Induction Of γH2AX Research Articles

Genotoxicity analysis of five particle matter toxicants from cigarette smoke based on γH2AX assay combined with Hill/Two-component model

To investigate the genotoxic characteristics of typical toxicants in particle phase of cigarette smoke, including B[a]P, nicotine, tar, NNN and NNK. The in vitro γH2AX assay was used to detect the DNA double-strand breaks (DSBs) in A549 cells using high content screening (HCS). The results showed all toxicants had a dose/time-dependent effects on induction of γH2AX except for NNN and NNK. Based on dose-response of γH2AX and Hill model, the ability to induce DSBs was evaluated: NNN-acetate > B[a]P > NNK-acetate > tar > nicotine. Based on time-course of γH2AX and two-component model, the complex DNA damage was the main subtypes of DNA damage induced by these toxicants. Overall, all toxicants were genotoxic in A549 cells in a dose- or time- dependent manner except for NNN and NNK based on the γH2AX HCS assay. NNN-acetate had more potential to induce DSBs, which was followed by B[a]P, NNK-acetate, tar and nicotine.

Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells.

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Assessment of genotoxicity of four volatile pollutants from cigarette smoke based on the in vitro γH2AX assay using high content screening

To evaluate the genotoxic effects of formaldehyde, acetaldehyde, acrolein and benzene on A549 cells, the in vitro γH2AX assay was used in combination with high content screening (HCS) technology. All aldehydes showed a significant genotoxicity in a dose/time-dependent effect on the induction of γH2AX. Benzene failed to show a significant genotoxicity based on the γH2AX assay. However, hydroquinone (one of metabolites of benzene) showed a significant genotoxicity in vitro. Based on the dose-response of γH2AX and Hill model, the ability to induce DNA double-strand break can be evaluated as acrolein>formaldehyde>acetaldehyde>benzene. The slow DNA damage/repair mechanism may be more important than the fast one for aldehydes based on time-course of γH2AX and two-component model. Overall, all toxicants were genotoxic in a dose- or time-dependent manner based on the in vitro γH2AX HCS assay, and acrolein had a strong potential to induce DNA damage followed by formaldehyde, acetaldehyde and benzene in sequence.

Abstract 5226: Novel class of potent and selective inhibitors efface MTH1 as broad-spectrum cancer target

Abstract Malignant transformation is accompanied by increased reactive oxygen species (ROS) known to promote carcinogenesis and damage free nucleotides and DNA. During replication, damaged nucleotides are incorporated into DNA resulting in DNA breaks and mutations, which can ultimately lead to cell death. Cancer cells may evade this process via overexpression of MTH1 (also known as NUDT1), a member of nudix phosphohydrolase protein family, which converts the oxidized nucleotides 8-oxo-dGTP and 2-OH-dATP into the corresponding monophosphates thus preventing their incorporation into DNA and avoiding cell death. Initial RNAi-mediated knockdown of MTH1 and tool compounds (TH588, (S)-crizotinib) inhibiting MTH1 supported this model. As MTH1 is not essential for non-transformed cell survival, MTH1 was hypothesized to be a non-oncogenic cancer addiction and a potential broad-spectrum cancer target. Attractive target rationale combined with previous success in identifying potent and cellularly active MTH1 inhibitors prompted us to develop new cancer therapeutics inhibiting MTH1. By using fragment-based screening and structure-based drug design, a series of 4-amino-2-carboxamide-7-azaindoles was identified. We developed biochemically potent and selective MTH1 inhibitors with good cell permeability and metabolic stability. These MTH1 inhibitors demonstrated target engagement in cellular thermal shift assay (CETSA), and a strong positive correlation between cellular and biochemical potency was observed. One promising MTH1 inhibitor from this structural class was BAY-707. Unexpectedly however, these properties did not translate into accumulation of oxidized nucleotides within DNA and consequent induction of γH2AX and DNA damage response. Moreover, while tool compounds (TH588, (S)-crizotinib) were confirmed to be biochemically potent MTH1 inhibitors which stunted the proliferation of a range of cancer cell lines, our more potent and cellularly active MTH1 inhibitors, including BAY-707, demonstrated no significant effect on cancer cell survival. Furthermore, we were unable to demonstrate in vivo efficacy using xenograft models of human cancers or syngeneic mouse tumor models. Finally, our in vitro and in vivo combination studies with pro-oxidants, standard-of-care drugs or radiation also failed to result in significant additive or synergistic growth inhibitory effects on cancer cells. Thus, our findings support the recently published observations made with other potent and selective MTH1 chemical probes (AZ compound 15, IACS-4759, NPD7155) and CRISPR/Cas9-mediated MTH1 knockout. Based on these observations and our additional target validation experiments, we concluded that MTH1 is not essential for cancer cell survival or for the sanitization of damaged nucleotides within cells and thus not a viable target for development of novel anticancer agents. Citation Format: Manuel Ellermann, Anja Giese, Ashley Eheim, Stefanie Bunse, Roland Neuhaus, Jörg Weiske, Maria Quanz, Andrea Glasauer, Fredrik Rahm, Jenny Viklund, Martin Andersson, Tobias Ginman, Rickard Forsblom, Johan Lindström, Lionel Trésaugues, Matyas Gorjanacz. Novel class of potent and selective inhibitors efface MTH1 as broad-spectrum cancer target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2017-5226

