DNA Double-strand Breaks Inducers Research Articles

Abstract CT194: ETCTN 10388: a first in human phase I trial of triapine and lutetium Lu 177 DOTATATE in well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs)

Abstract Background: Radiation is a potent inducer of DNA double-strand breaks, and ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis and repair of DNA, making RNR-targeted therapy a rationale therapeutic strategy for radiosensitization. ETCTN 10388 (NCT04234568) evaluated safety and efficacy of the combination of lutetium 177 DOTATATE, a beta-emitting radionuclide in combination with triapine, a ribonucleotide reductase (RNR) inhibitor. Method: This study was a multicenter phase 1 dose escalation trial [using the Bayesian optimal interval design (BOIN)] of triapine in combination with fixed dose lutetium Lu 177 DOTATATE for well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumor (GEP-NETs) after the failure of at least one line of prior systemic cancer treatment with an expansion cohort at the recommended phase 2 dose (RP2D). Oral triapine (100mg, 150mg, 200mg) was administered once daily on days 1-14 and Lu-177 DOTATATE [200 mCi] intravenously on day 1 of every 56-day cycle. A total of 4 cycles were administered. Response and adverse effects were assessed per RECIST and CTCAE 5.0, respectively. Exploratory correlative studies included tumor somatic and germline mutation testing, RNA sequencing, pharmacokinetics, deoxynucleosides and circulating cell free DNA analysis. Primary endpoints were safety and RP2D. Results: Overall, 31 patients were enrolled between 6 sites, 15 in the dose escalation phase and 16 in the dose expansion phase. Adverse events (AE) were assessed in all 31 patients per CTCAE 5.0. One DLT in dose level 1, seven DLTs in dose level 2, and one grade 5 DLT in dose level 3 were observed. The RP2D of the combination is triapine 150 mg QD (dose level 2) on days 1-14 in combination with Lu-177 DOTATATE on day 1 of every 56-day cycle. Detailed safety and adverse event data will be presented at the meeting. There were 28 patients evaluable for efficacy, of which 6 (21%) achieved a partial response. At 12 months, 6 patients had progressed, while 22 (86%) remained progression free. Median PFS has not been reached. PK data were available for 12 patients enrolled in the dose escalation cohort. The geometric mean (SD) AUC0-inf was 1159 (1.22) µg/L•h for the 100mg dose level and 1862 (1.76) µg/L•h for the 150 mg dose level, suggesting that exposure increased with dose, and inter-patient variability was as expected for an oral agent. Conclusion: The combination of triapine and Lu-177 DOTATATE was safe with preliminary efficacy signals, which will be further evaluated in ETCTN 10558, a randomized phase 2 study that is comparing the effectiveness of triapine and Lu-177 DOTATATE to Lu-177 DOTATATE alone. Citation Format: Aman Chauhan, Susanne Arnold, Jill Kolesar, William Carson, Heidi Weiss, Rani Jayswal, Donglin Yan, Riham El Khouli, Aman Khurana, Jan Beumer, Heloisa Soares, Mary Mulcahy, Thorvardur Halfdanarson, Daneng Li, Heather Jacene, Percy Ivy, Elise Kohn, John Wright, Larry Rubinstein, Zeta Chow, Piotr Rychahou, Mark B. Evers, Charles Kunos, Lowell Anthony, Bhavana Konda. ETCTN 10388: a first in human phase I trial of triapine and lutetium Lu 177 DOTATATE in well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT194.

Pharmacokinetics and RP2D analysis from ETCTN 10388: A phase I trial of triapine and lutetium Lu-177 dotatate in well-differentiated somatostatin receptor–positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs).

