CRISPR Screens Identify Novel Regulators of cFLIP Dependency and Ligand-Independent, TRAIL-R1-Mediated Cell Death

Abstract

Kaposi′s sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.

Expression Ratios of the Anti-Apoptotic BCL2 Family Members Dictate the Selective Addiction of KSHV-Transformed Primary Effusion Lymphoma Cell Lines to MCL1

“…In the case of PEL, the infected tumor cells neither express nor secrete immunoglobulin molecules. However, recent work from the Gottwein laboratory shows that PEL cell lines need to constitutively counteract death signals originating from the ER and Golgi compartments in the absence of any exogenous trigger (44). Consistent with this, PEL cell lines have elevated expression of genes involved in the unfolded protein response, often triggered by ER stress (4).…”

“…shRNAs were designed using the SplashRNA algorithm (31) using default parameters (http://splashrna.mskcc.org). sh2MCL1 was synthesized as a gene fragment (Twist Bioscience) while shREN was amplified from pZIP-ZsGreen-T2A-Hyg-shRen.713 (44). These shRNA cassettes were assembled in the 3’UTR of a synthetic eGFP gene (Twist Bioscience) via two-fragment Gibson assembly into the pSTZ backbone linearized by EcoRI and BamHI (NEB).…”