Effects Of DNA-damaging Agents Research Articles

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

C-Rel-dependent Chk2 signaling regulates the DNA damage response limiting hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.

Histone Deacetylase 1 Inhibition by Peptides Containing a DNA Damage-Induced, Nonenzymatic, Histone Covalent Modification.

Treatment of HeLa cells with the DNA damaging agent, bleomycin (BLM), results in the formation of a nonenzymatic 5-methylene-2-pyrrolone histone covalent modification on lysine residues (KMP). KMP is much more electrophilic than other N-acyllysine covalent modifications and post-translational modifications, including N-acetyllysine (KAc). Using histone peptides containing KMP, we show that this modification inhibits the class I histone deacetylase, HDAC1, by reacting with a conserved cysteine (C261) located near the active site. HDAC1 is inhibited by histone peptides whose corresponding N-acetylated sequences are known deacetylation substrates, but not one containing a scrambled sequence. The HDAC1 inhibitor, trichostatin A, competes with covalent modification by the KMP-containing peptides. HDAC1 is also covalently modified by a KMP-containing peptide in a complex milieu. These data indicate that peptides containing KMP are recognized by HDAC1 and are bound in the active site. The effects on HDAC1 indicate that KMP formation in cells may contribute to the biological effects of DNA damaging agents, such as BLM, that form this nonenzymatic covalent modification.

ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses.

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1’s ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.

The antiproliferative effects of ataxia-telangiectasia mutated and ATM- and Rad3-related inhibitions and their enhancements with the cytotoxicity of DNA damaging agents in cholangiocarcinoma cells.

To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines. Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis. The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells. Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

Short-term treatment with imetelstat sensitizes hematopoietic malignant cells to a genotoxic agent via suppression of the telomerase-mediated DNA repair process

Imetelstat is a specific and competitive inhibitor of telomerase enzymatic activity. We demonstrated that imetelstat could interfere with the DNA repair process and enhance the effect of DNA damaging agents using hematological tumor cell lines. Short-term administration of imetelstat enhanced growth suppression by anticancer agents and radiation. It also upregulated γH2AX expression induced by irradiation. Immunofluorescence staining showed that both human telomerase reverse transcriptase (hTERT) and γH2AX were upregulated and co-localized in the nucleus of peripheral blood mononuclear cells after irradiation, suggesting that hTERT was involved in the DNA-DSB repair process. Imetelstat enhanced growth inhibitory effect of cytotoxic agents in short-term culture without shortening of telomeres, indicating that this effect was attributed by telomere length independent mechanism. Our results suggest that the combination of short-term treatment with imetelstat and cytotoxic agent is a promising strategy to treat a wide variety of hematopoietic malignancies.

PARP inhibitor Olaparib Enhances the Apoptotic Potentiality of Curcumin by Increasing the DNA Damage in Oral Cancer Cells through Inhibition of BER Cascade.

Although Olaparib (Ola, a PARP-inhibitor), in combination with other chemotherapeutic agents, was clinically approved to treat prostate cancer, but cytotoxicity, off-target effects of DNA damaging agents limit its applications in clinic. To improve the anti-cancer activity and to study the detailed mechanism of anti-cancer action, here we have used bioactive compound curcumin (Cur) in combination with Ola. Incubation of Ola in Cur pre-treated cells synergistically increased the death of oral cancer cells at much lower concentrations than individual optimum dose and inhibited the topoisomerase activity. Short exposure of Cur caused DNA damage in cells, but more increased DNA damage was noticed when Ola has incubated in Cur pre-treated cells. This combination did not alter the major components of homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways but significantly altered both short patch (SP) and long patch (LP) base excision repair (BER) components in cancer cells. Significant reduction in relative luciferase activity, expression of BER components and PARylation after Cur and Ola treatment confirmed this combination inhibit the BER activity in cells. Reduction of PARylation, decreased expression of BER components, decreased tumor volume and induction of apoptosis were also noticed in Cur + Ola treated Xenograft mice model. The combination treatment of Cur and Ola also helped in recovering the body weight of tumor-bearing mice. Thus, Cur + Ola combination increased the oral cancer cells death by not only causing the DNA damage but also blocking the induction of BER activity.

Structure-Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System.

