Defective DNA Damage Response Research Articles

DNA polymerase β deficiency in the p53 null cerebellum leads to medulloblastoma formation

Defects in DNA damage response or repair mechanisms during neurogenesis result in genomic instability, which is causative for several neural defects. These include brain tumors, particularly medulloblastoma, which occurs in the cerebellum with a high incidence in children. We generated an animal model with defective base excision repair during brain development through selective inactivation of DNA polymerase β (Polb) in neuroprogenitor cells. All of Polb conditional knockout mice developed medulloblastoma in a p53 null background, similar to the Xrcc1 and p53 double deficient animal model. XRCC1 is a scaffolding protein which is involved in DNA damage repair and binds to POLB. In both animal models, the histopathological characteristics of the medulloblastoma were similar to those of human classic medulloblastoma. Brain tumor development was slower in the Polb and p53 double null animals than in the Xrcc1 and p53 double knockout animals. Molecular marker analysis suggested that Polb- and Xrcc1-deficient medulloblastomas belonged to the SHHα subtype, underscoring the important role of genomic stability in preventing this devastating pediatric cerebellar tumor.

Mutagenic assessment of chemotherapy and Smac mimetic drugs in cells with defective DNA damage response pathways

DNA damaging therapies can spur the formation of therapy-related cancers, due to mis-repair of lesions they create in non-cancerous cells. This risk may be amplified in patients with impaired DNA damage responses. We disabled key DNA damage response pathways using genetic and pharmacological approaches, and assessed the impact of these deficiencies on the mutagenicity of chemotherapy drugs or the “Smac mimetic” GDC-0152, which kills tumor cells by targeting XIAP, cIAP1 and 2. Doxorubicin and cisplatin provoked mutations in more surviving cells deficient in ATM, p53 or the homologous recombination effector RAD51 than in wild type cells, but suppressing non-homologous end joining (NHEJ) by disabling DNA-PKcs prevented chemotherapy-induced mutagenesis. Vincristine-induced mutagenesis required p53 and DNA-PKcs but was not affected by ATM status, consistent with it provoking ATM-independent p53-mediated activation of caspases and CAD, which creates DNA lesions in surviving cells that could be mis-repaired by NHEJ. Encouragingly, GDC-0152 failed to stimulate mutations in cells with proficient or defective DNA damage response pathways. This study highlights the elevated oncogenic risk associated with treating DNA repair-deficient patients with genotoxic anti-cancer therapies, and suggests a potential advantage for Smac mimetic drugs over traditional therapies: a reduced risk of therapy-related cancers.

Harnessing the Immune System in Pancreatic Cancer.

Managing patients with metastatic pancreatic adenocarcinoma (mPDA) is a challenging proposition for any treating oncologist. Although the potency of first-line therapies has improved with the approvals of FOLFIRINOX and gemcitabine plus nab-paclitaxel, many patients are unable to derive significant benefit from later lines of therapy upon progression. Enrollment on clinical trials remains among the best options for patients with mPDA in all lines of therapy. At our institution, we routinely check for microsatellite instability (MSI-H) and perform next-generation sequencing (NGS) at the time of diagnosis in all good performance status mPDA patients. Although MSI-H status is only found in 1% of patients with mPDA, given pembrolizumab's tissue-agnostic approval for MSI-H tumors in later-line settings, it is a viable option when deciding on subsequent lines of therapy. Any use of immune therapy in mPDA is investigational outside the MSI-H setting. NGS can identify BRCA or other DNA damage response (DDR) defects in patients which can predict sensitivity to platinum-based therapies and influence choice of both initial and later lines of therapy. It can also identify rare actionable genomic alterations such as HER2 (2%) and TRK fusions (0.1%) and offer patients the option of enrollment on clinical trials with agents targeting these or other identified alterations. We believe enrolling mPDA patients on clinical trials with immune-modulating agents is critical to determine if there are other patient subsets, outside of the MSI-H setting, who would benefit from these approaches. Immunotherapy's general tolerability and potential to generate durable responses make it particularly appealing for mPDA patients. Although single-modality immunotherapy such as checkpoint inhibitors or vaccines have not demonstrated efficacy in this disease, combinatorial strategies targeting unique aspects of PDA including the tumor microenvironment and desmoplastic stroma have shown preclinical or early-phase success. Validating these treatments with later-phase prospective studies is essential to making immunotherapy a routine component of the treatment armamentarium for mPDA patients.

