Defective DNA Damage Response Research Articles

The Inhibitory Effects of HYDAMTIQ, a Novel PARP Inhibitor, on Growth in Human Tumor Cell Lines With Defective DNA Damage Response Pathways.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

Multiple Malignancies in a Young Patient

SESSION TITLE: Non-Small Cell Lung Cancer SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: Fanconi anaemia (FA) is associated with chromosomal fragility and a high risk of cancers, including leukaemia and solid tumours. 3% of patients develop more than one cancer in their lifetime. We report a case of a 31 year old man with FA who was diagnosed concurrently with myelodysplastic syndrome and squamous cell carcinoma of the lung. CASE PRESENTATION: He presented to an acute hospital with cough and breathlessness of 1 month’s duration. A chest radiograph showed right lower lobe collapse and he also had thrombocytopenia with 2% blasts. Bone marrow aspiration showed myelodysplastic syndrome (deletion 20q). Bronchoscopy showed an endobronchial tumour obstructing the right lower lobe and an eventual diagnosis of Stage 3A squamous cell carcinoma was made. He had been healthy prior to his diagnosis of cancer with no developmental abnormalities. He was of normal height (167cm) and head circumference (53.5cm) and had no skeletal abnormalities. There were café au lait spots on his back and right neck. He worked as a delivery driver and had smoked for 5 pack years prior to his diagnosis. A review of his family history revealed significant history of malignancy. His sister had been diagnosed with skin cancer and lymphoma at the age of 35, and there was a history of lung cancer in both sides of his family. There was no history of consanguinity in his family. In view of his young age and family history, he underwent genetics testing and FA was eventually diagnosed. DISCUSSION: Squamous cell carcinomas have been described in FA, although these are more commonly of the head and neck. A review of the literature showed very few reported cases of FA with squamous cell carcinoma of the lung (1). These patients usually tolerate chemotherapy and radiotherapy poorly due to a defect in DNA damage response and prognosis is often poor (2). After extensive discussion with a multidisciplinary team, the patient was deemed not to be suitable for chemotherapy because of his myelodysplastic syndrome. He was treated with an attenuated dose of radical radiotherapy to the lung with palliative intent. This was complicated by 2 admissions for haemoptysis which were managed with bronchial artery embolization and platelet transfusions. Of date, he has tolerated 15 fractions of radiotherapy with twice a week platelet transfusions. The last staging scan showed mild reduction in the size of the endobronchial tumour. CONCLUSIONS: FA is a rare disease with an incidence of 1 in 200 000-400 000. Squamous cell carcinoma associated with FA is even rarer and there are currently no proven therapies. Reference #1: Cathart-Rake, Lopez-Chavaz. Young Male with Fanconi Anemia and EGFR-Mutant Non-Small-Cell Lung Cancer. J Thorac Oncol. 2014 Nov;9(11):e83-5 Reference #2: A.R. Venkitaraman. A growing network of cancer-susceptibility genes. N Engl J Med, 348 (19) (2003), pp. 1917-1919 DISCLOSURE: The following authors have nothing to disclose: Hui Zhong Chai, Qiao Li Tan No Product/Research Disclosure Information

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPC) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a pre-malignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation attenuated. We also document that CREBBP mutations may occur in HSPC from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid-transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status

AbstractThe role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)–p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised.

Mitochondrial dysfunction and potential anticancer therapy.

Mitochondrial dysfunction (MDF) has been identified as an important factor in various diseases ranging from neurological disorders, to diseases of the cardiovascular system and metabolic syndromes. MDF was also found in cancer as well as in cancer predisposition syndromes with defective DNA damage response (DDR) machinery. Moreover, a recent highlight arises from the detection of MDF in eukaryotic cells upon treatment with antibiotics. In this review, we focus on recent studies of MDF in pathological conditions with a particular emphasis on the effects of various classes of antibiotics on mitochondria. Special attention is given to the role of autophagy/mitophagy in MDF and repurposing antibiotics as anticancer drugs.

