DNA Damage And Repair Research Articles

Comprehensive Genetic Profiling and Clinical Outcomes in Gastrointestinal Cancers Treated with Radiotherapy

The clinical significance of many genetic mutations in gastrointestinal cancers is not well characterized. As next-generation sequencing (NGS) has become more commercially available, there is opportunity to better establish the role of mutations in DNA damage and repair (DDR), tumor suppressor (TS), cell cycle (CC) and immune checkpoint (IC) genes. This information may be used to better predict local control (LC), distant metastasis (DM) or overall survival (OS). Commercially available DNA sequencing was conducted on patients with gastrointestinal (GI) cancers to identify significant genetic mutations. A total of 86 solid tumor biopsy specimens from GI cancers were submitted for molecular profiling with NGS. Clinical outcomes were measured from time of radiotherapy (RT) to time of progression or last follow-up and included RT to primary lesions and distant metastases. DDR genes included MGMT, MSH 2/6, and ERCC1. CC genes included cMET, EGFR, ERBB2 and KRAS. TS genes included APC, PTEN, and TP53. IC genes included PD-1 and PDL-1. Patients with locally controlled versus progressive disease were compared with regards to prevalence of genetic mutations in order to identify predictors of LC and OS. Patients were treated locally or distantly for colorectal (CRC, n = 36), gastroesophageal (GE, n = 5), liver and bile duct (LBD, n = 14), pancreatic (PC, n = 26) and other (OC, n = 5) GI cancers. Overall, patients had at least one mutation in DDR (46.3%), CC (55.2%), TS (65.5%), or IC (25.3%). DDR and CC mutations were most common in PC (57.7% and 69.2%, respectively), TS mutations were most common in CRC (80.6%), and IC was most common in CE (80.0%). Patients with at least one mutation in DDR, CC, or IC did not have any difference in LC following RT compared to patients without mutations. Mutations in TS genes were associated with improved local control following RT versus those without mutations in TS genes (p = 0.035). Mutations in TS, DDR, CC, and IC were not significantly associated with overall survival. Genetic mutations may be used to predict better response to local radiotherapy in patients with gastrointestinal cancer. In our analysis, patients with mutations in tumor suppressor genes including APC, PTEN, and TP53 had better local control following radiotherapy than patients without mutations in tumor suppressor genes. This information may be useful in future studies to identify patients at increased risk of local failure.

Bone marrow mesenchymal stem cells from patients with SLE maintain an interferon signature during in vitro culture

BackgroundWe have previously shown that SLE BMSC have decreased proliferation, increased ROS, increased DNA damage and repair (DDR), a senescence associated secretory phenotype, and increased senescence-associated β-galactosidase. We have also shown SLE BMSC produce increased amounts of interferon beta (IFNβ), have increased mRNA for several genes induced by IFNβ, and have a pro-inflammatory feedback loop mediated by a MAVS. To better understand the phenotype of SLE BMSC we conducted mRNA sequencing. MethodsPatients fulfilling SLE classification criteria and age and sex matched healthy controls were recruited under an Institutional Review Board approved protocol. Bone marrow aspirates and peripheral blood samples were obtained. BMSC were isolated and grown in tissue culture. Early passage BMSC were harvested and mRNA samples were sent for RNAseq. Serum samples were assayed for IFNβ by ELISA. ResultsOn the basis of top differentially expressed genes between SLE and healthy controls, SLE patients with high levels of serum IFNβ clustered together while SLE patients with low levels of IFNβ clustered with healthy controls. Those genes differentially expressed in SLE patients generally belonged to known IFN pathways, and showed a strong overlap with the set of genes differentially expressed in IFNβ high subjects, per se. Moreover, gene expression changes induced by treating healthy BMSC with exogenous IFNβ were remarkably similar to gene expression differences in SLE IFNβ high vs low BMSC. ConclusionsBMSCs from SLE patients are heterogeneous. A subgroup of SLE BMSC is distinguished from other SLE BMSC and from controls by increased levels of mRNAs induced by type I interferons. This subgroup of SLE patients had increased levels of IFNβ in vivo.