Aberrant DNA Damage Response and DNA Repair Pathway in High Glucose Conditions

Higher cancer rates and more aggressive behavior of certain cancers have been reported in populations with diabetes mellitus. This association has been attributed in part to the excessive reactive oxygen species generated in diabetic conditions and to the resulting oxidative DNA damage. It is not known, however, whether oxidative stress is the only contributing factor to genomic instability in patients with diabetes or whether high glucose directly also affects DNA damage and repair pathways. Normal renal epithelial cells and renal cell carcinoma cells are more chemo- and radiation resistant when cultured in high concentrations of glucose. In high glucose conditions, the CHK1-mediated DNA damage response is not activated properly. Cells in high glucose also have slower DNA repair rates and accumulate more mutations than cells grown in normal glucose concentrations. Ultimately, these cells develop a transforming phenotype. In high glucose conditions, defective DNA damage responses most likely contribute to the higher mutation rate in renal epithelial cells, in addition to oxidative DNA damage. The DNA damage and repair are normal enzyme dependent mechanisms requiring euglycemic environments. Aberrant DNA damage response and repair in cells grown in high glucose conditions underscore the importance of maintaining good glycemic control in patients with diabetes mellitus and cancer.

USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

ABSTRACT Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair. Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.

Abstract LB-245: Multiple DNA-damage response pathways are modulated by RANBP9 protein in NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the most prominent cause of cancer-related deaths in the world. The most indicated treatment for over 50% of NSCLC patients is represented by platinum-based chemotherapy. These compounds exert their biological effects by inducing DNA damages. Differently from normal cells, cancer cells frequently display a defective DNA Damage Response (DDR), which results in cancer cell death upon treatment with DNA-damaging agents. However, cancer cells up-regulate specific DDR mechanisms to cope with genotoxic stress, leading to resistance to conventional chemotherapies. For these reasons, a better understanding of cellular DDR mechanisms in tumors might help to identify 1) biomarkers predictive of DNA-repair defects and 2) targeted agents to inhibit tumor specific DNA-repair pathways. We have previously demonstrated that RANBP9 is a new phosphorylation target and signaling facilitator of ATM, one of the most important kinases in DDR. We have recently shown that RANBP9 is upregulated in lung cancer vs normal adjacent tissues, and its protein levels correlate with tumor stage. Importantly, low RANBP9 levels were associated to enhanced progression free survival and overall survival upon treatment with platinum-based compounds, in a cohort of more than 130 NSCLC patients. To gain molecular insights in the role of RANBP9 in DDR and sensitivity to genotoxic stress, we generated NSCLC cell lines where its expression was abrogated by CRISPR/Cas9. These models confirmed that absence of RANBP9 results in enhanced therapeutic effects of cisplatin and reduced activation of ATM-dependent DDR. Notably, we also observed that RANBP9 abrogation resulted in increased levels of polyADP-ribosylated chromatin proteins, without affecting total levels of poly(ADP-ribose) polymerase 1 (PARP1), another critical mediator of DDR. Conversely, we found reduced levels of PAR-glycohydrolase (PARG), a negative regulator of nuclear PAR levels, in response DNA-damaging agents. Based on these results, we hypothesized that RANBP9 levels could dictate the preferential DDR mechanism activated by lung cancer cells in response to genotoxic stress. Accordingly, preliminary evidences show that RANBP9-negative cells (PARP-dependent) display higher sensitivity to PARP inhibition. On the other hand, RANBP9-positive cells (ATM-dependent) are more sensitive to ATM inhbition. In summary, our study suggests that RANBP9 could act both as a positive and as a negative regulator of different mechanisms of DDR. These findings pave the way to clinical studies where RANBP9 levels could be used to inform the choice of the most effective genotoxic therapy and prevent resistance. Citation Format: Anna Tessari, Kareesma Parbhoo, Meghan Pawlikowski, Matteo Fassan, Eliana Rulli, Claudia Foray, Alessandra Fabbri, Valerio Embrione, Monica Ganzinelli, Marina Capece, Massimo Broggini, Gabriella Farina, Mirko Marabese, Marina C. Garassino, Carlo Croce, Dario Palmieri, Vincenzo Coppola. Multiple DNA-damage response pathways are modulated by RANBP9 protein in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-245.

