bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A43: TFG-TEC oncogene modulates beta-enolase expression via both promoter activation and epigenetic modification
Authors: Kim Ah-young , Lim Bobae , Choi JeeHyun , Kim Jungho
Published: April 2017

Recurrent chromosome translocations are the hallmark of many human cancers. A proportion of human extraskeletal myxoid chondrosarcomas (EMCs) are associated with the characteristic chromosomal translocation t(3;9)(q11-12;q22), which results in the formation of a chimeric protein in which the N-terminal domain of the TRK-fused gene (TFG) is fused to the translocated in extraskeletal chondrosarcoma (TEC; also called CHN, CSMF, MINOR, NOR1, and NR4A3) gene. The oncogenic effect of this translocation may be due to the higher transactivation ability of the TFG-TEC chimeric protein; however, downstream target genes of TFG-TEC have not yet been identified. The results presented here demonstrate that TFG-TEC activates the human beta-enolase promoter. EMSAs, ChIP assays, and luciferase reporter assays revealed that TFG-TEC upregulates beta-enolase transcription by binding to two NGFI-B response element motifs located upstream of the putative transcription start site. In addition, northern blot, quantitative real-time PCR, and western blot analyses showed that overexpression of TFG-TEC up-regulated beta-enolase mRNA and protein expression in cultured cell lines. Finally, ChIP analyses revealed that TFG-TEC controls the activity of the endogenous beta-enolase promoter by promoting histone H3 acetylation. Overall, the results presented here indicate that TFG-TEC triggers a regulatory gene hierarchy implicated in cancer cell metabolism. This finding may aid the development of new therapeutic strategies for the treatment of human EMCs.

Citation Format: Ah-young Kim, Bobae Lim, JeeHyun Choi, Jungho Kim. TFG-TEC oncogene modulates beta-enolase expression via both promoter activation and epigenetic modification [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A43.
Abstract B13: Involvement of PAF1 complex in the DNA damage response
Authors: Park Garin , Kim Nari , Yoo Joo-Yeon
Published: April 2017

PAF1 complex is a transcriptional complex composed of PAF1, CTR9, CDC73, LEO1, RTF1 and SKI8 in human. It plays a key role in the regulation of every step of transcription, from initiation to termination. PAF1 complex associates with RNA polymerase ΙΙ during transcription, and regulates modification patterns of surrounding histones or chromatin structure via recruitment of histone modifying enzymes or chromatin remodeling factors to the actively transcribed site. In yeast, PAF1 complex has been also proposed to involve in other DNA associated processes, like DNA recombination, replication, and repair. However, detailed molecular mechanism of this regulation has not been fully understood. We recently observed significant DNA breakage in the PAF1 complex depleted cells. DNA repair of artificially introduced DNA break by I-SceI endonuclease was less efficiently repaired in these cells, indicating the connection between the DNA damage repair and active transcription. The molecular mechanism of this regulation will be discussed.

Citation Format: Garin Park, Nari Kim, Joo-Yeon Yoo. Involvement of PAF1 complex in the DNA damage response [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B13.
Abstract B34: Development of novel small molecule inhibitors targeting DNA repair proteins
Authors: Pawelczak Katherine S. , Gavande Navnath , VanderVere-Carozza Pamela , Turchi John
Published: April 2017

More than 1.6 million new cases of cancer will be diagnosed in the US in 2016 and a third of these will be from solid tumor of the lung, pancreas, breast, and ovary. These cancers represent a continuing clinical challenge in treatment and together account for over 250,000 deaths in the US alone, representing over 40% of all cancer deaths. There are limited therapeutic options for these patients, and targeted and combination therapies remain necessary for treating these aggressive cancers. The opportunity exists to exploit recent scientific advances in our knowledge of the underlying biology behind these cancers to create novel targeted therapeutics to dramatically enhance patient response to therapy and ultimately survival. To this end, we have developed a series of novel small chemical molecules that disrupt critical protein-DNA interactions in the nucleotide excision and non-homologous end joining DNA repair pathways. It is well understood that various cancer treatments like cisplatin, etoposide and ionizing radiation impart their chemotherapeutic effect by the formation of direct DNA damage which block DNA replication and transcription culminating in apoptosis. It is also well established that repair of this DNA damage by various DNA repair pathways reduces the effectiveness of chemo- or radio- therapy. It is our contention, borne out by analysis of clinical data to be presented on the development of our DNA repair inhibitors, that inhibition of these DNA repair pathways sensitizes these difficult to treat cancers to traditional DNA-damaging therapy. We anticipate both direct mechanisms of action on the repair pathways and synthetic lethal interactions can be exploited for therapeutic benefit. The series of novel small molecule inhibitors that we have developed targeting DNA repair proteins exhibit single-agent anti-cancer activity in cancer cell lines, and potentiate cellular sensitivity to chemotherapy and ionizing radiation treatment. Our data demonstrate that this class of inhibitors can be further developed as anti-cancer therapeutics with considerable potential to be used in conjunction with radiation therapy and other cancer therapies that induce DNA damage.