Plants are sessile organisms, and their DNA is particularly exposed to damaging agents. The integrity of plant mitochondrial and plastid genomes is necessary for cell survival. During evolution, plants have evolved mechanisms to replicate their mitochondrial genomes while minimizing the effects of DNA damaging agents. The recombinogenic character of plant mitochondrial DNA, absence of defined origins of replication, and its linear structure suggest that mitochondrial DNA replication is achieved by a recombination-dependent replication mechanism. Here, I review the mitochondrial proteins possibly involved in mitochondrial DNA replication from a structural point of view. A revision of these proteins supports the idea that mitochondrial DNA replication could be replicated by several processes. The analysis indicates that DNA replication in plant mitochondria could be achieved by a recombination-dependent replication mechanism, but also by a replisome in which primers are synthesized by three different enzymes: Mitochondrial RNA polymerase, Primase-Helicase, and Primase-Polymerase. The recombination-dependent replication model and primers synthesized by the Primase-Polymerase may be responsible for the presence of genomic rearrangements in plant mitochondria.

Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

Abstract 2814: Antitumor effects of WEE1 inhibitor, AZD1775 alone or in combination with PARP inhibitor in triple negative breast cancer cells

Abstract Background: Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. Especially, recent reports have shown that DNA replication stress and double strand breaks are induced by the activation of CDK1 following WEE1 inhibitor treatment. We investigated the effects and underlying mechanisms of WEE1 inhibitor, AZD1775, in Triple Negative Breast Cancer (TNBC). In addition, we examined whether AZD1775 would enhance the anti-tumor effect of PARP inhibitor in TNBC cells by modulating DNA repair activity. Methods: MTT assay was performed to investigate the antitumor effect of WEE1 inhibitor, AZD1775 alone or with PARP inhibitor in breast cancer cell lines. The regulation of DNA damage response activity by AZD1775 or/and PARP inhibitor was accessed by the comet and western blotting. The status of the S phase was evaluated by EdU-BrdU dual pulse labeling, and the drug effect was also confirmed in vivo by mouse xenograft model of TNBC. Results: AZD1775 showed heterogeneous antitumor effects among TNBC cells regardless of TP53 mutation statues. WEE1 inhibition led to accelerated S phase progression and mitotic entry in the sensitive MDA-MB-231 cells. In addition, increased DNA damage and caspase-3 dependent apoptosis were observed in sensitive cells following AZD1775 treatment. Furthermore, AZD1775 enhanced cellular sensitivity to PARP inhibitor through increase apoptosis and DNA damage accumulation. In a MDA-MB-231 xenograft model, AZD1775 impedes tumor growth as well. Conclusions: In the TNBC, the WEE1 inhibitor has a heterogeneous cytotoxic effect regardless of the p53 mutation status. In MDA-MB-231 cell, which is a sensitive TNBC cells to WEE1 inhibitor AZD1775, the progression of the cell cycle was accelerated and the aberrant DNA content was increased. This resulted in DNA damage, which led to cell death, and this effect could be applied in vivo. In addition, we investigated that WEE1 inhibition can increase PARP inhibitor sensitivity in TNBC cells via increasing accumulation of DNA damage. Our results provide a rationale for the future clinical trials of PARP inhibitor combined with WEE1 inhibition in triple negative breast cancer. Citation Format: Dong Hyeon Ha, Arrum Min, Seongyeong Kim, So Hyeon Kim, Hyemin Jang, Yu Jin Kim, Daeun Jung, YoonJung Park, Koung Jin Suh, Kyung-Hun Lee, Tae-Yong Kiim Kim, Seock-Ah Im. Antitumor effects of WEE1 inhibitor, AZD1775 alone or in combination with PARP inhibitor in triple negative breast cancer cells [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 2814.

Nanostructure of DNA repair foci revealed by superresolution microscopy.

Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (∼200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy ( dSTORM) with a lateral resolution of ∼20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of ∼45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ∼0.6 and ∼1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.-Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