Abstract 339: Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects

Abstract Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells. Citation Format: Robert Winqvist, Muthiah Bose, Juliane Sachsenweger, Niina Laurila, Ann Christin Parplys, Jonas Willmann, Leila Eshraghi, Thomas W. Dunlop, Marco Groth, Katrin Rapakko, Pentti Nieminen, Thomas W. Friedl, Lisa Heiserich, Felix Meyer, Hanna Tuppurainen, Ganapathy Raman Devarajan, Hellevi Peltoketo, Heli Nevanlinna, Katri Pylkäs, Kerstin Borgmann, Lisa Wiesmuller, Helmut Pospiech. Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 339.

Abstract 1364: Tip60 dependent DNA homologous recombination repair is impaired in VHL-deficient clear cell renal cell carcinoma

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability across all tumor stages, indicative of increased replicative stress and defects in DNA damage response (DDR) pathways including homologous repair (HR); however ccRCC does not display mutations in canonical DDR genes. We hypothesized that biallelic VHL loss is sufficient to cause HR deficiency (HRD) in ccRCC via direct regulation of DDR pathways. Experimental Procedures: We performed whole-exome (WES) sequencing of 15 small ccRCC tumors. We performed in silico genomic, transcriptomic, and proteomic analysis of tumors in KIRC TCGA to assess for HRD, using published HRD signatures. We assessed HR efficiency as a product of biallelic VHL loss in engineered cell line models. We studied the VHL-dependent DNA damage repair molecular mechanisms with cell line models, by HR and nonhomologous end joining (NHEJ) using reporter gene assays. We then assessed the status of TIP60 activation in Vhl deficient murine embryo fibroblasts, isogenic VHL deficient and proficient cell lines by determining DSB induced tip60 acetylation and foci formation. DSB induced HR and NHEJ activation were analyzed by assessing the induction of marker proteins including γH2AX, RAD51, and 53BP1. To examine VHL-protein interaction, green-fluorescent protein (GFP)-trap with GFP-tagged VHL and co-immunoprecipitation with specific antibodies were used. Results: 15/15 early stage ccRCC had biallelic VHL mutations and approximately 100 additional mutations per tumor, but no driver mutations typically associated with HRD. Nonetheless, in silico analysis showed 67% of KIRC TCGA displayed an HRD gene signature, and this signature was significantly higher in stage I disease (p=2.21e-08). Patients with VHL-mutated tumors were more commonly HRD than HRI (p=0.03), with frameshift/nonsense variants in VHL more likely to result in HRD than missense variants (p=0.02). Multivariate analysis showed HRD predicted for better overall survival compared to an HR intact (HRI) state (p<0.0001). Using Vhl knockout and VHL overexpressing cell models we demonstrated that VHL interacts with the acetyltransferase Tip60, and Tip60 is required for VHL-dependent HR but not for nonhomologous end joining (NHEJ). We also observed that during genotoxic stress, VHL-associated-Tip60 dissociated from histone H3 and increased its association with H4, consistent with VHL having a regulatory role on histone H4. Conclusions: Early stage, VHL-deficient ccRCC shows a strong HRD signature in the absence of additional driver mutations associated with DDR. Biallelic loss of VHL is sufficient to cause HRD by impairing Tip60 mediated DDR complex activation. Our findings provide a mechanistic explanation for the DDR defect in ccRCC, and provide a foundation for treatment strategies that target specific elements of the DDR pathway in VHL deficient ccRCC. Citation Format: Lijun Zhou, Patrick G. Pilie, Christine B. Peterson, Xian-de Liu, Xuesong Zhang, Eric Jonasch. Tip60 dependent DNA homologous recombination repair is impaired in VHL-deficient clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1364.

