either 1μM or 10μM of P053, however, induced apoptosis, as evidenced by elevated levels of cleaved caspase-3 and cleaved-PARP. CONCLUSIONS: PP5 inhibition with our novel small molecule inhibitor retains the ability to cause apoptosis in belzutifan-resistant cells. This suggests PP5 is a viable therapeutic target in advanced ccRCC even after belzutifan resistance. Citation Format: Sarah J. Backe, Rebecca Sager, Jennifer Heritz, John Chisholm, Mark Woodford, Dimitra Bourboulia, Gennady Bratslavsky, Mehdi Mollapour. Overcoming drug resistance in ccRCC through inhibition of protein phosphatase 5 (PP5) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1851.
Backe et al. (Fri,) studied this question.