Synthetic Lethal Combinations Research Articles

Abstract B37: Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma

Abstract Despite increasing prevalence and lethality for hepatocellular carcinoma (HCC), the need for effective treatment remains unmet. Metabolic reprogramming during hepatocarcinogenesis promotes cancer cell growth and survival, but also suggests the possibility of identifying as yet unexplored drug targets. In this study, we developed a synthetic lethal drug combination to treat HCC through the inhibition of the energy generation triangle. Most cancers, including HCC, increase glycolysis (the Warburg effect) to ensure sufficient supplies of energy (ATP), reducing equivalents (NADPH), and biochemical building blocks for cell growth and proliferation. Unlike most tumors, which express both hexokinase 1 (HK1) and HK2, many HCC tumors express only HK2, whereas normal hepatocytes only express HK4. We investigated the role of HK2 in HCC using a doxycycline (DOX)-inducible shRNA knockdown system. The HK4-to-HK2 isoform switch during hepatocarcinogenesis results in high sensitivity to HK2 silencing-induced glycolysis inhibition and cytostasis in HCC cells, in contrast to cancer cells expressing both HK1 and HK2. We performed a high throughput screen (HTS) of 3,205 drug-like small molecules and 119 FDA-approved oncology drugs to identify synthetic lethal partners of HK2 knockdown, using the CellTiter-Glo assay for primary HTS and confirmed with the alamarBlue assay. Diphenyleneiodonium (DPI), a mitochondrial complex-I inhibitor, is the best compound in our HTS that shows synthetic lethality in combination with HK2 silencing/inhibition in HCC cells that express only HK2. Cell proliferation was measured by the MTT assay and cell counting. Cytotoxicity was measured using Trypan blue to distinguish live and dead cells. HK2 activity in xenograft tumors was monitored non-invasively using 18F-fludeoxyglucose PET/CT scans. Perhexiline (PER), a fatty acid oxidation inhibitor, further sensitizes HCC cells to the complex-I/HK2-targeted combination. Cellular ATP levels were examined by a luciferase activity assay, oxygen consumption was examined by a Seahorse respirometry assay, and cell metabolism was examined by a metabolomics analysis. The inclusion PER significantly enhances the reduction of HCC cellular energy production caused by the combination of HK2 inhibition and DPI, and the efficacy of the complex-I/HK2-targeted combination in treating HCC tumors in vivo. Our findings indicate that selective inhibition of the energy generation triangle (glycolysis, oxidative phosphorylation, and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising for HCC therapy. Citation Format: Shili Xu, Arthur Catapang, Daniel Braas, Linsey Stiles, Jason T. Lee, Thomas G. Graeber, Robert Damoiseaux, Orian Shirihai, Harvey R. Herschman. Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B37.

Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs).

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.This type of metabolic Achilles’ heel will allow us and others to more effectively “starve” the CSC population.

Molecular characterization of TP53 mutations and copy number change in colorectal cancers.

e15143 Background: TP53 mutations (mut) are prevalent in colorectal cancer (CRC) patients (pts) and can result in oncogenic gain of function effect as well as loss of tumor suppressor function depending on the mut. Methods: The Cancer Genome Atlas (TCGA) data of 220 CRC pts with tumors sequenced and gene copy numbers (CN) annotated was obtained. Pts with CMS subtype data also available were included (n = 167). Chi square test was used to compare frequency of TP53 mut and CN change among CMS subtypes. TP53 mRNA and protein levels were compared by TP53 mut and CN change using independent sample t test. Results: 86 (51.5%) pts were TP53 mutant, with 35.3% missense and 16.2% loss of function (LOF) mut. LOF mut included 14 non-sense and 13 frameshift mut, and occurred mostly in codons 213 and 306. Missense mut mostly affected codons 175, 248 and 273 in 10, 9 and 8 pts respectively. TP53 mut were most frequent in CMS2 and CMS4 subtypes (62.7 and 62.5% respectively). TP53 mut in CMS2 were mostly missense and CMS 4 pts mostly had LOF mut. TP53 CN loss was seen in 74.7% CMS 2, 68.8% CMS 4, 43.5% CMS 3 and 6.9% CMS1 pts. 84.9% pts with TP53 mut had CN loss vs 28.4% of wild type pts (p < 0.001). Frequency of TP53 CN loss did not vary significantly by type of TP53 mut. Pts with TP53 missense mut had higher p53 mRNA and protein levels than those with LOF mut. Pts with TP53 CN loss had lower mRNA levels but no change in protein levels than those with normal CN. Conclusions: The higher TP53 mRNA and protein levels with missense mut suggest possible gain of function. Research on interactions of TP53 change with wnt, myc, and TGF beta pathway genes may reveal synthetic lethal treatment combinations. [Table: see text]

Transposon Insertion Site Sequencing for Synthetic Lethal Screening.