Abstract 336: Anti-tumor activity of the Chk1 inhibitor prexasertib (LY2606368) as a single agent in triple negative breast cancer models

Abstract Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is diagnosed in approximately 15% of all breast cancer patients and characterized by high level of genomic instability, defects in DNA damage response (DDR) and increased replication stress (RS). Currently the treatment options for TNBC are limited, and new approaches are needed. Checkpoint kinase 1 (Chk1) is a key kinase that regulates the cell cycle, DDR and RS through the modulation of cell cycle checkpoints and replication fork licensing; and has emerged as an attractive target for anti-cancer therapy. Prexasertib (LY2606368), an ATP-competitive inhibitor of Chk1 has demonstrated single agent activity in vitro and in vivo. Prexasertib is being evaluated in an ongoing TNBC Phase 2 trial sponsored by NCI; and preliminary results suggest modest single-agent activity in sporadic TNBC patients (https://academic.oup.com/annonc/article/27/suppl_6/231PD/2798970). To understand the biology and mechanisms underlying prexasertib's activity and/or identify the potential biomarkers that may predict response or identify novel combinations that could improve prexasertib's efficacy, we evaluated the anti-tumor activity of prexasertib as a single agent in TNBC cancer cell lines in vitro and in vivo, including xenograft, orthotopic and patient-derived xenograft (PDX) tumor models. Prexasertib inhibited cell proliferation in 12 TNBC cell lines with IC50 values ranging from 0.32 nM to 117.3 nM; and demonstrated inhibition of Chk1 auto-phosphorylation and activation of γH2AX, RPA32 and DNAPKc in TNBC cell lines including HCC1187, HCC1806, MX-1 and MDA-MB-231. Treatment with prexasertib inhibited tumor growth by 83.8%, 85.5% and 94.2% in HCC1187, MX-1 and HCC1806 xenograft models, respectively. Prexasertib also inhibited primary tumor growth by 74.4% and lung metastasis by 97.48% in a MDA-MB-231 mammary fat pad orthotopic model. The anti-tumor effect of prexasertib was further assessed in 40 TNBC PDX models. Prexasertib treatment resulted in 10% (4/40) of models achieving complete tumor regression, 40% (16/40) achieving partial tumor regression/stasis and 50% (20/40) not deriving benefit. Potential biomarkers of prexasertib were investigated. TNBC cells with higher phosphorylation levels of DNAPKc and RPA32 demonstrated higher sensitivity to prexasertib treatment. TNBC PDX tumors with higher RNA message levels of CyclinE1, Cyclin D1 and c-Myc showed better prexasertib response. Together these findings will inform subsets of TNBC and/or individual patient characteristics that should be considered for future clinical assessments and development of potential combination therapy strategies. Citation Format: Wenjuan Wu, Constance King, Gregory Donoho, Philip Iversen, Andrew Capen, Mark Castanares, Jennifer Stephens, Yan Ding, Susan Pratt, Ricardo Martinez, Sean Buchanan, Christoph Reinhard, Richard Beckmann, Aimee Lin. Anti-tumor activity of the Chk1 inhibitor prexasertib (LY2606368) as a single agent in triple negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 336.

