Abstract 3159: VHL1 activation with a CRISPR-based gene activation platform causes increased MLKL expression and triggers necroptosis in renal cell carcinoma

Abstract Background: Kidney cancer is a common and deadly disease. Over 90% of all kidney cancers are classified as renal cell carcinoma (RCC), of which clear-cell RCC (ccRCC) is the most predominant subtype. VHL is a tumor suppressor and plays a pivotal role in ccRCC development. While 50% of ccRCC patients present with pathogenic VHL mutations, deep deletions and low VHL expression are also common. Over the past two decades, therapeutic advances have been made for metastatic ccRCC; however, none directly target VHL, and the five-year overall survival for metastatic ccRCC remains ~12%. Here, we used a fusion protein, consisting of a deactivated Cas9 (dCas9) linked to the histone acetyltransferase protein p300, to determine if VHL activation initiates programmed cell death in a preclinical model of ccRCC. Methods: Caki-1 cells are a human ccRCC line, are wild-type VHL with low-moderate expression. Caki-1 cells were stably transduced with our dCas9-p300 system via lentivirus, and then transfected with either sgRNAs targeting VHL or a non-targeting (NT) sgRNA. Ten targeting sgRNAs were designed, with eight starting within -120 bp from the VHL1 transcriptional start site (chr3: 10141778), and two starting within +50 bp after the TSS. VHL gene expression changes from baseline were evaluated by RT-qPCR at 8h, 24h and 48h after sgRNA transfection. At 24-72h, Caki-1 cells were harvested and stained with Annexin V and propidium iodide; apoptosis and necrosis were then assessed by flow cytometry. Last, to determine whether VHL reactivation triggered necroptosis, MLKL gene expression changes were evaluated by RT-qPCR at 8h, 24h and 48h. Results: Four targeting sgRNAs met internal thresholds for evaluation within our dCas9-300 system: sgRNA3 bp), sgRNA4, sgRNA9, and sgRNA10. Significant increases in VHL expression were observed at 8h for sgRNA3 and sgRNA10 (62. 4% and 56. 5%, respectively; both P0. 0001, n=3), at 24h for all four sgRNAs (range 91. 1-236. 1%; all P0. 0001, n=3), and again at 48h for all four sgRNAs (range 84. 4-194. 4%; P0. 0001, n=3). At 48h and 72h, sgRNA4 (6. 78% and 7. 35%) and sgRNA9 (6. 71% and 6. 76%) showed evidence of necroptosis activation. sgRNA4 demonstrated significantly increased MLKL expression at 24h and 48h (199. 6% and 144. 6%; both P0. 0001, n=3) as did sgRNA9 (192. 7% and 235. 6%; both P0. 0001, n=3) suggesting necrosome activation. Conclusions: To our knowledge, this is the first study to associate VHL reactivation with increased MLKL expression and necroptosis initiation. These data support continued development of an experimental system we have pioneered (PMID: 31712774), which will leverage chemical-induced proximity to reactivate VHL. Citation Format: Manfred S. Meng, Ryan M. Kemper, Travis J. Nelson, Nathaniel A. Hathaway, Daniel James Crona. VHL1 activation with a CRISPR-based gene activation platform causes increased MLKL expression and triggers necroptosis in renal cell carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3159.