Defective DNA Damage Response Research Articles

Homologous repair deficiency score for identifying breast cancers with defective DNA damage response

Breast cancer (BC) in patients with germline mutations of BRCA1/BRCA2 are associated with benefit from drugs targeting DNA damage response (DDR), but they account for only 5–7% of overall breast cancer. To define the characteristics of these tumors and also to identify tumors without BRCA mutation but with homologous recombination deficiency (HRD) is clinically relevant. To define characteristic features of HRD tumors and analyze the correlations between BRCA1/BRCA2 and BC subtypes, we analyzed 981 breast tumors from the TCGA database using the signature analyzer. The BRCA signature was strongly associated with the HRD score top 10% (score ≥ 57) population. This population showed a high level of mutations in DDR genes, including BRCA1/BRCA2. HRD tumors were associated with high expression levels of BARD1 and BRIP1. Besides, BRCA1/2 mutations were dominantly observed in basal and luminal subtypes, respectively. A comparison of HRD features in BC revealed that BRCA1 exerts a stronger influence inducing HRD features than BRCA2 does. It reveals genetic differences between BRCA1 and BRCA2 and provides a basis for the identification of HRD and other BRCA-associated tumors.

The recent advances in non-homologous end-joining through the lens of lymphocyte development

Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.

Vitamin D3 Ameliorates DNA Damage Caused by Developmental Exposure to Endocrine Disruptors in the Uterine Myometrial Stem Cells of Eker Rats.

Early-life exposure of the myometrium to endocrine-disrupting chemicals (EDCs) has been shown to increase the risk of uterine fibroid (UF) prevalence in adulthood. Vitamin D3 (VitD3) is an unique, natural compound that may reduce the risk of developing UFs. However, little is known about the role and molecular mechanism of VitD3 on exposed myometrial stem cells (MMSCs). We investigated the role and molecular mechanism underlying VitD3 action on DNA damage response (DDR) defects in rat MMSCs due to developmental exposure to diethylstilbestrol (DES), with the additional goal of understanding how VitD3 decreases the incidence of UFs later in life. Female newborn Eker rats were exposed to DES or a vehicle early in life; they were then sacrificed at 5 months of age (pro-fibroid stage) and subjected to myometrial Stro1+/CD44+ stem cell isolation. Several techniques were performed to determine the effect of VitD3 treatment on the DNA repair pathway in DES-exposed MMSCs (DES-MMSCs). Results showed that there was a significantly reduced expression of RAD50 and MRE11, key DNA repair proteins in DES-exposed myometrial tissues, compared to vehicle (VEH)-exposed tissues (p < 0.01). VitD3 treatment significantly decreased the DNA damage levels in DES-MMSCs. Concomitantly, the levels of key DNA damage repair members, including the MRN complex, increased in DES-MMSCs following treatment with VitD3 (p < 0.01). VitD3 acts on DNA repair via the MRN complex/ATM axis, restores the DNA repair signaling network, and enhances DDR. This study demonstrates, for the first time, that VitD3 treatment attenuated the DNA damage load in MMSCs exposed to DES and classic DNA damage inducers. Moreover, VitD3 targets primed MMSCs, suggesting a novel therapeutic approach for the prevention of UF development.