Abstract CT135: A pre-surgical window of opportunity study to investigate the biomarker effects of DNA damage response (DDR) agents in patients (pts) with head and neck squamous cell carcinoma (HNSCC)

Abstract Background: The annual incidence of head and neck cancers is >550,000 cases worldwide and approximately 90% are HNSCC. Treatment for locoregionally advanced (stage III/IV) HNSCC involves primary surgery, radiation and/or chemotherapy. Cancer cells with DDR defects can activate the immune system and immunotherapy (IO) has shown therapeutic benefit in pts with advanced recurrent HNSCC. This study assesses the immunological effects of DDR agents in both tumor tissue and peripheral blood samples to inform optimal combinations with IO therapies. The overall hypothesis is that DDR agents will convert an “immunologically cold” tumor into an “immunologically hot” tumor that is both responsive to IO and improved prognosis. Objectives: The primary objective is to assess immune activation due to DDR inhibition by monitoring the induction of genes interacting with the immune system and potentially linked to prognosis. The secondary objective is to assess increasing prevalence of tumor infiltrating CD8+ and CD3+ T-cells also linked to prognosis. Exploratory measurements of tumour proliferative and DDR-relevant markers, peripheral T/B/NK and regulatory T cells, key immuno-regulatory cytokines, TCR repertoire, levels of circulating tumour cells and relevant genomic changes in both tumour and circulating tumour DNA will be conducted. Safety and tolerability of the investigational agents is also monitored. Methods: This ongoing, randomised multi-centre, window of opportunity biomarker study is enrolling patients with newly diagnosed, treatment naïve, HNSCC suitable for surgical resection followed by radiotherapy and/or chemotherapy (NCT03022409). Two oral DDR agents are currently under evaluation as monotherapy: AZD6738 is a potent selective inhibitor of the serine/threonine-specific protein kinase, ataxia telangiectasia and Rad3-related protein (ATR), and olaparib is a poly-ADP ribose polymerase (PARP) inhibitor. Eligible pts are randomised to receive a DDR agent for between 10 to 21 days (D), followed by surgery. After surgery, pts do not receive further investigational treatment and attend a follow-up visit at D31. Tumor tissue is collected pre- (archival diagnostic biopsy) and post-treatment (surgical specimen), and evaluated for changes in key biomarkers related to immune response and DNA damage inhibition. If surgery is scheduled between D11-21 (+3D) following three successive days of DDR agent, an on-treatment biopsy is also required between D10-12. Pts undergo a weekly assessment for adverse events, hematology, biochemistry, and electrocardiogram. Plasma samples are also included to characterise the pharmacokinetics of each agent, relative to any biomarker changes observed. Enrolment commenced in December 2017 and the study is designed to permit the addition of treatment arms, including different combinations, sequences and doses. Citation Format: Umamaheswar Duvvuri, Emma Dean, Paul Frewer, Alienor Berges, S. Y. Cheung, Christine Stephens, Musaddiq Khan, Simon J. Hollingsworth, Andrew J. Pierce. A pre-surgical window of opportunity study to investigate the biomarker effects of DNA damage response (DDR) agents in patients (pts) with head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT135.

PRIMUS-001: An adaptive phase II study of FOLFOX-A (FOLFOX and nab-paclitaxel) versus AG (nab-paclitaxel and gemcitabine) in patients with metastatic pancreatic cancer, with integrated biomarker evaluation (ISRCTN75002153) – Part of Precision-Panc.

TPS4158Background: Platinum-containing regimens (e.g. FOLFIRINOX) have shown survival benefit for pancreatic cancer (PC), with some “exceptional responders”; biomarkers (BM) of response are not well defined. Tumors with mutations in BRCA1/2 and other Fanconi Anemia genes, are preferentially sensitive to DNA-damaging agents (e.g. platinum) through synthetic lethality mechanisms, as they are defective in DNA damage response (DDR). We have shown that DDR deficiency may be present in 20% of PC. PRIMUS-001 aims to exploit defective DDR as a therapeutic vulnerability, with integrated study of BM of DNA-damaging agent response. Methods: PRIMUS-001 will enroll patients who are profiled molecularly using Precision-Panc NGS Diagnostic (BM via bespoke clinical grade assay). It is a multicentre, randomized (1:1), open-label, phase II trial comparing FOLFOX-A (nab-paclitaxel 150mg/m2 IV, oxaliplatin 85mg/m2, folinic acid 350mg flat dose, fluorouracil infusion 2400mg/m2 continuous IV infusion) vs AG (nab-paclitaxel 125...