Citation Format: Katherine S. Pawelczak, Navnath Gavande, Pamela VanderVere-Carozza, John Turchi. Development of novel small molecule inhibitors targeting DNA repair proteins [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B34.
Abstract B38: Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model
Authors: Wu Jeffrey , Ambady Prakash , Morris DreeAnna , Pagel Michael , Woltjer Randy , Walker Joshua , Muldoon Leslie , Neuwelt Edward
Published: April 2017

High levels of DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) in glioblastoma tumor are associated with temozolomide resistance and poor prognosis. MGMT provides a promising therapeutic target to overcome chemo-resistance and improve overall survival in cancer patients. In this study, we investigate the enhanced delivery of anti-MGMT morpholino oligonucleotide (AMON) using a sub-therapeutic dose of radiation to reduce MGMT expression of human cancer cells (T98G glioma and H460 and A549 non-small cell lung carcinoma) in vitro and in vivo. Compared to standard transfection techniques, sub-therapeutic dose of radiation enhanced intracellular AMON delivery and transiently reduced MGMT protein expression at 3 d in vitro. The optimal radiation dosage was cancer cell type dependent and ranged from 1-12 Gy. In addition, AMON delivered using sub-therapeutic dose of radiation increased cytotoxicity of T98G cells in response to temozolomide compared to radiation and temozolomide alone in vitro. In a H460 subcutaneous xenograft tumor model, intravenous AMON administration reduced MGMT protein level by 50% in irradiated tumors but not in contralateral non-irradiated lesions within the same animal. Tumor MGMT protein downregulation by AMON did not alter DNA promoter methylation status. Our results demonstrate for the first time the use of radiation to enhance the intracellular delivery of antisense oligonucleotides to modulate MGMT protein expression in vitro and in vivo. We have previously shown that antisense nucleotides can be delivered to the brain parenchyma using osmotic blood brain barrier disruption. Therefore, we believe that cancer patients with MGMT overexpression, unmethylated DNA promoter and/or resistance to DNA alkylating agents may benefit from this novel antisense therapeutic technology to improve overall survival.

Citation Format: Jeffrey Wu, Prakash Ambady, DreeAnna Morris, Michael Pagel, Randy Woltjer, Joshua Walker, Leslie Muldoon, Edward Neuwelt. Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B38.
Abstract B39: Tumor hypoxia induces DNA repair vulnerabilities through contextual “loss-of- heterozygosity”
Authors: Mahamud Osman , Chua Melvin L.K , Lo Winnie , Zafarana Gaetano , Bristow Robert G.
Published: April 2017

Introduction: Intratumoral hypoxia leads to decreased expression in DNA damage response (DDR) and repair pathways. Given the “two-hit” model for loss of gene function, we hypothesize that hypoxia-mediated down-regulation of gene expression and function, coupled with an already inactive allele may ultimately give rise to an exploitable contextual “loss-of-heterozygosity” phenotype.

Method: To interrogate this relationship, isogenic DLD-1 cells heterozygous and homozygous null for BRCA2, were placed under normoxic (21% O2) or hypoxic (0.2% O2) conditions for 72 hours. Hypoxia-mediated changes in DDR response and DNA repair were evaluated by cell proliferation, cell cycle analysis, western blots, qPCR, immunofluorescence, clonogenic assays and repair assays.

Results: No differences in proliferation and cell survival were observed between oxic and hypoxic cells (72 hours - 0.2% O2). Under chronic hypoxic conditions, confirmed by the up-regulation of VEGF and HIF1α, mRNA and protein expression of key homologous recombination (HR) genes (BRCA1, BRCA2, RAD51) were down-regulated. Functionally, BRCA2(-/-) null cells proved unable to recruit Rad51 foci and resultantly presented a profound sensitivity to PARP inhibition. Conversely, heterozygote BRCA2(+/-) cells retained the ability to recruit Rad51 foci under both oxic and hypoxic conditions, however, exposure to chronic hypoxia resulted in a reduction in the number of foci formed. Chronically hypoxic BRCA2(+/-) cells exhibited a 30 to 40% increase in sensitivity to PARP inhibition when compared to their oxic counterparts. Preliminary data shows a similar synthetically lethal relationship in genetic and tumor microenvironment induced HR deficient cells, when challenged with DNA damage response (DDR) kinase inhibitors ATRi and DNAPKi.

Conclusions: Herein we illustrate through a novel mechanism of contextual “loss-of heterozygosity”, which marries the tumor microenvironment and innate genetic alterations, increased sensitivity to DDR kinase inhibitors and PARPi.

