W. Lee Kraus

Cancer-related somatic histone mutants are present in 4% of diverse tumor types and display a similar genomic prevalence to that of other somatic mutations in cancer, but they are not well characterized. In this study, we curated and characterized 17 of the most frequently recurring missense mutations in histone H2A, H2B, and H3 genes in human breast cancers that occur at glutamate (E), aspartate (D), serine (S), and arginine (R) residues. Many of these mutants co-occur with PIK3CA mutations in breast cancer samples. We expressed these breast cancer–associated histone mutants in MCF-7 and MDA-MB-468 breast cancer cells or MCF-10A mammary epithelial cells and screened them for effects on oncogenic phenotypes in a variety of cell- and tumor-based assays (i.e., proliferation, migration, invasion, competitive outgrowth, and transformation). In addition, we examined the effects of selected mutants on DNA damage, gene expression, and histone modifications. Our results indicate varying phenotypic and functional effects. Some of the histone mutants promoted cancer-related phenotypes, with H2B-E76Q, H3-E97K, and H3-E105K eliciting strong oncogenic phenotypes, alterations in gene expression, and context-dependent functional enhancement in a PI3K-activated background. We further showed that H2B-E76Q and H3-E105K mutants exhibit reduced ADP-ribosylation (ADPRylation) levels compared with their cognate wild-type (WT) histones, resulting in altered H3S10 phosphorylation and H3K27 acetylation in cells. Our results indicate that some histone mutants can have oncogenic activity in breast cancers, contributing to breast cancer pathogenesis and tumor progression. Implications: These findings identify a subset of breast cancer–associated histone mutations with oncogenic activity, highlighting the role of histone mutations in promoting tumor phenotypes through epigenomic alterations and signaling pathway cross-talk.

Breast cancers are molecularly heterogeneous, with subtype-specific differences in transcriptional programs, chromatin architecture, and therapeutic responses. Although poly(ADP-ribose) polymerase (PARP) 1 has been extensively studied in the context of DNA repair, emerging evidence implicates its catalytic activity in a broader set of cellular processes, including the regulation of gene expression. In this study, we employed a nicotinamide adenine dinucleotide (NAD+) analogue–sensitive PARP1 chemical genetics approach combined with mass spectrometry to map the ADP-ribosylated proteome across 6 human breast cancer cell lines representing luminal and basal/triple-negative subtypes. We identified thousands of PARP1 substrates and hundreds of Glu/Asp ADP-ribosylation (ADPRylation) sites, revealing both shared and subtype-specific modifications in cell lines maintained under basal growth conditions. Luminal-specific substrates were enriched in chromatin and transcriptional regulators, whereas basal-specific substrates were preferentially linked to translation and RNA processing, highlighting lineage-dependent PARP1 activity. Transcription factors emerged as major substrates, with TFAP2A serving as a proof of concept; it is selectively ADPRylated in luminal cells, and inhibition of PARP1-mediated ADPRylation modulates its promoter occupancy in a subtype-specific manner. Our data provide a new resource for studying subtype-specific PARP1-mediated ADPRylation in breast cancer cells. Collectively, our findings expand the conceptual framework for PARP1 function beyond DNA repair, offering mechanistic insights into subtype-specific gene regulation and potential determinants of PARP inhibitor sensitivity in breast cancer. Implications:PARP1-driven ADPRylation encodes subtype-specific transcriptional programs in breast cancer, revealing a broader role for PARP1 in gene regulation that may influence therapeutic response.

Characterization of the total proteome of six breast cancer cell lines using mass spectrometry.

Characterization of the ADPRylated proteome of six breast cancer cell lines determined by an asPARP1 approach with mass spectrometry.

Transcription factors, histones, histone modifying enzmes, and ATP-dependent chromatin remodeling complexes that are ADPRylated by PARP1 in breast cancer cells. ADPRylation was determined using the asPARP1 approach in six different breast cancer cells lines.

Identification of TFAP2A PARylation sites by mass spectrometry.

Breast cancer cell lines exhibit different levels of PARP1 activity.

Purification of asPARP proteins.

Comparison of ADPRylation substrates and site IDs across studies.