The differential regulation of poly(ADP-ribose) in cancer and its role in protein

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DESCRIPTION (provided by applicant): Dr. Krukenberg's background is in chemistry and biochemistry, and she completed her Ph.D. in Chemistry and Chemical Biology at the University of San Francisco, California. Under the supervision of Dr. David Agard, she worked on the structural and functional characterization of the molecular chaperone Hsp90. As a postdoctoral fellow at Harvard Medical School, she is training in cell biology under the guidance of Dr. Timothy Mitchison. Dr. Krukenberg's ultimate goal is to lead an academic research group focused on understanding the function and regulation of proteins in signaling pathways and their impacts on cancer. The NIH Pathway to Independence Award would provide necessary training in cell-based assays including microscopy, genomics, proteomics, and bioinformatics as well as in cancer pharmacology and the application of basic science to translational research. In pursuit of her goals, Dr. Krukenberg is investigating poly(ADP-ribose) polymerases (PARPs) and their relevance to cancer. PARPs catalyze the addition of poly(ADP-ribose) onto acceptor proteins involved in a variety of processes including the DNA damage response, inflammation, and transcription. PARPs also regulate the stability of a handful of proteins with diverse biological functions. Multiple PARP inhibitors are currently in clinical trials, either in combination with DN damaging agents or as single agents in cancers deficient in DNA damage repair (BRCA1/2 deficient cancers). Recent studies suggest that PARP inhibitors may have utility in treating some cancers independent of DNA damage. Understanding the broader roles of PARPs and pADPr may provide new therapeutic targets and expand the clinical uses of PARP inhibitors. Using an assay, which Dr. Krukenberg developed, for quantitating pADPr levels, she found that pADPr levels vary widely between breast cancer cells. Preliminary data suggests that this results from PARP1 hyperactivation in some cells. The aims of this proposal are to 1) investigate the mechanism and functional consequences of PARP1 hyperactivation, including its role in drug sensitivity and to 2) explore the role of pADPr modification in regulating protein stability. Cancer signaling pathways regulated by pADPr will be elucidated, and the relationship between pADPr levels and chemotherapeutic responsiveness will be investigated. During the mentored phase (K99), mechanistic studies of PARP1 regulation and initial identification of pADPr modified targets in different cell lines will be completed. A survey of proteins whose stability is influenced by pADPr modification and an exploration of the relationship between pADPr levels and drug sensitivity will also be completed. If the data suggest pADPr levels have potential as a biomarker this exciting direction will be further developed in the independent phase with the initiation of additional clinical collaborations. Also during the independent phase, the biological consequences of pADPr modification on selected pathways will be investigated. Significant progress in understanding the biological function of pADPr and its role in cancer is anticipated. As a co-mentor, Dr. Judith Steen will provide mass spectrometry and proteomics expertise and instrumentation. As a contributor, Dr. Cyril Benes will provide cell lines for analysis along with data on their drug sensitivity and genotype. Dr. Benes will also provide critical expertise in cancer pharmacology. The proposed experiments will further define the biological roles and regulation of PARPs. This study will also determine the potential of pADPr levels in predicting drug sensitivity and identify possible new strategies for the use of PARP inhibitors in the clinic.