Prevalence of DNA damage response and repair, cellular signaling, tumor suppressor, and immune checkpoint gene mutations in gastrointestinal cancers.

e15149 Background: The prevalence of genetic mutations in gastrointestinal (GI) cancers is not well characterized. Molecular targeted therapies have substantial potential to improve outcomes. As next-generation sequencing (NGS) has become more commercially available, there may be additional opportunities to find novel targets for therapy or identify predictors of response to chemotherapy and radiotherapy. Commercially available DNA sequencing was conducted on patients with GI cancers to identify novel therapeutic and prognostic factors. Methods: A total of 86 solid tumor biopsy specimens from GI cancers were submitted for molecular profiling with NGS. Tumor molecular profiles were reviewed to identify frequently occurring pathogenic mutations in DNA damage and repair (DDR), cell cycle (CC), tumor suppressor (TS) and immune checkpoint (IC) pathways. DDR genes included MGMT, MSH 2/6 and ERCC1. CC genes included cMET, EGFR and KRAS. TS genes included APC, PTEN and TP53. IC genes included PD-1 and PDL-1. The overall frequency of mutations as well as the frequency of mutations for each primary site was analyzed. Results: Samples from colorectal (CRC, n=36), gastroesophageal (GE, n=5), liver and bile duct (LBD, n=14), pancreatic (PC, n=26) and other (OC, n=5) GI cancers were analyzed. Overall 46.3% of samples had at least one DDR mutation and commonly occurred in PC (57.7%) and CRC (50.0%).The most commonly mutated DDR genes were MGMT, TOPO1 and TOP2A. 55.2% of samples had at least one CC mutation and were most common in PC (69.2%), CRC (61.1%) and LBD (42.9%). The most common CC mutations were PTEN, P53 and KRAS. 65.5% of samples had at least one TS mutation with TP52 and PTEN being the most common. TS mutations occurred commonly in CRC (80.6%) and GE (60.0%). Mutations in PD-1 and PD-L1 (IC) were present in 25.3% of samples and occurred most frequently in GE (80.0%) and LBD (28.6%). Conclusions: Mutations were commonly seen in DDR (46.3%), CC (55.2%), TS (65.5%) and IC (25.3%) genes in GI primaries, providing a path to explore the role of novel therapeutic agents and how these mutations might be used to predict clinical outcomes.

Oncogenic activation of the HRR pathway drives RAD54L expression in early stage lung adenocarcinoma.

e20037 Background: In recent years, there has been a better understanding of ways to use DNA damage and repair (DDR) mechanisms to improve overall sensitivity and/or overcome resistance to traditional DNA damage treatments. The DDR network is quite complex and highly dynamic with as many as 450 proteins integral to the DNA repair. The current study is to identify the key dysregulated genes and its related pathways especially DDR pathways in early progression of lung adenocarcinoma. Methods: TCGA dataset of lung adenocarcinoma (LUAD) including 59 healthy lung tissues and 517 tumor tissues was utilized to detect the differentially expressed mRNAs and lncRNAs. Gene ontology (GO) analysis was conducted with DAVID, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes was performed using gene set enrichment analysis (GSEA) methods. Results: 41 lncRNAs and 2,047 mRNAs were screened out to be differentially expressed in LUAD. Besides, 38 lncRNAs and 1,801 mRNAs were found to be differentially expressed in T1 stage LUAD. The homologous recombination repair (HRR) pathway was found to be significantly up-regulated in LUAD, with four genes in this pathway up-regulated. In these genes of HRR pathway, PPP4R4 and RAD54L were recognized to be significantly differential expressed in T1 stage, compared with T2 stage and T3 stage, which were putative biomarkers of early stage LUAD. The survival analysis revealed that the expression of RAD54L was significantly related to the survival rate of patients with tumor of T1 stage. Conclusions: HRR pathway was up-regulated in lung adenocarcinoma, in which the expression of PPP4R4 and RAD54L were found to be tumor stage specific and RAD54L was related with survival rate of T1 stage patients. This study provided a further insight into the mechanism of the progression in early stage lung adenocarcinoma.