648 Background: Radiation is a potent inducer of DNA double-strand breaks, and ribonucleotide reductase (RNR) is the rate-limiting enzyme for conversion of ribonucleoside diphosphate to deoxyribonucleotide diphosphate, and thus repair of DNA in this setting. ETCTN 10388 evaluated safety of combination Lu-177 DOTATATE, a beta-emitting radionuclide in combination with triapine, a ribonucleotide reductase (RNR) inhibitor. Methods: This investigator initiated, NCI sponsored, multicenter phase 1 trial, enrolled a total of 31 patients in the dose escalation [using the Bayesian optimal interval design (BOIN)] and dose expansion cohorts. Oral triapine was administered on days 1-14 and Lu-177 DOTATATE [200 mCi] intravenously on day 1 of every 56-day cycle. A total of 4 cycles were administered. All enrolled patients had blood samples collected for triapine pharmacokinetic (PK) analysis in EDTA tubes prior to and at 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after oral administration during cycle 1. Results: Five patients were enrolled in triapine Dose Level 1 (100 mg/day), twenty-five to dose level two (150 mg/day), and one patient to dose level three (200 mg/day). PK data were available for 12 patients enrolled in the dose escalation cohort. The geometric mean (SD) AUC0-inf was 1159 (1.22) µg/L•h for the 100mg dose level and 1862 (1.76) µg/L•h for the 150 mg dose level, suggesting that exposure increased with dose, and inter-patient variability was as expected for an oral agent. Triapine PK parameter values observed in this trial, were comparable to previous reports that used a previous formulation [ 1 ]. While exposure was similar, variability appeared smaller with the current oral formulation. Adverse events (AE) were assessed in all 31 patients per CTCAE 5.0. A total of one DLT in dose level 1, seven DLTs (Transient cytopenia; primarily neutropenia and rarely thrombocytopenia) in dose level 2, and one grade 5 DLT (Death probably from progressive cancer and carcinoid heart disease but possibly from trial drugs) in dose level 3 were observed. Detailed AE profile will be presented at the meeting. Conclusions: The RP2D of triapine is 150 mg QD on days 1-14 in combination with Lu-177 DOTATATE on day 1 of every 56-day cycle. Clinical trial information: 04234568 .

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

Abstract 674: A phase I trial of Triapine and Lutetium Lu 177 Dotatate in combination for well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs)

Abstract Radiolabeled somatostatin analogues provide a means of delivering targeted radiation with a high therapeutic index to NETs that express somatostatin receptors (SSTRs). Radiolabeled somatostatin analogue Lutetium Lu 177 Dotatate (Lutathera) is a beta- and gamma-emitting radionuclide. Lutathera has been recently FDA approved for use in SSTR positive gastroenteropancreatic neuroendocrine tumors (GEPNETS) in the US based on NETTER-1 Phase III trial. Despite favorable PFS, and safety profile, the drug has limited cytoreductive capability. NETTER-1 reported 17% ORR. PRRT also doesn't seem to be very effective in treating peritoneal disease. We hypothesize that addition of an effective radiation sensitizer can help improve antitumor activity of Lutathera. Rationale: Triapine is a ribonucleotide reductase (RNR) inhibitor. Radiation is a potent inducer of DNA double-strand breaks (DSBs). Targeting signaling networks involved in DSB repair is a promising approach for enhancing cellular radio sensitivity. RNR is the rate-limiting enzyme in the synthesis and repair of DNA, and it is directly involved in the cellular response to radiation, making RNR-targeted therapy to enhance radiation treatment a rational therapeutic strategy. RNR is the only enzyme responsible for conversion of ribonucleoside diphosphate to deoxyribonucleotide diphosphate (dNDP), the key building blocks for DNA synthesis. This study will test the hypothesis that triapine is an effective radiation sensitizer which can be safely combined with peptide receptor radionuclide therapy and can improve antitumor activity of Lutetium Lu 177 Dotatate. Method: This study is an investigator initiated, NCI sponsored, multicenter phase 1 trial of triapine and Lutetium Lu 177 Dotatate in combination for well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumor (GEP-NETs) after the failure of at least one line of prior systemic cancer treatment. A total of 29 patients will be enrolled in the dose escalation (BOIN design) and dose expansion cohorts. The study will be open through the entire ETCTN. Patients will be treated with 177 lutetium dotatate in combination with Triapene. Triapene will be administered orally from D1-14 with each dose of PRRT. Primary endpoint is to evaluate RP2D. Secondary endpoints are looking into clinical activity (ORR, PFS and OS). We are also evaluating NETEST, a novel blood based predicting as well as prognosticating test. In addition, the study will evaluate baseline somatostatin receptor density, somatic tumor mutations and germline mutations and correlate with clinical outcome. ClinicalTrials.gov Identifier: NCT04234568 Citation Format: Aman Chauhan, Charles Kunos, Riham El Khouli, Jill Kolesar, Heidi L. Weiss, Bill Carson, Mark Kidd, Jan Beumer, Susanne Arnold, Elise Kohn, Lowell Anthony. A phase I trial of Triapine and Lutetium Lu 177 Dotatate in combination for well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 674.

