Abstract C01: Systematic blueprinting of genetic vulnerabilities using ultra-complex shRNA libraries identifies genes synergizing with proteasome inhibition or blockade of IRE1 in multiple myeloma cells
Authors: Acosta-Alvear Diego , Kampmann Martin , Bassik Michael C. , Lee Crystal P. , Cho Min Y. , Aftab Blake T. , Li Xiaokai , Gestwicki Jason E. , Shuman Marc A. , Weissman Jonathan S. , Walter Peter 
Published: August 2014

Abstract
Multiple myeloma (MM) remains a devastating incurable disease. Because of their secretory nature, MM cells rely on proper endoplasmic reticulum (ER) function. The unfolded protein response (UPR) oversees the folding capacity of the ER and adjusts it according to need. Paraprotein production by MM cells imposes an ER burden that provides an opportunity for therapeutic intervention. High ER function exceeds the buffering capacity of the cell for protein degradation, rendering MM cells exquisitely sensitive to proteasome inhibitors. While successful, standard-of-care proteasome inhibitors, like bortezomib, remain palliative, as patients ultimately become refractory, suggesting the selection of genetic escape routes. ER and proteasome functions are intimately connected. Proteasomal degradation of unfolded ER client proteins is accomplished after dislocation and ubiquitination of unfolded proteins from the ER. Therefore, genetic escape routes can exist within the ubiquitin-proteasome system and the UPR. The most conserved branch of the UPR, overseen by the sensor kinase IRE1 and its downstream effector, XBP1, has been a primary focus of the MM research community in recent years. In healthy cells IRE1/XBP1 allow adaptation to rises in ER function, while in MM cells IRE1/XBP1 are thought to confer a survival advantage.
For these reasons, we hypothesized that modulation of the UPR, alone or in combination with proteasome inhibitors, while therapeutically beneficial, might also provide the driving force for acquired drug resistance. Therefore, a single agent is unlikely to lead to long-term remission. Here we show that the therapeutic benefit of blocking the proteasome or IRE1 in MM cells can be enhanced when combined with additional therapies targeting synthetic-lethal interactions, and that these can be systematically identified using an shRNA screening method developed by our groups. Employing an ultra-complex shRNA library, we performed pooled shRNA screens in RPMI-8226 and MM1-S MM cells and identified genes whose diminished function impact the cell's response to proteasome or IRE1 inhibition. Importantly, we show that unlike proteasome inhibition, blocking IRE1 function does not lead to substantial MM cell death, strongly suggesting that IRE1 alone is not a fate determinant. Instead, we posit IRE1 is a handicap that might be therapeutically exploited through the identification of synthetic-lethal combinations. We applied the same rationale to proteasome inhibition and found several synthetic-lethal pairs. Some of these genes can be targeted pharmacologically, which allowed us to query the susceptibility of a panel of MM cells to combinations of bortezomib and several drug-like molecules, including novel Hsp70 inhibitors. Our results inform which pathways may be selectively blocked to shut down potential escape routes that may lead to drug resistance. Notably, retrospective analyses on publicly available gene expression datasets of MM patients indicate that several of the genes we found on our screens are predictors of poor survival. Taken together, our preliminary results indicate our screening platform is a powerful tool for the discovery of (i) novel therapy targets, (ii) synthetic-lethal pairs that can be exploited in potential combination therapies, and (iii) diagnostics biomarkers. Currently, we continue to validate our results in additional cell lines prior to verification in primary patient samples and in an orthotopic xenograft model developed by our groups (DAA and PW, unpublished).
Citation Format: Diego Acosta-Alvear, Martin Kampmann, Michael C. Bassik, Crystal P. Lee, Min Y. Cho, Blake T. Aftab, Xiaokai Li, Jason E. Gestwicki, Marc A. Shuman, Jonathan S. Weissman, Peter Walter. Systematic blueprinting of genetic vulnerabilities using ultra-complex shRNA libraries identifies genes synergizing with proteasome inhibition or blockade of IRE1 in 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 C01.