Inhibition of TACC3 by a small molecule inhibitor in breast cancer

Studies have shown that transforming acidic coiled-coil protein 3 (TACC3), a key component of centrosome-microtubule dynamic networks, is significantly associated with various types of human cancer. We have recently reported that high levels of TACC3 are found in breast cancer, lead to the accumulation of spontaneous DNA damage due to defective DNA damage response signaling, and confer cellular sensitivity to radiation and poly(ADP-ribose) polymerase (PARP) inhibitors. Although our study suggests a potential role of TACC3 as a biomarker in breast cancer detection and prediction of therapy outcome, its role as a therapeutic target in breast cancer is not well studied. In this study, we show that a small molecule TACC3 inhibitor, KHS101, suppresses cell growth, motility, epithelial-mesenchymal transition (EMT), and breast cancer cell stemness while it induces apoptotic cell death. Quantitative multiplexed proteomic analysis using tandem mass tags (TMTs) revealed that KHS101 alters multiple biological processes and signaling pathways, and significantly reduces the expression of mitotic kinases Aurora A and Polo-like kinase 1 (PLK1), which are closely associated with TACC3. Our findings therefore provide a new insight into the potential mechanisms of the action of KHS101 and suggest its possible use as a dual or multi-targeting mitotic inhibitor in breast cancer.

ERRATUM.

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.

Homologous repair deficiency in VHL-mutated clear cell renal cell carcinoma.

585 Background: Clear cell renal cell carcinoma (ccRCC) displays genomic instability across all tumor stages, indicative of increased replicative stress and defects in DNA damage response (DDR) pathways including homologous repair (HR); however ccRCC does not display mutations in canonical DDR genes and advanced ccRCC is not traditionally sensitive to DNA damaging agents. We hypothesized that biallelic VHL loss is sufficient to cause HR deficiency (HRD) and that early stage ccRCC is more likely to display HRD than late stage disease. Methods: We performed whole-exome (WES) sequencing of 15 small ccRCC tumors to assess for mutational burden, mutational signature, and cancer-related driver gene mutations. We performed in silico genomic, transcriptomic, and proteomic analysis of tumors in KIRC TCGA to assess for HRD. Finally, we assessed HR efficiency as a product of biallelic VHL loss in engineered cell line models. Results: 15/15 early stage ccRCC had VHL mutations, approximately 100 additional mutations per tumor, but no common mutations found in KIRC TCGA. In silico analysis showed 67% of KIRC TCGA displayed HRD gene signature, and was significantly higher in stage I disease (p = 2.21e-08). Patients with VHL-mutated tumors are more commonly HRD than HR intact (HRI) (p = 0.03), with frameshift/nonsense variants in VHL more likely to result in HRD than missense variants (p = 0.02). Multivariate analysis showed HRD predicted for better overall survival compared to HRI (p < 0.0001). Proteomic analysis of KIRC TCGA revealed HRI samples had significantly higher protein expression of Rad51 and RAPTOR when compared to HRD samples. Lastly, etoposide-treated MEF Vhl-/- cell lines displayed reduced HR efficiency when compared to Vhl intact controls. Conclusions: Biallelic loss of VHL is sufficient to cause HRD. HRD gene signature predicts for significantly better OS in ccRCC patients, and early stage ccRCC is more likely to be HRD. mTOR/PI3K signaling pathway proteins are differentially expressed in HRI patient samples from TCGA compared to HRD, suggesting a possible role for these oncogenes driving a HRI phenotype. Treatment strategies combining PARP inhibitors with PI3K/mTOR inhibitors should be tested in advanced VHL-mutated ccRCC.

