OR16-02 Histone ADP-Ribosylation Site Mutations As Oncogenic Drivers In Breast And Ovarian Cancers

Disclosure: D. Huang: None. C.V. Camacho: None. A. Nagari: None. W.L. Kraus: Other; Self; - Founder and stockholder for Ribon Therapeutics, Inc. - Founder, stockholder, scientific advisory board member, member of the board of directors for ARase Therapeutics, Inc. - Coholder of U.S. Patent. Recent studies have identified oncohistones, which are encoded by histone genes that harbor missense mutations, as a new class of cancer drivers. We found that many of the oncohistone mutations identified with the highest frequencies in human cancers occur on glutamate (Glu, E) and aspartate (Asp, D) residues, which are potential sites of ADP-ribosylation (ADPRylation). ADPRylation is a post-translational modification, which is catalyzed by the PARP family of ADP-ribosyl transferases and results in the covalent attachment of ADP-ribose on protein substrates. Unlike other well characterized histone modifications, the functions of histone ADPRylation are poorly characterized. Mass spectrometry analyses confirmed that 20 Glu or Asp residues subjected to oncohistone mutation are bona fide sites of ADPRylation by PARP-1. Ectopic expression of each of six different oncohistone mutants from three different core histones (H2B, H3, and H4) altered the proliferation of at least 2 out of 3 cancer cell lines, including MCF-7 and MDA-MB-231 breast cancer cells, as well as OVCAR-3 ovarian cancer cells. We focused on H2B-D51, whose mutation resulted in enhanced cell proliferation in both of the breast cancer cell lines for further characterization of its potential transcription regulatory effects. Proteomic (histone PTMs) and genomic (ATAC-Seq, ChIP-Seq and RNA-Seq) analyses revealed that loss of ADPRylation on H2B-D51 by mutation to alanine (Ala, A) or asparagine (Asn, N) dramatically enhanced p300-mediated acetylation of H2B, altered chromatin accessibility, and changed the pattern of gene expression in MDA-MB-231 cells. To determine the effects of H2B-D51 ADPRylation on cancer cell growth in vivo and their responses to the FDA-approved PARP inhibitor Niraparib, we performed xenograft tumor growth assays in immunocompromised mice using MDA-MB-231 cells ectopically expressing wild-type or D51A mutant histone H2B. Ectopic expression of the H2B-D51A mutant led to a significant increase in tumor growth versus wild-type H2B. Interestingly, these effects were inhibited by treatment with Niraparib, suggesting that the H2B-D51A mutant alone does not confer resistance to PARP inhibitor. Taken together, this study demonstrates that functional sites of Glu/Asp ADPRylation on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of ADPRylation. (This work was supported by grants from the NIH/NIDDK and CPRIT, and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K.) Presentation: Friday, June 16, 2023