Citation Format: Osman Mahamud, Melvin L.K Chua, Winnie Lo, Gaetano Zafarana, Robert G. Bristow. Tumor hypoxia induces DNA repair vulnerabilities through contextual “loss-of- heterozygosity” [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B39.
Abstract B40: [10]-gingerol as a pro-apoptotic molecule in triple negative breast cancer
Authors: Martin Ana Carolina B. M. , Tomasin Rebeka , Becceneri Amanda Blanque , Fuzer Angelina , Vieira Paulo Cezar , Cominetti Marcia Regina
Published: April 2017

The aim of this work was to study the pro-apoptotic effects of [10]-gingerol and connect them to anti-tumor and anti-metastatic activities in triple negative breast cancer (TNBC) cell lines and in an spontaneous triple negative breast cancer model in vivo.

Breast cancer has the highest incidence among women and metastasis is the major cause of death in cancer. TNBC affects 20% of women with breast cancer. This cancer type is very aggressive and has a high propensity to form lung and brain metastases. Nowadays, there is no effective treatment for TNBC and therefore, new therapies are required. Studies to discovery new molecules to be used in more efficient treatments for this disease are essential. Molecules that can interfere with cell proliferation, migration, invasion, apoptosis and cell cycle can be used as a model or tools to develop new anti-metastatic drugs. Epidemiologic studies have shown that Asian populations have lower incidence of breast cancer comparing to western populations. This has been attributed in part to differences in dietary habits. In particular, gingerols, natural compounds extracted from ginger rhizomes, have been shown to present anti-cancer properties against various tumor cell types in vitro and in vivo.

MTT assay was performed to verify the inhibitory effects of [10]-gingerol on cell proliferation. To investigate [10]-gingerol mechanisms of action, apoptosis, cell cycle assays and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) were performed. For the in vivo data an spontaneous model of breast cancer metastasis was used.

Results: [10]-gingerol was able to inhibit cell proliferation in different cell lines. In the murine cell line (4T1Br4), [10]-gingerol induced apoptosis, cell cycle arrest at sub-G0 phase and also induced DNA damage demonstrated by TUNEL assay, therefore interfering with DNA and provoking its damage. [10]-gingerol inhibited lung, bone and brain metastases in an intra-mammary fat pad spontaneous breast cancer model.

Conclusion: [10]-gingerol showed to be a non-toxic compound with anti-proliferative and DNA damaging effects, inducing cell apoptosis in vitro and also inhibiting lung, bone and brain metastases in a spontaneous TNBC model in vivo. Patent deposit: BR 10 2015 024093 7. Approved by ethical committee – E507 and 3224-1.

Citation Format: Ana Carolina B. M. Martin, Rebeka Tomasin, Amanda Blanque Becceneri, Angelina Fuzer, Paulo Cezar Vieira, Marcia Regina Cominetti. [10]-gingerol as a pro-apoptotic molecule in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B40.
Abstract B41: REV7 is a possible prognostic predictor and a potential therapeutic target in human malignancy
Authors: Murakumo Yoshiki , Niimi Kaoru , Okina Sosei
Published: April 2017

Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription and carcinogenesis, and is a key protein in translesion DNA synthesis. Here, we present our study to evaluate the significance of REV7 expression in human malignancy and its possibility to be a molecular target for cancer therapy. REV7 expression was assessed in epithelial ovarian cancer (EOC) and diffuse large B-cell lymphoma (DLBCL) by immunohistochemical staining. REV7 expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in ovarian clear cell carcinomas (CCCs) (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stages II to IV) EOC. REV7 expression was also assessed in DLBCL, the most common type of non-Hodgkin lymphoma, in which high REV7 expression was associated with significantly shorter overall survival and progression-free survival. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. REV7 knockdown in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that high REV7 expression is associated with poor prognosis in EOC and DLBCL, and depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate for molecular target in human malignancy.

Citation Format: Yoshiki Murakumo, Kaoru Niimi, Sosei Okina. REV7 is a possible prognostic predictor and a potential therapeutic target in human malignancy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B41.
Abstract B43: RECQ1, a breast cancer susceptibility gene, is upregulated by cancer therapeutics in a p53-dependent manner
Authors: Lu Xing , Parvathaneni Swetha , Sharma Sudha
Published: April 2017

Sensitivity of cancer cells to DNA-damaging chemotherapeutics is determined by DNA repair processes. Consequently, cancer cells may upregulate expression of certain DNA repair genes as a mechanism to promote chemoresistance. Here, we report identification of RECQ1, a breast cancer susceptibility gene that encodes the most abundant RecQ helicase in humans, as a p53-regulated target potentially acting as a defense against DNA-damaging agents. Quantitative RT-PCR analysis in a variety of cancer cell lines revealed RECQ1 to be upregulated following DNA damage, which was confirmed by Western blot experiments. Significant increase in RECQ1 mRNA level was observed following treatment with alkylating agent methylmethanesulfonate (MMS) as well as the representative chemotherapeutic drugs doxorubicin, temozolomide and fotemustine. Upregulation of RECQ1 expression also appeared to depend on the nature of DNA lesion. Induction of RECQ1 by genotoxins requires p53 since it was observed in p53 wild-type but not in p53 null cells. Consistent with that, the RECQ1 promoter is bound by endogenous p53 and is responsive to p53 in luciferase assay. Knocking down RECQ1 expression in cancer cells resulted in increased sensitivity to MMS, fotemustine and temozolomide supporting the hypothesis that RECQ1 is involved in the recovery of cells from DNA damage. Notably, RECQ1 is frequently overexpressed and amplified in a variety of cancers. As RECQ1 efficiently protects cells from genomic instability through repair of DNA lesions including those induced by alkylating and other chemotherapeutic agents, RECQ1 overexpression may provide resistance to anti-cancer drugs. RECQ1 expression level may therefore serve to predict chemotherapeutic response of cancer patients and timely implementation of alternative therapeutic strategies.