Synthetic Lethal Interaction between Apto-253 and Impaired BRCA1/2 Function

APTO-253 is a novel indolyl-phenanthroline-imidazole small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies including leukemia, lymphoma, and colon, non-small cell lung, renal and prostate carcinomas. Of particular significance, APTO-253 did not elicit myelosuppression in its initial Phase 1 trial in patients with solid tumors even at the maximum tolerated dose. The goal of this project was to provide fundamental information on the mechanism of action and resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination drug studies.The human Burkitt'slymphoma cell line Rajiwas exposed to progressively increasing concentrations of APTO-253 over a period of 6 months to generate a resistant subline Raji/253R with 16.7 ± 3.9-fold resistance (IC50: Raji IC50 91.9 ± 22.3 nM; Raji /253R IC50 1387.7 ± 98.5 nM). RNA-seq analysis of the parental and resistant cells demonstrated marked up-regulation of ATP-binding cassette sub-family G member 2 (ABCG2) in the Raji/253R cells that was confirmed by qRT-PCR and Western blot analysis. Ko143 is a specific inhibitor of ABCG2 that was not toxic to either Raji or Raji/253R when used as a single agent. When Raji/253R cells were concurrently treated with either 5 nM or 50 nM Ko143 and APTO-253, resistance to APTO-253 was reversed by 1.6- and 6.8-fold, respectively, confirming that overexpression of ABCG2 is one mechanism of resistance to APTO-253. Raji/253R was found to be cross-resistant to topotecan, a known ABCG2 substrate, and Ko143 also reversed topotecan resistance in Raji/253R. Intriguingly, Raji/253R was also significantly cross-resistant to carboplatin even though carboplatin is not thought to be an ABCG2 substrate; treatment with Ko143 did not reduce the carboplatin IC50 in the Raji/253R cells. The curious finding that Raji/243R cells were cross-resistant to carboplatin led to the discovery that APTO-253 causes DNA damage as documented by the neutral comet assay and gH2AX phosphorylation and foci formation assays. Surprisingly, Raji/253R cells were hypersensitive to etoposide, an ABCG2 substrate and a potent DNA double strand break inducer. GO and pathway analysis from the RNA-seq data revealed that DNA repair pathways were downregulated in Raji/253R, which partially explained the hypersensitivity to etoposide. We then asked whether cells deficient in homologous recombination repair pathways were hypersensitive to APTO-253 and if there was synthetic lethality between APTO-253 and BRCA1 deficiency (using isogenic pairs of BRCA1-proficient and -deficient human cell lines). BRCA1 deficiency was documented by treating the BRCA1-proficient and -deficient isogenic cell lines with the PARP inhibitor olaparib. BRCA1-deficient sublines of MCF10A, MCF7 and hTERT -IMEC were all hypersensitive to both olaparib and APTO-253. We also found that the BRCA2deficient ovarian cancer cell line PEO4 and two colorectal HCT116 BRCA2 -/- subclones were hypersensitive to both olaparib and APTO-253.We conclude that AP-253 causes DNA damage and that it exhibits a degree of synthetic lethality comparable to olaparib in cells deficient in BRCA1 and BRCA2-function. The ability of APTO-253 to exploit homologous recombination deficiency is of particular interest because, unlike all the other drugs for which loss of this repair function results in hypersensitivity, APTO-253 does not produce myelosuppression even at the maximum tolerated dose. The observations reported here also identify gH2AX as a potential biomarker of clinical drug effect and open the window more detailed studies of how APTO-253 causes DNA damage. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees.

