Inhibition Of Homologous Recombination Repair Research Articles

1G6-D7 Inhibits Homologous Recombination Repair by Targeting Extracellular HSP90α to Promote Apoptosis in Non-Small Cell Lung Cancer.

Despite recent advances in treatment, non-small cell lung cancer (NSCLC) continues to have a high mortality rate. Currently, NSCLC pathogenesis requires further investigation, and therapeutic drugs are still under development. Homologous recombination repair (HRR) repairs severe DNA double-strand breaks. Homologous recombination repair deficiency (HRD) occurs when HRR is impaired and causes irreparable double-strand DNA damage, leading to genomic instability and increasing the risk of cancer development. Poly(ADP-ribose) polymerase (PARP) inhibitors can effectively treat HRD-positive tumors. Extracellular heat shock protein 90α (eHSP90α) is highly expressed in hypoxic environments and inhibits apoptosis, thereby increasing cellular tolerance. Here, we investigated the relationship between eHSP90α and HRR in NSCLC. DNA damage models were established in NSCLC cell lines (A549 and H1299). The activation of DNA damage and HRR markers, apoptosis, proliferation, and migration were investigated. In vivo tumor models were established using BALB/c nude mice and A549 cells. We found that human recombinant HSP90α stimulation further activated HRR and reduced DNA damage extent; however, eHSP90α monoclonal antibody, 1G6-D7, effectively inhibited HRR. HRR inhibition and increased apoptosis were observed after LRP1 knockdown; this effect could not be reversed with hrHSP90α addition. The combined use of 1G6-D7 and olaparib caused significant apoptosis and HRR inhibition in vitro and demonstrated promising anti-tumor effects in vivo. Extracellular HSP90α may be involved in HRR in NSCLC through LRP1. The combined use of 1G6-D7 and PARP inhibitors may exert anti-tumor effects by inhibiting DNA repair and further inducing apoptosis of NSCLC cells.

Inhibition of homologous recombination repair by Mirin in ovarian cancer ameliorates carboplatin therapy response in vitro

Abstract Chemoresistance poses one of the most significant challenges of cancer therapy. Carboplatin (CbPt) is one of the most used chemotherapeutics in ovarian cancer (OVC) treatment. MRE11 constitutes a part of homologous recombination (HR), which is responsible for the repair of CbPt-induced DNA damage, particularly DNA crosslinks. The study’s main aim was to address the role of HR in CbPt chemoresistance in OVC and to evaluate the possibility of overcoming CbPt chemoresistance by Mirin-mediated MRE11 inhibition in an OVC cell line. Lower expression of MRE11 was associated with better overall survival in a cohort of OVC patients treated with platinum drugs (TCGA dataset, P < 0.05). Using in vitro analyses, we showed that the high expression of HR genes drives the CbPt chemoresistance in our CbPt-resistant cell line model. Moreover, the HR inhibition by Mirin not only increased sensitivity to carboplatin (P < 0.05) but also rescued the sensitivity in the CbPt-resistant model (P < 0.05). Our results suggest that MRE11 inhibition with Mirin may represent a promising way to overcome OVC resistance. More therapy options will ultimately lead to better personalized cancer therapy and improvement of patients’ survival.

