Ultra-complex shRNA Libraries Research Articles

High-Complexity shRNA Libraries and PI3 Kinase Inhibition in Cancer: High-Fidelity Synthetic Lethality Predictions

Deregulated signal transduction is a cancer hallmark, and its complexity and interconnectivity imply that combination therapy should be considered, but large data volumes that cover the complexity are required in user-friendly ways. Here, we present a searchable database resource of synthetic lethality with a PI3 kinase signal transduction inhibitor by performing a saturation screen with an ultra-complex shRNA library containing 30 independent shRNAs per gene target. We focus on Ras-PI3 kinase signaling with Tcell leukemia as a screening platform for multiple clinical and experimental reasons. Our resource predicts multiple combination-based therapies with high fidelity, ten of which we confirmed with small molecule inhibitors. Included are biochemical assays, as well as the IPI145 (duvelisib) inhibitor. We uncover the mechanism of synergy between the PI3 kinase inhibitor GDC0941 (pictilisib) and the tubulin inhibitor vincristine and demonstrate broad synergy in 28 cell lines of 5 cancer types and efficacy in preclinical leukemia mouse trials.

The mTOR Complex Controls HIV Latency

A population of CD4T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and invitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Abstract B22: Screening and validation of combination therapy in T cell leukemia

Abstract Oncogenic Ras mutations occur in more than 30% of metastatic cancers. There are no effective inhibitors to block oncogenic K- or N-RAS, however, blockade of Ras’ downstream RAF-MEK-ERK and PI3K-Akt-mTOR-S6 signaling cascades is currently being explored as therapy in clinical trials through the use of potent kinase inhibitors. Nonetheless, many cancers have a complex make-up of cooperating oncogenic lesions and it is therefore questionable if inhibition of single molecules in pathways will provide sufficient, long-term efficacy. To explore effective combination therapies to treat Ras-driven cancers we chose synthetic lethal screens (SLS), focusing on PI3K due to its frequent involvement in oncogenic signaling. We performed a set of SLS on T cell leukemia (T-ALL) cell lines combining specific PI3K inhibitors with high coverage shRNA libraries, initially aiming at kinases. We employed the UCSF EXPANDed RNAi library resource, which dramatically improves RNAi screening compared to commercial resources. This ultra-complex shRNA library targets each gene with >25 independent shRNAs, thus drastically minimizing experimental noise and allowing us to overcome both the common problems of high false-negative rates and high false-positive rates. Using deep sequencing of shRNAs recovered from our screening samples, we identified shRNAs that synergize with inhibitors in these drop-out SLS conditions and which represent key molecules for the survival of T-ALL lines. In addition, we also identified enriched shRNAs that confer inhibitor resistance or enhanced proliferation. To verify our screening results we used combinations of chemical inhibitors, targeting PI3K and molecules identified by our screens. To date, all inhibitor combinations tested caused strong synthetic lethality in T-ALL cells, confirming the robustness of the ultra-complex shRNA libraries used for SLS. The most promising results will be further verified in vivo, and we will expand our efforts to different leukemia and also solid cancer lines. Altogether, our high-troughput platform is a promising approach to identify effective partners for already established inhibitors, leading to novel combination therapies for the treatment of cancer. Citation Format: Marsilius Mues, Marthe F. Lindenbergh, Michael T. McManus, Jeroen P. Roose. Screening and validation of combination therapy in T cell leukemia. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B22.

Abstract A2-57: Identification of genetic vulnerabilities within the proteostasis network of multiple myeloma

