Overview

Part of my dissertation entitled “The Role of FLICE-Inhibitory Proteins in Primary Effusion Lymphoma (PEL).”

This paper is published in the prestigious journal Cell Death & Differentiation – the first and current top journal specific to the vast and important field of cell death. Notably, this paper was selected for the “CDDpress Monthly Reader’s Choice.” CDDpress is amalgamation of three cell death journals published under a collective arm of the world-renowned Nature Publishing Group.

In this work I make use of high-throughput CRISPR-based methods to understand why a human protein is needed to promote the survival of PEL (a cancer caused by the virus KSHV) cells despite the presence of a viral mimic of this protein. In the process I uncover novel regulators and contributors to a recently identified and enigmatic, “non-canonical” cell death process. This has relevance to understanding key aspects of a range of diseases beyond PEL, including other types of cancer, cardiac ischaemia (heart attack), and the rare genetic disorder Charcot-Marie-Tooth neuropathy. Beyond this, my offer the first evidence that can point towards a deeper understandig of general cell biology, in particular how secreted and transmembrane proteins are synthesized.

Access

Download the open access pre-print (bioRxiv)

Full manuscript available online Cell Death and Differntiation

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.