Abstract P5-03-10: CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer

Abstract CYC065 is a novel CDK inhibitor, which inhibits CDK2 and 9 with IC50 values of 5 and 26 nM, respectively. Following completion of IND-enabling studies, CYC065 has been cleared by FDA for first-in-human Phase 1 clinical trials. Triple-negative breast cancers (TNBC), particularly the basal subtype, often exhibit aggressive characteristics. Despite good initial responses to chemotherapy, patients experience early relapse and diminished 5 year survival. Molecular features of basal-like TNBC include amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor such as CYC065. CYC065 is effective in cyclin E-overexpressing tumors, such as uterine serous carcinoma1 and trastuzumab-resistant Her2+ breast cancer2. Moreover, CDK inhibition has also been reported to be synthetic lethal with overexpressed MYC3. This led us to assess the potency and mechanism of action of CYC065 in basal-like TNBC models to evaluate the potential for CYC065 development in this indication. In vitro cell-based experiments support twice weekly pulse dosing using submicromolar concentrations of CYC065 to achieve maximum impact on cell growth in the majority of breast cancer cell lines tested. Preclinical toxicology data indicate that such levels and durations of exposure are achievable and well tolerated. As a single agent, CYC065 treatment in breast cancer cells resulted in inhibition of RNA-Pol II phosphorylation, down-regulation of Mcl-1, up-regulation of p53 and rapid induction of apoptosis. The impact of CYC065 on CDK2 targets, cyclin E and MYC was also explored. Interestingly immortalized cell lines obtained from non-malignant tissue displayed similar effects on RNA Pol II, Mcl-1, and p53 but did not undergo apoptosis and consequently exhibited relative resistance to CYC065, indicative of a potential therapeutic window. Cell cycle analysis demonstrated that CYC065 treatment induced an increase in G1 population with no significant induction of cell death in non-malignant derived cell lines, compared to cancer cell lines, in which there was significant induction of cell death. CDKs have a role in DNA repair which can be exploited to enhance the effectiveness of DNA damaging agents. Seliciclib, an oral, first generation CDK2/9 inhibitor, can be effectively combined with DNA damaging agents, such as the oral nucleoside analogue sapacitabine, or its active metabolite CNDAC. A Phase 1 clinical trial is currently underway to evaluate this combination (NCT00999401). Similarly to seliciclib, we demonstrate that CYC065 is synergistic in combination with CNDAC when given sequentially across multiple breast cancer cell lines. CNDAC-induced double strand breaks persisted for longer when cells were subsequently treated with CYC065, supporting the conclusion that these CDK inhibitors suppress DNA double-strand break repair capacity, which may contribute to the observed synergy. Taken together the data establish CYC065 as a promising anti-cancer agent in basal-like TNBC, with the potential to be combined effectively in this indication with DNA damaging agents. 1. Cocco, E. et al. AACR, 2015, Abstract 3103. 2. Scaltriti, M. et al. Proc Natl Acad Sci USA. 201, 108, 3761-6. 3. Horiuchi, D. et al. J Exp Med. 2012, 209, 679-96. Citation Format: Blake DG, MacKay C, Frame S, Zheleva D. CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-10.

Abstract LB-165: Antiproliferative effects of AZD6738 and the inhibition of DDR activity

Abstract Background Anti-tumor effect of DNA damaging agents was reduced by the activation of DNA damage repair (DDR), which was led to resistance. Therefore, the inhibition of DNA repair could lead to induce the accumulation of errors which is becoming an attractive strategy. The ataxia telangiectasia and Rad3-related (ATR) proteins play a role of sensor for DNA damage, which induces homologous recombination-dependent repair. ATR is a master regulator of DDR, signaling to control DNA replication, DNA repair, and apoptosis. Therefore, the ATR pathway might be useful target for new drug development and it is important that the effects of many current cancer treatments are modulated by DDR. Materials and Methods The growth inhibitory effects of ATR inhibitor, AZD6738 were studied on human breast cancer cell lines using MTT assay. Cell cycle analysis and western blotting were also performed to determine molecular changes. Immunofluorescence assay and comet assay were conducted to understand the action mechanisms of AZD6738. Results This research identified the anti-proliferative effects and the inhibition of DDR activity by AZD6738 on human breast cancer cell lines. AZD6738 induces cell cycle arrest and apoptosis, which impaired DDR function and promoted cell death by damage accumulation. Results of MTT showed that the heterogeneous response and two cell lines were chosen for the focus on the HER2-positive breast cell lines: SKBR-3 and BT-474. In sensitive cell line, SKBR-3 cell, the expression of phosphorylated CHK1 was reduced with the other repair markers; RAD51, MRE11 and ERCC1 as opposed to less sensitive breast cancer cell BT-474. The decreased functional CHK1 leads to the accumulation of DNA damage due to homologous recombination inactivation. And it was also identified that ATR inhibitor played a role of sensitizer to increase the efficacy of cytotoxic chemotherapeutic agents, cisplatin and paclitaxel in breast cancer cell lines. Conclusion Understanding the antitumor efficacy and the mechanisms of ATR inhibitor in the breast cancer cell lines open up the possibility of future clinical trial targeting DNA damage repair in breast cancer Keywords: DNA damage response, ATR inhibitor, Breast cancer, Homologous recombination Citation Format: Hee Jun Kim, Ahrum Min, Seock-Ah Im, Hyemin Jang, Miso Lee, Seongyeong Kim, Jungen Kim, Kyung-Hun Lee, Sae-Won Han, Tae-Yong Kim, Do-Youn Oh, Tae-You Kim, Yung-Jue Bang. Antiproliferative effects of AZD6738 and the inhibition of DDR activity. [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 LB-165. doi:10.1158/1538-7445.AM2015-LB-165

The Role of DNA Mismatch Repair and Recombination in the Processing of DNA Alkylating Damage in Living Yeast Cells.

