(656) Genome-wide functional genomics analyses of F508del-CFTR degradation

Background: Over 80% of people with cystic fibrosis (CF) carry a mutation (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) that causes the ion channel to fold inefficiently and be targeted by the endoplasmic reticulum associated degradation (ERAD) system. Even though ERAD was implicated in CF twenty-five years ago, the mechanism by which ERAD recognizes F508del-CFTR is not well-understood. Nevertheless, studies have shown that pharmacologically inhibiting ERAD components rescues F508del-CFTR [1] and thus identifying F508del-CFTR ERAD factors is an important avenue for designing new modulators. Importantly, a mechanism-based strategy for drug design has the potential to treat people who express misfolded CFTR but do not respond to elexacaftor/tezacaftor/ivacaftor. Our goal is to conduct genome-wide CRISPR/Cas9 knockout (CRKO) screens to define the pathways responsible for F508del-CFTR ERAD.

Methods: We employed our established CRKO pipeline to identify client-specific ERAD pathways that target different folding-defective proteins [2] to investigate F508del-CFTR ERAD. Our platform uses fluorescence activated cell sorting to isolate cells with sgRNAs that increase the expression of a fluorescent reporter from a population carrying a genome-wide sgRNA library. We designed an mNeonGreen(mNG)-F508del-CFTR K562 cell line, and our reporter faithfully recapitulated the glycosylation patterns, degradation kinetics, and response to correctors observed in cells endogenously expressing F508del-CFTR.

Results: We determined the effect of knocking out ~20,000 different genes on F508del-CFTR expression. We observed ~780 genes with a significant response, including chaperones, ubiquitin ligases, and other ERAD machinery. Notably, the top E3 ligase, RNF5, was implicated in F508del-CFTR ERAD previously [1], confirming the efficacy of our platform. Gene effect scores were lower than those we observed with simple ERAD substrates [2], leading us to hypothesize that complex misfolded proteins like F508del-CFTR are targeted by multiple pathways. Indeed, knocking out RNF5 and the top E2 ubiquitin-conjugating enzyme, UBE2D3, revealed that the two genes act in independent pathways. Gene-drug interaction studies with RNF5 knockouts and correctors revealed that RNF5-mediated degradation is not mitigated by lumacaftor, Corr-4a, or elexacaftor, suggesting that these drugs do not stabilize the folding intermediate recognized by RNF5. Thus inhibiting ERAD factors like RNF5 is a viable strategy for modulating F508del-CFTR expression beyond that of current correctors.

Conclusions: Our work demonstrates that F508del-CFTR ERAD is a complex process involving multiple pathways. Our platform provides us with a launching point for double-knockout and gene-drug CRKO screens to identify novel gene-gene and gene-drug interactions. We hope to build a comprehensive model for F508del-CFTR ERAD that can inform the next generation of CF therapeutics.

Acknowledgements: This work was supported by NIH/NIGMS 5R01-GM074874-13A1 and the CFF PTAC Postdoctoral Fellowship.

References: [1] Sondo E, Falchi F, Caci E, Ferrera L, Giacomini E, Pesce E, et al. Pharmacological inhibition of the ubiquitin ligase RNF5 rescues F508del-CFTR in cystic fibrosis airway epithelia. Cell Chem Biol. 2018 Jul;25(7):891-905.e8.

[2] Leto DE, Morgens DW, Zhang L, Walczak CP, Elias JE, Bassik MC, et al. Genome-wide CRISPR analysis identifies substrate-specific conjugation modules in ER-associated degradation. Mol Cell. 2019 Jan 17;73(2):377-389.e11.