Targeting DNA Repair Research Articles

Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes

Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Ofnine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations.

Abstract 1069: Inhibition of the nuclear import receptor, KpnB1 synergizes with cisplatin toxicity in cervical cancer cells

Abstract Elevated expression of members of the nuclear transport protein family has been reported in multiple cancers and presents as novel anticancer therapeutic targets. Using a cervical cancer model system, we have previously shown that inhibition of the nuclear import receptor, KpnB1 by siRNA and a novel small molecule, Inhibitor of Nuclear Import (INI-43) resulted in cancer cell death via apoptosis. In this study, we investigated the cancer cell killing effects of KpnB1 inhibition in combination with Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. KpnB1 siRNA and INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP. Our data shows that the combination treatment of INI-43 and CDDP significantly decreases CDDP IC50 compared to CDDP treatment alone. Increased PARP-cleavage was observed in combination treated cells and this correlated with increased γH2A.X, indicating increased apoptosis and DNA damage. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response transcription factor known to be a KpnB1 cargo protein that promotes cancer cell survival. Decreased levels of downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and XIAP, which correlated with increased DNA damage and apoptosis. Taken together, results suggest increased Cisplatin sensitivity of cervical cancer cells when nuclear import via KpnB1 is inhibited. Citation Format: Ru-pin Chi, Wei Wei, Michael Birrer, Virna D. Leaner. Inhibition of the nuclear import receptor, KpnB1 synergizes with cisplatin toxicity in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2017-1069

Targeting DNA repair and replication stress in the treatment of ovarian cancer.

Approximately half of high-grade serous epithelial ovarian cancers incur alterations in genes of homologous recombination (BRCA1, BRCA2, RAD51C, Fanconi anemia genes), and the rest incur alterations in other DNA repair pathways at high frequencies. Such cancer-specific gene alterations can confer selective sensitivity to DNA damaging agents such as cisplatin and carboplatin, topotecan, etoposide, doxorubicin, and gemcitabine. Originally presumed to inhibit DNA repair, PARP inhibitors that have recently been approved by the FDA for the treatment of advanced ovarian cancer also act as DNA damaging agents by inducing PARP-DNA complexes. These DNA damaging agents induce different types of DNA lesions that require various DNA repair genes for the repair, but commonly induce replication fork slowing or stalling, also referred to as replication stress. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. Hence, targeting DNA repair genes or DNA repair checkpoint genes augments the anti-tumor activity of DNA damaging agents. This review describes the rational basis for using DNA repair and DNA repair checkpoint inhibitors as single agents. The review also presents the strategies combining these inhibitors with DNA damaging agents for ovarian cancer therapy based on specific gene alterations.

NAMPT inhibition is a novel synthetic lethal therapeutic approach exploiting nuclear-mitochondrial crosstalk in ERCC1-deficient populations.

e23159 Background: ERCC1 (Excision Repair Cross-Complementation group 1) deficiency is the most frequent DNA repair defect in non-small cell lung cancers (NSCLC), making this enzyme an attractive therapeutic target for synthetic lethal (SL) approaches. Previous data support that DNA damage response is involved in regulation of metabolic homeostasis. We hypothesized that ERCC1 deficiency resulted in metabolic reprogramming of cancer cells that in turn generated specific vulnerabilities. Methods: High-throughput proteomic SILAC (stable isotope labeling by aminoacids in cell culture) and metabolomic (LC-MS, GC-MS, LC-QTOF) profiling were performed on a in-house generated NSCLC model of ERCC1 deficiency. Nicotinamide phosphoribosyltransferase (NAMPT) was selected as main hit and revalidated in low-throughput. Sensitivity to NAMPT inhibition was assessed in several NSCLC models. Potential SL interactions were investigated using functional DNA repair and metabolic assays, and transmission electronic microscopy (TEM). Results: We found marked metabolic rewiring in ERCC1-deficient populations, including decreased NAMPT and NAD+ levels and defects in the tricarboxylic acid cycle. These caused exquisite selective sensitivity to NAMPT inhibition in several in vitro and in vivoNSCLC models; these effects were reversed by the restoration of a functional ERCC1 isoform, establishing a causal link with the ERCC1 defective state. TEM, functional DNA repair and metabolic studies revealed structural and functional defects in the mitochondria of ERCC1-deficient cells, and allowed us to propose a model for this nuclear-mitochondrial SL link. A correlation between NAMPT expression and ERCC1 expression was observed in NSCLC patient samples. Conclusions: These findings open novel therapeutic opportunities that exploit a previously undescribed nuclear/mitochondrial SL link between ERCC1 deficiency and NAMPT inhibition in NSCLC. This highlights the potential for targeting DNA repair / metabolic crosstalk for cancer therapy, and support the evaluation of NAMPT inhibitors in ERCC1-deficient tumors.