Transposon insertion site sequencing (TIS) permits genome-wide, quantitative fitness assessment of individual genomic loci. In addition to the identification of essential genes in given growth conditions, TIS enables the elucidation of genetic networks such as synthetic lethal or suppressor gene combinations. Therefore, TIS becomes an exceptionally powerful tool for the high-throughput determination of genotype-phenotype relationships in bacteria. Here, we describe a protocol for the generation of high-density transposon insertion libraries and subsequent preparation of DNA samples for Illumina sequencing using the Gram-negative bacterium Vibrio cholerae as an example.

234 - A large-scale siRNA kinome screen identifies novel synthetic lethal combinations in colorectal cancer

234 - A large-scale siRNA kinome screen identifies novel synthetic lethal combinations in colorectal cancer

Synthetic Lethality in PTEN-Mutant Prostate Cancer Is Induced by Combinatorial PI3K/Akt and BCL-XL Inhibition.

Activation of the PI3K pathway through PTEN loss represents a major molecular pathway in the progression of prostate and other cancers. This study defines a synthetic lethal therapeutic combination with significant translational potential. Synthetic lethality in PTEN-mutant prostate cancer cells with combined PI3K/Akt and BCL-XL inhibition. PTEN-mutant prostate cancer cells expressing ITGA5 bind to fibronectin in the putative bone marrow niche and transduce survival signals to BCL-XL Additional PTEN-regulated signals independent of the PI3K/Akt pathway likely feed into the BCL-XL-regulated survival program to explain synthetic lethality observed with the combination.Visual Overview: http://mcr.aacrjournals.org/content/early/2016/12/02/1541-7786.MCR-16-0202/F1.large.jpg. Mol Cancer Res; 14(12); 1176-81. ©2016 AACR.

Synthetic lethal approaches for assessing combinatorial efficacy of chemotherapeutic drugs.

The recent advances in pharmacogenomics have made personalized medicine no longer a pipedream but a precise and powerful way to tailor individualized cancer treatment strategies. Cancer is a devastating disease, and contemporary chemotherapeutic strategies now integrate several agents in the treatment of some types of cancer, with the intent to block more than one target simultaneously. This constitutes the premise of synthetic lethality, an attractive therapeutic strategy already demonstrating clinical success in patients with breast and ovarian cancers. Synthetic lethal combinations offer the potential to also target the hitherto "undruggable" mutations that have challenged the cancer field for decades. However, synthetic lethality in clinical cancer therapy is very much still in its infancy, and selecting the most appropriate combinations-or synthetic lethal pairs-is not always an intuitive process. Here, we review some of the recent progress in identifying synthetic lethal combinations and their potential for therapy and highlight some of the tools through which synthetic lethal pairs are identified.

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.

Small molecule inhibition of the CHFR-PARP1 interaction as novel approach to overcome intrinsic taxane resistance in cancer.

The mitotic checkpoint protein CHFR has emerged as a major mediator of taxane resistance in cancer. Here we show that CHFR's PAR-binding zinc finger domain (PBZ) mediates a protein interaction with poly-ADP ribosylated PARP1 leading to stabilization of CHFR. Disruption of the CHFR-PARP1 interaction through either PARP1 shRNA-mediated knockdown or overexpression of a PBZ domain peptide induces loss of CHFR protein expression. In an attempt to exploit this observation therapeutically, and to develop compounds with synthetic lethality in combination with taxanes, we performed a high-throughput computational screen of 5,256,508 chemical structures against the published crystal structure of the CHFR PBZ domain to identify candidate small molecule CHFR protein-protein interaction inhibitors. The 10 compounds with the best docking scores (< -9.7) were used for further in vitro testing. One lead compound in particular, termed 'A3', completely disrupted the protein-protein interaction between CHFR and PARP1, resulting in the inhibition of mitotic checkpoint function, and led to therapeutic synergy with docetaxel in cell viability and colony formation assays. In mouse xenografts, i.p. administration of 'A3' led to a significant reduction in nuclear CHFR protein expression with a maximal effect 4 hours after administration, confirming relevant pharmacodynamics following the peak of 'A3' plasma concentration in vivo. Furthermore, combination of A3 and taxane led to significant reduction of implanted tumor size without increase in hematological, hepatic or renal toxicity. These findings provide a proof-of-principle that small molecule inhibition of CHFR PBZ domain interaction is a novel potential therapeutic approach to increase the efficacy of taxane-based chemotherapy in cancer.

