Unbiased CRISPR Synthetic Lethal Screening for Genetic Vulnerabilities in Succinate Dehydrogenase (SDH)-loss Model of Paraganglioma

Abstract Succinate dehydrogenase (SDH)-deficient paraganglioma and pheochromocytoma (PPGL) are rare neuroendocrine tumors for which no effective targeted therapies currently exist. To uncover new potential therapeutic targets, we performed an unbiased CRISPR-Cas9 genetic screen in immortalized mouse chromaffin cells (imCCs) with and without Sdhb loss. Our screen identified genes that differentially affect cell proliferation in Sdhb-deficient versus normal imCCs. Notably, several subunits of the transcriptional Mediator complex emerged as potential tumor suppressors, as their loss selectively promoted growth of Sdhb-deficient cells. Most strikingly, we found that the neddylation pathway—required for ubiquitin-mediated selective protein degradation—plays a critical role in controlling cell growth and survival in Sdhb-deficient imCCs. Specifically, loss of the neddylation regulator Ube2m led to increased proliferation, while loss of Ube2f suppressed growth of Sdhb-deficient imCCs. Consequently, global neddylation inhibitor MLN4924 (Pevonedistat) and UBE2F-CRL5 axis inhibitor HA-9104 were shown to downregulate neddylation, suppressing UBE2F activity and selectively inhibiting growth of Sdhb-deficient imCCs. This unexpected result highlights the neddylation pathway as a promising druggable vulnerability in this cell culture model of SDH-deficient PPGL. Competing Interest Statement The authors have declared no competing interest. Abbreviations - caRpools - analysis pipeline for pooled CRISPR screens - CRISPR - clustered regularly interspaced short palindromic repeats - DepMap - dependency map - FDR - false discovery rate - GRC - genome reference consortium - imCCs - immortalized mouse chromaffin cells - iMEFs - immortalized mouse embryonic fibroblasts - LOH - loss of heterozygosity - MAGeCK - model-based analysis of genome-wide CRISPR-Cas9 knockouts - MEFs - mouse embryonic fibroblasts - NT - none-targeted - PCR - polymerase chain reaction - PGL - paraganglioma - PHEO - pheochromocytoma - PPGL - pheochromocytoma and paraganglioma - PRRT - peptide receptor radionuclide therapy - RRA - robust rank aggregation - SDH - succinate dehydrogenase - shRNA - short hairpin RNA