Olaparib-induced Adaptive Response Is Disrupted by FOXM1 Targeting that Enhances Sensitivity to PARP Inhibition

FOXM1 transcription factor network is activated in over 84% of cases in high-grade serous ovarian cancer (HGSOC), and FOXM1 upregulates the expression of genes involved in the homologous recombination (HR) DNA damage and repair (DDR) pathway. However, the role of FOXM1 in PARP inhibitor response has not yet been studied. This study demonstrates that PARP inhibitor (PARPi), olaparib, induces the expression and nuclear localization of FOXM1. On the basis of ChIP-qPCR, olaparib enhances the binding of FOXM1 to genes involved in HR repair. FOXM1 knockdown by RNAi or inhibition by thiostrepton decreases FOXM1 expression, decreases the expression of HR repair genes, such as BRCA1 and RAD51, and enhances sensitivity to olaparib. Comet and PARP trapping assays revealed increases in DNA damage and PARP trapping in FOXM1-inhibited cells treated with olaparib. Finally, thiostrepton decreases the expression of BRCA1 in rucaparib-resistant cells and enhances sensitivity to rucaparib. Collectively, these results identify that FOXM1 plays an important role in the adaptive response induced by olaparib and FOXM1 inhibition by thiostrepton induces "BRCAness" and enhances sensitivity to PARP inhibitors.Implications: FOXM1 inhibition represents an effective strategy to overcome resistance to PARPi, and targeting FOXM1-mediated adaptive pathways may produce better therapeutic effects for PARP inhibitors. Mol Cancer Res; 16(6); 961-73. ©2018 AACR.

DNA damage and repair (DDR) gene variants and outcomes in localized and metastatic prostate cancer (mPC).

304 Background: Moderate frequencies of DDR gene variants exist in mPC. Their prognostic role in localized and mPC is unclear. This study aims to characterize DDR variants in a cohort of hormone sensitive localized and mPC and assess for association with outcome. Methods: A retrospective cohort of men with PC and ≥ 2 follow up visits at DFCI and targeted next generation sequencing of prostate or metastatic tissue was identified. Biomarker (BM) positive was defined as pathogenic changes in the following DDR genes: ATM BAP1 BRCA1 BRCA2 BRIP1 CHEK2 MSH2 MSH6 PALB2 PMS2. For localized PC, outcome endpoints included event-free survival (EFS) (time from prostate biopsy to PSA relapse/metastasis/death), metastasis-free survival (MFS) (time to metastasis/death), time to castration resistance (CRPC), and death (OS); for mPC, outcome events included time from androgen deprivation start to CRPC and OS. BM status was descriptively summarized. Kaplan-Meier method and Cox regression model assessed associations of BM status with clinical outcomes. Results: Of the 237 men included, 16 (7%) were BM positive; 12 (6%) in 205 localized PC, 6 (14%) in 43 mPC. BM positive genes included 9 (4%) ATM, 6 (3%) BRCA2, and 2 (0.8%) CHEK2. In men with localized PC, EFS events occurred in 58% (7/12) BM positive vs 34% (66/193) BM negative at median follow up of 3.1 years; BM positive trended toward a shorter EFS (median 1.9 vs 5 years) than BM negative (HR 2.17, 95%CI 0.99-4.76). MFS events were observed in 17% (2/12) BM positive vs 7% (15/193) BM negative. CRPC events occurred in 17% (2/12) BM positive vs 6% (11/193) BM negative. In men with mPC, there were CRPC events in 100% (6/6) BM positive vs 67% (25/37) BM negative at median follow up of 3.3 years; BM positive trended toward a shorter time to CRPC (median 4 vs 8.8 months) compared to BM negative (HR 1.87, 95%CI 0.76-4.64). There was no association between BM status and OS. Conclusions: DDR variants appear infrequently in localized PC but may portend a shorter time to PSA relapse and metastasis. Similarly, DDR variants in mPC may be associated with a shorter time to CRPC. Low frequency of variants and short follow up limit these analyses. The prognostic value of DDR variants remains unclear and requires further research.