Abstract 2335: Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing

Abstract MCL-1 is a a pro-survival BCL2 protein family member which is over-expressed in drug resistant cancer cells. Our purpose was to assess whether MCL-1 increases genomic instability along with its anti-apoptotic function in cancer cells. Depletion of MCL-1 by treating human small cell lung cancer (SCLC) lines with immunotoxin sensitizes these cells to increased killing by BH3 mimetic - Navitoclax (ABT-263) both in vitro and in mice xenograft models. These SCLC cell lines are resistant to both single agent Immunotoxin or ABT-263. In addition, inhibition of NANOG or NANOGP8 by lentivrus delivered shRNA depletes MCL-1 in human colorectal cancer cells and sensitizes these cells to increased cell killing by venetaclox (ABT-199). This increased killing depended on loss of MCL-1, was caspase-dependent and reversed by re-expression of MCL-1. Depletion of MCL-1 in cancer cells , increases cell sensitivity to ionizing radiation (IR) induced death, which was reversed by expression of MCL-1. In response to IR, MCL-1 depleted cells showed reduced survival even at radiation doses less than 6 Gy, however an increase in caspases 3/7 activity was observed at 6 Gy and above, suggesting that apoptosis is activated only when MCL-1 depleted cells are irradiated with higher IR doses. In order to explain the decreased survival post- irradiation the genomic instability and DNA repair pathways were analyzed in MCL-1 depleted cells. Post-irradiation MCL-1 depleted cells exhibited increased genomic instability as measured by increase in chromosome aberrations at different phases of the cell cycle. The increase in aberrations were significantly higher in G2 and S- phases of the cell cycle suggesting defect in Homologous recombination repair pathway (HR). Moreover, the MCL-1 depleted cells show decreased gamma-H2AX foci at earlier time points (30 and 90 minutes) post-irradiation indicating defect in DNA Damage response (DDR) and higher residual foci at 360 minutes, indicating defective DNA repair. These observations were further confirmed by decreased phosphorylation of ATR suggesting defect in DNA Damage response and higher levels of residual 53BP1 and RIF1 foci in MCL-1 depleted cells , confirming DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells exhibited a reduced frequency of IR-induced MRE11, BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after Hydoxyurea (HU) induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. In summary, MCL-1 is an important therapeutic target in cancer cells and its depletion increases cell killing by either increase in apoptosis or suppression of HR and increased replication stress. Citation Format: Abid Mattoo, J Milburn Milburn Jessup, Tej K. Pandita. Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing [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 2335. doi:10.1158/1538-7445.AM2017-2335

Abstract 1281: Germline mutations in NBN conferring DNA damage response defects are found in patients with multiple cancer types

Abstract Nibrin, the protein encoded by the NBN gene forms a complex with Mre11 and Rad51 (MRN complex) that is crucial for DNA damage repair. Mutations in NBN are found in >90% of patients with Nimjegen breakage syndrome (NBS), an autosomal recessive disorder characterized by growth retardation, microcephaly, radiosensitivity, immunodeficiency and increased cancer risk. Most of NBS patients harbor the common founder mutation c.657del5 that leads to expression of a hypomorphic 70kDa C-terminal fragment produced by alternative translation initiation. Cell lines derived from these patients show increased sensitivity to DNA-damaging agents, chromosome instability, and abnormal cell cycle checkpoint function. Several studies have addressed cancer incidence in individuals with germline NBN mutations, showing increased cancer risk for individuals harboring the c.657del5 founder mutation and for carriers of the R215W missense mutation across multiple cancer types. For other NBN mutations conflicting reports exist as to their association with cancer risk. Here, we report newly identified NBN germline frameshift and truncating mutations in patients with multiple cancer types, including prostate, lung, breast cancer, acute lymphocytic leukemia, and chronic lymphocytic leukemia. Modeling these mutations in an NBN-deficient cellular background showed expression of a novel C-terminal truncated fragment that can bind to Mre11. Cells expressing these mutant proteins display attenuated DNA damage repair function and decreased overall survival following induction of DNA damage. Impaired Chk2 phosphorylation was also observed, indicating cell cycle checkpoint deficiencies. Thus, the NBN germline mutations identified here could contribute to genomic instability predisposing to tumorigenesis. Further in vitro studies with these and additional germline mutations occurring in cancer patients are ongoing, in order to better understand the role of this pathway of DNA damage repair in susceptibility to malignancies. Citation Format: Sabine Topka, Michael F. Walsh, Ann Maria, Annie Lincoln, Diana Mandelker, Liying Zhang, Marc Ladanyi, Michael F. Berger, Mark E. Robson, Joseph Vijai, Kenneth Offit. Germline mutations in NBN conferring DNA damage response defects are found in patients with multiple cancer types [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 1281. doi:10.1158/1538-7445.AM2017-1281

Neurodegeneration in ataxia-telangiectasia: Multiple roles of ATM kinase in cellular homeostasis.