Recent advances in targeting DNA repair pathways for the treatment of ovarian cancer and their clinical relevance.

Poly (ADP-ribose) polymerase (PARP) inhibitors have attracted much attention as one of the major molecular-targeted therapeutics for inhibiting DNA damage response. The PARP inhibitor, olaparib, has been clinically applied for treating certain recurrent ovarian cancer patients with BRCA1/2 mutations in Europe and the United States. It was also designated on 24 March 2017 as an orphan drug in Japan for similar clinical indications. In this review, we discuss (i) the prevalence of BRCA1/2 mutations in ovarian cancer, (ii) clinical trials of PARP inhibitors in ovarian cancer, (iii) genetic counseling for hereditary breast and ovarian cancer patients, and (iv) non-BRCA genes that may be associated with homologous recombination deficiency.

Clinical-pharmacogenetic models for personalized cancer treatment: application to malignant mesothelioma

Large interindividual differences in treatment outcome are observed in cancer patients undergoing chemotherapy. Our aim was to develop and validate clinical-pharmacogenetic prediction models of gemcitabine/cisplatin or pemetrexed/cisplatin treatment outcome and develop an algorithm for genotype-based treatment recommendations in malignant mesothelioma (MM). We genotyped 189 MM patients for polymorphisms in gemcitabine, pemetrexed and cisplatin metabolism, transport and drug target genes and DNA repair pathways. To build respective clinical-pharmacogenetic models, pharmacogenetic scores were assigned by rounding regression coefficients. Gemcitabine/cisplatin model was based on training group of 71 patients and included CRP, histological type, performance status, RRM1 rs1042927, ERCC2 rs13181, ERCC1 rs3212986, and XRCC1 rs25487. Patients with higher score had shorter progression-free (PFS) and overall survival (P < 0.001). This model’s sensitivity was 0.615 and specificity 0.812. In independent validation group of 66 patients the sensitivity and specificity were 0.667 and 0.641, respectively. Pemetrexed/cisplatin model was based on 57 patients and included CRP, MTHFD1 rs2236225, and ABCC2 rs2273697. Patients with higher score had worse response and shorter PFS (P < 0.001). This model’s sensitivity was 0.750 and specificity 0.607. In independent validation group of 20 patients the sensitivity and specificity were 0.889 and 0.500, respectively. The proposed algorithm based on these models could enable the choice of the most effective chemotherapy for 85.5% of patients and lead to improved treatment outcome in MM.