Susceptibility to DNA damage caused by abrogation of Rad54 homolog B: A putative mechanism for chemically induced cleft palate

Exposure to environmental toxicants such as all-trans retinoic acid (atRA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may cause cleft palate (CP), which process is related to DNA damage. Rad54B, an important DNA damage repaired protein, has been proved to be associated with non-syndromic cleft lip with palate (NSCLP). In the present study, we sought to clarify the role of Rad54B in palatal development and environment-induced CP. atRA (100 mg/kg) and TCDD (40 μg/kg) were used to induce CP in mice (C57BL/6 J mice). In this study, mouse embryonic heads were collected on embryonic day (E) 13.5∼16.5. The expression level of DNA repair protein Rad54 homolog B (Rad54B) was significantly decreased while those of the DNA double-strand breaks (DSBs) marker γ-H2A.X, apoptosis marker caspase-3 and p53 were significantly increased in the palatal shelves upon exposure to atRA and TCDD relative to the control. Primary mouse embryonic palatal mesenchymal cells (MEPMs) were cultured and transfected with siRNA or adenovirus in vitro to knock down or increase the level of Rad54B. Rad54B knockdown resulted in increased cellular S-phase arrest and apoptosis as well as decreased cell proliferation. Rad54B overexpression also increased apoptosis and reduced cell proliferation. Western blotting was used to detect the level of γ-H2A.X in transfected cells stimulated with etoposide (ETO, a DSBs inducer), and after 5 μM ETO stimulation of transfected MEPMs, the expression of γ-H2A.X was increased in Rad54B-knockdown cells. The expression of Mdm2, Mdmx and p53 with changes in Rad54B was also detected and coimmunoprecipitation was performed to analyze the combination of Mdm2 and p53 when Rad54B was changed in MEPMs. Knockdown of Rad54B inhibited the expression of Mdm2 and Mdmx, while the level of p53 increased. The coimmunoprecipitation results showed a decreased combination of Mdm2 and p53 when Rad54B was knocked down. Therefore, Rad54B can regulate the cell cycle, proliferation, and apoptosis of MEPMs. The loss of Rad54B increased the sensitivity of MEPMs to DSBs inducers, promoted apoptosis, and suppressed the proliferation of MEPMs by inhibiting the degradation of p53. Taken together, these findings suggest that Rad54B may play a key regulatory role in environment-induced CP.

Etctn 10388: A phase I trial of triapine and lutetium Lu 177 dotatate in well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs).

TPS4660 Background: Radiolabeled somatostatin analogues provide a means of delivering targeted radiation with a high therapeutic index to NETs that express somatostatin receptors (SSTRs). Radiolabeled somatostatin analogue Lutetium Lu 177 Dotatate (Lutathera) is a beta-emitting radionuclide, recently FDA approved for use in SSTR positive gastroenteropancreatic neuroendocrine tumors (GEPNETS) in the US based on the NETTER-1 Phase III trial. Despite favorable PFS and safety profile, the drug has limited cytoreductive capability with a 17% ORR. We hypothesize that addition of an effective radiation sensitizer could help improve antitumor activity of Lutathera. Ribonucleotide reductase (RNR) is the only enzyme responsible for conversion of ribonucleoside diphosphate to deoxyribonucleotide diphosphate (dNDP), the key building blocks for DNA synthesis. Radiation is a potent inducer of DNA double-strand breaks (DSBs), and RNR is the rate-limiting enzyme in the repair of DNA in this setting. Triapine is an inhibitor of RNR. This study will test the hypothesis that radiation sensitizer triapine can be safely combined with peptide receptor radionuclide therapy and ultimately may improve antitumor activity of Lutetium Lu 177 Dotatate. Methods: This study is an investigator initiated, NCI sponsored, multicenter phase 1 trial of triapine and Lutetium Lu 177 Dotatate in well-differentiated somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumor (GEP-NETs) after the failure of at least one line of prior systemic cancer treatment. A total of 29 patients will be enrolled in the dose escalation with help of Bayesian optimal interval design (BOIN) and dose expansion cohorts. The study will be open through the NCI ETCTN (National Cancer Institute Experimental Therapeutics Clinical Trials Network) program. Patients will be treated with 177 lutetium dotatate in combination with triapine. Triapine will be administered orally (100 mg once a day starting dose) from D1-14 with each dose of PRRT [200 mCi]. Primary endpoint is to evaluate recommended phase II dose (RP2D). Secondary endpoints are to evaluate safety, pharmacokinetics, and clinical activity (ORR and PFS). We are also evaluating NETEST, a novel blood based test that evaluates levels of 51 neuroendocrine tumor gene transcripts. In addition, the study will correlate clinical outcome with baseline somatostatin receptor density, somatic tumor mutations and germline mutations. Clinical trial information: 04234568 .