Abstract 3072: A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death

Abstract The effectiveness of certain classes of cytotoxic cancer therapeutics likely depends on whether drug-induced DNA damage is successfully repaired or not, with the latter situation leading to mutations and strand breaks. However, double strand breaks (DSBs) also occur independently of genotoxic insults during apoptotic cell death caused by many drug classes, as well as natural biological processes. We have developed an immunofluorescent confocal microscopy assay that uses a biomarker profile suitable for individual cell analysis designed to distinguish between DSBs caused by apoptosis and those caused by direct DNA damage from cytotoxic drug action. γ-H2AX is an established biomarker for DSBs and activated cleaved caspase 3 is an executioner caspase important for apoptosis, which leads to nuclear condensation, DNA fragmentation, plasma membrane blebbing, and subsequent cell death. Our assay defines the DSBs of apoptosis by co-localized γ-H2AX and cleaved caspase 3 in individual cells, while defining the DSBs from early drug effects of DNA damaging chemotherapeutics by γ-H2AX induction in the absence of cleaved caspase 3. Building on our published findings that topotecan strongly induces γ-H2AX and DSBs within 1-4 hours in vitro and in vivo, we observed exposure-dependent increases in γ-H2AX /cleaved caspase 3 double positive cells at later time points, both in an HT29 in vitro spheroid model and an MDA-MB-231 xenograft model. Fit-for-purpose studies in the MDA-MB-231 xenograft model treated with birinipant, a SMAC mimetic and IAP deregulator that does not directly produce lethal DSBs, demonstrated a dose-dependent increase in cellular co-localization of γ-H2AX/cleaved caspase 3 consistent with birinipant induced apoptosis and the established mechanism of action of this compound. Clinical feasibility was established in a canine clinical trial using formalin-fixed paraffin-embedded (FFPE) 18-gauge needle biopsies: two novel indenoisoquinolines, indotecan (LMP400) and indimitecan (LMP776), increased tumor cell co-localization of γ-H2AX/cleaved caspase 3 in tumor samples obtained on day 5 of qdx5 treatment. This PD biomarker assay of early and late DSB response to drug exposure could have important applications for elucidation of mechanisms of action of anticancer drugs and the development of investigational agents. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Angie B. Dull, Deborah Wilsker, Robert J. Kinders, Ralph E. Parchment, David Evans, Beverly A. Teicher, James H. Doroshow. A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2017-3072

Abstract LB-319: Development and characterization of ADC999: A novel, potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo

Abstract Wee1 is a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDC2) to inactivate the CDC2/cyclinB complex. Wee1 is therefore a key regulator of the cell cycle, maintaining the G2 checkpoint arrest to allow for pre-mitotic repair upon genomic stress. Unlike normal cells, most p53-deficient tumors lack a functional G1 checkpoint and instead rely on the G2 checkpoint for DNA repair. Abrogation of the G2 checkpoint by a Wee1 inhibitor has been shown to sensitize p53-deficient tumors to a variety of DNA-damaging anticancer agents. Strong chemosensitization has also been demonstrated in multiple pre-clinical models using targeted agents including CHK1 or PARP inhibitors. These studies combined with recent clinical data demonstrating robust anti-tumor activity in combination with carboplatin or as a single agent in tumors under high replicative stress make Wee1 a promising target for anticancer therapy. With only one inhibitor (AZD1775) in clinical development and very few reports of compounds at the preclinical stage, the development of alternative Wee1 inhibitors is of significant medical relevance. In this study, we describe the development of a novel and highly potent small molecule inhibitor of Wee1 (ADC999) and subsequent analogs. These inhibitors exhibit single digit nM IC50 values against Wee1 and good overall selectivity profiles against the kinome. In cells, target engagement was demonstrated through the inhibition of both CDC2- and CDK2-dependent Wee1 phosphorylation. In line with the mechanism of action, γH2AX and apoptosis induction was also observed in a dose-dependent manner. Further profiling in a panel of cell lines indicated strong anti-proliferative activity both in combination with cytotoxics and in monotherapy. Single agent activity was observed in multiple cancer types, including gynecological, lung and breast. In vivo, oral administration of ADC999 as a single agent led to excellent antitumor activity in a KRAS-mutant non-small cell lung carcinoma xenograft model. Responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. ADC999 was well tolerated following a daily dosing regimen and over a prolonged period with no adverse effects observed. Evaluation of ADC999 anti-tumor activity will also be discussed in a panel of genetically-characterized patient-derived xenografts (PDXs) both as monotherapy (mini-trial design) and in combination with PARP inhibitors. In summary, we describe the development and profiling of a novel, highly potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo. This inhibitor and potential back-up compounds will provide new opportunities to fully exploit the therapeutic potential of Wee1 inhibition both as standalone intervention and in combination modalities. Citation Format: Gerald Gavory, Colin O'Dowd, Ewelina Rozycka, Caroline Boyd, Beronia Gorges, Estelle McLean, Julien Daubriac, Shane Rountree, Steven Shepherd, Stephanie Burton, Mary McFarland, Adam Treder, Andy Wilkinson, Frank Burkamp, Tim Harrison. Development and characterization of ADC999: A novel, potent orally available Wee1 inhibitor with robust antitumor efficacy in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-319. doi:10.1158/1538-7445.AM2017-LB-319