Analysis of DNA damage response (DDR) genes and tumor mutational burden (TMB) across 17,486 carcinomas of the tubular GI tract: Implications for therapy.

43 Background: The DDR pathway is important in tumor biology, allowing cancer cells a mechanism to resist damage by chemotherapy and radiotherapy. BRCA1/2 are the most well described genes in the pathway, but several others ( ATM, ATR, PALB2, etc.) are involved with DDR and are mutated in many cancers. Tumors with a DDR defect are susceptible to PARP inhibition (PARPi) in some cases, but also potentially to immune checkpoint inhibitors (ICPIs), given immunomodulatory effects of PARPi and the hypermutated phenotype commonly associated with these genomic alterations (GA). We looked at the prevalence of select DDR defects in tubular GI cancers and the correlation between DDR and TMB. Methods: We prospectively analyzed samples from 17,486 unique patients with advanced colorectal (CRC), esophageal, gastric, or small bowel carcinomas using hybrid-capture based comprehensive genomic profiling. TMB (mutations/Mb) was calculated from 1.11 Mb of sequenced DNA. We looked at GA in 8 genes- ARID1A, ATM, ATR, BRCA1, BRCA2, CDK12, PALB2, RAD51 across these tumors types. Results: DDR GA were found in 16% of cases: gastric 464/1,750 (27%), small bowel 141/666 (21%), esophageal 441/2,501 (18%), and CRC 1,824/12,569 (15%). ARID1A (9.0%) and ATM (5%) were the most commonly altered DDR genes in this series, followed by BRCA2 (2.3%), BRCA1 (1.1%), ATR (0.8%), CDK12 (0.7%), PALB2 (0.6%), and RAD51 (0.1%), with 24% (675/2,870) of DDR-altered cases having GA in more than one DDR gene. Among DDR-altered cases, 21% had high TMB (≥20 mut/Mb) compared to just 1.4% high TMB in DDR-wild-type cases (p < 0.001). Microsatellite instability (MSI) status was available for a subset of cases and 19% (419/2,154) of those with a DDR GA were MSI high. CDK12 and ATR altered cases had the highest proportion of high TMB: CDK12 (55%, median TMB 31.5 mut/Mb) and ATR (55%, median TMB 35.1 mut/Mb), followed by cases with GA in BRCA2 (40%), BRCA1 (28%), ARID1A (27%), ATM (22%), and RAD51 (20%). Conclusions: DDR defects are relatively common across tubular GI tumor types and are associated with a hypermutated phenotype in over 20% of cases. This may be important to identify likely responders to both PARPi and ICPIs.

The replicative senescent mesenchymal stem / stromal cells defect in DNA damage response and anti-oxidative capacity.

Replicative senescence and potential malignant transformation are great limitations in the clinical application of bone marrow-derived mesenchymal stem / stromal cells (MSCs). An abnormal DNA damage response may result in genomic instability, which is an integral component of aging and tumorigenesis. However, the effect of aging on the DNA damage response in MSCs is currently unknown. In the present study, we evaluated the DNA damage response induced by oxidative stress and DNA double-strand breaks in human bone marrow-derived MSCs. After long-term cell culture, replicative senescent MSCs (sMSCs) were characterized by a poor proliferation rate, high senescence-associated β-galactosidase activity, and enhanced expression of P53 and P16. Features of the DNA damage response in these sMSCs were then compared with those from early-passage MSCs. The sMSCs were more sensitive to hydrogen peroxide and bleomycin treatment with respect to cell viability and apoptosis induction. Combined with the comet assay, γH2AX foci characterization and reactive oxygen species detection were used to demonstrate that the antioxidant and DNA repair ability of sMSCs are attenuated. This result could be explained, at least in part, by the downregulation of anti-oxidation and DNA repair genes, including Cu/Zn-SOD, GPX, CAT, OGG1, XRCC1, Ku70, BRCA2 and XRCC4. In conclusion, MSCs aging is associated with a reduction in the DNA repair and anti-oxidative capacity.