Abstract A116: Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival

Abstract Although loss of MGMT expression is used as a predictive biomarker for response to temozolomide (TMZ), some patients do not benefit from treatment, suggesting that additional criteria for patient selection remain to be identified. We used a panel of newly developed patient derived models and well-characterized cancer lines to evaluate potential determinants of TMZ response and identify strategies to overcome TMZ resistance in MGMT negative cancers. There was 100% association between microsatellite instability (MSI) and TMZ resistance in the lines tested. Moreover, only a fraction of MGMT/MSI double-negative lines were sensitive to TMZ at clinically relevant concentrations (37.6µM). TMZ-induced DNA damage and repair (DDR) were compared in a bladder line sensitive to TMZ (BLX1, IC50 11.4µM) and a resistant large neuroendocrine cancer line (LNX1, IC50>80 µM). We observed DDR activation in both models, but only BLX1 had measurable DNA damage (PARP cleavage and γH2AX accumulation, starting 24 h post treatment). An extended G2/M arrest occurred in BLX1 but not in LNX1, suggesting that LNX1 efficiently repaired TMZ-induced damage. ATR inhibition (VE821, VX970 or AZD6738), but not ATM inhibition, in LNX1 cells increased response to TMZ (IC50 27.5µM), PARP cleavage, γH2AX accumulation, and protracted G2/M arrest. BLX1 had minimal homologous recombination (HR) capacity (measured by pDR-GFP/SceI assay) compared to LNX1; however, ATR inhibition abrogated HR activity in LNX1 cells, suggesting HR importance in repair of TMZ-induced damage. To assess whether HR defects sensitize cells to TMZ, we used MGMT positive BRCA1 null cells (UWB1.289) and their BRCA1 reconstituted counterpart line. BRCA1 status did not affect TMZ response in the presence of an MGMT inhibitor (TMZ IC50>80µM); however, BRCA1 null cells were sensitive to TMZ in combination with an ATR inhibitor (VE821 0.3µM, TMZ IC50 10µM). BRCA1 reconstituted cells were sensitized to TMZ+VE821 using a Rad51 inhibitor (B02 2.5µM, TMZ IC50 22.5µM), suggesting that when HR is compromised, cells rely on ATR signaling for repair of TMZ-induced damage. Our data suggest that MGMT/MSI double-negative cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects could be used as additional selection criteria for TMZ+ATRi treatment. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, Erik Harris, Curtis Hose, John Connelly, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A116.

Highlights of This Issue

Androgen receptor splice variants (AR-Vs) are implicated in castration-resistant prostate cancer (CRPC). When expressed alone, some AR-Vs localize primarily to the nucleus, whereas others localize to the cytoplasm. The clinical relevance of the latter is often dismissed due to their cytoplasm localization. This study demonstrates a significant role of these cytoplasmic-localized AR-Vs in castration resistant disease through dimerizing with the nucleus-predominant AR-Vs and AR. The findings reveal important consequences for clinical castration resistance due to simultaneous expression of AR and different AR-Vs in patient tumors and suggest that dissecting their interactions will lead to effective strategies to disrupt AR-V signaling.While therapeutic benefit of bromodomain and extra-terminal (BET) protein inhibitors for hematological malignancies has been reported, the identification of sensitive solid tumor indications has been challenging. Castration-resistant prostate cancer (CRPC) dependence on transcription factor drivers rendered it exquisitely sensitive to ABBV-075, a novel BET inhibitor currently in clinical trials. ABBV-075 disrupted the functional interaction of AR and BRD4 at enhancers to inhibit the androgen receptor (AR) gene expression signature in both androgen-dependent and enzalutamide-resistant models. ABBV-075 activity also converged on parallel transcription factor drivers of CRPC progression, MYC and TMRPSS2-ETS. Thus, this proof-of-concept study supports the development of ABBV-075 in CRPC and similar transcription-factor dependent tumors.In this issue, Balaji and colleagues define a novel role for the AXL receptor tyrosine kinase in modulating the DNA damage and repair (DDR) response across three different cancer model systems. Higher AXL levels associate with higher expression of homologous recombination (HR) genes critical for DNA repair. Furthermore, inhibition of AXL decreases HR gene expression, impairs DNA repair function, and sensitizes to PARP therapy. Given the current clinical development of AXL and PARP inhibitors for both lung and breast cancer, these findings demonstrate that combinatorial inhibition of AXL and PARP significantly increases the therapeutic response beyond either inhibitor alone by inducing a type of synthetic lethality through AXL blockade.Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin A2 (AnxA2) as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal-Annexin A2 (exo-AnxA2) are still lacking. Using various in vitro and in vivo techniques, Maji and colleagues illustrate that exo-AnxA2 is functionally active and is capable of promoting angiogenesis in a tPA-dependent manner. Furthermore, exo-AnxA2 causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 signaling pathways and increases secretion of IL-6 and TNF-α to promote breast cancer metastasis. Collectively, these data establish for the first time that exo-AnxA2 is an important mediator in breast cancer pathogenesis.