TTF-1/NKX2-1 binds to DDB1 and confers replication stress resistance to lung adenocarcinomas.

TTF-1, also known as NKX2-1, is a transcription factor that has indispensable roles in both lung development and physiology. We and others have reported that TTF-1 frequently exhibits high expression with increased copy number in lung adenocarcinomas, and also has a role as a lineage-survival oncogene through transcriptional activation of crucial target genes including ROR1 and LMO3. In the present study, we employed a global proteomic search for proteins that interact with TTF-1 in order to provide a more comprehensive picture of this still enigmatic lineage-survival oncogene. Our results unexpectedly revealed a function independent of its transcriptional activity, as TTF-1 was found to interact with DDB1 and block its binding to CHK1, which in turn attenuated ubiquitylation and subsequent degradation of CHK1. Furthermore, TTF-1 overexpression conferred resistance to cellular conditions under DNA replication stress (RS) and prevented an increase in consequential DNA double-strand breaks, as reflected by attenuated induction of pCHK2 and γH2AX. Our findings suggest that the novel non-transcriptional function of TTF-1 identified in this study may contribute to lung adenocarcinoma development by conferring tolerance to DNA RS, which is known to be inherently elicited by activation of various oncogenes.

Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase.

Chk1 kinase is a critical component of the DNA damage response checkpoint and Chk1 inhibitors are currently under clinical investigation. Chk1 suppresses oncogene-induced replication stress with Chk1 inhibitors demonstrating activity as a monotherapy in numerous cancer types. Understanding the mechanism by which Chk1 inhibitors induce DNA damage and cancer cell death is essential for their future clinical development. Here we characterize the mechanism by which the novel Chk1 inhibitor (V158411) increased DNA damage and cell death in models of human cancer. V158411 induced a time- and concentration-dependent increase in γH2AX-positive nuclei that was restricted to cells actively undergoing DNA synthesis. γH2AX induction was an early event and correlated with activation of the ATR/ATM/DNA-PKcs DNA damage response pathways. The appearance of γH2AX positive nuclei preceded ssDNA appearance and RPA exhaustion. Complete and sustained inhibition of Chk1 kinase was necessary to activate a robust γH2AX induction and growth inhibition. Chk1 inhibitor cytotoxicity correlated with induction of DNA damage with cells undergoing apoptosis, mitotic slippage and DNA damage-induced permanent cell cycle arrest. We identified two distinct classes of Chk1 inhibitors: those that induced a strong increase in γH2AX, pChk1 (S317) and pRPA32 (S4/S8) (including V158411, LY2603618 and ARRY-1A) and those that did not (including MK-8776 and GNE-900). Tumor cell death, induced through increased DNA damage, coupled with abrogation of cell cycle checkpoints makes selective inhibitors of Chk1 a potentially useful therapeutic treatment for multiple human cancers.