OR16-06 Age-Associated Local GH Promotes Colon Neoplasia

Colon polyp and cancer frequency increase with age, yet little is known about age-related mechanisms underlying development of these neoplasms. Defective DNA damage response and accumulation of unrepaired DNA damage can trigger genomic instability and cellular transformation.Patients with acromegaly have a higher prevalence of colon polyps and, arguably, colon adenocarcinoma, while those with GH signaling deficiency do not develop cancer. We showed that APC+/- mice that all develop colon adenomas exhibit a significant decrease in the number and volume of colon tumors with deletion of the GH transcription factor Prop1. Further, DNA damage response triggered GH expression in colon cells, and GH, in turn, altered DNA damage repair, resulting in DNA damage accumulation and cell transformation both in vitro and in vivo. These findings prompted us to hypothesize that accumulated DNA damage in aging colon induces local GH, suppressing DNA damage repair and creating a milieu consistent with genomic instability favoring neoplastic development.In human colon tissue we now show increased expression of γH2AX, a marker of DNA breaks, associated with increased GH transcription (detected by RNA scope) and translation (assessed by immunohistochemistry) as well as GH induction in both human and murine colon after DNA damaging radiotherapy. In vitro studies support these results, showing GH induction in normal human colon epithelial cells, fibroblasts, and 3D human intestinal organoids after DNA damage. Of note, local GH was secreted in the medium, indicating a paracrine effect. Paracrine/autocrine GH expression in these cellular models resulted in suppression of p53, induction of EMT, and attenuation of DNA damage response, and accumulated unrepaired DNA damage in human colon cells and in human intestinal organoids. In vivo, colon cells infected with lentivirus expressing GH generated more metastases than did cells expressing control vector. Co-culturing of human normal colon fibroblasts expressing GH together with normal human colon cells led to increased motility and accumulation of DNA damage as well as increased proliferation of epithelial cells on a gut-on-a-chip microfluidic device, confirming paracrine GH effects. In an in vitro model of aging, culturing human intestinal organoids for up to 2 months resulted in decreased telomere length and increased GH mRNA and protein expression associated with suppressed DNA damage response evident by decreased phosphorylation of ATM and DNA-PKcs, both kinases involved in DNA repair, and DNA damage accumulation assessed by Comet assay. Suppression of GH in these aging organoids led to increased phospho-p53 and reduced DNA damage. Although somatotroph axis endocrine activity decreases with age, local GH induced in response to age-related DNA damage may trigger a “field change,” creating a milieu favorable for colon neoplastic development.

Tumor cell-intrinsic PD-L1 signals promote DNA damage responses that mediate resistance to Chk1 and PARP inhibitors in vivo

Abstract Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (&amp;gt;15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Replication Stress, DNA Damage, Inflammatory Cytokines and Innate Immune Response.

Complete and accurate DNA replication is essential to genome stability maintenance during cellular division. However, cells are routinely challenged by endogenous as well as exogenous agents that threaten DNA stability. DNA breaks and the activation of the DNA damage response (DDR) arising from endogenous replication stress have been observed at pre- or early stages of oncogenesis and senescence. Proper detection and signalling of DNA damage are essential for the autonomous cellular response in which the DDR regulates cell cycle progression and controls the repair machinery. In addition to this autonomous cellular response, replicative stress changes the cellular microenvironment, activating the innate immune response that enables the organism to protect itself against the proliferation of damaged cells. Thereby, the recent descriptions of the mechanisms of the pro-inflammatory response activation after replication stress, DNA damage and DDR defects constitute important conceptual novelties. Here, we review the links of replication, DNA damage and DDR defects to innate immunity activation by pro-inflammatory paracrine effects, highlighting the implications for human syndromes and immunotherapies.

Targeting the DNA Damage Response for the Treatment of High Risk Neuroblastoma.

Despite intensive multimodal therapy, the survival rate for high risk neuroblastoma (HR-NB) remains <50%. Most cases initially respond to treatment but almost half will subsequently relapse with aggressive treatment resistant disease. Novel treatments exploiting the molecular pathology of NB and/or overcoming resistance to current genotoxic therapies are needed before survival rates can significantly improve. DNA damage response (DDR) defects are frequently observed in HR-NB including allelic deletion and loss of function mutations in key DDR genes, oncogene induced replication stress and cell cycle checkpoint dysfunction. Exploiting defects in the DDR has been a successful treatment strategy in some adult cancers. Here we review the genetic features of HR-NB which lead to DDR defects and the emerging molecular targeting agents to exploit them.

Atm deficiency in the DNA polymerase β null cerebellum results in cerebellar ataxia and Itpr1 reduction associated with alteration of cytosine methylation.

Genomic instability resulting from defective DNA damage responses or repair causes several abnormalities, including progressive cerebellar ataxia, for which the molecular mechanisms are not well understood. Here, we report a new murine model of cerebellar ataxia resulting from concomitant inactivation of POLB and ATM. POLB is one of key enzymes for the repair of damaged or chemically modified bases, including methylated cytosine, but selective inactivation of Polb during neurogenesis affects only a subpopulation of cortical interneurons despite the accumulation of DNA damage throughout the brain. However, dual inactivation of Polb and Atm resulted in ataxia without significant neuropathological defects in the cerebellum. ATM is a protein kinase that responds to DNA strand breaks, and mutations in ATM are responsible for Ataxia Telangiectasia, which is characterized by progressive cerebellar ataxia. In the cerebella of mice deficient for both Polb and Atm, the most downregulated gene was Itpr1, likely because of misregulated DNA methylation cycle. ITPR1 is known to mediate calcium homeostasis, and ITPR1 mutations result in genetic diseases with cerebellar ataxia. Our data suggest that dysregulation of ITPR1 in the cerebellum could be one of contributing factors to progressive ataxia observed in human genomic instability syndromes.