Targeting Oxidatively Induced DNA Damage Response in Cancer: Opportunities for Novel Cancer Therapies

Cancer is a death cause in economically developed countries that results growing also in developing countries. Improved outcome through targeted interventions faces the scarce selectivity of the therapies and the development of resistance to them that compromise the therapeutic effects. Genomic instability is a typical cancer hallmark due to DNA damage by genetic mutations, reactive oxygen and nitrogen species, ionizing radiation, and chemotherapeutic agents. DNA lesions can induce and/or support various diseases, including cancer. The DNA damage response (DDR) is a crucial signaling-transduction network that promotes cell cycle arrest or cell death to repair DNA lesions. DDR dysregulation favors tumor growth as downregulated or defective DDR generates genomic instability, while upregulated DDR may confer treatment resistance. Redox homeostasis deeply and capillary affects DDR as ROS activate/inhibit proteins and enzymes integral to DDR both in healthy and cancer cells, although by different routes. DDR regulation through modulating ROS homeostasis is under investigation as anticancer opportunity, also in combination with other treatments since ROS affect DDR differently in the patients during cancer development and treatment. Here, we highlight ROS-sensitive proteins whose regulation in oxidatively induced DDR might allow for selective strategies against cancer that are better tailored to the patients.

Signature-guided approaches for targeting DNA damage response defects in cancer

Signature-guided approaches for targeting DNA damage response defects in cancer

Maintenance of telomere length in AML.

The importance of telomere length to human health, aging, and cancer continues to be underappreciated. This review examines some basics of telomere biology and relates how telomere function, telomerase activity, and mutations in TERC or TERT are involved in bone marrow failure, leukemias, and other cancers. Given the challenge to obtain accurate data on telomerase activity and telomere length in specific cell types, the situation in acute myeloid leukemia (AML) remains puzzling. In most cancers, telomerase levels are increased after cells have encountered a "telomere crisis," which is typically associated with poor prognosis. Cells emerging from "telomere crisis" have defective DNA damage responses, resulting, for example, from loss of p53. Such cells often express elevated telomerase levels as a result of point mutations in the TERT promoter or amplification of the TERT gene. While telomeres in AML blasts are typically shorter than expected for normal leukocytes, most AML cells do not show evidence of having gone through a "telomere crisis." In chronic myeloid leukemia (CML), the difference between the telomere length in nonmalignant T cells and malignant blasts from the same patient was found to correlate with the remaining duration of the chronic phase. This observation supports that a mitotic clock is ticking in CML stem cells and that disease progression in CML heralds the onset of a "telomere crisis." The presence of very short telomeres in tumor cells was found to predict disease progression in chronic lymphocytic leukemia, myeloma, and various solid tumors. In view of these findings longitudinal studies of telomere length in AML appear worthwhile.

The expression of miRNAs is associated with tumour genome instability and predicts the outcome of ovarian cancer patients treated with platinum agents

miRNAs, a class of short but stable noncoding RNA molecules, have been revealed to play important roles in the DNA damage response (DDR). However, their functions in cancer genome instability and the consequent clinical effect as the response to chemotherapy have not been fully elucidated. In this study, we utilized multidimensional TCGA data and the known miRNAs involved in DDR to identify a miRNA-regulatory network that responds to DNA damage. Additionally, based on the expression of ten miRNAs in this network, we developed a 10-miRNA-score that predicts defects in the homologous recombination (HR) pathway and genome instability in ovarian cancer. Importantly, consistent with the association between HR defects and improved response to chemotherapeutic agents, the 10-miRNA-score predicts the outcome of ovarian cancer patients treated with platinum agents, with a surprisingly better performance than the indexes of DNA damage. Therefore, our study demonstrates the implication of miRNA expression on cancer genome instability and provides an alternative method to identify DDR defects in patients who show the best effect with platinum drug treatment.