EAF2 regulates DNA repair through Ku70/Ku80 in the prostate

Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

Inhibition of ATR-dependent feedback activation of Chk1 sensitises cancer cells to Chk1 inhibitor monotherapy

The Chk1 and ATR kinases are critical mediators of the DNA damage response pathway and help protect cancer cells from endogenous and oncogene induced replication stress. Inhibitors of both kinases are currently being evaluated in clinical trials. Chk1 inhibition with V158411 increases DNA damage and activates the ATR, ATM and DNA-PKcs dependent DNA damage response pathways. Inhibiting ATR, ATM and/or DNA-PKcs has the potential to increase the therapeutic activity of Chk1 inhibitors. ATR inhibition but not ATM or DNA-PKcs inhibition potentiated the cytotoxicity of V158411 in p53 mutant and wild type human cancer cell lines. This increased cytotoxicity correlated with increased nuclear DNA damage and replication stress in a dose and time dependent manner. γH2AX induction following Chk1 inhibition protected cells from caspase-dependent apoptosis. Inhibition of ATR increased Chk1 inhibitor induced cell death independently of caspase activation. The effect of ATR, ATM and/or DNA-PK inhibition on Chk1 inhibitor induced replication stress was dependent on the concentration of Chk1 inhibitor. ATR inhibition potentiated Chk1 inhibitor induced replication stress and cytotoxicity via the abrogation of ATR-dependent feedback activation of Chk1 induced by Chk1 inhibitor generated replication stress. This study suggests that combining an ATR inhibitor to lower the threshold by which a Chk1 inhibitor induces replication stress, DNA damage and tumour cell death in a wide range of cancer types may be a useful clinical approach.

Abstract 1260: Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell

Abstract Resistance is one the main reason for overall decrease in survival of cancer patient treated with cisplatin in different types of cancer. Cisplatin kills cancer cells by various mechanisms, but mainly through formation of inter- and intra stand crosslinks of DNA. Different types of translesion polymerase including Polymerase kappa (POLK) are involved in repair of DNA lesions. We observed high expression levels of POLK in cisplatin resistant bladder and ovarian cancer cells compared to parental cells. Due to its low proof-reading activity POLK can incorporate 8-oxo-dGTP into DNA. The MTH1 protein (Nudix hydrolase- NUDT1) sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Recently we have generated MTH1 inhibitors that damage the DNA and induce cancer specific cell death through incorporation of more oxidized dNTPs. We found cisplatin resistant bladder cancer cells (NTUB1/P) were more sensitive to MTH1 inhibitors in comparison to parental NTUB1 cells. As POLK is involved in incorporation of 8-oxo-dGTP into DNA, we hypothesized that high expression levels of POLK in cisplatin resistant cells make them more sensitive to MTH1 inhibitors as more 8-oxo-dGTP would be incorporated into DNA, resulting in more DNA damage and cell death in comparison to parental cells. Indeed, we observes higher induction of cleaved-PARP, γH2AX, cleaved-Caspase 3 and more annexin v positive cells in cisplatin resistant NTUB1/P cells in comparison to parental NTUB1 cells upon treatment with MTH1 inhibitors. MTH1 inhibitor also significantly delays the NTUB1/P xenograft tumor growth in comparison to vehicle treatment in immunosuppressive mice. Knocking down POLK in cisplatin resistant NTUB1/P cells by siRNA resulted in decreased incorporation of 8-oxo-dGTP and sensitivity to MTH1 inhibitors compared to non target control cells. Overexpression of POLK in NTUB1 and NTUB1/P cells results in further sensitization to MTH1 inhibitors. In conclusion elevated levels of POLK in cisplatin resistance cells determines increased sensitivity towards MTH1 inhibitors. Thus MTH1 inhibitors can be a potential promising therapy for the treatment of cisplatin resistant tumors in patients. Citation Format: Kumar Sanjiv, Helge Gad, Sean Rudd, Rachel Hurley, Patric Herr, José Manuel Calderón Montaño, Oliver Mortusewicz, Tobias Koolmeister, Sylvain Jaques, Estefanía Burgos Morón, Andreas Hoglund, Te-Chang Lee, Martin Scobie, Scott Kaufmann, John Weroha, Ulrika Warpman Berglund, Andrea Wahner Hendrickson, Thomas Helleday. Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell. [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 1260.

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9.

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546-558, 2016. © 2016 Wiley Periodicals, Inc.

Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors.

A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells.

Our prior study utilized both invitro and invivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6h and 24h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells.

A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.

A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.