Patient-Level DNA Damage and Repair Pathway Profiles and Prognosis After Prostatectomy for High-Risk Prostate Cancer.

A substantial number of patients diagnosed with high-risk prostate cancer are at risk for metastatic progression after primary treatment. Better biomarkers are needed to identify patients at the highest risk to guide therapy intensification. To create a DNA damage and repair (DDR) pathway profiling method for use as a prognostic signature biomarker in high-risk prostate cancer. A cohort of 1090 patients with high-risk prostate cancer who underwent prostatectomy and were treated at 3 different academic institutions were divided into a training cohort (n = 545) and 3 pooled validation cohorts (n = 232, 130, and 183) assembled for case-control or case-cohort studies. Profiling of 9 DDR pathways using 17 gene sets for GSEA (Gene Set Enrichment Analysis) of high-density microarray gene expression data from formalin-fixed paraffin-embedded prostatectomy samples with median 10.3 years follow-up was performed. Prognostic signature development from DDR pathway profiles was studied, and DDR pathway gene mutation in published cohorts was analyzed. Biochemical recurrence-free, metastasis-free, and overall survival. Across the training cohort and pooled validation cohorts, 1090 men were studied; mean (SD) age at diagnosis was 65.3 (6.4) years. We found that there are distinct clusters of DDR pathways within the cohort, and DDR pathway enrichment is only weakly correlated with clinical variables such as age (Spearman ρ [ρ], range, -0.07 to 0.24), Gleason score (ρ, range, 0.03 to 0.20), prostate-specific antigen level (ρ, range, -0.07 to 0.10), while 13 of 17 DDR gene sets are strongly correlated with androgen receptor pathway enrichment (ρ, range, 0.33 to 0.82). In published cohorts, DDR pathway genes are rarely mutated. A DDR pathway profile prognostic signature built in the training cohort was significantly associated with biochemical recurrence-free, metastasis-free, and overall survival in the pooled validation cohorts independent of standard clinicopathological variables. The prognostic performance of the signature for metastasis-free survival appears to be stronger in the younger patients (HR, 1.67; 95% CI, 1.12-2.50) than in the older patients (HR, 0.77; 95% CI, 0.29-2.07) on multivariate Cox analysis. DNA damage and repair pathway profiling revealed patient-level variations and the DDR pathways are rarely affected by mutation. A DDR pathway signature showed strong prognostic performance with the long-term outcomes of metastasis-free and overall survival that may be useful for risk stratification of high-risk prostate cancer patients.

Mitochondria-Mediated Oxidative Stress: Old Target for New Drugs.

Oxidative stress, one of the crucial factors of genomic instability, is involved in many illnesses - from DNA damage and repair (DDR) related diseases to neurological abnormalities and cancer. Patients with defective DDR pathways display high level of cancer predisposition and at the same time - reveal hydrocephalia, dementias and even diabetes mellitus - all representing common hallmarks of mitochondria-related disorders. Since mitochondria are responsible both for the cell energetic metabolism and for reactive oxygen/nitrogen species (RO/NS) formation, mitochondrial dysfunction (MDF) play a pivotal role in the above disorders. Not surprisingly, RO/NS are considered to be a primary target for a large spectrum of compounds aiming to eliminate these adverse species or, in contrary, enhance their presence in order to amplify cellular death pathways. Yet, only few chemicals have received medical appreciation mainly because of their questionable therapeutic values in healthy states. As a result, recent efforts have been focused on finding the drugs that improve mitochondrial functions or chemoprevent MDF rather than being applied as RO/NS scavengers. This review addresses the most recent progress in the development and application of such chemicals and outlines some future perspectives.