Opportunities and challenges provided by crosstalk between signalling pathways in cancer

During evolution, connections between the major signalling pathways were established to provide cells with an ability to deal with perturbations of homeostasis. However, these feedback and crosstalk mechanisms can become a liability in the treatment of cancer, as the inhibition of one cancer-relevant signalling pathway can lead to the activation of a secondary survival pathway that interferes with cancer drug efficacy. In this review, we discuss connections between signalling pathways in relation to cancer therapy and we evaluate the use of genetic approaches to identify pathway crosstalk. We also discuss how insight into connections between signalling pathways can be exploited to design powerful synthetic lethal drug combination therapies for the treatment of cancer.

Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6

Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB-231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB-231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6.

DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma.

The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis. We have combined genome-wide DNA methylation analyses and gene expression profiling after pharmacological DNA demethylation with functional screening to identify novel tumor suppressors in diffuse large B cell lymphoma (DLBCL). We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases. The DUSP4 genomic locus is further deleted in up to 13% of aggressive B cell lymphomas, and the lack of DUSP4 is a negative prognostic factor in three independent cohorts of DLBCL patients. Ectopic expression of wild-type DUSP4, but not of a phosphatase-deficient mutant, dephosphorylates c-JUN N-terminal kinase (JNK) and induces apoptosis in DLBCL cells. Pharmacological or dominant-negative JNK inhibition restricts DLBCL survival in vitro and in vivo and synergizes strongly with the Bruton's tyrosine kinase inhibitor ibrutinib. Our results indicate that DLBCL cells depend on JNK signaling for survival. This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling.

Metabolic and hypoxic adaptation to anti‐angiogenic therapy: a target for induced essentiality

Anti-angiogenic therapy has increased the progression-free survival of many cancer patients but has had little effect on overall survival, even in colon cancer (average 6–8 weeks) due to resistance. The current licensed targeted therapies all inhibit VEGF signalling (Table1). Many mechanisms of resistance to anti-VEGF therapy have been identified that enable cancers to bypass the angiogenic blockade. In addition, over the last decade, there has been increasing evidence for the role that the hypoxic and metabolic responses play in tumour adaptation to anti-angiogenic therapy. The hypoxic tumour response, through the transcription factor hypoxia-inducible factors (HIFs), induces major gene expression, metabolic and phenotypic changes, including increased invasion and metastasis. Pre-clinical studies combining anti-angiogenics with inhibitors of tumour hypoxic and metabolic adaptation have shown great promise, and combination clinical trials have been instigated. Understanding individual patient response and the response timing, given the opposing effects of vascular normalisation versus reduced perfusion seen with anti-angiogenics, provides a further hurdle in the paradigm of personalised therapeutic intervention. Additional approaches for targeting the hypoxic tumour microenvironment are being investigated in pre-clinical and clinical studies that have potential for producing synthetic lethality in combination with anti-angiogenic therapy as a future therapeutic strategy.

Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.

Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants.

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a CanR mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the CanR mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

The Drosophila Werner Exonuclease Participates in an Exonuclease-Independent Response to Replication Stress

Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM, cause Werner and Bloom syndromes, which are diseases characterized by genome instability and an increased risk of cancer. While WRN contains both a helicase and an exonuclease domain, the Drosophila melanogaster homolog, WRNexo, contains only the exonuclease domain. Therefore the Drosophila model system provides a unique opportunity to study the exonuclease functions of WRN separate from the helicase. We created a null allele of WRNexo via imprecise P-element excision. The null WRNexo mutants are not sensitive to double-strand break-inducing reagents, suggesting that the exonuclease does not play a key role in homologous recombination-mediated repair of DSBs. However, WRNexo mutant embryos have a reduced hatching frequency and larvae are sensitive to the replication fork-stalling reagent, hydroxyurea (HU), suggesting that WRNexo is important in responding to replication stress. The role of WRNexo in the HU-induced stress response is independent of Rad51. Interestingly, the hatching defect and HU sensitivity of WRNexo mutants do not occur in flies containing an exonuclease-dead copy of WRNexo, suggesting that the role of WRNexo in replication is independent of exonuclease activity. Additionally, WRNexo and Blm mutants exhibit similar sensitivity to HU and synthetic lethality in combination with mutations in structure-selective endonucleases. We propose that WRNexo and BLM interact to promote fork reversal following replication fork stalling and in their absence regressed forks are restarted through a Rad51-mediated process.

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.