Abstract Multiple myeloma (MM) remains an incurable disease afflicting more than 20,000 patients yearly in the US alone. Because of their secretory nature, MM cells rely on the proteostasis network and re-wire it to their advantage. We focused on two interconnected essential pathways within the proteostasis network to query genetic vulnerabilities yielding synthetic lethality. These pathways are the ubiquitin-proteasome system (UPS) and the unfolded protein response (UPR). Standard-of-care proteasome inhibitors, like bortezomib, remain non-curative in MM patients, suggesting the selection of genetic escape routes. The same rationale is applicable to the UPR, a conserved regulatory network overseeing the processing capacity of the endoplasmic reticulum (ER). Since proteasomal degradation of unfolded ER client proteins is accomplished after their dislocation and ubiquitination, we reasoned that common genetic escape routes might exist within the UPS and the UPR. Because single agents provide the driving force for acquired drug resistance and do not lead to long-term remission, we employed a systems-level shRNA screening method that systematically identified synthetic-lethal interactions that can enhance the therapeutic benefit of proteasome inhibition or modulation of the UPR. In the latter, we focused on the UPR branch overseen by the sensor kinase/nuclease IRE1 because it is thought to confer a survival advantage to MM. Applying chemical-genetics approaches using bortezomib or novel chemical inhibitors of IRE1, and an ultra-complex shRNA library we developed, we performed pooled shRNA screens in RPMI-8226 and MM1-S MM cells and identified genes whose diminished function impact the response to proteasome or IRE1 inhibition. Unlike proteasome inhibition, blocking IRE1 did not lead to considerable MM cell death, suggesting that IRE1 alone is not a fate determinant but a weakness that can be exploited through the identification of synthetic-lethal combinations. We applied the same rationale to proteasome inhibition and found synthetic-lethal pairs. Because some of these genes can be targeted pharmacologically, we explored the susceptibility of a panel of MM cells to combinations of bortezomib and drug-like molecules, including novel Hsp70 inhibitors. Retrospective analyses on publicly available gene expression datasets of MM patients treated with bortezomib indicated that several of the genes we found in our screen also predicted clinical outcomes. Together, our preliminary results indicate our approach is a powerful tool for the discovery of synthetic-lethal pairs that can be exploited in combination therapies. Citation Format: Diego Acosta-Alvear, Martin Kampmann, Min Y. Cho, Blake T. Aftab, Xiaokai Li, Jason E. Gestwicki, Marc A. Shuman, Jonathan S. Weissman, Peter Walter. Identification of genetic vulnerabilities within the proteostasis network of multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-57.

Abstract A2-12: Opposing roles of the 19S regulatory and 20S core proteasomal subunits in controlling sensitivity to proteasome inhibitors

Abstract Targeted therapies for cancer are often hindered by the variable genetic makeup within and between tumors. This hurdle makes the identification of therapeutic targets challenging. Genetic susceptibilities found within networks encoded in the genome of a cancer cell can provide such therapeutic opportunities. Essential cellular networks overseeing homeostasis are often rewired in tumors, providing a landscape where synthetic lethal pairs may exist. A prominent example is the susceptibility of multiple myeloma (MM) cells to proteasome inhibitors. Because of their highly secretory nature, the proteasomes of MM cells can be burdened in a load-versus-capacity tug-of-war. This renders MM cells sensitive to proteasome inhibitors. Unfortunately, single agent therapies against the proteasome do not provide a cure, as tumor cells often acquire resistance. Because of this, we sought to understand how MM cancer cells respond and adapt to proteasome inhibition. Utilizing ultra-complex shRNA libraries, we screened several MM cell lines exposed to the proteasome inhibitor bortezomib. In these screens, we identified several genes that either sensitize or protect MM cells towards bortezomib intoxication. Unexpectedly we found that, contrary to knockdown of genes encoding subunits of the 20S core particle of the proteasome, knockdown of genes encoding subunits of the 19S regulatory particle of the proteasome lead to bortezomib desensitization. To understand the nature of this phenotype, we queried proteome changes in bortezomib treated cells where we ablated a single 19S subunit. By performing ubiquitin-remnant profiling and total proteome SILAC experiments, we identified several proteins involved in autophagy and NF-κB signaling as potential mediators of the protective phenotype. We are currently investigating the roles of components of these pathways in tailoring the response towards bortezomib. We surmise that some of these will be amenable for pharmacological targeting in developing novel combination therapies. Citation Format: Min Y. Cho, Diego Acosta-Alvear, Martin Kampmann, Thomas Wild, Chuna Ram Choudhary, Jonathan S. Weissman, Peter Walter. Opposing roles of the 19S regulatory and 20S core proteasomal subunits in controlling sensitivity to proteasome inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-12.

Abstract A43: Opposing roles of the 19S regulatory- and 20S core-proteasomal subunits in controlling sensitivity of multiple myeloma cells to proteasome inhibition.