Abstract B25: “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery

Abstract Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Inhibition of DSB repair sensitizes cells to these therapies. Mutations have been reported in nearly every DNA repair pathway and these pathways often exhibit redundancy. Inhibition of functional repair factors can induce synthetic lethality in repair-deficient tumors even in the absence of exogenous DNA damage and whilst sparing healthy tissue. We demonstrate a compound and target discovery platform comprising the integration of isogenic cell line screening, high content repair foci assays and relative DSB repair pathway reporting cells, along with chemical biology and medicinal chemistry. Two diverse drug-like compound library screens have been conducted. Screening for synthetic lethality with deficiency in FANCD2 and BRCA2 led to a hit series broadly active against a range of homologous recombination (HR) repair deficiencies, which is now undergoing medicinal chemistry optimization and target deconvolution studies. Outcome of these initial chemoproteomic target pulldown experiments will be presented. A second cellular screen, (50k compounds) for inhibition of BRCA1 and Rad51 foci formation following radiomimetic drug induced DNA damage, has led to identification of compounds that inhibit damage response after ionizing radiation and selectively inhibit HR repair. Focusing on therapeutic targeting of proliferating tumor cells, our research platform has enabled novel hit compound identification, mechanistic profiling, hit-to-lead optimization chemistry and proof of concept efficacy in in vitro tumor models. Effects on tumor cell clonogenic survival and sensitization towards chemotherapeutics and ionizing radiation will be presented, along with comparisons to known DNA repair inhibitors that demonstrate these compounds' differentiated mechanisms of action. TargetDBR has created a technology platform focused on DNA repair inhibitor discovery and has identified differentiated hits and lead series with potential for development and opportunity to meet the need for new druggable targets in oncology. Citation Format: Jonathan J. Hollick, Laura Abriola, Francoise Bono, Denise Hegan, Pamela Klingbeil, Yanfeng Liu, Ranjini Sundaram, Yulia V. Surovtseva, Mark Whittaker, Ranjit S. Bindra, Peter M. Glazer. “TargetDBR”—A DNA repair drug and target discovery collaboration: Exploiting synthetic lethal, high content, and functional cellular reporter assays to accelerate DNA repair targeted drug discovery [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 B25.

Abstract A21: Enhancing chemotherapeutic responses in CNS malignancy through suppression of hyperactive DNA damage repair pathways

Abstract Introduction: We are targeting DNA repair pathways to enhance existing chemoradiotherapeutic strategies against medulloblastoma (MB) and malignant glioma (MG), two highly invasive tumors of the central nervous system (CNS). Current methods to treat childhood medulloblastoma are highly intrusive and lead to poor quality of life while the three-year survival rate of patients afflicted with malignant glioma remains abysmal (&amp;lt;5%). Recurrence of these exceedingly malignant tumors is pervasive as they can adopt several mechanisms to resist anti-cancer therapeutics including activation and up-regulation of DNA repair pathways that act to resolve DNA damage elicited by radiation and chemotherapeutic agents (chemo-radiotherapy). DNA repair inhibitors like Poly (ADP-Ribose) Polymerase (PARPi), DNA-dependent protein kinase (DNA-PKi) and Ataxia Telengiectasia Mutated (ATMi) have shown promise to sensitize tumors to DNA damaging chemo-radiotherapeutics as these inhibitors specifically target single yet highly critical DNA repair response pathway enzymes. In combination with anti-tumor agents, these sensitizers can significantly augment anti-cancer therapeutic success. However, differing tumors have variable expression/activity of these enzymes and their corresponding repair pathway(s), therefore; their identification, characterization and the development of strategies to modulate their expression can enhance current anti-tumor treatment efficacy. Methods: I will identify specific differentially-regulated DNA repair enzymes/pathways by gene expression methodology. From these findings, I will inhibit these DNA repair enzymes via shRNA or enzyme-specific inhibitors (if available), to chemosensitize MB and MG cells to DNA damaging therapeutics in an effort to reduce DNA repair in these cells thereby boosting tumor genotoxicity and cell death. Extent of DNA damage will be measured using novel high-throughput DNA repair assays combined with unique cellular DNA damage reporters/sensors to facilitate these studies. Results: In comparing subsets of MB and MG cell lines with existing DNA repair inhibitors, I have found differing DNA repair pathways as potential targets to sensitize these CNS tumors to chemo-radiotherapeutics. MB cells rely on DNA-PK/SSBR (single strand break repair) dependent pathways to resolve induced genotoxicity, while MG utilizes the ATM/SSBR pathways. My focus is now to expand this dataset in additional pertinent tumors and to perform a detailed comparative analysis to identify specific highly active tumor-specific DNA repair enzymes with which to target in order to enhance tumor cell death. These include the use of DNA damage repair response PCR arrays to compare the expression levels of individual DNA repair enzyme/pathway members amongst these CNS tumors. Conclusion: I have identified unique DNA repair enzymes, which may mediate specific differential chemo-radioresistant phenotypes in MB and MG. An expanded analysis is currently underway to further differentiate DNA repair mechanisms and therapeutic responses between these two brain tumor types. I hope to translate these findings into pre-clinical models whereby my data may lead to identifying next-generation brain cancer treatments with improved patient survival and quality-of-life. Citation Format: Marina Mostafizar, Sachin Katyal. Enhancing chemotherapeutic responses in CNS malignancy through suppression of hyperactive DNA damage repair pathways [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 A21.