Predicting effective pro-apoptotic anti-leukaemic drug combinations using co-operative dynamic BH3 profiling.

The BH3-only apoptosis agonists BAD and NOXA target BCL-2 and MCL-1 respectively and co-operate to induce apoptosis. On this basis, therapeutic drugs targeting BCL-2 and MCL-1 might have enhanced activity if used in combination. We identified anti-leukaemic drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism using the technique of dynamic BH3 profiling, whereby cells were primed with drugs to discover whether this would elicit mitochondrial outer membrane permeabilisation in response to BCL-2-targeting BAD-BH3 peptide or MCL-1-targeting MS1-BH3 peptide. We found that a broad range of anti-leukaemic agents–notably MCL-1 inhibitors, DNA damaging agents and FLT3 inhibitors–sensitise leukaemia cells to BAD-BH3. We further analysed the BCL-2 inhibitors ABT-199 and JQ1, the MCL-1 inhibitors pladienolide B and torin1, the FLT3 inhibitor AC220 and the DNA double-strand break inducer etoposide to correlate priming responses with co-operative induction of apoptosis. ABT-199 in combination with pladienolide B, torin1, etoposide or AC220 strongly induced apoptosis within 4 hours, but the MCL-1 inhibitors did not co-operate with etoposide or AC220. In keeping with the long half-life of BCL-2, the BET domain inhibitor JQ1 was found to downregulate BCL-2 and to prime cells to respond to MS1-BH3 at 48, but not at 4 hours: prolonged priming with JQ1 was then shown to induce rapid cytochrome C release when pladienolide B, torin1, etoposide or AC220 were added. In conclusion, dynamic BH3 profiling is a useful mechanism-based tool for understanding and predicting co-operative lethality between drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism. A plethora of agents sensitised cells to BAD-BH3-mediated mitochondrial outer membrane permeabilisation in the dynamic BH3 profiling assay and this was associated with effective co-operation with the BCL-2 inhibitory compounds ABT-199 or JQ1.

ELAS1 induces apoptotic death in adenocarcinoma DU145 and squamous-cell carcinoma SAS cancer cells, but not in normal KD cells.

We previously reported that an ELAS1 peptide containing 29 amino acids induces apoptotic death in U2OS human osteosarcoma cells following DNA double-strand break insults. Here, we show that ELAS1 also caused apoptosis in prostate adenocarcinoma DU145 cells and tongue squamous-cell carcinoma SAS cells. ELAS1 appears to be safe because it induced apoptosis only in cancer cells, not in normal KD cells. Because the effect of ELAS1 is dependent on increased stability of p53 and enhanced phosphorylation of p53-S46, we exogenously expressed wild-type p53 protein to fully promote ELAS1-mediated induction of apoptosis in SAS cells. Interestingly, simultaneous expression of Myc-ELAS1 and FLAG-p53 mediated by an internal ribosome entry site efficiently induced apoptosis in SAS cells. Moreover, we prepared a recombinant adenovirus that simultaneously expressed Myc-ELAS1 and FLAG-p53. This adenovirus also killed SAS cells, as determined by a cell viability assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 in vivo. Notably, Cy5-tagged ELAS1-t, which contained only ten amino acids, also efficiently induced apoptosis in both DU145 and SAS cells, suggesting the usefulness of ELAS1-t as a peptide. Taken together, our results suggest that ELAS1 is therapeutically useful as a peptide drug.