Checkpoint kinase inhibition in prostate cancer cells resistant to poly ADP-ribose polymerase inhibitors.

150 Background: Poly ADP-ribose polymerase inhibitors (PARPi) have shown promise in the treatment of metastatic castration-resistant prostate cancer patients with DNA damage response defects . The phase 3 PROfound trial showed olaparib delayed the time to radiographic progression or death as compared with abiraterone or enzalutamide. In addition to olaparib, three other PARPi are in Phase 3 trials in prostate cancer (PC): rucaparib, talazoparib, and niraparib. Despite responses, resistance is common and treatment options for PARPi-resistant patients are limited. In this study, we observed de novo activation of checkpoint kinases (CHEK) in talazoparib-resistant (TR) PC cells. Therefore, we hypothesized that targeting CHEK may mitigate resistance to PARPi in PC. Methods: We developed TR human prostate cancer PC3 (low BRCA2 protein due to heterozygous deletion of BRCA2) cells. We performed phosphoproteomic analysis to identify possible mechanisms of talazoparib resistance in PC3 cells and validated the results with Western blot. Results: TR-PC3 cells proliferated slower and had a significant increase in the phosphorylation of CHEK2 compared to parental (p) PC3. Treatment with a CHEK2-selective inhibitor, CCT241533, did not affect cell growth in TR-PC3 cells. Conversely, treatment with a CHEK 1/2 inhibitor, prexasertib, led to significant cell growth inhibition in TR-PC3 at a much lower IG 50% concentration compared to pPC3. RNAi-mediated knockdown validated the superior efficacy of combined CHEK1 and CHEK2 inhibition since this combination produced the greatest cell growth inhibition seen in both TR-PC3 and de novo PARPi-resistant p22RV1. Treatment of pPC-3 and p22RV1 with combinations of talazoparib and prexasertib showed greater cell growth inhibition compared to either drug alone. Conclusions: Resistance to PARPi in PC cells with deletion of BRCA2 may potentially be overcome with CHEK inhibition. Moreover, our preliminary data suggested that the effect of PARPi and CHEK inhibitors on PARPi/CHEK inhibitor-naïve PC cells was greatest when used together, indicating that patients with PC may experience greatest anti-tumor activity of the two drugs when they are used in combination.

Prospective use of the single-mouse experimental design for the evaluation of PLX038A.

Defining robust criteria for drug activity in preclinical studies allows for fewer animals per treatment group, and potentially allows for inclusion of additional cancer models that more accurately represent genetic diversity and, potentially, allows for tumor sensitivity biomarker identification. Using a single-mouse design, 32 pediatric xenograft tumor models representing diverse pediatric cancer types [Ewing sarcoma (9), brain (4), rhabdomyosarcoma (10), Wilms tumor (4), and non-CNS rhabdoid tumors (5)] were evaluated for response to a single administration of pegylated-SN38 (PLX038A), a controlled-release PEGylated formulation of SN-38. Endpoints measured were percent tumor regression, and event-free survival (EFS). The correlation between response to PLX038A was compared to that for ten models treated with irinotecan (2.5mg/kg × 5days × 2cycles), using a traditional design (10 mice/group). Correlations between tumor sensitivity, genetic mutations and gene expression were sought. Models showing no disease at week 20 were categorized as 'extreme responders' to PLX038A, whereas those with EFS less than 5weeks were categorized as 'resistant'. The activity of PLX038A was evaluable in 31/32 models. PLX038A induced > 50% volume regressions in 25 models (78%). Initial tumor volume regression correlated only modestly with EFS (r2 = 0.238), but sensitivity to PLX038A was better correlated with response to irinotecan when one tumor hypersensitive to PLX038A was omitted (r2 = 0.6844). Mutations in 53BP1 were observed in three of six sensitive tumor models compared to none in resistant models (n = 6). This study demonstrates the feasibility of using a single-mouse design for assessing the antitumor activity of an agent, while encompassing greater genetic diversity representative of childhood cancers. PLX038A was highly active in most xenograft models, and tumor sensitivity to PLX038A was correlated with sensitivity to irinotecan, validating the single-mouse design in identifying agents with the same mechanism of action. Biomarkers that correlated with model sensitivity included wild-type TP53, or mutant TP53 but with a mutation in 53BP1, thus a defect in DNA damage response. These results support the value of the single-mouse experimental design.