Ataxia-telangiectasia (A-T) is characterized by neuronal degeneration, cancer, diabetes, immune deficiency, and increased sensitivity to ionizing radiation. A-T is attributed to the deficiency of the protein kinase coded by the ATM (ataxia-telangiectasia mutated) gene. ATM is a sensor of DNA double-strand breaks (DSBs) and signals to cell cycle checkpoints and the DNA repair machinery. ATM phosphorylates numerous substrates and activates many cell-signaling pathways. There has been considerable debate about whether a defective DNA damage response is causative of the neurological aspects of the disease. In proliferating cells, ATM is localized mainly in the nucleus; however, in postmitotic cells such as neurons, ATM is mostly cytoplasmic. Recent studies reveal an increasing number of roles for ATM in the cytoplasm, including activation by oxidative stress. ATM associates with organelles including mitochondria and peroxisomes, both sources of reactive oxygen species (ROS), which have been implicated in neurodegenerative diseases and aging. ATM is also associated with synaptic vesicles and has a role in regulating cellular homeostasis and autophagy. The cytoplasmic roles of ATM provide a new perspective on the neurodegenerative process in A-T. This review will examine the expanding roles of ATM in cellular homeostasis and relate these functions to the complex A-T phenotype. Developmental Dynamics 247:33-46, 2018. © 2017 Wiley Periodicals, Inc.

Defining DDR defectiveness in pancreatic cancer.

4115 Background: Recent whole genome sequencing analysis of Pancreatic Cancer (PC) revealed that up to 24% of PC may harbor defects in DNA damage response (DDR). There is increasing evidence that DDR defective tumors preferentially respond to DNA damaging agents, representing novel therapeutic strategy for PC using a synthetic lethality approach. The aim of this study is to define and refine DDR defective phenotypes in PC using next-generation preclinical model systems. Methods: From a panel of 40 patient-derived cell lines (PDCL) and 64 patient-derived xenografts (PDX), generated and extensively characterized as part of the International Cancer Genome Initiative (ICGC), we identified DDR defective models using recently described putative biomarkers of DDR defectiveness. Cytotoxic viability assays were performed using a panel of DNA damaging agents and inhibitors of key molecules in DDR pathway, including Cisplatin, PARP inhibitors, ATR inhibitor (AZD6738); and ATM inhibitor (AZD0156). Appropriate subcutaneous PDX models were also generated to test the hypothesis using various therapeutic regimens. Results: DDR defective PDCLs were selected based on a combination of an unstable genome, and/or a high BRCA mutational signature, and/or deleterious mutations in BRCA1/2, PALB2 or ARID1A. DDR defective PDCLs were significantly more sensitive to Cisplatin, PARP and ATR inhibitors. The ATR inhibitor AZD6738, and ATM inhibitor AZD0156 sensitized PDCLs with no putative biomarkers of DDR defectiveness to Cisplatin, demonstrating a ‘fabricated’ synthetic lethality. A BRCA1 mutant PDX model responded exceptionally to Cisplatin and the PARP inhibitor Olaparib monotherapy. Conclusions: This study provides proof of concept data that DDR deficiency represents an attractive segment to target in PC using a variety of DNA damaging agents and novel agents targeting key molecules of the DDR pathway in PC. In addition, the DDR defective segment may be significantly larger than just germline BRCA1/2 mutants, which is current clinical trial recruitment criteria. Robust molecular assays with clinical utility to define DDR defectiveness is urgently needed.

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion.

The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome. Despite these insights, specific roles for SUMO chains and STUbL in the DDR remain poorly defined. Notably, fission yeast defective in SUMO chain formation exhibit near wild-type resistance to genotoxins and moreover, have a greatly reduced dependency on STUbL activity for DNA repair. Based on these and other data, we propose that a critical role of STUbL is to antagonize DDR-inhibitory SUMO chain formation at DNA lesions. In this regard, we identify a SUMO-binding Swi2/Snf2 translocase called Rrp2 (ScUls1) as a mediator of the DDR defects in STUbL mutant cells. Therefore, in support of our proposal, SUMO chains attract activities that can antagonize STUbL and other DNA repair factors. Finally, we find that Taz1TRF1/TRF2-deficiency triggers extensive telomeric poly-SUMOylation. In this setting STUbL, together with its cofactor Cdc48p97, actually promotes genomic instability caused by the aberrant processing of taz1Δ telomeres by DNA repair factors. In summary, depending on the nature of the initiating DNA lesion, STUbL activity can either be beneficial or harmful.