Citation Format: Xing Lu, Swetha Parvathaneni, Sudha Sharma. RECQ1, a breast cancer susceptibility gene, is upregulated by cancer therapeutics in a p53-dependent manner [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B43.
Abstract B44: Dual targeting of PI3K and MEK impairs DNA double-strand break repair as a relevant mechanism for radioresistance of K-RAS mutated non-small cell lung cancer
Authors: Toulany Mahmoud , Ilda Mari , Wheeler Deric L. , Rodemann H. Peter
Published: April 2017

Introduction: One of the major mechanisms of radioresistance in solid tumors is the activa-tion of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through stimulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). As it is known that the PI3K/Akt pathway is hyperactivated in K-RAS mutated (K-RASmut), we investigated if targeting PI3K would be a potential approach for enhancing radiosensitivity of K-RASmut cells non-small lung cancer (NSCLC).

Methods: The K-RASmut NSCLC cell lines A549 and H460 were utilized to examine the effect of PI3K inhibition on Akt signaling, non-homologous end joining (NHEJ) repair of DSBs, and post-irradiation cell survival.

Results: Short-term (1-2 h) pre-treatment of K-RASmut cells with the PI3K inhibitor PI-103 (1 µM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect in K-RASmut cells after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with PI-103 together MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through NHEJ leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts in vivo. Akt and P-DNA-PKcs activity was inhibited 30 min post-irradiation in xenografts, which were pre-treated by PI-103 30 min before irradiation. However, after a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation.

Conclusion: Due to ERK-dependent reactivation of Akt in K-RAS mutated NSCLC cells, dual targeting of PI3K and MEK is an effective approach to induce radiosensitization.

Acknowledgement: This work was supported by grants from the Deutsche Forschungsge-meinschaft (DFG, RO 527/7-1) awarded to HPR /MT and GRK 1302/2 (T11) awarded to MT/HPR.

Citation Format: Mahmoud Toulany, Mari Ilda, Deric L. Wheeler, H. Peter Rodemann. Dual targeting of PI3K and MEK impairs DNA double-strand break repair as a relevant mechanism for radioresistance of K-RAS mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B44.
Abstract IA01: Advancing the field of mutational signatures: Mechanisms and clinical applications
Authors: Nik-Zainal Serena
Published: April 2017

Mutational signatures are the imprints of the biological processes that have gone awry in human cells. We previously outlined the methods for identifying and quantifying base substitution mutational signatures present in primary human cancers (http://cancer.sanger.ac.uk/cosmic/signatures). Here, using a highly-curated cohort of 560 whole genome sequenced breast cancers, we extend the understanding of mutational signatures to include six novel rearrangement signatures. Diving into the detail of individual mutational signatures, we reveal intriguing mechanistic insights into the DNA damage and repair processes that mark the landscape of cancer genomes. Furthermore, we highlight how these mutational signatures distinguish clinical cohorts in breast cancer and have potential clinical applications.

Citation Format: Serena Nik-Zainal. Advancing the field of mutational signatures: Mechanisms and clinical applications [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA01.
Abstract IA02: Defects in DNA repair genes revealed by clinical sequencing of advanced cancer patients
Authors: Chinnaiyan Arul
Published: April 2017

In 2011, we introduced the Mi-Oncoseq program, an IRB-approved protocol to carry out integrative sequencing of advanced cancer patients. It was one of the first comprehensive DNA and RNA sequencing programs offered for cancer patients. The purpose of this program was to determine the utility of genomic sequencing of tumors and germline coupled with a multi-disciplinary Precision Medicine Tumor Board (PMTB) in the management of advanced cancer patients. Collectively across all patient cohorts we have enrolled over 1500 patients since inception of Mi-Oncoseq where molecular results for both somatic and germline aberrations are returned to physicians. Both common and rare mutations were identified in numerous cellular pathways that are involved in carcinogenesis including those in the DNA repair pathway. We identified homozygous deletions, deleterious mutations and/or LOH in genes including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2 that are potentially actionable. For example, a multi-institutional, collaborative SU2C-PCF study of men with metastatic castrate-resistant prostate cancer (mCRPC) found that treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate (Mateo et al. NEJM, 2015). Additionally, a follow-up study found 11.8% of mCRPC patients had a germline mutation in a DNA-repair gene, including moderate risk genes (ATM, CHEK2, PALB2, RAD51D) for which clinical testing criteria do not exist (Pritchard et al, NEJM, 2016). Most of these patients would not qualify for germline genetic testing under current clinical care models. Our studies demonstrate that DNA repair mutations are common in mCRPC patients who may benefit from therapies such as PARP inhibitors and when identified in the germline, could have a significant impact on their family members where early cancer screening could be implemented as appropriate.

