Abstract 3495: The CCT chaperonin is a regulator of mutagenic APOBEC3A activity

Abstract

APOBEC3 (A3) enzymes deaminate DNA cytosine bases and are implicated as the cause of a prevalent mutational signature found in cancer genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes. A3 enzymes normally function as part of the innate immune system by deaminating viral genomes to restrict infection. Basal expression of A3 enzymes is low in healthy tissues, but is increased by type I interferon signaling. Aside from interferon induction, the mechanisms regulating expression and activity of A3 enzymes are unknown. Thus the circumstances that allow for off-target activity of A3 on cellular DNA, leading to mutations and genome instability, cannot be predicted. We sought to determine protein interactors of A3 enzymes to identify potential regulators of expression and/or activity. Using a doxycycline-inducible expression system, we introduced A3 genes into isogenic cell lines. Each A3 enzyme was immunoprecipitated (IP) and lysates were analyzed by mass spectrometry (MS). MS analysis of A3A interactors revealed all 8 subunits of the TriC/CCT (TCP-1 ring complex or chaperonin containing TCP1) complex. The CCT complex is a molecular chaperone that assists in folding of many newly-synthesized proteins, preventing aggregation and facilitating protein function. Validation of MS results by A3A IP followed by immunoblotting confirmed interaction with CCT complex subunits. Endogenous A3A levels are increased significantly by interferon in peripheral blood mononuclear cells (PBMCs). We performed IP of CCT in PBMCs followed by immunoblot for A3A, which demonstrated an interaction between endogenous A3A and the CCT complex. To evaluate the impact of the CCT-A3A interaction, we used siRNA to knock down the CCT complex. Although CCT is an essential chaperonin, transient knockdown of individual subunits resulted in minimally decreased cell viability. However, knockdown of CCT subunits combined with induction of A3A expression led to significant decrease in cell proliferation and increase in cell death. To determine the mechanism by which CCT knockdown with A3A expression results in cytotoxicity, we performed an in vitro deamination assay on lysates from cells that were transfected with siRNA targeting CCT subunits and/or treated with doxycycline to induce A3A expression. Quantification of deamination activity demonstrated that CCT knockdown resulted in increased in A3A activity relative to expression levels. Finally, we evaluated cancer genome sequences in TCGA and found an enrichment of the mutational signature attributed to A3 deamination in cancers with deleterious mutations in CCT subunit genes. Together, these data suggest that the CCT complex regulates A3A activity, and disruption of CCT function results in increased A3A mutational activity. Citation Format: Abby M. Green, Ariel S. Dineen, Katarzyna Kulej, Julia H. Szeto, Matthew D. Weitzman. The CCT chaperonin is a regulator of mutagenic APOBEC3A activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3495.