Imatinib enchances the sensitivity of gastrointestinal stromal tumors to topoisomerase II inhibitors

Objective: to study the sensitivity of gastrointestinal stromal tumors (GISTs) to the topoisomerases type II inhibitors and ability of imatinib to enhance GISTs sensitivity to the chemotherapeutic drugs indicated above. Subjects and Methods. We studied the sensitivity of gastrointestinal stromal tumors (GISTs) to the topoisomerases II inhibitors and ability of imatinib to enhance GISTs sensitivity to these chemotherapeutic agents. The expression of DNA damage and repair (DDR) markers was examined by western-blotting. Cleaved forms of poly (ADP-rybose) polymerase and caspase-3 were served as an apoptotic markers measured by western blotting. Amount of apoptotic cells was counted by flow cytometry analysis by using a propidium iodide DNA staining procedure and counting the numbers of hypodiploid cells. Results. We observed the sensitivity of GISTs to topoisomerase II inhibitors – doxorubicine and etoposide inducing DNA double-strand breaks and apoptotic cell death. Imatinib enhances GISTs sensitivity to topoisomerase II inhibitors. This might be due to reduced ability of GISTs to repair DNA damage by homologous recombination. Imatinib-induced reduction of Rad51 recombinase might be due to increased proteasome-dependent degradation. Conclusion. GIST cells are sensitive to topoisomerase II inhibitors (etoposide and doxorubicin) in vitro. Imatinib enhances GISTs sensitivity to the chemotherapeutic agents indicated above.

Abstract A12: Viability screens in leukemic and breast cancer cells with pooled lentiviral shRNA libraries identify potential therapeutic targets and synthetic or synergistic lethal interactions

Abstract One strategy to circumvent problems with conventional chemotherapy is to develop drugs against more specific cancer targets. Another is to use molecularly targeted agent (MTA) combinations to circumvent tumor resistance and increase the therapeutic index. Such synergistically lethal (SL) MTA combinations, however, are not easily predicted based on our rudimentary knowledge of cancer biology and drug action mechanisms. In the first strategy, our aim was to identify genes modulating proliferation and survival in leukemic cell lines (K562, Jurkat, and Raji) using a pooled lentiviral library expressing 27,500 shRNA targeting 5,043 human pathway-associated genes. Cells were transduced by the viral library and collected at several time points. Bar-codes were amplified from genomic DNA and sequenced (Illumina GAIIx). In the second strategy, we have adapted the same approach to combinatorially screen shRNA sequences targeting 40 DNA Damage and Repair (DDR) genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Human mammary epithelial cancer (HMEC) cells were transduced with a 27K shRNA SL DDR lentiviral library comprised of a redundant set of 16 binary shRNA constructs for each possible gene-gene combination (1,600). The library-transduced HMEC cells were grown for ten days, then bar-codes amplified and sequenced (Illumina HiSeq2000). The viability screen with leukemic cell lines identified more than 250 essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that in each screen, about 80% of shRNAs identified did indeed lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed several non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. The SL screen in HMEC cells identified 10 SL shRNA pair candidates including known SL shRNA pair PARP1/BRCA1. Additional analysis of lethal combinations indicated redundant, complementary, and compensatory responses in cancer cells. We believe that newly discovered hematopoietic-specific genes represent potentially novel drug targets. Moreover, they can be used to develop and establish both novel cancer targeted therapies and myeloablative conditioning regimens with decreased toxicity. Based on SL screen results in HMEC cells, we believe that comprehensive experimental annotation of SL gene-gene interactions in a wider range of cancer and normal cells will not only predict the most promising synergistic lethal combinations but also allow the development of a new generation of multi-specific, highly effective anti-cancer therapeutics with unique mechanisms of action. Citation Format: Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Karim Hyder, Paul Diehl, Costas G. Frangou, Alex Chenchik. Viability screens in leukemic and breast cancer cells with pooled lentiviral shRNA libraries identify potential therapeutic targets and synthetic or synergistic lethal interactions. [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 A12.

Abstract 876: Viability screens in leukemic and breast cancer cells with pooled lentiviral shRNA libraries identify potential therapeutic targets and synergistic lethal interactions