Abstract LB-170: A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent

Abstract UAB30, a synthetic analog of 9-cis-RA is a potent retinoid X receptors (RXR) agonist. It was found to have significantly low retinoic acid receptor (RAR)-binding activity. This unique property of UAB30 is responsible for its low lipid toxicity. Thus, it does not cause elevation in serum triglycerides both in humans and in experimental animals. These properties with favorable pharmacokinetics make it a suitable candidate for chronic use as chemopreventive agent. Here, we show that UAB30 is highly effective in inhibiting UVB-induced pathogenesis of skin cancers in Ptch1+/-/SKH-1 hairless mice. Similar to humans, this unique genetically engineered murine model develops both basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin following chronic UVB irradiation. Compared to controls, the UAB30-treated group showed a 48% reduction in tumor number and a 78% reduction in tumor volume. The number of papillomas, BCCs, and SCCs were reduced by 33%, 74%, and 76%, respectively. UAB30 also diminished UVB-induced skin inflammation, hyperplasia, and enhanced differentiation of suprabasal epidermal cells. The expression of cyclins A, D1, D2, and D3, and cdc25A were decreased in UAB30 treated SCCs, while pro-apoptotic proteins, Bax and cleaved caspase 3 were increased. UAB30 when topically applied 30 min after each UVB irradiation for 30 weeks, it showed significant reduction in cancer development. A 57% reduction in tumor number and 72% reduction in tumor volume were observed. BCCs and SCCs were reduced by 55% and 60%, respectively. We also demonstrate that UAB30 acts by a unique molecular mechanism. Induction of lethal DNA double strand breaks (DSBs) characterized by the presence of p-γH2AX was observed following UAB30 treatment. These effects were more prominent in cancer cells than normal keratinocytes. Induction of DSBs was associated with the activation of DNA damage response (DDR) signaling leading to increased phosphorylation of ATM, Rad50, and Chk2. These effects paralleled with the reduction in cell cycle proteins and cell cycle arrest in G2/M phase followed by apoptosis of arrested cells. UAB30-induced DSBs and DDR were not dependent on ROS as we did not observe significant induction of ROS in UAB30-treated cells and did not notice any protective effect of N-acetyl cysteine. These effects were similar to DDR and changes in proliferation/apoptosis response observed following treatment with cisplatin which is known inducer of DSBs. Moreover, other RXR agonists were significantly less potent in inducing DSBs and DDR responses. The underlying mechanism for the accumulation of DSBs was the diminished ability of cancer cells to repair these DNA lesions. Reduction in FANCD2 and FANCC, the Fanconi anemia core complex proteins which are known to be involved in DSBs repair appear to be the cause of extensive DNA damage associated tumor cell death. Citation Format: Sandeep C. Chaudhary, Craig A. Elmets, Mohammad Athar. A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent [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-170. doi:10.1158/1538-7445.AM2017-LB-170

Non-SMC elements 1 and 3 are required for early embryo and seedling development in Arabidopsis.

Early embryo development from the zygote is an essential stage in the formation of the seed, while seedling development is the beginning of the formation of an individual plant. AtNSE1 and AtNSE3 are subunits of the structural maintenance of chromosomes (SMC) 5/6 complex and have been identified as non-SMC elements, but their functions in Arabidopsis growth and development remain as yet unknown. In this study, we found that loss of function of AtNSE1 and AtNSE3 led to severe defects in early embryo development. Partially complemented mutants showed that the development of mutant seedlings was inhibited, that chromosome fragments occurred during anaphase, and that the cell cycle was delayed at G2/M, which led to the occurrence of endoreduplication. Further, a large number of DNA double-strand breaks (DSBs) occurred in the nse1 and nse3 mutants, and the expression of AtNSE1 and AtNSE3 was up-regulated following treatment of the plants with DSB inducer compounds, suggesting that AtNSE1 and AtNSE3 have a role in DNA damage repair. Therefore, we conclude that AtNSE1 and AtNSE3 facilitate DSB repair and contribute to maintaining genome stability and cell division in mitotic cells. Thus, we think that AtNSE1 and AtNSE3 may be crucial factors for maintaining proper early embryonic and post-embryonic development.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

