Genome-wide CRISPR Screens Reveal Genetic Mediators of Cereblon Modulator Toxicity in Primary Effusion Lymphoma

Genome-wide CRISPR/Cas9 screens represent a powerful approach to study mechanisms of drug action and resistance. Cereblon modulating agents (CMs) have recently emerged as candidates for therapeutic intervention in primary effusion lymphoma (PEL), a highly aggressive cancer caused by Kaposis sarcoma-associated herpesvirus. CMs bind to cereblon (CRBN), the substrate receptor of the cullin-RING type E3 ubiquitin ligase CRL4CRBN, and thereby trigger the acquisition and proteasomal degradation of neosubstrates. Downstream mechanisms of CM toxicity are incompletely understood, however. To identify novel CM effectors and mechanisms of CM resistance, we performed positive selection CRISPR screens using three CMs with increasing toxicity in PEL: lenalidomide (LEN), pomalidomide (POM), and CC-122. Results identified several novel modulators of the activity of CRL4CRBN. The number of genes whose inactivation confers resistance decreases with increasing CM efficacy. Only inactivation of CRBN conferred complete resistance to CC-122. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 also conferred robust resistance against LEN and POM. Inactivation of additional genes, including the Nedd8-specific protease SENP8, conferred resistance to only LEN. SENP8 inactivation indirectly increased levels of unneddylated CUL4A/B, which limits CRL4CRBN activity in a dominant negative manner. Accordingly, sensitivity of SENP8-inactivated cells to LEN is restored by overexpression of CRBN. In sum, our screens identify several novel players in CRL4CRBN function and define pathways to CM resistance in PEL. These results provide rationale for increasing CM efficacy upon patient relapse from a less efficient CM. Identified genes could finally be developed as biomarkers to predict CM efficacy in PEL and other cancers.\n\nKey PointsO_LIGenome-wide CRISPR/Cas9 screens identify novel mediators of resistance to lenalidomide, pomalidomide and CC-122 in PEL cells.\nC_LIO_LIUBE2G1 and SENP8 are modulators of CRL4CRBN and their inactivation drives resistance to CMs in PEL-derived cell lines.\nC_LI