Homocysteine aggravates DNA damage by impairing the FA/Brca1 Pathway in NE4C murine neural stem cells.

There is existing evidence that elevated homocysteine (Hcy) levels are risk factors for some neurodegenerative disorders. The pathogenesis of neurological diseases could be contributed to excessive cell dysfunction and death caused by defective DNA damage response (DDR) and accumulated DNA damage. Hcy is a neurotoxic amino acid and acts as a DNA damage inducer. However, it is not clear whether Hcy participates in the DDR. To investigate the effects of Hcy on DNA damage and the DDR, we employed mitomycin C (MMC) to cause DNA damage in NE4C murine neural stem cells (NSCs). Compared to treatment with MMC alone, we found that co-treatment with MMC and Hcy worsened DNA damage and increased death in NE4C cells. Intriguingly, in this DNA damage model mimicked by MMC, immunoblotting results showed that the monoubiquitination levels of Fanconi anemia complementation group I (Fanci) and Fanconi anemia complementation group D2 (Fancd2) were decreased to about 60.3% and 55.7% by supplementing cell culture medium with Hcy, indicating Hcy inactivates the function of Fanci and Fancd2 in DNA damage conditions. Given Breast Cancer 1 (BRCA1) is an important downstream of FANCD2, we next detected the interaction between Fancd2 and Brca1 in NE4C cells. Compared to treatment with MMC alone, the Fancd2-Brca1 interaction and the amount of Brca1 on chromatin were decreased when cells were co-exposed to MMC and Hcy, suggesting Hcy could impair the Fanconi anemia (FA)/Brca1 pathway. Taken together, our study demonstrates that Hcy may enhance cell death, which contributes to the accumulation of DNA damage and promotion of hypersensitivity to cytotoxicity by impairing the FA/Brca1 pathway in murine NSCs in the presence of DNA damage.

Harnessing DNA Double-Strand Break Repair for Cancer Treatment.

DNA double-strand breaks (DSBs) are highly deleterious, with a single unrepaired DSB being sufficient to trigger cell death. Compared to healthy cells, cancer cells have a higher DSB burden due to oncogene-induced replication stress and acquired defects in DNA damage response (DDR) mechanisms. Consequently, hyperproliferating cancer cells rely on efficient DSB repair for their survival. Moreover, augmented DSB repair capacity is a major cause of radio- and chemoresistance and, ultimately, cancer recurrence. Although inherited DDR defects can predispose individuals to develop certain cancers, the very same vulnerability may be therapeutically exploited to preferentially kill tumor cells. A paradigm for DNA repair targeted therapy has emerged in cancers that exhibit mutations in BRCA1 or BRCA2 tumor suppressor genes, conferring a strong defect in homologous recombination, a major and error-free DSB repair pathway. Clinical validation of such approaches, commonly described as synthetic lethality (SL), has been provided by the regulatory approval of poly(ADP-ribose) polymerase 1 inhibitors (PARPi) as monotherapy for BRCA1/2-mutated breast and ovarian tumors. In this review, we will describe the different DSB repair mechanisms and discuss how their specific features could be exploited for cancer therapy. A major emphasis is put on advances in combinatorial treatment modalities and SL approaches arising from DSB repair pathway interdependencies.

Abstract C001: Prospective use of the single mouse experimental design for evaluation of PLX038A against pediatric solid tumor models