Citation Format: Arul Chinnaiyan. Defects in DNA repair genes revealed by clinical sequencing of advanced cancer patients [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA02.
Abstract IA05: Prospective identification of vulnerabilities to DNA repair inhibitors
Authors: Durocher Daniel
Published: April 2017

The advent of CRISPR/Cas9 as a gene editing tool has enable the development of genome-scale libraries of single-guide RNAs that can be used to probe biological processes. CRISPR/Cas9 screens can be used to profile the essential gene complement of cancer cell lines, map genetic interactions and chart the response to drugs. During my presentation, I will present our efforts to map the genetic networks that govern the DNA damage response and will discuss our work aimed at mapping the genetic architecture of the response to PARP inhibitors. Our work revealed new types of genetic vulnerabilities to PARP inhibition and in particular, I will discuss a type DNA lesion that could act as the elusive inhibited-PARP “trap”.

Citation Format: Daniel Durocher. Prospective identification of vulnerabilities to DNA repair inhibitors [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA05.
Abstract IA06: The Fanconi anemia-BRCA pathway and cancer
Authors: Taniguchi Toshiyasu
Published: April 2017

The Fanconi anemia (FA)-BRCA pathway has emerged as an important pathway in cancer biology. FA is a rare genetic disease characterized by chromosomal instability, cancer-susceptibility and cellular sensitivity to interstrand DNA crosslink (ICL)-inducing agents. The FA proteins including breast/ovarian cancer susceptibility proteins, BRCA1(FANCS) and BRCA2(FANCD1), cooperate in a common pathway (the FA-BRCA pathway) required for cellular resistance to ICL-inducing agents. The main function of the pathway is to coordinate multiple DNA repair mechanisms during ICL repair, although many of the components of the pathway also play critical roles in protecting replication forks.

Inactivation of this pathway in cancer cells can lead to sensitivity to anti-cancer ICL-inducing agents, such as cisplatin and carboplatin, while reactivation of this pathway is implicated in acquired resistance to ICL-inducing agents. Therefore, inhibition of the pathway is an attractive therapeutic strategy to overcome DNA-crosslinker resistance of tumor cells.

In this talk, I will introduce what the FA-BRCA pathway is and how this pathway is inactivated in human cancer. I will also mention some of the mechanisms of acquired resistance to cisplatin and PARP inhibitors in cancer cells, related to the FA-BRCA pathway. I will also discuss our recent findings about the regulation of the FA-BRCA pathway by FANCI protein and a deubiquitinating enzyme, USP28.

Citation Format: Toshiyasu Taniguchi. The Fanconi anemia-BRCA pathway and cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA06.
Abstract IA07: Genetic determinants of tumor development, therapy response and resistance in mouse models of BRCA-deficient breast cancer
Authors: Jonkers Jos
Published: April 2017

Heterozygous germline mutations in BRCA1 or BRCA2 strongly predispose to development of breast and ovarian cancer (as well as other cancer types) via loss of the remaining wildtype allele. BRCA1/2-deficient cancers are defective in DNA double-strand break (DSB) repair via homologous recombination (HR) and therefore hypersensitive to DNA-damaging agents, including platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors. However, these treatments do not result in tumor eradication and eventually resistance develops. To maximize therapeutic efficacy of these drugs and achieve durable remissions, it is important to unravel the mechanisms by which these tumors acquire resistance to platinum drugs and PARP inhibitors, in order to develop combination therapies that prevent development of resistance or re-sensitize resistant tumors.

To study therapy response and resistance in a realistic in vivo setting, we have established several genetically engineered mouse models (GEMMs) and patient-derived tumor xenograft (PDX) models for BRCA-deficient breast cancer. These mice develop mammary tumors that are characterized by genomic instability and hypersensitivity to DNA-damaging agents, including platinum drugs and PARP inhibitors. Using cross-species oncogenomics and reverse genetics, we have identified several cancer genes including p53, MYC and RB as critical drivers in BRCA1-associated breast cancer. In addition, we have used these mammary tumor models for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. Using functional genetic screens, reverse genetics and genomic analysis of therapy-resistant tumors, we found that therapy response and resistance of BRCA1-deficient mammary tumors to cisplatin and the clinical PARP inhibitor olaparib is affected by several factors, including drug efflux transporter activity, type of BRCA1 founder mutation and restoration of HR repair via loss of 53BP1 or REV7. Also BRCA1 re-activation via genetic or epigenetic mechanisms contributes to therapy resistance in PDX models of BRCA1-deficient breast cancer. Importantly, pharmacokinetic or HR-related mechanisms underlie olaparib-resistance in only a fraction of BRCA1/2-deficient mammary tumors, indicating the existence of additional, unknown mechanisms.