TORC2 Signaling Pathway Guarantees Genome Stability in the Face of DNA Strand Breaks

A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage.

Abstract A24: Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers

Abstract Introduction: Gene expression signatures from large cohorts of lung tumors suggest that distinct neuroendocrine (NE) cancers with poor prognosis appear in ~10% of otherwise pathologically unremarkable NSCLCs. However, a complete molecular characterization is lacking because a similar subtype has yet to be identified in a significant number of lung cancer cell lines. Our goal is to develop a pre-clinical model for NSCLC-NE and use it to rationale targeted therapy for this important subtype of lung cancer. We are developing targeted siRNA-based synthetic lethal screens to identify acquired vulnerabilities in lung cancers by using “mini-libraries” of putative cancer stem cell genes (CSC, N = 40), nuclear receptors and their co-regulators (NR/CoReg, N = 120), chromatin remodelers (CR, N = 102), and ASCL1-regulated druggable genes (N = 40). The siRNA libraries were derived from multiple studies while the ASCL1-targeted library was developed from our own ChIP-Seq data. Identifying NSCLC-NE Cell Lines: Using mRNA expression signatures from 207 lung cell lines (NSCLC/SCLC/HBEC) we identified a cluster of NSCLC cell lines (9% of NSCLC) that express genes indicative of a neuroendocrine phenotype (NSCLC-NE). A highly expressed gene in this class of cell lines is the lineage-specific transcription factor ASCL1, which is required for the development of pulmonary neuroendocrine cells. ASCL1 loss-of-function studies demonstrate dramatic cell death compared to controls, suggesting these cells are addicted to ASCL1 for survival. ASCL1 ChIP-Seq Analysis: To better understand the molecular pathogenesis of ASCL1-dependent NSCLC-NEs and identify druggable therapeutic downstream targets, we performed ChIP-Seq analysis on six ASCL1+ and two ASCL1- cell lines. A 125-member “ASCL1-signature” was generated and tested on multiple resected NSCLC datasets (N &amp;gt; 800 patients) for prognostic utility and tumors that exhibited the “ASCL1 signature” had significantly worse prognosis. A target of ASCL1 as determined by ChIP-Seq analysis is the anti-apoptotic regulator BCL2. BCL2 knockdown using siRNA as well as inhibition of BCL2 using a small molecule (ABT-263) results in cell death that is specific to ASCL1+/BCL2+ cells. ASCL1 Upstream Regulation: A separate therapeutic avenue is to determine upstream regulators of ASCL1 and we have demonstrated that ASCL1 participates in a double-negative feedback loop with the MEK/ERK pathway. Pharmacological activation of the MEK/ERK pathway results in loss of ASCL1 mRNA and protein, and induces apoptosis in ASCL1-dependent NSCLC-NE lines. Additionally, siRNA-mediated knockdown of ASCL1 results in reciprocal activation of the MEK/ERK pathway even in the presence of an siRNA targeting MEK1. Combining MEK/ERK activators with ABT-263 results in dramatic induction of apoptosis in NSCLC-NE while sparing normal immortalized HBEC cells suggesting the possibility of a synthetic lethal combination therapy for this subset of tumors. Synthetic Lethal Screens: We have completed synthetic lethal siRNA screens for the CSC, NR/CoReg, and CR mini-libraries using a panel of lung cancer lines. Optimal transfection conditions were established for each line and siRNA pools were used with multiple (n = 9) replicates providing great reproducibility (r &amp;gt; 0.9). From these screens, preliminary data demonstrates that the NSCLC-NE tumor lines have response phenotypes distinct from all other classes of lung cancer. Conclusions: We show that NE gene expression in NSCLC is of clinical relevance, and ASCL1 appears to be a lineage-dependent oncogene for NSCLC-NE. ASCL1 ChIP-Seq and siRNA screen data provide a roadmap for systematically searching for therapeutic targets such as BCL2 while work focused on targeting pathways upstream of ASCL1 also demonstrates clinical potential for NSCLC with neuroendocrine features. Our results indicate that lung cancers driven by ASCL1 have distinct acquired vulnerabilities. (Supported by NCI SPORE P50CA70907, CPRIT) Citation Format: Alexander Augustyn, Mark Borromeo, Tao Wang, Chunli Shao, Patrick Dospoy, Kenneth Huffman, Ryan Carstens, Luc Girard, Carmen Behrens, Ignacio Wistuba, Yang Xie, Jane Johnson, Adi Gazdar, John Minna. Targeted synthetic lethal screens to identify acquired vulnerabilities in a novel class of ASCL1-dependent neuroendocrine non-small cell lung cancers. [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 A24.