Abstract Purpose: In a previous analysis of &amp;gt;2100 in vivo pediatric xenograft tumor/drug studies (Murphy et al. Cancer Research, 2016), we reported that results from one mouse/treatment group gave essentially similar results for 67 drugs as 10 mice (solid tumor models) or 8 mice (leukemia models). The use of fewer animals per treatment group allows for inclusion of more cancer models that more accurately represent genetic diversity within an histology or between histology’s. Further, this increase in genetic diversity allows for tumor sensitivity biomarker identification, either within a tumor type (e.g. neuroblastoma) or independent of tumor lineage. Here we have used the single mouse design to evaluate a nanoformulated camptothecin analog, PLX038A, where SN-38 (the active metabolite of irinotecan) is released at a controlled rate. Additional objectives of the study were to determine the relationship between initial tumor volume regression and Event-Free Survival (EFS), and to mine genomics data on each tumor model with a goal to identify potential biomarkers that relate to drug sensitivity. Experimental Procedures: Pediatric patient derived xenografts (PDX) were grown subcutaneously, and treatment was administered when tumors were ~300mm3(mean 297 ±34 mm3; range 260-370 mm3). Models tested included rhabdomyosarcoma (Rh10, Rh18, Rh28, Rh30, Rh30R, Rh41, Rh65), Wilms tumors (KT-5, KT-10, KT-11, KT-13); and non-CNS rhabdoid tumors (KT-12, KT-14, KT-16, RBD1, RBD2) and cell line-derived xenografts (Ewing sarcoma lines ES-1, ES-4, ES-5, ES-6, EW-8, CHLA258, TC-71, SK-NEP-1). New models include RBD1 an atypical teratoid rhabdoid tumor established from a metastatic lung lesion, NCH-EWS-1, a Ewing sarcoma from a lung lesion, and S12-6321 was established from a patient with metastatic pleiomorphic xanthoastrocytoma. All tumors were used at low passage and authenticated by STR analysis against reference profiles developed by this group. Tumor models were selected for testing without reference to genetic or molecular characteristics, or sensitivity to irinotecan in previous testing. PLX038A was administered one time at a dose of 120 umol/kg by intraperitoneal injection. For 13 models, with data for the single agent irinotecan (2.5 mg/kg daily x 5 with cycle repeated at day 21), the correlation between tumor responsiveness to PX038A and irinotecan was examined. Results: The activity of single-dose PLX038A was evaluable in 30/31 models. PLX038A induced &amp;gt;50% volume regressions in 25 models (83%). EFS varied from 30 to &amp;gt;120 days dependent on the xenograft model. For 13 tumor models having complete regression, EFS times varied from 38 to &amp;gt;120 days, hence initial tumor volume regression correlated only modestly with EFS (r2= 0.453). Sensitivity to PLX038A was highly correlated with response to irinotecan (r2=0.729). Mutations in TP53BP1 were enriched in 6 sensitive tumor models compared to 4 resistant models. Conclusions: PLX038A was highly active in most xenograft models, and tumor sensitivity to PLX038A was highly correlated with sensitivity to irinotecan. Biomarkers that correlated with model sensitivity included wild type TP53, or mutant TP53 but with a mutation in TP53BP1, thus a defect in DNA damage response. These results support the value of the single mouse experimental design, and also support further development of PLX038 for pediatric clinical evaluation. Support: UO1CA199297, CA169368 and CA165995 (PJH) from NCI and CPRIT RR170055 (SZ). Citation Format: Samson Ghilu, Qilin Li, Shaun D Fontaine, Daniel V. Santi, Raushan T. Kurmasheva, Siyuan Zheng, Peter J. Houghton. Prospective use of the single mouse experimental design for evaluation of PLX038A against pediatric solid tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C001. doi:10.1158/1535-7163.TARG-19-C001

DNA repair functional analyses of NBN hypomorphic variants associated with NBN-related infertility.

Nijmegen breakage syndrome caused by biallelic pathogenic variants of the DNA-damage response gene NBN, is characterized by severe microcephaly, cancer proneness, infertility, and karyotype abnormalities. We previously reported NBN variants in siblings suffering from fertility defects. Here, we identify a new founder NBN variant (c.442A>G, p.(Thr148Ala)) in Lebanese patients associated with isolated infertility. Functional analyses explored preserved or altered functions correlated with their remarkably mild phenotype. Transcript and protein analyses supported the use of an alternative transcript with in-frame skipping of exons 4-5, leading to p84-NBN protein with a preserved forkhead-associated (FHA) domain. The level of NBN was dramatically reduced and the MRN complex delocalized to the cytoplasm. Interestingly, ataxia-elangiectasia mutated (ATM) also shifted from the nucleus to the cytoplasm, suggesting some interaction between ATM and the MRN complex at a steady state. The ATM pathway activation, attenuated in typical patients with NBS, appeared normal under camptothecin treatment in these new NBN-related infertile patients. Cell cycle checkpoint defect was present in these atypical patients, although to a lesser extent than in typical patients with NBS. In conclusion, we report three new NBN-related infertile patients and we suggest that preserved FHA domain could be responsible for the mild phenotype and intermediate DNA-damage response defects.