Taking a Bad Turn: Compromised DNA Damage Response in Leukemia.

Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB) and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK) and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2), key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS), and isocitrate dehydrogenase 1 and 2 (IDH1/2). A detailed understanding of the basis for defective DNA damage response (DDR) mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients.

A role of human RNase P subunits, Rpp29 and Rpp21, in homology directed-repair of double-strand breaks

DNA damage response (DDR) is needed to repair damaged DNA for genomic integrity preservation. Defective DDR causes accumulation of deleterious mutations and DNA lesions that can lead to genomic instabilities and carcinogenesis. Identifying new players in the DDR, therefore, is essential to advance the understanding of the molecular mechanisms by which cells keep their genetic material intact. Here, we show that the core protein subunits Rpp29 and Rpp21 of human RNase P complex are implicated in DDR. We demonstrate that Rpp29 and Rpp21 depletion impairs double-strand break (DSB) repair by homology-directed repair (HDR), but has no deleterious effect on the integrity of non-homologous end joining. We also demonstrate that Rpp29 and Rpp21, but not Rpp14, Rpp25 and Rpp38, are rapidly and transiently recruited to laser-microirradiated sites. Rpp29 and Rpp21 bind poly ADP-ribose moieties and are recruited to DNA damage sites in a PARP1-dependent manner. Remarkably, depletion of the catalytic H1 RNA subunit diminishes their recruitment to laser-microirradiated regions. Moreover, RNase P activity is augmented after DNA damage in a PARP1-dependent manner. Altogether, our results describe a previously unrecognized function of the RNase P subunits, Rpp29 and Rpp21, in fine-tuning HDR of DSBs.

DNA damage response defect in Williams-Beuren syndrome

Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non-Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ-H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient-derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea-induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR-dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation.

Nucleotide Excision Repair: From Neurodegeneration to Cancer.

DNA damage poses a constant threat to genome integrity taking a variety of shapes and arising by normal cellular metabolism or environmental insults. Human syndromes, characterized by increased cancer pre-disposition or early onset of age-related pathology and developmental abnormalities, often result from defective DNA damage responses and compromised genome integrity. Over the last decades intensive research worldwide has made important contributions to our understanding of the molecular mechanisms underlying genomic instability and has substantiated the importance of DNA repair in cancer prevention in the general population. In this chapter, we discuss Nucleotide Excision Repair pathway, the causative role of its components in disease-related pathology and recent technological achievements that decipher mutational landscapes and may facilitate pathological classification and personalized therapy.

Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

Use of poly ADP-ribose polymerase [PARP] inhibitors in cancer cells bearing DDR defects: the rationale for their inclusion in the clinic.

BackgroundDNA damage response (DDR) defects imply genomic instability and favor tumor progression but make the cells vulnerable to the pharmacological inhibition of the DNA repairing enzymes. Targeting cellular proteins like PARPs, which cooperate and complement molecular defects of the DDR process, induces a specific lethality in DDR defective cancer cells and represents an anti-cancer strategy. Normal cells can tolerate the DNA damage generated by PARP inhibition because of an efficient homologous recombination mechanism (HR); in contrast, cancer cells with a deficient HR are unable to manage the DSBs and appear especially sensitive to the PARP inhibitors (PARPi) effects.Main bodyIn this review we discuss the proof of concept for the use of PARPi in different cancer types and the success and failure of their inclusion in clinical trials.The PARP inhibitor Olaparib [AZD2281] has been approved by the FDA for use in pretreated ovarian cancer patients with defective BRCA1/2 genes, and by the EMEA for maintenance therapy in platinum sensitive ovarian cancer patients with defective BRCA1/2 genes. BRCA mutations are now recognised as the molecular targets for PARPi sensitivity in several tumors. However, it is noteworthy that the use of PARPi has shown its efficacy also in non-BRCA related tumors. Several trials are ongoing to test different PARPi in different cancer types. Here we review the concept of BRCAness and the functional loss of proteins involved in DDR/HR mechanisms in cancer, including additional molecules that can influence the cancer cells sensitivity to PARPi. Given the complexity of the existing crosstalk between different DNA repair pathways, it is likely that a single biomarker may not be sufficient to predict the benefit of PARP inhibitors therapies. Novel general assays able to predict the DDR/HR proficiency in cancer cells and the PARPi sensitivity represent a challenge for a personalized therapy.ConclusionsPARP inhibition is a potentially important strategy for managing a significant subset of tumors. The discovery of both germline and somatic DNA repair deficiencies in different cancer patients, together with the development of new PARP inhibitors that can kill selectively cancer cells is a potent example of targeting therapy to molecularly defined tumor subtypes.

