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2008 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Jeffrey P. MacKeigan, Ph.D. Laboratory of Systems Biology Dr. MacKeigan received his Ph.D. in microbiology and immunology at the University of North Carolina Lineberger Comprehensive Cancer Center in 2002. He then served as a postdoctoral fellow in the laboratory of John Blenis in the Department of Cell Biology at Harvard Medical School. In 2004, he joined Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, as an investigator and project leader in the Molecular and Developmental Pathways expertise platform. Dr. MacKeigan joined VARI in June 2006 as a Scientific Investigator. Staff Brendan Looyenga, Ph.D. Christina Ludema, B.S. 34 Students Katie Sian, B.S. Natalie Wolters, B.S. Joe Church Halley Crissman Alyse DeHaan Sara Herman Geoff Kraker Matthew McElliott

VARI | 2008 Research Interests The primary focus of the Systems Biology laboratory is identifying and understanding the genes and signaling pathways that, when mutated, contribute to the pathophysiology of cancer. We take advantage of RNA interference (RNAi) and novel proteomic approaches to identify the enzymes that control cell growth, proliferation, and survival. For example, after screening the human genome for more than 600 kinases and 200 phosphatases—called the “kinome” and “phosphatome”, respectively—that act with chemotherapeutic agents in controlling apoptosis, we identified several essential kinases and phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these survival enzymes regulated at the molecular level? What signaling pathway(s) do they regulate? Does changing the number of enzyme molecules present inhibit waves of compensatory changes at the cellular level (system-level changes)? What are the system-level changes after reduction or loss of each gene? Novel modulators of chemotherapeutic sensitization Kinases and phosphatases play an integral role in balancing the survival and apoptotic signals within a cell. In an attempt to define proteins with a major role in these processes, we tested an RNAi library against all known kinases and phosphatases in the human genome and assayed various phenotypes, including sensitization to apoptosis and chemoresistance. A group of apoptosis sensitizers was identified whose siRNA knock-out conferred a marked increase in cell survival as well as a striking chemoresistant phenotype (Figure 1). One of these proteins, MK-STYX, resembles the dual-specificity phosphatases implicated in MAP kinase signaling, but it is catalytically inactive due to a cysteine-to-serine mutation at its active site. When MK-STYX is knocked down via RNAi, the cells display a profound decrease in apoptosis; MK-STYX-overexpressing cells, on the other hand, are sensitized to apoptotic signals. We propose that MK-STYX could function as a dead phosphatase, sequestering potential phosphoproteins that promote survival. Through further experiments, we plan to characterize MK-STYX and elucidate its mechanism of apoptotic sensitization; these studies may identify a survival signal that would constitute a novel target for chemotherapy. Figure 1 Figure 1. Human kinase and phosphatase siRNA library screen. HeLa cells were transfected with siRNAs directed against all known and putative human phosphatases and kinases. Cells were incubated for 72 h to allow target knockdown, and apoptosis was measured by a DNA-fragmentation ELISA. The graph shows relative apoptosis for 600 kinase and 200 phosphatase siRNA targets. 35

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