It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensitivity of cells defective in homologous recombination (HR) to DNA alkylation is reduced by defects in MMR genes. We have assessed the contribution of different MMR genes to the processing of alkylation damage in vivo. We have directly visualized recombination complexes formed upon DNA damage using fluorescent protein (FP) fusions. We find that msh6 mutants are more resistant than wild type cells to MNNG, and that an msh6 mutation rescues the sensitivity of rad52 strains more efficiently than an msh3 mutation. Analysis of RAD52-GFP tagged strains indicate that MNNG increases repair foci formation, and that the inactivation of the MHS2 and MSH6 genes but not the MSH3 gene result in a reduction of the number of foci formed. In addition, in the absence of HR, NHEJ could process the MNNG-induced DSBs as indicated by the formation of NHEJ-GFP tagged foci. These data suggest that processing of the alkylation damage by MMR, mainly by MSH2-MSH6, is required for recruitment of recombination proteins to the damage site for repair.

Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Efficacy of Combined Histone Deacetylase and Checkpoint Kinase Inhibition in a Preclinical Model of Human Burkitt Lymphoma.

Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies.

Full Donor Myeloid Engraftment with Minimal Toxicity in Dyskeratosis Congenita Patients Undergoing Allogeneic Bone Marrow Transplantation without Radiation or Alkylating Agents

Dyskeratosis congenita (DC) is an inherited bone marrow failure (BMF) syndrome caused by defects in telomere biology genes. BMF occurs in approximately 80% of patients by 30 years of age and, in addition to pulmonary failure and malignancies, remains a major contributor to mortality. Hematopoietic cell transplantation (HCT) cures BMF in DC patients but is associated with a high incidence of graft failure and transplant-related mortality. Radiation and/or alkylating agents (e.g. cyclophosphamide, busulfan, melphalan) used in conditioning may have a role in the poor outcomes in DC patients following HCT. Recently, reduced-intensity conditioning approaches have improved short-term outcomes, but concerns remain about the long-term effects of DNA damaging agents in these regimens, given the multisystem disease and lifelong predisposition to malignancy conferred by telomere dysfunction.Radiation and alkylating agents are mainstays of allogeneic HCT preparative regimens: their highly effective myeloablative and immunosuppressive properties provide a niche for donor hematopoietic progenitors and decrease graft rejection. Based on a rationale that presumes (1) niche availability in DC patients with BMF, and (2) an intrinsic replicative defect in DC hematopoietic cells, we hypothesized that engraftment would be feasible in DC patients using a regimen of immunosuppressive therapy alone.Here, we report that in a single-institution prospective study, 4 consecutive patients with DC have undergone successful HCT using a radiation- and alkylator-free preparative regimen. Patients received alemtuzumab and fludarabine, followed by bone marrow transplantation (BMT) from unrelated donors. Cyclosporine A and mycophenolate mofetil were used for graft versus host disease (GVHD) prophylaxis. The primary outcome measures were neutrophil engraftment at day +30 and overall survival at day +100. Secondary outcome measures included acute toxicities and rates of graft failure, infections, and GVHD.The first two patients were 18 month-old twins with TINF2 mutations and transfusion-dependent BMF who received a BMT from a fully HLA-matched unrelated donor. The third patient was an 18 year-old female with CTC1 mutations and transfusion-dependent BMF, who underwent a B-mismatched unrelated donor BMT. The fourth patient, a 22 year-old male with clinical DC and short telomeres, had significant pulmonary disease (diffusing capacity of lung for carbon monoxide 28% of predicted) and BMF, and underwent a fully HLA-matched unrelated donor BMT.All 4 patients engrafted neutrophils by day +30, and are alive and well with follow-up ranging from 6 months (1 patient) to 2 years (3 patients). Platelet transfusion-independence occured between day +4 to +18, and red cell transfusion-independence occured between day 0 to +90. No patients required transfusions thereafter. All patients showed full donor myeloid chimerism by day +60. 3 of 4 patients showed full donor lymphoid chimerism by year 2; the fourth patient has high and increasing mixed donor lymphoid chimerism (60%) at day +180. There were no significant, unexpected toxicities, bacterial or fungal bloodstream or tissue infections. All patients were monitored weekly by serum PCR for CMV, EBV, adenovirus and HHV6 until day +100; only one patient showed CMV reactivation which was controlled with pre-emptive ganciclovir therapy. There was no acute GVHD. Only one patient developed chronic GVHD with limited skin involvement controlled with topical steroids.These results provide promising early data for engraftment and a favorable short-term toxicity profile using a radiation- and alkylator-free conditioning regimen in HCT for BMF due to DC. This approach could spare DC patients the acceleration of non-hematologic complications and malignancies, which has been attributed to HCT, and improve long-term survival. The limited toxicity of the alemtuzumab/fludarabine regimen may also allow patients with severe disease-related co-morbidities such as pulmonary dysfunction to safely undergo HCT. To our knowledge, this is the first prospective HCT study to achieve full donor myeloid engraftment in a series of patients following conditioning without radiation or alkylating agents. DisclosuresOff Label Use: alemtuzumab - conditioning for bone marrow transplantation fludarabine - conditioning for bone marrow transplantation cyclosporine A - graft versus host disease prophylaxis in bone marrow transplantation mycophenolate mofetil - graft versus host disease prophylaxis in bone marrow transplantation.