Abstract B34: Development of novel small molecule inhibitors targeting DNA repair proteins

Abstract 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 A40: Profile of DNA repair status in a recently established panel of patient-derived ovarian carcinoma xenografts

Abstract Background and Purpose: A xenobank of patient derived (PDX) ovarian tumor samples has recently been established and shown to recapitulate the biological, histological, molecular and pharmacological features of human epithelial ovarian cancer (EOC). This EOC-xenobank consists of tumors with different sensitivity to cisplatin (DDP) from very responsive, responsive to resistant tumors. As DNA repair pathway represents an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in Nucleotide Excision Repair (NER), in Fanconi Anemia (FA), in Homologous Recombination (HR), in Base Excision Repair (BER), in Mismatch Repair (MMR) and Translesion Repair (TLR) pathways and the methylation pattern of some of these genes. The correlation with the response to a platinum-based therapy was also investigated. Methods: The genes selected have a key role in the BER (OGG1 and PARP1); in the NER pathway (ERCC1, XPA, XPF, XPD and XPG); the FA pathway (BRCA1, FANCA, FANCC, FANCD2 and FANCF); in TLR (PolEta), in MMR (MLH1), in MMEJ (PolQ) and in the transcription of some genes (CDK12). The mRNA levels were evaluated by RT-PCR with ad hoc validated primers. The promoter methylation of BRCA1, XPA, ERCC1 and MLH1 was studied by pyrosequencing, while for XPG and FANCF no predesigned assays were available and we evaluated their methylation status following reported assays. Results: We demonstrated: i) all the DNA genes considered were variably expressed in all the 44 PDX analyzed, with no specific istotype-specific cluster of expression; ii) in high grade seros/endometriod PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and PolB; iii) high grade serous/endometriod PDXs with TP53 mutation had statistically significant higher levels of PolQ, FANC-D2, RAD51 and Pol beta than high grade TP53 wild type PDXs; iv) BRCA1 was found to be hypermethylated in 48% of the xenografts. However, no correlation between mRNA levels and methylation status of the considered CpG islands was found; v) only the mRNA levels of CDK12, PALB2 and XPF inversely correlated with the in vivo DDP antitumor activity. Conclusions: These data suggest the mRNA expression levels of targeted DNA repair pathway-related genes as PALB2, XPF and CDK12 predict the response to a platinum based therapy in ovarian cancer, even if they need to be prospectively validated in cohort of ovarian cancer patients. Citation Format: Federica Guffanti, Maddalena Fratelli, Monica Ganzinelli, Francesca Ricci, Maria Rosa Cappelletti, Damiele Generali, Francesca Bizzaro, Raffaella Giavazzi, Giovanna Damia. Profile of DNA repair status in a recently established panel of patient-derived ovarian carcinoma xenografts [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 A40.