Abstract Multiple myeloma (MM) remains a devastating incurable disease. Because of their secretory nature, MM cells heavily rely on proteostasis networks to maintain homeostasis. Indeed, paraprotein production by MM cells imposes a protein-folding burden that provides an opportunity for therapeutic intervention: because protein production in MM cells can exceed the buffering capacity of the cell for protein degradation, they are exquisitely sensitive to proteasome inhibitors. While successful, standard-of-care proteasome inhibitors, like bortezomib, remain not curative, as patients ultimately become refractory, suggesting the selection of genetic escape routes. Employing an ultra-complex shRNA library, we performed pooled shRNA screens in RPMI-8226, U-266, and JJN-3 MM cells, and in K-562 leukemia cells, and identified genes whose diminished function impact the cell's response to proteasome inhibition. Unexpectedly, we found that, contrary to knockdown of subunits in the 20S proteasome core-particle, the genetic ablation of subunits within the 19S regulatory-particle led to partial desensitization towards bortezomib. Proteome-wide analyses of in vivo ubiquitylated sites in K562 cells, in which we constitutively ablated a component of the 19S regulatory-particle, revealed potential candidate substrates that were differentially degraded in knockdown cells. We are investigating the mechanistic basis for this differential degradation to pinpoint key substrates mediating proteasome inhibitor resistance. Our results reveal potential escape routes in MM tumor cells to proteasome inhibitors, suggesting new promising targets to pre-empt resistance. Citation Format: Diego Acosta-Alvear, Martin Kampmann, Min Y. Cho, Thomas Wild, Chunaram Choudhary, Marc A. Shuman, Jonathan S. Weissman, Peter Walter. Opposing roles of the 19S regulatory- and 20S core-proteasomal subunits in controlling sensitivity of multiple myeloma cells to proteasome inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A43.

Next-Generation NAMPT Inhibitors For ALL Identified By Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens

Abstract There is a critical need for new agents with novel therapeutic targets and improved safety profiles in high-risk acute lymphoblastic leukemia (ALL), which is a significant cause of morbidity and mortality in pediatric and adult populations. Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of their mechanistically relevant targets remains a major experimental challenge. We applied a chemical genetics approach involving sequential unbiased high-throughput chemical and ultra-complex, genome-scale shRNA screens to address this challenge and identify novel agents in ALL. A cell-based phenotypic high-throughput chemical screen of 115,000 compounds identified 640 compounds that inhibited growth of one or both ALL cell lines with high-risk Mixed Lineage Leukemia (MLL) genetic abnormalities, but did not inhibit the growth of a cell line lacking MLL rearrangement. The most potent and selective 64 were tested on an expanded panel of eight human B-ALL cell lines to identify lead compound STF-118804. STF-118804 inhibited the growth of most B-ALL cell lines with high potency demonstrating IC50 values in the low nanomolar range. Leukemic samples from five pediatric ALL patients were also sensitive to STF-118804 in the low nanomolar range. STF-118804 displayed 5–10 fold more potency against most leukemias in comparison to cycling human (lineage-negative cord blood) and murine (c-kit+ bone marrow) progenitor cells, demonstrating a therapeutic index. STF-118804 displays distinctive cytotoxicity by inducing apoptosis without causing a phase-specific cell cycle arrest. To discover the molecular target of STF-118804, a functional genomic screen was performed to identify shRNAs that conferred sensitivity or resistance to STF-118804, utilizing an ultra-complex (∼25 shRNAs per gene) library targeting in total ∼9300 human genes and 1000s of negative control shRNAs. NAMPT was the most statistically significant gene to confer sensitivity to STF-118804, suggesting that STF-118804 functioned as a NAMPT inhibitor. NAMPT encodes nicotinamide phosphoribosyl transferase, a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. STF-118804 was confirmed as a novel class of NAMPT inhibitor through metabolic rescue, enzymatic, and genetic studies. STF-118804 displayed strong inhibitory activity in in vitro NAMPT enzymatic assays. Over-expression of wild-type or mutant NAMPT in cells indicated that STF-118804 cytotoxicity is a result of its ability to inhibit NAMPT, and that STF-118804 does not have significant off-target effects on cell viability. The potential efficacy of STF-118804 in vivo was assessed in an orthotopic xenograft model of ALL. Sublethally irradiated immunodeficient mice were transplanted with human ALL cells engineered to constitutively express firefly luciferase. Dosing of STF-118804 was initiated two weeks post-transplant when ALL cells had engrafted and bioluminescent signal was detectable. Mice treated with STF-118804 showed regression of leukemia by bioimaging and significantly extended survival. The leukemia initiating cell (LIC) frequency in STF-118804 treated mice was significantly lower (∼8 fold) than vehicle treated mice, showing that STF-118804 was effective in reducing LICs. In summary, tandem high-throughput screening identified a highly-specific, potent, and structurally novel small molecule inhibitor of NAMPT that is active in ALL. Tandem high throughput screening using chemical and ultra-complex shRNA libraries provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation. Disclosures: No relevant conflicts of interest to declare.

