Project Summary/Abstract The molecular heterogeneity of cancers poses a major hurdle for treatment and drug discovery efforts. Previous studies have addressed this challenge by characterizing distinct molecular subtypes of specific cancers (e.g., breast cancers), based on cell type-specific patterns of gene expression. To interrogate the molecular underpinnings of cancer subtypes, the Kraus Lab has developed a robust and multi-faceted computational pipeline that integrates data from various genomic assays to define a Total Functional Score of Enhancer Elements (TFSEE) for each subtype. One outcome of this method is the identification of cancer subtype- enriched transcription factors (TFs) that promote subtype-specific enhancer formation and drive downstream transcriptional outcomes. In breast cancers, several TFSEE-identified subtype-specific TFs are uniquely required for the growth of the cognate breast cancer subtype, but do not affect the proliferation or viability of other subtypes. Recent studies have also shown that ADP-ribosylation (ADPRylation), a post-translational modification of proteins, varies dramatically across the different subtypes of breast cancers. ADPRylation is mediated by the Poly(ADP-ribose) polymerase (PARP) family of enzymes, including PARP-1, a nuclear enzyme which is the target of FDA-approved PARP inhibitor drugs. PARPs are well known for the roles in DNA repair, but recent studies suggest an important BRCA1/2-independent role in transcriptional regulation as well. In preliminary analyses, we have identified a cohort of cancer-related TFs that are ADPRylated in breast cancers. The long-term objective of these studies is to achieve a better understanding of the molecular and biochemical mechanisms underlying the regulation of breast cancer subtype-specific TFs by ADPRylation, as well as the responses of distinct breast cancer subtypes to PARP inhibitors. Our hypothesis is that ADPRylation of subtype-specific TFs dictates their function and may influence the response of breast cancer cells to PARP inhibitors. We have proposed a project that will use an integrated set of biochemical, molecular, cell-based, mouse-based, genomic, and proteomic assays to test our overarching and specific mechanistic hypotheses. Specifically, we will: (1) Identify TFs that are ADPRylated in breast cancers (Aim 1), (2) Determine how ADPRylation of TFs affects their molecular and biochemical functions (Aim 2), and (3) Determine the effects of TF ADPRylation on the responses of breast cancer cells to clinically used PARP inhibitors (Aim 3). These studies will take advantage of the expertise of the PI’s lab in PARPs, ADPRylation, enhancer function, and gene regulation in cancer. Although focused initially on breast cancers, our results should be broadly applicable across a variety of cancer types. Our integrative approach using ‘omics’ and functional assays will provide new insights into the regulation of TF ADP-ribosylation in breast cancers that will serve as a model for how to explore PARP function and ADP-ribosylation in cancer cells. The use of mouse-based models and patient samples will allow us to explore the clinical relevance of our mechanistic results. We anticipate that our studies will suggest new avenues for the therapeutic potential of PARP inhibitors in cancers beyond DNA repair pathways.

Project Narrative/Relevance Understanding the molecular mechanisms that drive cancer formation and responses to therapeutic interventions will improve our ability to diagnose and treat cancer, which is one of the leading causes of death in the U.S. We have proposed an integrative set of studies that will provide new information about the role of transcription factor ADP-ribosylation by nuclear PARPs in breast cancers, as well as their role in determining the unique biology and therapeutic responses of different breast cancer subtypes to PARP inhibitors. We anticipate that these studies will suggest new avenues for the therapeutic potential of PARP inhibitors in cancers beyond DNA repair pathways.