Abstract 1375: Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells

Abstract Introduction: PARP inhibitors (PARPi) effectively target BRCA mutated cancers by exploiting defects in homologous recombination repair (HRR) by synthetic lethality. PARPi cause replication stress, activating ATR, which signals to HRR and to S and G2/M cell cycle checkpoints via CHK1 and WEE1. Our aim was to determine if a CHK1 inhibitor (CHK1i) would be synthetically lethal with PARPi and to identify the underlying mechanism. Methods: Effects of the PARPi rucaparib and the CHK1i PF-477736 were investigated in V-C8 (BRCA2 mutant Chinese hamster fibroblasts) and V-C8.B2 (BRCA2 restored) cell lines. PARP activity was determined by immunoblot densitometry of the PAR product. CHK1, ATR and WEE1 activity was measured by CHK1S296, CHK1S345 and CDK1y15 phosphorylation, respectively, by Western blot. HRR activity was determined by γH2AX and RAD51 foci formation by immunofluorescence microscopy. Cytotoxicity was determined by colony formation assay and cell cycle analysis by flow cytometry using propidium iodide. Results: V-C8 cells had higher baseline PARP activity than V-C8.B2 cells (1.6 and 0.7 nmol/106 cells, respectively), and in both cell lines PARP activity was suppressed 99% by 1 µM rucaparib. ATR, CHK1 and WEE1 were activated by rucaparib (10 µM) in both V-C8 cells (2.1, 1.5 and 1.8-fold) and V-C8.B2 cells (1.7, 1.4 and 1.5-fold) respectively. Conversely, activation of CHK1 and WEE1 was completely inhibited by 50 nM PF-477736 in both cell lines, while upstream activation of ATR by PF-477736 was almost 2-fold in both cell lines. Rucaparib was 550x more cytotoxic to HRR-defective V-C8 cells compared to HRR-competent V-C8.B2 cells (LC50 <0.1 µM vs 8.8 µM; p<0.001 respectively). Combination treatment with PF-477736 (50 nM) sensitised V-C8.B2 cells to rucaparib cytotoxicity 4.9-fold but did not sensitise V-C8 cells. In V-C8.B2 cells, 10 µM rucaparib caused a 6-fold increase in γH2AX and a 4.8-fold increase in RAD51 foci in γH2AX positive cells, demonstrating both increased replication stress and HRR activity in response to DNA damage. Combination treatment with PF-477736 (50 nM) increased γH2AX foci 2-fold compared to rucaparib single agent and inhibited the rucaparib induced RAD51 foci formation entirely, showing complete inhibition of HRR in V-C8.B2 γH2AX positive cells. Conclusion: The CHK1i PF-477736 sensitised HRR competent, but not HRR defective V-C8 cells to rucaparib suggesting that the primary mechanism was through PF-477736 abrogation of HRR mediated by its direct inhibition of CDK1. Cell cycle analysis is ongoing to determine the impact of rucaparib plus PF-477736 modulation of CHK1 and WEE1 activities on S and G2/M checkpoint control. Citation Format: Hannah L. Smith, Lisa Prendergast, Nicola J. Curtin. Investigating synergy between CHK1 and PARP inhibitors in BRCA2 mutant and restored cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1375.

RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.

ABSTRACTRIF1 controls both DNA replication timing and the DNA double-strand break (DSB) repair pathway to maintain genome integrity. However, it remains unclear how RIF1 links these two processes following exposure to ionizing radiation (IR). Here, we show that inhibition of homologous recombination repair (HRR) by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 after a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR, even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.This article has an associated First Person interview with the first author of the paper.

Prolonged exposure to particulate Cr(VI) is cytotoxic and genotoxic to fin whale cells

BackgroundHexavalent chromium [Cr(VI)] is a human lung carcinogen and global marine pollutant. High Cr concentrations, resembling the ones observed in occupationally exposed workers, have been observed in fin whales (Balaenoptera physalus) in the Gulf of Maine. This outcome suggests Cr might be disrupting the health of fin whale populations. Indeed, Cr in acute (24 h) exposure does cause toxicity in fin whale cells. However, human cell culture data indicate prolonged exposures (120 h) induce a higher amount of toxicity compared to 24 h exposure due to an inhibition of homologous recombination repair. However, whether prolonged exposure causes similar outcomes in fin whale cells is unknown. ObjectiveDue to the importance of assessing prolonged exposure toxicity, this study focuses on characterizing acute and prolonged exposure of Cr(VI) in male and female fin whale cells. MethodsCytotoxicity was measured by the clonogenic assay, also known as colony forming assay, which measures the ability of cells to proliferate and form colonies after the treatment. DNA double strand breaks were analyzed by neutral comet assay. Clastogenicity was measured using the chromosome aberration assay. Intracellular Cr levels were measured with Graphite Furnace Atomic Absorption Spectrometry (GFAAS) with Syngistix Software. ResultsIn this study, we demonstrate that particulate Cr(VI) induces cytotoxicity and genotoxicity in a treatment-dependent manner after 24 h and 120 h exposures. Cytotoxicity levels were generally low with relative survival above 64 %. DNA double strand break data and chromosome aberration data were elevated after a 24 h exposure, but decreased after a 120 h exposure. While cytotoxicity was similar after 24 h and 120 h exposures, less DNA double strand breaks and chromosomal instability occurred with prolonged exposure. ConclusionParticulate Cr(VI) is cytotoxic and genotoxic to fin whale cells after acute and prolonged exposures. The reduction of genotoxicity we have observed after 120 h exposure may be partly explained by lower intracellular Cr levels after 120 h. However, the decrease in intracellular levels is not reflected by a similar decrease in chromosome aberrations suggesting other mechanisms may be at play. Male fin whale cells appear to be more susceptible to the genotoxic effects of particulate Cr(VI) while female cells are less susceptible possibly due to increased cell death of damaged cells, but more work is needed to clarify if this outcome reflects a sex difference or interindividual variability. Overall, the study shows particulate Cr(VI) does induce toxicity at both acute and prolonged exposures in fin whales cells indicating Cr(VI) exposure is a health risk for this species.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 μM–300 μM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

Exploring the Synergy between PARP and CHK1 Inhibition in Matched BRCA2 Mutant and Corrected Cells.