Drugging the Cancers Addicted to DNA Repair.

Defects in DNA repair can result in oncogenic genomic instability. Cancers occurring from DNA repair defects were once thought to be limited to rare inherited mutations (such as BRCA1 or 2). It now appears that a clinically significant fraction of cancers have acquired DNA repair defects. DNA repair pathways operate in related networks, and cancers arising from loss of one DNA repair component typically become addicted to other repair pathways to survive and proliferate. Drug inhibition of the rescue repair pathway prevents the repair-deficient cancer cell from replicating, causing apoptosis (termed synthetic lethality). However, the selective pressure of inhibiting the rescue repair pathway can generate further mutations that confer resistance to the synthetic lethal drugs. Many such drugs currently in clinical use inhibit PARP1, a repair component to which cancers arising from inherited BRCA1 or 2 mutations become addicted. It is now clear that drugs inducing synthetic lethality may also be therapeutic in cancers with acquired DNA repair defects, which would markedly broaden their applicability beyond treatment of cancers with inherited DNA repair defects. Here we review how each DNA repair pathway can be attacked therapeutically and evaluate DNA repair components as potential drug targets to induce synthetic lethality. Clinical use of drugs targeting DNA repair will markedly increase when functional and genetic loss of repair components are consistently identified. In addition, future therapies will exploit artificial synthetic lethality, where complementary DNA repair pathways are targeted simultaneously in cancers without DNA repair defects.

SA 5.1 - Targeting DNA repair

SA 5.1 - Targeting DNA repair

Abstract PR13: Inactivation of DNA repair triggers dynamic neoantigen evolution and impairs cancer growth

Abstract Colorectal, ovarian, endometrial and other tumors carrying defects in DNA mismatch repair often show favorable prognosis and indolent progression. The genomes of these tumors -also known as microsatellite unstable (MSI) cancers- bear hundreds of thousands of somatic mutations, a feature which fosters cancer progression and rapid evolution of resistance to targeted therapies. The molecular bases for the favorable outcome of MMR deficient cancers have long remained a mystery. Recent evidences that a subset of MSI tumors respond prominently to immune checkpoint blockade led to the seminal hypothesis that the presence of high number of somatic mutations may be responsible for effective immune-surveillance. However, several reports indicate that a relevant fraction of hyper-mutated tumors have unfavorable prognosis and do not respond to immune-modulators. Intrigued by these contradictory findings, we used the CRISPR system to genetically inactivated MutL homolog 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR deficient cells was comparable to their proficient counterparts in vitro and upon transplantation in immune-compromised mice. Strikingly however, isogenic MMR deficient colorectal, breast and pancreatic cancer cells were largely unable to form tumors when injected subcutaneously or orthotopically in syngeneic mouse models. MMR deficient tumors initially established in immune-deficient mice continued to grow exponentially when transplanted in syngeneic animals but regressed promptly when immune checkpoint inhibitors (anti PD-1 and anti CTLA-4) were administered. Exome sequencing of MMR proficient cells revealed mutational loads and neo-antigen profiles that were stable over time. MMR inactivation further increased the mutation burden and led to highly dynamic mutational profiles, resulting in persistent renewal of neoantigens. These results led us hypothesize that enforced increase of the number of mutations in cancer cells could be paradoxically- beneficial for therapeutic purposes. We therefore performed a pharmacological screen to identify agents capable of permanent inactivation of MMR in colorectal, breast and PDAC cancer cells. We found that temozolomide triggers MLH1 inactivation and leads to rapid clonal evolution and dynamic neoantigen profiles. Temozolomide-treated cells were unable to form tumors in syngeneic animals, while cells treated with other alkylating agents did. Genomic analysis of these tumor models revealed that fluctuating levels of neoantigens, rather than the absolute number of mutations is critical to provoke immune surveillance. These results provide the rationale for developing innovative anticancer therapies that target DNA repair proteins. This abstract is also being presented as Poster B11. Citation Format: Giovanni Germano, Simona Lamba, Giuseppe Rospo, Alessandro Magrì, Federica Maione, Mariangela Russo, Nabil Amirouchene Angelozzi, Ludovuc Barault, Monica Montone, Federica Di Nicolantonio, Enrico Giraudo, Alberto Bardelli. Inactivation of DNA repair triggers dynamic neoantigen evolution and impairs cancer growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR13.

