Abstract PR07: Systematic genetic interaction maps reveal rewiring of the stress response network and resulting vulnerabilities in leukemia and multiple myeloma cells

Abstract

Abstract Systematic, high-density mapping of genetic interactions is a powerful approach to elucidate functional pathways and reveal synthetic lethal gene pairs, and has successfully been applied in microorganisms. We have recently developed a functional genomics platform that enables the construction of high-density genetic interaction maps in mammalian cells. In a first step, we conduct pooled primary screens using an ultracomplex shRNA library that targets each protein-coding gene with 25 independent shRNAs and contains >1,000 negative control shRNAs. This strategy enables us to robustly identify hit genes and shRNAs that target them effectively, while minimizing the identification of false-positive hits, which has plagued many genome-wide RNAi screens. In a second step, we construct and screen a double-shRNA library targeting all pairwise combinations of hit genes of interest to construct a high-density genetic interaction map. Thus, our approach enables us to determine 100,000s of genetic interactions in a single experiment. Here, we present the application of our platform to identify adaptations and vulnerabilities in the stress response network of leukemia and multiple myeloma cells. Stress response pathways, including the unfolded protein response, starvation, hypoxia and oxidative stress responses, and the associated induction of autophagy, play important roles in cancer cell survival, drug resistance and tumor progression. The goal of the research presented here is to systematically characterize vulnerabilities in the stress response network of cancer cells, and in particular, to identify synthetic-lethal vulnerabilities that are potential new targets for combination drug therapy. We conducted our first experiments in two human cell lines derived from hematologic malignancies, K562 (leukemia) and RPMI-8226 (multiple myeloma), for which we determined genetic vulnerabilities and their genetic interactions in the absence and presence of stress-inducing agents. We discovered several genetic vulnerabilities that can be targeted pharmacologically. An important class of factors we detected as vulnerabilities in our screens are Hsp70 proteins, which are commonly upregulated under stress conditions and in cancer cells. We have recently synthesized a series of small-molecule Hsp70 inhibitors, which we have successfully used to selectively kill cancer cells. Using a chemical-genetics approach, we have probed the genetic factors affecting the sensitivity of cancer cells to two of these inhibitors with selectivity for Hsp70 proteins in different subcellular compartments. We externally validated several of the synthetic-lethal vulnerabilities identified in our screens by demonstrating synergistic effects of drug combinations targeting these gene pairs in panels of cancer cell lines. Comparison of drug sensitivities across our cell line panel, in conjunction with our experimentally derived set of genetic vulnerabilities, has generated testable hypotheses for the role of the genetic background in determining vulnerabilities related to the stress response network. Intriguingly, expression levels of several genes we identified as vulnerabilities in multiple myeloma cells are prognostic of patient survival in a published multiple myeloma clinical trial. This abstract is also presented as poster B25. Citation Format: Martin Kampmann, Diego Acosta-Alvear, Min Cho, Yuwen Chen, Xiaokai Li, Luke Gilbert, Blake T. Aftab, Jason Gestwicki, Peter Walter, Jonathan S. Weissman. Systematic genetic interaction maps reveal rewiring of the stress response network and resulting vulnerabilities in leukemia and multiple myeloma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR07.