Next-Generation NAMPT Inhibitors Identified by Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

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

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.

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

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.

Abstract A08: Systematic identification of genetic vulnerabilities synergizing with proteasome and IRE1 inhibition in multiple myeloma cells using ultra-complex shRNA libraries

Abstract Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow. MM remains incurable; thus, effective therapies remain an unmet clinical need. MM cells produce large amounts of paraproteins, which impose a sustained demand on the endoplasmic reticulum (ER) resulting in chronic ER stress. Standard-of-care therapies, like the proteasome inhibitor bortezomib, take advantage of the reliance of MM cells on ER function. By blocking the proteasome, client protein load exceeds the capacity of the proteasome, inducing unmitigatable ER stress and apoptosis. However, most MM cells develop resistance to bortezomib, indicating that genetic escape routes are activated upon selective pressure. Adaptive measures to counteract ER stress are controlled by the unfolded protein response (UPR), which is orchestrated by a network of three intertwined signaling pathways, the most conserved of which is activated by the ER stress sensor IRE1 and its effector, the transcription factor XBP1. IRE1/XBP1 are part of a cell-protective response that, in healthy cells, permits adaptation in response to upsurges in ER function, while in MM, they are thought to promote survival. We show that genetic susceptibilities of MM cells can be identified to guide combination therapies using bortezomib or a chemical inhibitor of IRE1. To this end, we systematically interrogated genetic vulnerabilities involving the ER stress response in MM cells using an unbiased genome-wide RNA interference approach that employs screening of ultra-complex shRNA libraries. Conducting pooled shRNA screens in conventional and microcarrier-based cell culture systems, we show that, unlike bortezomib, IRE1 inhibition alone does not lead to tumor cell death, suggesting that IRE1 is not a single fate determinant. Rather, we show that IRE1 represents an Achilles heel that allows identification of combinatorial genetic vulnerabilities resulting in synthetic lethality. The same rationale was applied to proteasome inhibition, where we identified several genes whose diminished function resulted in synthetic lethality. In this way, we identified novel pathways that when modulated in parallel with proteasome inhibition block escape routes leading to drug resistance. By applying our screening method, we validated known genetic susceptibilities of MM cells, identified new ER-associated MM survival factors, and uncovered vulnerabilities that result in synthetic lethality when combined with pharmacological inhibition of IRE1 or the proteasome. Analyses of published gene expression datasets from MM patient samples suggest that the expression levels of some of the identified genes are predictive of poor survival. Taken together, our data validate the screening method as a powerful tool for the discovery of diagnostic biomarkers and novel therapy targets. Citation Format: Diego Acosta-Alvear, Martin Kampmann, Michael C. Bassik, Crystal P. Lee, Marc A. Shuman, Jonathan S. Weissman, Peter Walter. Systematic identification of genetic vulnerabilities synergizing with proteasome and IRE1 inhibition in multiple myeloma cells using ultra-complex shRNA libraries. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A08.

Abstract PR06: Systematic genetic interaction maps reveal synthetic-lethal vulnerabilities in leukemia and multiple myeloma

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 (Bassik*, Kampmann* et al. Cell 152, Feb. 14, 2013 *Equal contribution). 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 that targets all pairwise combination of hit genes to contruct a high-density genetic interaction map. 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 synthetic-lethal vulnerabilities in the stress response network of leukemia and multiple myeloma cells. Stress response pathways, including the endoplasmic reticulum stress response (unfolded protein response), heat shock, hypoxia and oxidative stress responses, and the associated induction of autophagy, have been shown to play important roles in cancer cell survival, drug resistance and disease 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 novel targets for combination drug therapy. We conducted our first experiments in two human cell lines derived from hematologic cancers, 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 previously unknown genetic vulnerabilities, several of which are considered “druggable”. We externally validated several of the synthetic-lethal vulnerabilities we identified by demonstrating synergistic effects of drug combinations targeting these gene pairs. Intriguingly, expression levels of several vulnerabilities we identified are prognostic of patient survival in a published multiple myeloma clinical trial. We are in the process of testing the therapeutic potential of targeting the synthetic-lethal vulnerabilities we identified in the multiple myeloma stress response network in primary patient cells and in multiple myeloma mouse models. This abstract is also presented as Poster A15. Citation Format: Martin Kampmann, Diego Acosta-Alvear, Michael C. Bassik, Yuwen Chen, Peter Walter, Jonathan S. Weissman. Systematic genetic interaction maps reveal synthetic-lethal vulnerabilities in leukemia and multiple myeloma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR06.