Citation Format: Jos Jonkers. Genetic determinants of tumor development, therapy response and resistance in mouse models of BRCA-deficient breast cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA07.
Abstract IA10: DNA repair mutations in ovarian carcinoma and relationship to therapeutic response
Authors: Swisher Elizabeth M.
Published: April 2017

Ovarian carcinomas with germline or somatic mutations in BRCA1 and BRCA2 are sensitive to platinum chemotherapy and to PARP inhibitors. The therapeutic role of damaging mutations in other homologous recombination genes is less certain. We will review various measures of homologous recombination deficiency in ovarian cancer and their correlation with efficacy of PARP inhibitor therapy in the relapsed setting.

Citation Format: Elizabeth M. Swisher. DNA repair mutations in ovarian carcinoma and relationship to therapeutic response [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA10.
Abstract IA12: Poisoning cancer with oxidized nucleotides by targeting MTH1
Authors: Helleday Thomas
Published: April 2017

DNA damaging agents, i.e., radio- and chemotherapy, constitute the backbone for treatment of a wide variety of cancers and may result in complete cure from the disease. Here, I will give an overview on how DNA repair can be targeted using completely novel inhibitors and more specifically how cancer cells may require a specific DNA repair pathway to mediate survival to the high load of endogenous DNA damage. Cancers have deregulated levels of reactive oxygen species (ROS), damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools, converting 8-oxo-dGTP to 8-oxo-dGMP, to prevent incorporation of damaged bases during DNA replication. MTH1 overexpression reverses the mutator phenotype caused by mismatch repair defects and prevents Ras-induced senescence by suppressing the overall level of DNA damage. These data suggest that a majority of damage in cancer cells occur on the free dNTP pool and that this need sanitation for cancer cell survival. Here we show that cancer cells are dependent on MTH1 activity for survival, due to the effects of MTH1 in preventing incorporation of oxidized dNTPs into DNA to avoid ATM and p53 mediated apoptosis. As MTH1-/- mice are viable and MTH1 is not required for survival of non-transformed cells, targeting MTH1 may selectively cause DNA damage to cancer cells. We validate MTH1 as an anti-cancer target in vivo and describe small molecules, TH287 and TH588 that potently and selectively inhibit MTH1. Protein co-crystal structures demonstrate that the compounds bind as inhibitors in the enzymatic pocket of MTH1. These first-in-class inhibitors of the Nudix hydrolase family cause increased incorporation of oxidized dNTPs in cells subject to high ROS levels, causing DNA damage and cytotoxicity to cancer cells. This study exemplifies a new general therapeutic approach to convert oxidative stress to cytotoxic DNA damage and cancer cell death. Here, I will present the progression of MTH1 inhibitors from pre-clinical development into early clinical trials.

References:

Gad et al 2014 Nature. 508(7495):215-21.

Citation Format: Thomas Helleday. Poisoning cancer with oxidized nucleotides by targeting MTH1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA12.
Abstract IA13: Replication stress in cancer pathogenesis: Mechanisms and treatment opportunities
Authors: Bartek Jiri , Bartkova Jirina
Published: April 2017

Replication stress (RS) induced by activated oncogenes and loss of some tumor suppressors is emerging as one of the hallmarks of cancer. The RS-induced DNA damage and the ensuing activation of cell cycle checkpoints commonly induce cellular senescence or cell death of the nascent tumor cells, providing an inducible intrinsic barrier to cancer progression. On the other hand, this scenario creates an environment that favors outgrowth of tumor cell clones featuring p53 mutations and other checkpoint defects such as those in ATM or Chk2 kinases, events that allow tumor growth at the expense of enhanced genomic instability. The ongoing enhanced RS, on the other hand, unmasks higher dependence of tumor cells on RS-support pathways such as those provided by the ATR-Chk1 axis and replication fork protective mechanisms, a vulnerability that can be targeted by inhibitors of ATR, Chk1, MK2 and Wee1 kinases, for example.

The lecture will briefly outline this concept and then focus on our new data relevant for three of the open questions in this field:

i) How are the molecular obstacles such as RNA-DNA hybrids and aberrant intermediates resulting from RS-causing collisions between replication and transcription resolved in cells?;

ii) What is the impact of RS and the ensuing ATR signaling on the mutation spectra (mutation signatures) in major types of human solid tumors such as breast carcinomas?; and

iii) How do human aggressive cancers such as glioblastomas cope with the excessive endogenous RS, how do such mechanisms promote tumor cell survival and how could this knowledge help in designing innovative treatment strategies.