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

BackgroundThe Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Methodology/Principal FindingsHere we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).Conclusions/SignificanceThese data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

Abstract 3047: MK1775, a selective Wee1 inhibitor shows antitumor activity against sarcoma cells

Abstract Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10-20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 has been reported to to enhance the cytotoxic effect of DNA damaging agents. However, its role in the treatment of mesenchymal tumors has not been explored. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo both alone and in combination with other chemotherapeutic drugs frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of the DNA damaging agents, gemcitabine and doxorubicin. In MK1775 treated cells CDC2 activity was enhanced and G2/M ceheckpoint was impaired as assessed by increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to treatment. Additionally, in patient-derived bone and soft tissue sarcoma samples we showed that MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. In patient-derived xenograft mouse models of sarcoma we are currently analyzing the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3047. doi:1538-7445.AM2012-3047

Abstract 4922: Patterns of tumor evolution in high grade serous ovarian carcinoma

Abstract BACKGROUND High grade serous ovarian cancer (HGS-OvCa) is the fifth leading cause of cancer death amongst women in the United States and is characterized by a 25% rate of recurrence within six months after the end of treatment. To gain insight into the mechanisms used by tumor cells to evade the toxic effects of DNA damaging therapy, we genomically characterized sixteen triplets of tumor primary, tumor recurrence and normal samples using whole exome sequencing, DNA copy number arrays, methylation arrays and gene expression arrays. RESULTS Fifteen out of sixteen recurrent samples acquired both somatic mutations as well as DNA copy number alterations, and the number of novel abnormalities acquired was correlated with the time elapsed between primary and recurrence. Interestingly, nine of sixteen recurrence samples harbored only a subset of the mutations identified in the primary tumor. Similarly, these cases appeared to have lost a subset of the copy number alterations with which the primary sample originally presented, suggesting that primary and recurrent tumors were derived from an ancestral clone rather than to have evolved from the dominant clone present at diagnosis. The remaining seven recurrent samples contained all abnormalities found at time of diagnosis and are thus likely to have evolved from the dominant diagnostic clone. The time to recurrence was shorter for ‘diagnostic clone’ patients than for ‘ancestral clone’ patients, with borderline significance (likelihoodratio test p-value 0.08). Analysis of mutation allele fractions suggested a higher degree of tumor heterogeneity in primary tumor samples than in recurrent tumor samples. Recurrence was not associated with a specific mutation or copy number alteration. Tumors from patients whose interval between end of platinum therapy and recurrence was less than six months were not characterized by specific patterns of abnormalities. DISCUSSION Analysis of genomic alterations in primary and recurrent ovarian cancer tumor samples suggests that recurrences can be classified into two dominant groups: recurrences derived from an ancestral clone and recurrences originating from the same dominant clone present at time of diagnosis. Analysis of mutation allele fractions provided further insights into the clonality of primary and recurrent tumor samples. These findings shed light on the mechanism by which tumors overcome the effects of DNA damaging agents and may lead to development of therapies that specifically address the clonal origin properties of recurrent tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4922. doi:1538-7445.AM2012-4922