Abstract MS2-3: MS2-3 Clinical implications of DNA repair and genome instability research

Abstract Breast cancer has now been subjected to extensive molecular characterisation. Although much attention has focused on transcriptional profiling, with consequent identification of a number of biological sub-types, it is clear that breast cancers show significant variability in their somatic genome. Breast cancers are diseases that are largely dominated by copy number aberrations (CNAs) rather than single nucleotide variants (SNVs). Although HER2 is the poster child CNA driven therapeutic target a number of other mutation and CNA candidates are being pursued. The mutational processes that drive the different forms of genomic instability in breast cancer are increasingly understood and have been described in the last year. These mutational and rearrangement signatures may reveal aberrations in the DNA damage response that themselves represent a vulnerability and a therapeutic target. I will discuss a range of strategies and clinical trials being employed to target DNA repair, genome instability and consequent replicative stress in breast cancer. I will also discuss the state of development of assays that categorise a range of forms of mutational signature and genome instability which may act in future as predictive biomarkers of therapy response or resistance to DNA damage response targeting therapies with utility in the clinic. Citation Format: Tutt A. MS2-3 Clinical implications of DNA repair and genome instability research [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr MS2-3.

Germline mutations in seemingly sporadic pancreatic cancer.

312 Background: Genetic predisposition underlies approximately 10% of pancreatic cancer (PC). Germline mutations in BRCA1, BRCA2, PALB2, ATM, TP53, MLH1, MSH2, MSH6, PMS2, PRSS1, APC, BMPR1A, STK11, CDKN2A, and SMAD4 are associated with PC susceptibility. Since young onset is not a hallmark of hereditary PC, we have hypothesized that seemingly sporadic cases may carry germline mutations in these genes. Methods: To investigate the prevalence of germline mutations in these 15 genes among sporadic PC cases, we analyzed lymphocyte DNA next generation sequencing data from 296 PC cases ascertained over 1 year from two PC referral centers in Toronto and Montreal. Results: We identified 21 mutations in 5 of the 15 genes (10 in BRCA2, 2 in BRCA1, 5 in ATM, 1 in p16, 3 in PRSS1) in 19 individuals. One individual carried two BRCA2 mutations that are usually observed together, and another individual carried both a PRSS1 and ATM mutation, however, in the absence of pancreatitis, PRSS1 mutations are not known to increase risk of PC. Incidences of 1.7% and 4.1% in ATM and BRCA-1 or -2 were observed. Approximately half of these mutations (4/9 for BRCA2, 1/2 for BRCA1, and 1/1 for p16) were known founder mutations in the French Canadian, Ashkenazi Jewish, Dutch or Asian populations. Since the role of ATMin PC predisposition remains under investigation, we obtained further evidence by testing for loss of the wild-type allele in cases with available tumor tissue. We observed loss of the wild-type allele in the 2 cases tested. Interestingly, the final pathology for the resected specimen from one of these two cases favored an ampullary cancer rather than a PC diagnosis. Conclusions: Since there are screening implications for the relatives of carriers and potentially treatment considerations for patients using therapies targeting DNA repair defects, these findings suggest a role for reflex testing of incident cases for at least population-specific recurrent mutations. Our findings also suggest that ATM germline mutations may give rise to ampullary cancer, underlie 1.4% of incident PC cases and imply broader testing considerations. For patients, testing may direct therapy choices if these tumors are targetable using agents that exploit DNA repair defects.

Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway.

Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC.

Origins and Applications of CRISPR-Mediated Genome Editing.

In this graphical review we highlight key milestones in the discovery and development of CRISPR and cas9 technology, as well as illustrate mechanisms of cas9:sgRNA target binding and DNA repair.

Abstract IA12: Targeting DNA repair in cancer therapy

Abstract Human cancer cells have genomic instability resulting from germline or acquired defects in DNA repair. One DNA repair pathway—the Fanconi Anemia/BRCA pathway (D'Andrea, A., N Engl J Med 362:1909, 2010)—is defective in many human cancers, including breast, ovarian, and pancreatic neoplasms. Disruption of the FA/BRCA Pathway results in the characteristic chromosome instability and radiation/crosslinker hypersensitivity of these tumors. In general, loss of one DNA repair pathway often leads to hyperdependence on another pathway for tumor cell survival. This hyperdependence offers a unique opportunity for the development of anticancer therapeutics. For instance, FA/BRCA pathway deficient tumors are hyperdependent on Base Excision Repair (BER) and, accordingly, these tumors are hypersensitive to single agent treatment with PARP1 inhibitors, which block BER, or with ATM inhibitors. Our research program is focused on profiling the FA/BRCA pathway and the other five major DNA repair pathways in tumor cells (Kennedy and D'Andrea, JCO 24: 3799, 2006). Each DNA repair pathway has a characteristic biomarker and repairs a specific type of DNA lesion. By profiling these pathways in primary tumor samples with activation-specific antibodies to DNA repair proteins, we have been able (1) to predict the sensitivity of tumors to conventional chemotherapy and radiation (2) to subset tumors for their sensitivity to novel classes of DNA repair inhibitors, (i.e., PARP1, Chk1, ATM, and proteasome inhibitors) and (3) to screen for new small molecule inhibitors of other DNA repair pathways. This combination of novel DNA repair inhibitors, conventional DNA damaging agents, and DNA repair biomarkers offers new opportunities for developing more effective anticancer therapy. In my presentation, I will also discuss novel approaches to sensitizing Triple Negative Breast Cancers (TNBCs) and Ovarian Tumors (OCs) to PARP1 inhibitors. We have previously shown that exposure of these tumors to either a CDK inhibitor or a proteasome inhibitor can block homologous recombination (HR) repair and thereby sensitize the tumor cells to PARP1 inhibitor. The CDK inhibitor/PARP1 inhibitor combination has been examined in orthotopic primary TNBC and OC xenograft models. Finally, we have identified an additional PARP1-dependent DNA repair mechanism in TNBCs and OCs. This pathway is driven by a novel alternative end-joining polymerase, POLθ. These tumors are hypersensitive to inhibitors of this new pathway, suggesting a new target for DNA repair therapies. Citation Format: Alan D. D'Andrea. Targeting DNA repair in cancer therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA12.

Targeting DNA Repair.

Genomic instability is a characteristic of most human cancers and plays critical roles in both cancer development and progression. There are various forms of genomic instability arising from many different pathways, such as DNA damage from endogenous and exogenous sources, centrosome amplification, telomere damage, and epigenetic modifications. DNA-repair pathways can enable tumor cells to survive DNA damage. The failure to respond to DNA damage is a characteristic associated with genomic instability. Understanding of genomic instability in cancer is still very limited, but the further understanding of the molecular mechanisms through which the DNA damage response (DDR) operates, in combination with the elucidation of the genetic interactions between DDR pathways and other cell pathways, will provide therapeutic opportunities for the personalized medicine of cancer.

Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.

Various inhibitors of DDR components are in preclinical and clinical development. A thorough understanding of DDR pathway complexities must now be combined with strategies and lessons learned from the successful registration of PARP inhibitors in order to fully exploit the potential of DDR inhibitors and to ensure their long-term clinical success. Cancer Discov; 7(1); 20-37. ©2016 AACR.