Citation Format: Jiri Bartek, Jirina Bartkova. Replication stress in cancer pathogenesis: Mechanisms and treatment opportunities [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA13.
Abstract IA19: PARP trapping and Schlafen 11
Authors: Pommier Yves , Murai Junko
Published: April 2017

Poly(ADP-ribose) polymerase inhibitors (PARPIs) kill cancer cells by trapping PARP1 and PARP2. Talazoparib, the most potent PARP inhibitor (PARPI), exhibits remarkable selectivity among the NCI-60 cancer cell lines beyond BRCA inactivation. Our genomic analyses reveal high correlation between response to talazoparib and Schlafen 11 (SLFN11) expression. Causality was established in four isogenic SLFN11-positive and -negative cell lines and extended to olaparib. Response to the talazoparib-temozolomide combination was also driven by SLFN11 and validated in 36 small cell lung cancer cell lines, and in xenograft models. Resistance in SLFN11-deficient cells was caused neither by impaired drug penetration nor by activation of homologous recombination. Rather, SLFN11 induced irreversible and lethal replication inhibition, which was independent of ATR-mediated S-phase checkpoint. The resistance to PARPIs by SLFN11 inactivation was overcome by ATR inhibition, mechanistically because SLFN11-deficient cells solely rely on ATR activation for their survival under PARPI treatment. Our study reveals that SLFN11 inactivation, which is common (~45%) in cancer cells, is a novel and dominant resistance determinant to PARPIs.

Citation Format: Yves Pommier, Junko Murai. PARP trapping and Schlafen 11 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA19.
Abstract IA26: Dual functions of PARP1 in prostate cancer: mechanisms and implications for therapeutic intervention
Authors: Knudsen Karen E.
Published: April 2017

Prostatic adenocarcinoma (PCa) is the 2nd leading cause of cancer death in US men. Organ-confined PCa can be effectively managed, but there is no durable treatment for advanced disease. Advanced PCa is treated through androgen deprivation therapy, often coupled with direct AR antagonists, as PCa is exquisitely dependent on androgen receptor (AR) activity for survival. Furthermore, recent studies identified AR as a major effector of DNA repair, manifest through the ability of the receptor to regulate DNAPK expression and activity. While AR directed therapeutics effectively suppress the pro-proliferative, pro-survival, and pro-DNA repair functions of AR and result in tumor remission, relapse is common. Recurrent disease arises largely due to resurgent AR activity with 2-3 years, and there is no cure for this castration-resistant phase (CRPC, castration-resistant PCa). Thus, there is a significant need to develop new means for targeting recurrent AR activity or develop adjuvant therapies in advanced PCa.

The function of PARP1 in DNA repair has been cultivated as a therapeutic target for tumors incurring alterations of specific DNA repair pathways. However, it is now clear that factors beyond DNA repair alterations play a role in the response to PARP1 inhibitors-- as noted in the TO-PARP trial, not all patients with DNA repair alterations responded to PARP1 inhibitors as single agents; conversely, a significant number tumors lacking BRCA1/2 or other DNA repair alterations show objective responses to PARP1 inhibitors in PCa and other cancers. PARP1 is known to harbor a second major cellular function on chromatin as a transcriptional co-regulator, capable of modulating chromatin structure and selected transcription factor activity. Notably, previous studies demonstrated that PARP1 regulates AR residence on chromatin, and downstream AR-dependent tumor progression. Recent unpublished studies to be discussed demonstrate that PARP1 activity is significantly enriched in lethal disease, and that a newly identified gene signature of PARP1-regulated networks is associated with poor outcome. Further, mechanistic investigation revealed new insight into the means by which PARP1 inhibitors likely act function as single agents in advanced prostate cancer, manifest through both DNA repair and transcriptional regulatory functions.

Citation Format: Karen E. Knudsen. Dual functions of PARP1 in prostate cancer: mechanisms and implications for therapeutic intervention [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA26.
Abstract IA27: Exploiting the inhibition of cullin-RING-ligases in DSB repair as a therapeutic strategy
Authors: Morgan Meredith
Published: April 2017