PARP inhibition results in the accumulation of DNA SSBs, causing replication stress (RS) and lesions that can only be resolved by homologous recombination repair (HRR). Defects in HRR, e.g., due to BRCA2 mutation, confer profound sensitivity to PARP inhibitor (PARPi) cytotoxicity. In response to RS, CHK1 is activated to signal to S and G2/M cell cycle checkpoints and also to HRR. To determine the relative contribution of these two functions of CHK1 to survival following PARPi exposure, we investigated the effects of rucaparib (a PARPi) and PF-477736 (a CHK1 inhibitor) alone and in combination in cells with mutated and corrected BRCA2. The BRCA2 mutated V-C8 cells were 1000× more sensitive to rucaparib cytotoxicity than their matched BRCA2 corrected V-C8.B2 cells, but no more sensitive to PF-477736 despite having seven-fold higher levels of RS. PF-477736 caused a five-fold enhancement of rucaparib cytotoxicity in the V-C8.B2 cells, but no enhancement in the V-C8 cells. This differential sensitivity was not due to a difference in PARP1 or CHK1 expression or activity. PF-477736 increased rucaparib-induced RS (γH2AX foci) and completely inhibited RAD51 focus formation, indicating a profound suppression of HRR. Our data suggested that inhibition of HRR was the main mechanism of sensitisation to rucaparib, compounded with an inhibition of cell cycle checkpoints by PF-477736.

Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

DNA repair via homologous recombination (HR) is indispensable for genome integrity and cell survival but if unrestrained can result in undesired chromosomal rearrangements. The regulatory mechanisms of HR are not fully understood. Cyclic GMP‐AMP synthase (cGAS) is best known as a cytosolic innate immune sensor critical for the outcome of infections, inflammatory diseases, and cancer. Here, we report that cGAS is primarily a chromatin‐bound protein that inhibits DNA repair by HR, thereby accelerating genome destabilization, micronucleus generation, and cell death under conditions of genomic stress. This function is independent of the canonical STING‐dependent innate immune activation and is physiologically relevant for irradiation‐induced depletion of bone marrow cells in mice. Mechanistically, we demonstrate that inhibition of HR repair by cGAS is linked to its ability to self‐oligomerize, causing compaction of bound template dsDNA into a higher‐ordered state less amenable to strand invasion by RAD51‐coated ssDNA filaments. This previously unknown role of cGAS has implications for understanding its involvement in genome instability‐associated disorders including cancer.

Discovery of a novel DNA polymerase inhibitor and characterization of its antiproliferative properties

ABSTRACTChromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silico screening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces “BRCAness” in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

Synthetic Lethality of PARP Inhibition and Ionizing Radiation is p53-dependent.

PARP inhibitors (PARPi) are potentially effective therapeutic agents capable of inducing synthetic lethality in tumors with deficiencies in homologous recombination (HR)-mediated DNA repair such as those carrying BRCA1 mutations. However, BRCA mutations are rare, the majority of tumors are proficient in HR repair, and thus most tumors are resistant to PARPi. Previously, we observed that ionizing radiation (IR) initiates cytoplasmic translocation of BRCA1 leading to suppression of HR-mediated DNA repair and induction of synthetic PARPi lethality in wild-type BRCA1 and HR-proficient tumor cells. The tumor suppressor p53 was identified as a key factor that regulates DNA damage-induced BRCA1 cytoplasmic sequestration following IR. However, the role of p53 in IR-induced PARPi sensitization remains unclear. This study elucidates the role of p53 in IR-induced PARPi cytotoxicity in HR-proficient cancer cells and suggests p53 status may help define a patient population that might benefit from this treatment strategy. Sensitization to PARPi following IR was determined in vitro and in vivo utilizing human breast and glioma tumor cells carrying wild-type BRCA1 and p53, and in associated cells in which p53 function was modified by knockdown or mutation. In breast and glioma cells with proficient HR repair, IR-induced BRCA1 cytoplasmic sequestration, HR repair inhibition, and subsequent PARPi sensitization in vitro and in vivo was dependent upon functional p53.Implications: Implications: p53 status determines PARP inhibitor sensitization by ionizing radiation in multiple BRCA1 and HR-proficient tumor types and may predict which patients are most likely to benefit from combination therapy. Mol Cancer Res; 16(7); 1092-102. ©2018 AACR.