A Phase 1 Study of the PARP Inhibitor Veliparib in Combination with Temozolomide in Acute Leukemias

Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009).Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals.Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs.Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03).Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment.Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. [Display omitted] DisclosuresOff Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

Abstract C59: Role of MTH1 (NUTD1) in cancer cell survival

Abstract Human mutT homolog MTH1 (also known as NUDT1) is a purine nucleoside triphosphatase which hydrolyses oxidised nucleotides (8-oxo-dGTP and 2-OH-dATP) into mono-phosphate forms to prevent these damaged bases from being incorporated into DNA. Recent studies have suggested a key role of MTH1 in the survival of cancer cells. It was hypothesized that in cancer cells with high levels of reactive oxygen species (ROS), small molecule inhibition or loss of MTH1 would lead to accumulation of oxidised nucleotides in DNA, increased genome instability and loss of cell viability. In order to validate MTH1 as a potential cancer therapeutic target we have developed additional potent small molecule inhibitors of MTH1 and generated siRNA knock-down and CRISPR-mediated knock-out cell lines. Using these tools we evaluated the impact of either MTH1 inhibition or loss of expression on cell viability, proliferation, induction of DNA damage and cell cycle arrest across multiple cell lines in vitro. While we have confirmed that the previous study compounds (TH588, TH287, (S)-crizotinib) are potent inhibitors of MTH1 and have broad cancer cell line growth inhibition activity, we were unable to demonstrate a corresponding induction of γH2AX, 53BP1 or phospho-ATM DNA damage response (DDR). In addition none of our MTH1 inhibitors were able to show significant growth inhibitory activity across cell lines panels or induce a DDR despite being able to potently engage MTH1 in cells as evidenced by cell thermal stability assay (CETSA). Due to the lack of DNA damage induction and inconsistent anti-proliferative activity between MTH1 inhibitors, we investigated the effects of loss of protein expression using additional, independent siRNA and CRISPR knockout cell lines. Despite potent and long lasting siRNA knockdown of MTH1 protein expression in U2OS cells, we did not observe significant differences in cell viability or induction in DNA damage when compared to control cell lines. Consistent with these findings we were also able to generate SW480 clones with complete knockout of all MTH1 alleles and this did not impair growth rates when compared to parental cells. Finally, to probe the specificity of MTH1 inhibitors we assessed their ability to impact the growth of these cells which do not express MTH1. We did not observe a differential response in U2OS cells with or without MTH1 siRNA or in the SW480 parental versus MTH1 knockout clones. Here we show through the use of novel small-molecule MTH1 inhibitors, with further experiments with RNAi-mediated knockdown of MTH1 and the ability to generate stable CRISPR-mediated MTH1 knockouts, that the cellular effects previously ascribed to MTH1 inhibition may be driven through alternative mechanisms. In summary, our results do not support an essential role for MTH1 in cancer cell survival. Citation Format: Husam Alwan, Kay Eckersley, Louise Goodwin, Alan Lau, David Jones, Jason Kettle, Willem M. Nissink, Jon Read, James S. Scott, Benjamin J.M. Taylor, Graeme E. Walker, Kevin M. Foote. Role of MTH1 (NUTD1) in cancer cell survival. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C59.

Abstract LB-C23: A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

Abstract The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anti-cancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibitor treatment and undercover potential resistance mechanisms, identification of these currently unknown genes and underappreciated functions of Wee1 is necessary. The aim of this study was to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1 and CDK2. Stable depletion of SKP2, CUL1 and CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1 and CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patient. Conversely, loss of the identified S-phase genes could serve as a resistance mechanism and coincides with development of severe genomic instability. Citation Format: Anne Margriet Heijink, Vincent Blomen, Fabian Degener, Felipe Yu Matsushita, Floris Foijer, Marcel A. Van Vugt. A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C23.

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.

Comparison of methods to quantify histone H2AX phosphorylation and its usefulness for prediction of radiosensitivity

Purpose: The number of radio-induced double-strand breaks is correlated with the number of histone gamma-H2AX (γ-H2AX) foci. For this reason, foci quantification is a useful tool to measure radiation-induced DNA damage and the number of foci has been suggested as a predictive biomarker of radiosensitivity. The aim of the present study was to evaluate the reproducibility of different microscopic methodologies and flow cytometry analysis to score γ-H2AX induction, and its suitability to distinguish a radiosensitive (RS) cell line from a radioresistant (RR) one.Materials and methods: γ-H2AX analyses were performed by semi-automated and automated microscopic methods and by flow cytometry before and after irradiation in two human lymphoblastoid cell lines and in lymphocytes from three healthy donors.Results: Reproducible results were obtained by all the methodologies tested, although not all showed the same sensitivity. The RS cell line always showed higher foci counts and higher levels of immunofluorescence intensity after irradiation than the RR cell line.Conclusions: Our results suggest that microscopic methodologies with z-stage capacity give the most accurate results after 1 Gy irradiation. However, for high doses of ionizing radiation, flow cytometry gives reliable results. Further studies will be necessary to determine the usefulness of γ-H2AX analysis to predict adverse side reactions in radiotherapy patients.