Targeting DNA Replication Stress for Cancer Therapy.

The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

Abstract 2745: A genetic screen to identify tumor suppressive DDR genes in breast cancer

Abstract To maintain genomic integrity, cells utilize a DNA damage response (DDR) mechanism that 1) functions to repair damaged DNA efficiently and 2) commits cells to apoptosis if damage is irreparable. Failure of this mechanism results in genomic instability and cancer predisposition. Defects in DDR have been implicated in both sporadic and familial breast and ovarian cancer predisposition syndromes (e.g. BRCA1, BRCA2, and PALB2), although the specific pathway components that are disrupted have not been elucidated. In particular, an impaired DDR may contribute to the pathophysiology of triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) due to the extent and complexity of the genomic instability that is observed. We identified the DNA damage sensing Mre11- Rad50-Nbs1 (MRN) complex as an essential mediator of the oncogene-induced DDR. Disrupting the MRN complex in mouse models promotes the development of genomically unstable, metastatic breast cancer. The clinical relevance of this pathway is highlighted by the observation that components of the Mre11 complex are perturbed in a significant fraction of human TNBCs, which correlates with clinical outcomes. However, lack of MRN suppression in a majority of TNBCs indicates that alternative DDR components may also be inactivated to promote breast cancer development. Our goal is to identify additional DDR genes that, when perturbed, promote the development of genomically unstable breast cancers. In order to accurately distinguish driver genes from bystander genes we have developed a platform to identify DDR genes that mediate oncogene-induced senescence in mammary epithelial cells using both shRNA and CRISPR pooled libraries. Analysis of newly identified tumor suppressor genes, compared to what is already known about Mre11 and p53 (positive controls), will enable us to determine causality of specific DDR mutations in breast cancer development and importantly, the genomic instability phenotype that is typical for TNBC. The insights gleaned from this study may ultimately provide the means to determine the optimal treatment regimen for each patient with TNBC, and to prevent over-treatment of patients where certain chemotherapies may not be effective. Citation Format: Katerina D. Fagan-Solis, Gaorav P. Gupta. A genetic screen to identify tumor suppressive DDR genes in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2745.

DNA damage to human genetic disorders with neurodevelopmental defects

Although some mutations are beneficial and are the driving force behind evolution, it is important to maintain DNA integrity and stability because it contains genetic information. However, in the oxygen-rich environment we live in, the DNA molecule is under constant threat from endogenous or exogenous insults. DNA damage could trigger the DNA damage response (DDR), which involves DNA repair, the regulation of cell cycle checkpoints, and the induction of programmed cell death or senescence. Dysregulation of these physiological responses to DNA damage causes developmental defects, neurological defects, premature aging, infertility, immune system defects, and tumors in humans. Some human syndromes are characterized by unique neurological phenotypes including microcephaly, mental retardation, ataxia, neurodegeneration, and neuropathy, suggesting a direct link between genomic instability resulting from defective DDR and neuropathology. In this review, rare human genetic disorders related to abnormal DDR and damage repair with neural defects will be discussed.

Roles of PTEN with DNA Repair in Parkinson's Disease.

Oxidative stress is considered to play key roles in aging and pathogenesis of many neurodegenerative diseases such as Parkinson’s disease, which could bring DNA damage by cells. The DNA damage may lead to the cell apoptosis, which could contribute to the degeneration of neuronal tissues. Recent evidence suggests that PTEN (phosphatase and tensin homolog on chromosome 10) may be involved in the pathophysiology of the neurodegenerative disorders. Since PTEN expression appears to be one dominant determinant of the neuronal cell death, PTEN should be a potential molecular target of novel therapeutic strategies against Parkinson’s disease. In addition, defects in DNA damage response and DNA repair are often associated with modulation of hormone signaling pathways. Especially, many observations imply a role for estrogen in a regulation of the DNA repair action. In the present review, we have attempted to summarize the function of DNA repair molecules at a viewpoint of the PTEN signaling pathway and the hormone related functional modulation of cells, providing a broad interpretation on the molecular mechanisms for treatment of Parkinson’s disease. Particular attention will be paid to the mechanisms proposed to explain the health effects of food ingredients against Parkinson’s disease related to reduce oxidative stress for an efficient therapeutic intervention.