DNA double-strand breaks induce the expression of flavin-containing monooxygenase and reduce root meristem size in Arabidopsis thaliana.

Plants use various mechanisms to cope with environmental stresses, which often threaten genome integrity. In Arabidopsis, DNA double-strand breaks (DSBs) reduce root meristem size in a SOG1-dependent manner. SOG1 is a key transcription factor controlling the response to DNA damage. However, the underlying mechanism remains largely unknown. In this study, we found that treatment with the DSB inducer zeocin increased the accumulation of H2O2 in root tips. Chromatin immunoprecipitation analysis showed that SOG1 directly binds to the promoter of FMO1, which encodes a flavin-containing monooxygenase and is associated with the production of reactive oxygen species (ROS), H2O2 in particular. Indeed, zeocin induced the expression of FMO1 in a SOG1-dependent manner, and neither the sog1 nor the fmo1 knockout mutant exhibited higher H2O2 accumulation in root tips. Consequently, both sog1 and fmo1 could tolerate exposure to zeocin, in terms of root growth and the maintenance of the meristem size. However, transgenic plants over-expressing FMO1 also accumulated H2O2 in response to zeocin exposure, suggesting that other ROS-synthesis genes are also involved in the regulation of ROS production. We conclude that SOG1-mediated regulation of ROS homeostasis plays a key role in the reduction of root meristem size under DNA stress conditions.

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.

Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination inPyricularia oryzae

To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

The p53 tumor-suppressor protein has a key role in the induction of cellular senescence, an important barrier to cancer development. However, very little is known about the physiological mediators of cellular senescence induced by p53. CEACAM1 is an immunoglobulin superfamily member whose expression is frequently lost in human tumors and exhibits tumor-suppressor features in several experimental systems, including Ceacam1 knockout mice. There is currently little understanding of the pathways and mechanisms by which CEACAM1 exerts its tumor-suppressor function. Here we report that CEACAM1 is strongly upregulated during the cellular response to DNA double-strand breaks (DSBs) starting from the lowest doses of DSB inducers used, and that upregulation is mediated by the ataxia telangiectasia mutated (ATM)/p53 pathway. Stable silencing of CEACAM1 showed that CEACAM1 is required for p53-mediated cellular senescence, but not initial cell growth arrest, in response to DNA damage. These findings identify CEACAM1 as a key component of the ATM/p53-mediated cellular response to DNA damage, and as a tumor suppressor mediating cellular senescence downstream of p53.

Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks

In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.

Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay

Comet assay under neutral conditions allows detection of DNA double-strand breaks (DSBs), which has consequence to genome instability and carcinogenesis. The present study aims to validate the neutral Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R +) with three well known DSBs inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and subsequently its efficacy in detecting DSBs in the organism exposed to a well known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D. melanogaster were fed different concentrations of BLM, CPT and CP (50.0–200.0 μg/ml) or Cr(VI) (5.0–20.0 μg/ml) mixed standard Drosophila food for 48 h. Neutral Comet assay was performed in cells of mid gut and brain from control and treated larvae. Our results show a dose-dependent increase in the migration of DNA in cells of the exposed organisms. A comparison among DNA lesions per mole number of the test chemical in the exposed groups showed that both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0 μg/ml was found to induce significantly increased ( p < 0.001) DSBs in the exposed organism as compared to the control. The study while validating neutral Comet assay in D. melanogaster suggests its use for in vivo assessment of environmental chemical induced DSBs.

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2–MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.