Abstract Background: One strategy to circumvent problems with conventional chemotherapy is to develop drugs against more specific cancer targets. Another is to use molecularly targeted agent (MTA) combinations to circumvent tumor resistance and increase the therapeutic index. Such synergistically lethal (SL) MTA combinations, however, are not easily predicted based on our rudimentary knowledge of cancer biology and drug action mechanisms. Method: In the first strategy, our aim was to identify genes modulating proliferation and survival in leukemic cell lines (K562, Jurkat, and Raji) using a pooled lentiviral library expressing 27,500 shRNA targeting 5,043 human pathway-associated genes. Cells were transduced by the viral library and collected at several time points. Bar-codes were amplified from genomic DNA and sequenced (Illumina GAIIx). In the second strategy, we have adapted the same approach to combinatorially screen shRNA sequences targeting 40 DNA Damage and Repair (DDR) genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Human mammary epithelial cancer (HMEC) cells were transduced with a 27K shRNA SL DDR lentiviral library comprised of a redundant set of 16 binary shRNA constructs for each possible gene-gene combination (1,600). The library-transduced HMEC cells were grown for ten days, then bar-codes amplified and sequenced (Illumina HiSeq2000). Results: The viability screen with leukemic cell lines identified more than 250 essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that in each screen, about 80% of shRNAs identified did indeed lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed several non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. The SL screen in HMEC cells identified 10 SL shRNA pair candidates including known SL shRNA pair PARP1/BRCA1. Additional analysis of lethal combinations indicated redundant, complementary, and compensatory responses in cancer cells. Conclusion: We believe that newly discovered hematopoietic-specific genes represent potentially novel drug targets. Moreover, they can be used to develop and establish both novel cancer targeted therapies and myeloablative conditioning regimens with decreased toxicity. Based on SL screen results in HMEC cells, we believe that comprehensive experimental annotation of SL gene-gene interactions in a wider range of cancer and normal cells will not only predict the most promising synergistic lethal combinations but also allow the development of a new generation of multi-specific, highly effective anti-cancer therapeutics with unique mechanisms of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 876. doi:1538-7445.AM2012-876

Abstract A36: Viability screens in leukemic and breast cancer cells with pooled lentiviral shRNA libraries identify potential therapeutic targets and synergistic lethal interactions

Abstract Background: One strategy to circumvent problems with conventional chemotherapy is to develop drugs against more specific cancer targets. Another is to use molecularly targeted agent (MTA) combinations to circumvent tumor resistance and increase the therapeutic index. Such synergistically lethal (SL) MTA combinations, however, are not easily predicted based on our rudimentary knowledge of cancer biology and drug action mechanisms. Method: In the first strategy, our aim was to identify genes modulating proliferation and survival in leukemic cell lines (K562, Jurkat, and Raji) using a pooled lentiviral-based library expressing 27,500 shRNA targeting 5,043 human pathway-associated genes. Cells were transduced by the viral library, selected using puromycin, and collected at several time points. Genomic DNA was extracted from cell populations, and shRNA/bar-codes PCR amplified and subjected to HT sequencing (lllumina GAIIx). In the second strategy, we have adapted the same approach to combinatorially screen shRNA sequences targeting 40 rationally selected DNA Damage and Repair (DDR) genes to discover additive and synergistic combinations that generate a synthetic-lethal phenotype. Human mammary epithelial cancer (HMEC) cells were tranduced with a 27,000 (27K) shRNA SL DDR lentiviral library comprised of a redundant set of 16 binary shRNA constructs for each possible gene-gene combination (1,600). The library-transduced HMEC cells were grown for three days in the presence of puromycin to select for infected cells and then grown for one week (without puromycin). Genomic DNA was extracted from cell populations, and bar-codes were PCR amplified and subjected to HT sequencing (Illumina HiSeq 2000). Results: The viability screen with leukemic cell lines identified more than 250 essential genes for each panel of cells. Subsequent validation using single shRNA-expressing constructs showed that in each screen, about 80% of shRNAs identified did indeed lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed several non-random clusters of interacting proteins that provide some insight into signaling pathways and protein networks specific to these cancers. The SL screen in HMEC cells identified approximately 10 SL shRNA pair candidates including known SL shRNA pair PARP1/BRCA1. Additional analysis of lethal combinations indicated redundant, complementary, and compensatory responses in cancer cells. Conclusion: We believe that newly discovered hematopoietic-specific genes represent potentially novel drug targets. Moreover, they can be used to develop and establish both novel cancer targeted therapies and myeloablative conditioning regimens with decreased toxicity. Based on SL screen results in HMEC cells, we believe that comprehensive experimental annotation of SL gene-gene interactions in a wider range of cancer and normal cells will not only predict the most promising synergistic lethal combinations but also allow the development of a new generation of multispecific, highly effective anticancer therapeutics with unique mechanisms of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A36.