Abstract IA21: Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer

Abstract Pancreas cancer is projected to cause approximately 42,000 deaths in 2016, making it the third greatest cause of cancer mortality (behind lung and colorectal, but greater even than breast). Systemic failure remains the dominant cause of death, although local failure is responsible for up to 1/3 of the pancreatic cancer related mortality. We have focused on improving the treatment of locally advanced pancreatic cancer, defined as disease which cannot be resected but has not overtly metastasized, where the issue of local control becomes even more important, and have hypothesized that the treatment of locally advanced pancreatic cancer will require control of both the gross primary tumor as well as the microscopic disease that exists in virtually every patient. Gemcitabine has been a mainstay of treatment for pancreatic cancer, and we have standardized combining it with radiation for locally advanced disease. Because our initial studies revealed that activation of the DNA damage response, particularly activation of CHK1 and WEE1, were crucial in the defense pancreatic cancers mount against chemotherapy and radiation, we have studied the effect of CHK1 and WEE1 inhibitors on gemcitabine and gemcitabine-radiation mediated cytotoxicity. These inhibitors abrogate the G2 checkpoint after DNA damage. The initial hypothesis for using CHK1 and WEE1 inhibitors was that cancer cells lacking a p53 checkpoint would be selectively sensitized, compared to normal cells, because normal cells would be protected by an intact G1 checkpoint. After initial studies with several agents, we have focused on the WEE1 inhibitor AZD1775, chiefly because it was available for a clinical trial. We have carried out both mechanistic and clinical studies using AZD1775. Mechanistically, we have evaluated both the contributions of homologous recombination repair (HRR) inhibition and G2 checkpoint abrogation by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistoneH3 flow cytometry, respectively in pancreatic cancer model systems. AZD1775 causes inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. Sensitization by AZD1775 is associated with persistent γH2AX and inhibition of RAD51 focus formation. Using BRCA2 isogenic cells, we have found that AZD1775 sensitizes to gemcitabine-radiation in BRCA2 wild type cells, while in BRCA2 null cells AZD1775 did not sensitize despite significant G2 checkpoint abrogation. In vivo, in patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibits tumor growth and impairs RAD51 focus formation in response to gemcitabine-radiation. We've concluded that, whereas sensitization is accompanied by inhibition of CDK1 phosphorylation and abrogation of the G2 checkpoint, this mechanism is not sufficient for sensitization. Our findings suggest that sensitization to chemoradiation by WEE1 inhibition also requires inhibition of HRR. The importance of HRR has also supported studies combining CHK1 or WEE1 inhibition with a PARP inhibitor, which would be anticipated to potentiate the effects of HRR inhibition. Based on these studies, we have launched a clinical trial combining gemcitabine and gemcitabine-radiation, at standard doses, with dose escalating AZD1775 for patients with locally advanced pancreatic cancer. Patients receive one cycle of gemcitabine with AZD1775, then 2 cycles of gemcitabine-radiation with AZD1775, then another cycle of AZD1775-gemcitabine, followed by assessment for response and toxicity. Optional cycles of AZD1775 and gemcitabine are then given to patients who have not progressed. AZD1775 has been escalated according to a Time-to-Event Continual Reassessment Model, in which the dose for each patient is based on the toxicity profile of all prior patients, and the goal is to establish a tolerable dose of the combination therapy and to estimate the response and survival. With 21 patients entered onto the study, the median survival has not been reached, and the 64% survival at the 18-month landmark point exceeds the 18-month median survival that was the agreed on endpoint for triggering a larger trial. The trial also includes a pharmacodynamic endpoint of a hair follicle biopsy to assess target engagement. In summary, these findings suggest that WEE1 inhibition may act through both G2 checkpoint abrogation but, more importantly, inhibition of HRR, and is a promising potentiator of gemcitabine-radiation in the treatment of locally advanced pancreatic cancer. Supported by P50 CA130810, R01 CA163895, P30 CA46592, and the Taubman Institute Citation Format: Theodore S. Lawrence, Meredith A. Morgan. Targeting WEE1 kinase to potentiate chemoradiation in the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA21.

