Assistant Professor of Surgery
The major effort in the lab is directed towards investigating how tumor-specific dysregulation of the pRB signaling pathway affects downstream gene expression and the cellular response to DNA damage. Four projects are currently underway. First, we are utilizing a modified chromatin immunoprecipitation approach to capture and identify genomic DNA target sequences conditionally associated with pRB-containing complexes recovered from intact chromatin in untransformed primary human cells. Second, we are investigating functional heterogeneity amongst closely related components in the pRB pathway. Specifically, we are conducting comparative analyses of the INK4 proteins p16INK4a and p18INK4c and their preferred target kinases, the cyclin dependent kinases cdk4 and cdk6. Third, in collaboration with Dr. Jeff Marks, we are developing a mammary gland organoid approach to quantitate and analyze parity-dependent DNA damage checkpoint responses in the context of the primary human mammary tissue. Finally, we are engaged in a collaborative effort with Dr. Francis Ali-Osman to investigate the role of the glutathione-S-transferase protein P1 (GSTP1) in conferring chemotherapeutic drug resistance in human gliomas. To date, we have made significant progress in these projects. We have isolated a collection of genomic clones that are preferentially bound by pRB in senescent primary human mammary epithelial cells, and we intend to characterize these candidate regulatory elements as potential downstream targets of pRB-mediated gene regulation. Interestingly, the majority of the captured sequences do not contain canonical E2F binding sites, a finding that supports our approach of seeking pRB-bound sequences without the limitation of prior assumptions regarding the identity of the DNA-binding transcription factor bound by the pRB complex. In our comparative study of the closely related INK4 proteins p16INK4a and p18INK4c, we have found that differential substrate kinase preference may provide a molecular explanation for why p16INK4a but not p18INK4c is selectively targeted for inactivation in human tumors. In an extension of our previously published studies in T-cell acute lymphoblastic leukemias, we have determined that p18INK4c is highly expressed in a series of medulloblastoma cell lines (derived by Dr. Hai Yan), and that these same cell lines have selectively lost p16INK4a expression. Re-introduction of p16INK4a into these cells induces complete cell cycle arrest, but exogenous expression of p18INK4c has no effect on proliferation. Molecular analysis of p16INK4a and p18INK4c complexes in these cells indicates that p16INK4a associates preferentially with cdk4 whereas p18INK4c binds cdk6. Although cdk4 and cdk6 are highly similar (71% amino acid identity) and are generally assumed to be functionally redundant, we have found that these two kinases differ in their ability to bypass INK4-protein induced cell cycle arrest. In the context of medulloblastomas, we are currently testing the hypothesis that cdk4 and cdk6 execute opposing functions, with cdk4 activity driving proliferation and cdk6 activity inducing differentiation and cell cycle exit. A manuscript describing these findings is currently in preparation.
Previously, we have observed that mammary glands isolated from age-matched parous and nulliparous mice differed in their response to gamma-irradiation. Essentially, glands isolated from nulliparous animals failed to undergo a DNA damage checkpoint arrest, whereas glands from parous animals ceased cellular proliferation following exposure to mutagenic insult. In collaboration with Dr. Jeffrey Marks, we will analyze luminal and basal epithelial primary cells isolated from human reduction mammoplasty tissue with the goal of identifying parity- and compartment-dependent differences in checkpoint functional response.
In collaboration with Dr. Francis Ali-Osman, we are investigating the role of GSTP1-containing protein complexes in mediating drug resistance in gliomas. Our approach exploits Dr. Ali-Osman’s extensive background in chemotherapeutic drug resistance and GSTP1 activity and my laboratory’s expertise in identifying and characterizing protein:protein interactions and functional determinants of checkpoint response and apoptosis. Through biochemical enrichment and utilization of the new Duke Proteomics facility, we have begun systematically identifying GSTP1-associated proteins from extracts of cultured human glioma cells. We will then determine how the interacting proteins contribute to GSTP1-mediated chemotherapeutic drug resistance and other functional readouts of GSTP1 activity. Using this approach, we have found that the tissue transglutaminase TGM2 forms a dynamic, non-covalent complex with GSTP1 in actively dividing gliomas, and that this complex confers resistance to clinically important DNA-damaging drugs such as cisplatin (manuscript in preparation). In the coming year, we plan to extend these results to in vivo systems and investigate whether interfering peptides that disrupt the complex could serve as sensitizing agents to improve chemotherapeutic response to cisplatin.
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