Telomere Shortening By Terc Knockout in the Eµ-TCL1 Transgenic Murine Model of CLL: Characterization of Disease Development and Survival

In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations. Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice. Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells. We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p&lt;0.001) as compared to TCL1+ mice. The aggressiveness of the TCL1+ mTerc+/- G4 tumors was further verified using adoptive transfer into syngeneic mice, and mice transplanted with these tumors showed a significantly shorter survival as compared to TCL1+ or G3 tumors. In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Schneider:Celgene: Other: travel grant. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Veliparib in ovarian cancer: a new synthetically lethal therapeutic approach.

Epithelial ovarian cancer (EOC) accounts for nearly 90% of all ovarian malignancies. The standard therapeutic strategy includes cytoreductive surgery and neo (adjuvant) platinum-based chemotherapy. Relapse of advanced high grade serous ovarian cancer (HGSOC) is related to the development of drug resistance. A defective DNA damage response is a defining hallmark of HGSOC. Poly (ADP-ribose) polymerase (PARP) inhibitors exploit this deficiency through synthetic lethality and have emerged as promising anticancer therapies, especially in breast cancer gene (BRCA1 or BRCA2) mutation carriers. Apart from inducing synthetic lethality, PARP inhibitors have also been shown to trap PARP1 and PARP2 on DNA, leading to PARP-DNA complexes. This "PARP trapping" potentiates synergism between PARP inhibition and both alkylating agents and platinum-based chemotherapy. However, there are remarkable differences in the ability of PARP inhibitors to trap PARP, based on the size and structure of each separate molecule. Since monotherapy with PARP inhibitors is unlikely to induce cancer cell death in BRCA-proficient tumors, the efficacy of PARP inhibitors could be potentially optimized when combined with DNA-damaging agents, or with molecular targeted agents that also impair mechanisms of DNA repair. Olaparib, rucaparib, and niraparib have all obtained US Food and Drug Administration (FDA) and/or European Medicines Agency (EMA) approval in ovarian cancer in different settings. Veliparib does not yet have an approved label; nevertheless, there are currently promising results available in preclinical and early clinical settings. This comprehensive review summarizes the mechanism of action of veliparib and provides an overview of its early and ongoing clinical investigations.

The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas.

Defective DNA damage response (DDR) is frequently associated with tumorigenesis. Abrogation of DDR leads to genomic instability, which is one of the most common characteristics of human cancers. TP53 mutations with gain-of-function activity are associated with tumors under high replicative stress, high genomic instability, and reduced patient survival. The BRCA1 and RAD17 genes encode two pivotal DNA repair proteins required for proper cell-cycle regulation and maintenance of genomic stability. We initially evaluated whether miR-205-5p, a microRNA (miRNA) highly expressed in head and neck squamous cell carcinoma (HNSCC), targeted BRCA1 and RAD17 expression. We found that, in vitro and in vivo, BRCA1 and RAD17 are targets of miR-205-5p in HNSCC, leading to inefficient DNA repair and increased chromosomal instability. Conversely, miR-205-5p downregulation increased BRCA1 and RAD17 messenger RNA (mRNA) levels, leading to a reduction in in vivo tumor growth. Interestingly, miR-205-5p expression was significantly anti-correlated with BRCA1 and RAD17 targets. Furthermore, we documented that miR-205-5p expression was higher in tumoral and peritumoral HNSCC tissues than non-tumoral tissues in patients exhibiting reduced local recurrence-free survival. Collectively, these findings unveil miR-205-5p’s notable role in determining genomic instability in HNSCC through its selective targeting of BRCA1 and RAD17 gene expression. High miR-205-5p levels in the peritumoral tissues might be relevant for the early detection of minimal residual disease and pre-cancer molecular alterations involved in tumor development.

Niraparib Before Surgery in Treating Patients With High Risk Localized Prostate Cancer and DNA Damage Response Defects

Niraparib Before Surgery in Treating Patients With High Risk Localized Prostate Cancer and DNA Damage Response Defects

Abstract 2567: TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability

Abstract TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in “trunk” clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing. Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution. Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed. Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Thomas Webber, Nnennaya Kanu, Charles Swanton. TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2567.