While single component E3 ubiquitin ligases have established roles in DNA double-strand break (DSB) repair, the function of multicomponent cullin-RING-ligases (CRL) in DSB repair is only beginning to emerge. FBXW7 is a substrate recognition component of Skp1-Cullin1-F-box E3 ubiquitin ligases previously known only to regulate the proteasomal degradation of substrates such as Cyclin E and MCL1. Given that FBXW7 loss promotes genomic instability, we hypothesized that FBXW7 may have a direct function in DSB repair. To establish the functions of FBXW7 in DSB repair, we first demonstrated the rapid localization of FBXW7 to DSB sites in an ATM-dependent manner. Subsequently, we found that FBXW7 depletion impaired nonhomologous end-joining (NHEJ), but not homologous recombination (HR) repair, resulting in persistent radiation-induced DSBs. Investigation of the molecular mechanisms of FBXW7 in NHEJ revealed that FBXW7 interacts with and promotes K63-linked ubiquitination of XRCC4. This ubiquitination of XRCC4 promotes interaction between the XRCC4/XLF/LIG4 and DNAPK/KU70/KU80 complexes to facilitate NHEJ. To begin to therapeutically leverage this mechanism, strategies to both pharmacologically inhibit FBXW7-CRLs and to exploit FBXW7 mutations occurring in human cancers are being developed. To address the former, pevonedistat (MLN4924), an agent which inhibits CRLs via inhibition of cullin-neddylation, inhibits FBXW7-mediated XRCC4 ubiquitination and NHEJ. This activity leads to increased sensitivity of tumor cells to chemotherapy and radiation and represents a strategy that may be particularly effective in cancers with other DSB repair defects. As another strategy to leverage the mechanisms of FBXW7 in NHEJ, loss-of-function mutations in the WD domain of FBXW7 which occur frequently in human cancers are being explored. These mutations render cells defective in NHEJ and more sensitive to DNA damage. Thus, we hypothesize that inhibition of other DSB repair pathways (e.g. alternative-end-joining) may be an effective therapeutic strategy for FBXW7 mutant cancers. These novel mechanisms of NHEJ regulation as well as their implications in the treatment of human cancers with an emphasis on pancreatic and colorectal cancers will be discussed.

This work was supported by NIH grants R01CA163895, P50CA130810, R01CA118762, R01CA156744, and R01CA171277.

Citation Format: Meredith Morgan. Exploiting the inhibition of cullin-RING-ligases in DSB repair as a therapeutic strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA27.
Abstract PR04: Mechanism for PARPi resistance: Homologous recombination without BRCA1
Authors: He Yizhou Joseph , Meghani Khyati , Chowdhury Dipanjan
Published: April 2017

Approximately 15-20% of all epithelial ovarian cancers harbor germline or somatic mutations in BRCA1/2. Despite PARP inhibitor induce synthetic lethality in non-cancerous mouse embryo fibroblast cells that harbor BRCA1/2 mutations, a substantial fraction of BRCA1/2-mutated tumors patients do not respond to PARPi treatment, and a large fraction of BRCA1/2-mutated tumors patients eventually develop resistance to PARPi suggest significant frequency of acquired mutation lead to PARPi resistance preventing complete cure.

We use CRISPR based library screen identified ATMIN/DYNLL1 loss as strong candidate for PARPi and cisplatin resistance mechanism. Targeted mutation on ATMIN or its transcription target DYNLL1 lead to increase of Cisplatin IC50 up to 200% and Olaparib IC50 up to 400%. More interestingly, ATMIN/DYNLL1 loss lead to restoration of Homologous recombination in BRCA1 mutant cells. ATMIN is under-expressed in about 30% ovarian cancer patient and low ATMIN expression correlate with shorter survival. Since ATMIN is important for DNA base damage repair but not Double Strand Break repair, we hypothesis ATMIN play important role in DNA damage repair pathway choice to suppress DSB repair while promote DNA base damage repair. Cells with mutant BRCA1 and loss ATMIN/DYNLL1 may result in abnormal DSB repair activity and lead to resistance to DNA damage agent such as Olaparib and Cisplatin.

This abstract is also being presented as Poster A18.

Citation Format: Yizhou Joseph He, Khyati Meghani, Dipanjan Chowdhury. Mechanism for PARPi resistance: Homologous recombination without BRCA1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR04.
Abstract PR17: The transcriptional repressor Slug promotes the DNA damage response
Authors: Zhou Wenhui , Ouyang Jian , Huber Kathryn , Kuperwasser Charlotte
Published: April 2017

The transcriptional repressor Slug/SNAI2 orchestrates epigenetic programs indispensable for tissue self-renewal and tumorigenesis. Although Slug-deficient animals are highly sensitive to lethal irradiation, the direct biological relationship between Slug and the DNA damage response remains largely unexplored. Here we report that Slug interacts with DNA repair proteins, including FANCI, BCCIP and PARP1, in a mass spectrometry screen for Slug binding partners. In response to double-strand breaks (DSBs) induced by irradiation, Slug-deficient cells exhibited a marked delay in the resolution of γH2ax foci. Furthermore, we showed that Slug inhibition significantly impaired DSBs repair that is mediated by homologous recombination. Accordingly, Slug-deficient mouse tissue accumulates DNA damage markers and showed altered nuclear morphology. Mechanistically, Slug interacts with and is stabilized by ATM upon DNA damage. Finally, we demonstrated that Slug depletion sensitizes aggressive triple-negative breast cancer cells to irradiation and chemotherapy. These findings suggest that Slug may be an important regulator of the DNA damage response and an attractive therapeutic target for aggressive cancer types.

This abstract is also being presented as Poster B16.

Citation Format: Wenhui Zhou, Jian Ouyang, Kathryn Huber, Charlotte Kuperwasser. The transcriptional repressor Slug promotes the DNA damage response [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR17.