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90–95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

XPO1 Inhibition using Selinexor Synergizes with Chemotherapy in Acute Myeloid Leukemia by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus.

Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy. Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC-dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142-52. ©2016 AACR.

Late DNA Damage Mediated by Homologous Recombination Repair Results in Radiosensitization with Gemcitabine.

Gemcitabine (dFdCyd) shows broad antitumor activity in solid tumors in chemotherapeutic regimens or when combined with ionizing radiation (radiosensitization). While it is known that mismatches in DNA are necessary for dFdCyd radiosensitization, the critical event resulting in radiosensitization has not been identified. Here we hypothesized that late DNA damage (≥24 h after drug washout/irradiation) is a causal event in radiosensitization by dFdCyd, and that homologous recombination repair (HRR) is required for this late DNA damage. Using γ-H2AX as a measurement of DNA damage in MCF-7 breast cancer cells, we demonstrate that 10 or 80 nM dFdCyd alone produced significantly more late DNA damage compared to that observed within 4 h after treatment. The combination of dFdCyd treatment followed by irradiation did not produce a consistent increase in DNA damage in the first 4 h after treatment, however, there was a synergistic increase 24-48 h later relative to treatment with dFdCyd or radiation alone. RNAi suppression of the essential HRR protein, XRCC3, significantly decreased both radiosensitization and late DNA damage. Furthermore, inhibition of HRR with the Rad51 inhibitor B02 prevented radiosensitization when added after, but not during, treatment with dFdCyd and radiation. To our knowledge, this is the first published study to show that radiosensitization with dFdCyd results from a synergistic increase in DNA damage at 24-48 h after drug and radiation treatment, and that this damage and radiosensitization require HRR. These results suggest that tumors that overexpress HRR will be more vulnerable to chemoradiotherapy, and treatments that increase HRR and/or mismatches in DNA will enhance dFdCyd radiosensitization.

Abstract 519: Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair

Abstract Mutant KRAS is found in approximately 30% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Despite the use of radiation (RT) therapy for the treatment of locally advanced disease, local recurrence remains an issue. The observation that mutations in KRAS lead to both replication stress and DNA double-strand breaks (DSBs) suggests these cancers may have a greater reliance on DNA damage response pathways and therefore may be preferentially radiosensitized by therapies targeting DNA repair. In this study, we investigated the combined inhibition of WEE1 and PARP1 using the small molecule inhibitors, AZD1775 and olaparib, respectively as a radiosensitizing strategy in KRAS mutant NSCLC. We began by comparing radiosensitization by AZD1775+olaparib, in KRAS isogenic H1703 lung cancer cells and found that KRAS mutant cells were preferentially radiosensitized (ER, enhancement ratio 1.8) compared to KRAS wild type cells (ER 1.4). Combined WEE1 and PARP1 inhibition also radiosensitized KRAS mutant Calu-6 and NCI-H23 lung cancer cells (ER 1.9 and 1.5, respectively). These findings were further confirmed in vivo: Calu-6 tumor xenografts were significantly radiosensitized by AZD1775+olaparib, as evidenced by an 11 day delay in tumor volume doubling time relative to RT treatment. Given that WEE1 and PARP1 function to prevent and manage replication stress, respectively, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent replication stress. While replication stress did contribute to AZD1775-mediated radiosensitization in Calu-6 cells, as evidenced by pan-nuclear γH2AX staining, and the ability of exogenous nucleosides to protect cells from radiosensitization by AZD1775 alone, nucleosides had little effect on radiosensitization by AZD1775+olaparib. These results suggest that replication stress is not required for radiosensitization by AZD1775+olaparib. As WEE1 and PARP1 both promote repair of radiation-induced DNA damage, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent, unrepaired DSBs. Assessment of the kinetics of DSB repair by γH2AX flow cytometry demonstrated that while total γH2AX levels in cells treated with RT alone had returned to control levels within 24 h, AZD1775+olaparib treatment significantly delayed the resolution of γH2AX following RT, with 44.8% or 46.2% of Calu-6 or KRAS mutant H1703 cells, respectively remaining γH2AX-positive 24 h post-RT. This delay corresponded with the inhibition of radiation-induced RAD51 foci by AZD1775, indicating inhibition of homologous recombination repair. Taken together these data demonstrate the efficacy of combined inhibition of WEE1 and PARP1 with radiation in KRAS mutant lung cancer. Furthermore, these results suggest that although replication stress occurs in response to AZD1775 and olaparib, persistent DSBs are the cause of radiosensitization. Citation Format: Leslie A. Parsels, David Karnak, Joshua D. Parsels, Zachery Reichert, Jonathan Maybaum, Theodore S. Lawrence, Meredith Ann Morgan. Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair. [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 519.

Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Hyperthermia-induced radiosensitization in CHO wild-type, NHEJ repair mutant and HR repair mutant following proton and carbon-ion exposure.

The DNA repair mechanisms involved in hyperthermia-induced radiosensitization with proton and carbon ion radiation exposure were investigated in the present study. In a previous study, Chinese hamster ovary (CHO) cells were exposed to low linear energy transfer (LET) photon radiation. These cells can be sensitized by hyperthermia as a result of inhibition of homologous recombination (HR) repair. The present study used wild-type, non-homologous end joining (NHEJ) and HR repair-deficient CHO cells to define the contributions of each repair pathway to cellular lethality following hyperthermia-induced hadron radiation sensitization. The cells were exposed to ionizing radiation, followed by hyperthermia treatment (42.5°C for 1 h). Hyperthermia-induced radiosensitization was determined by the colony formation assay and thermal enhancement ratio. HR repair-deficient cells exhibited no hyper-sensitization to X-rays, protons, or low and high LET carbon ions when combined with hyperthermia. Wild-type and NHEJ repair-deficient cells exhibited significant hyperthermia-induced sensitization to low LET photon and hadron radiation. Hyperthermia-induced sensitization to high LET carbon-ion radiation was less than at low LET radiation. Relative biological effectiveness (RBE) between radiation alone and radiation combined with hyperthermia cell groups was not significantly different in any of the cell lines, with the exception of wild-type cells exposed to high LET radiation, which exhibited a lower RBE in the combined group. The present study investigated additional cell lines to confirm the lower RBE observed in DNA repair-deficient cell lines. These findings suggested that hyperthermia-induced hyper-sensitization to hadron radiation is also dependent on inhibition of HR repair, as was observed with photon radiation in a previous study.

Abstract 3629: Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs

Abstract Nucleoside analogs (NAs) represent a large group of anticancer drugs, several members of which have been shown to sensitize cells to ionizing radiation (IR). Among the lesions induced in cells by IR, the DNA double-strand break (DSB) is considered to be the most detrimental. Therefore we focused on interactions between NAs and pathways of DSB repair. We opted for post-irradiation treatment with NAs to avoid complications arising from redistribution of cells throughout the cycle prior to IR exposure. We compared the effects of 4h treatments post-IR with vidarabine (ara-A), fludarabine (F-ara-A) or gemcitabine (dFdC) on the survival of A549 lung carcinoma cells. We found that post-irradiation treatment with ara-A and F-ara-A, but not dFdC, resulted in cell radiosensitization to killing. Radiosensitization by ara-A was superior to that by F-ara-A. We employed immunofluorescence detection of IR-induced foci and I-SceI-based reporter cell lines to identify the pathways of DSB repair that are inhibited by ara-A and cause the observed radiosensitization. We found that the formation of IR-induced Rad51-foci was compromised by ara-A suggesting inhibition of homologous recombination repair (HRR). Repair of I-SceI-induced DSBs by HRR was also suppressed by ara-A. Furthermore, A549 cells deprived of Rad51 by RNAi could not be efficiently radiosensitized by ara-A. On the other hand, we could not detect impairment by ara-A of non-homologous end-joining (NHEJ), as measured by pulsed-field gel electrophoresis (PFGE) at high doses of IR (20 Gy). Notably, we observed an increase in the frequency of mutagenic NHEJ events in several I-SceI-based repair reporter systems, indicative of processing by error-prone repair pathways such as alternative/backup NHEJ (B-NHEJ). In addition, we could show that treatment with ara-A induces a dramatic increase in B-NHEJ in plateau phase Lig4-/- MEFs. Treatment with F-ara-A also inhibited HRR, but was less proficient in promoting mutagenic NHEJ. We conclude that inhibition of HRR and promotion of B-NHEJ represent two important mechanisms of radiosensitization by NAs. The strong impact of ara-A on both of these repair pathways underpins its superior radiosensitizing potential. Targeting of both HRR and B-NHEJ may provide a promising new strategy for radiosensitization. Citation Format: George Iliakis, Simon Magin. Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3629. doi:10.1158/1538-7445.AM2015-3629

Regulation of DNA Damage Response by Estrogen Receptor β-Mediated Inhibition of Breast Cancer Associated Gene 2.

Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER)-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα) in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2), correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC) irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM) activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells.

Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors.