Activity Of DNA-damaging Agents Research Articles

The NAD + Metabolic Dependency Restriction Overcomes Drug-Resistance Phenotype of Multiple Myeloma CELLS

Introduction: We previously reported that Nicotinamide Adenine Dinucleotide (NAD +), a master regulator of catabolic metabolism and several enzymatic activities, plays a crucial role for Multiple Myeloma (MM) cells growth; indeed, small molecules targeting the rate-limiting NAD + producer enzyme NAMPT, have potent anti-MM activity. However, targeting dysregulated metabolic landscape of tumors has proven to be a major challenge as suggested by existence of alternative NAD + production routes which confer resistance to NAMPT inhibitors. As result here we focused on whole NAD + metabolic dependency of MM cells by investigating relevance of a comprehensive NAD + metabolic restriction as strategy to enhance activity of currently used anti-MM strategies. Methods: whole transcriptomic analysis of the NADome signature was performed on MM patients by probing MMRF CoMMpass study; its correlation with clinical data (OS and PFS) was also investigated. Next, GSEA algorithm on biological modules was run on MM patients carrying low- vs high-NADome signature. Chemical and genome editing-approaches using CRISPR-Cas9-based strategy were both used to investigate relevance of our findings. Then, an anti-MM drugs screen was used to challenge fully-NAD depleted MM cells using different viability tools. Mechanistic studies were also performed using qPCR, western blot, immunofluorescence analyses, targeted enzymatic assays and in-vivo xenograft MM models. Finally, clinical benefits of our findings were accomplished by challenging publicly available dataset on ASCT-treated MM patients. Results: RNAseq analysis on CoMMPass dataset sharply divided MM patients according to their comprehensive NADome signaling landscape: high-NAD producers showed poorer outcome than those expressing low mRNA levels, both in terms of PFS and OS as well. In line with these data, a panel of MM cell lines showed that pervasive NAPRT levels reduced NAMPT inhibitors activity thus supporting a complete NAD +-depletion for more effective strategies also in in-vivo xenograft models. PROGENy analysis revealed an increased NF-KB activity in NAPRT-depleted cells which in turn resulted in higher oxygen reactive species generation from these cells. Indeed, NAPRT KO cells showed oxidative damage accumulation, with increased malondialdehyde (MDA) activity and lower endogenous antioxidant defenses as suggested by glutathione reductase, glutathione peroxidase and ratio of reduced GSH to oxidized glutathione (GSSG) ratio measurements. Next, mitochondrial-energetic status was also tested by measuring electron transport chain (ETC) Complex I and Complex II activities: a shift towards oxidative metabolism was observed. Overall these data phenocopy the energetic status observed in drug-resistant cells; as result NAPRT-KO makes MM cells resistant to Melphalan. In such a context, the use of NAMPT-inhibitors was able to overcome drug-resistance by re-sensitizing cells to anti-MM activity of DNA damaging agents. Finally, a focused analysis on ASCT-treated MM patients, revealed poor outcome for “high-NAD + producers” thus supporting clinical relevance for NAD + metabolic restriction in these patients. Conclusions. Our data provide evidence that NAD + biosynthesis shapes metabolic phenotypes of MM cells, with its impairment resulting in metabolic reprogramming and cells viability injury. As result, a comprehensive NAD + depletion with dual NAPRT and NAMPT inhibition primes MM cells to alkylating agents which still represent the backbone strategy for ASCT-eligible patients.

Abstract 2594: Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor

Abstract DNA-damaging poisons and replicative stress commonly result in increased double-strand breaks (DSBs) in tumor cells. Non-homologous end joining (NHEJ), driven by the enzymatic function of DNA-PK, is one of the critical pathways employed to repair such DSBs. Inhibition of DNA-PK kinase activity impairs NHEJ, potentiates the activity of DNA-damaging agents, and inhibits survival of tumor cells incapable of repairing DSBs via other means, including homologous recombination (HR). We have developed an orally bioavailable, highly potent, and selective pharmacological inhibitor of DNA-PK activity, ZL-2201, for the treatment of cancer. ZL-2201 demonstrates enzymatic inhibition of DNA-PK in the single-digit nM range, with nearly 500-fold or greater selectivity against other phosphatidylinositol 3-kinase-related kinases (PIKKs). In addition, ZL-2201 showed high selectivity for DNA-PK kinase activity in a kinase panel screening study and a clean off-target profile with no appreciable inhibition against a panel of diverse, safety-relevant targets. We demonstrate that ZL-2201 effectively inhibits DNA-PK autophosphorylation and its downstream target phospho-RPA in multiple cell lines in a concentration- and time-dependent manner. DNA-PK has been shown previously to have synthetically lethal interactions with ATM. Therefore, we profiled the impact of ATM expression on the antiproliferative effects of ZL-2201. Cancer cell lines possessing defects in ATM displayed greater sensitivity to ZL-2201 compared to ATM-WT cancer cell lines. These observations were recapitulated in two ATM knockout isogenic cell lines. Moreover, ZL-2201 displayed minimal effects in a DNA-PK deficient model or in normal human and canine colon and kidney cell lines. Independent of ATM status, ZL-2201 showed strong synergy in vitro with topoisomerase II inhibitors. Following oral administration of ZL-2201 in vivo, ZL-2201 demonstrated dose-dependent and exposure-dependent antitumor activity against multiple models, with the greatest activity against ATM deficient human cancer xenografts. Antitumor activity correlated with duration of ZL-2201 exposure and level of target engagement. In addition, ZL-2201 significantly enhanced the activity of DNA-damaging agents in vivo, even in human cancer xenograft models with limited monotherapy antitumor activity for ZL-2201. The projected human PK properties, based on the ADME properties across species and PK properties in preclinical species of ZL-2201, suggest that ZL-2201 is suitable for oral administration. In conclusion, ZL-2201 demonstrates potent and selective inhibition of DNA-PK enzymatic activity, strong synergy with DNA damaging agents in vitro and in vivo, antitumor activity against multiple human xenograft models, and a favorable projected human PK profile. ZL-2201 is expected to enter clinical development in 2022. Citation Format: Shruti Lal, Bee-Chun Sun, Yuping Chen, Tom Huang, Neil Bhola, Vivian Morton, Kevin Chen, Shanghua Xia, Haoyu Zhang, Qiuping Ye, Petter Veiby, David I. Bellovin, Yuhua Ji. Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2594.

Targeting DNA repair and replication stress in the treatment of ovarian cancer.

Approximately half of high-grade serous epithelial ovarian cancers incur alterations in genes of homologous recombination (BRCA1, BRCA2, RAD51C, Fanconi anemia genes), and the rest incur alterations in other DNA repair pathways at high frequencies. Such cancer-specific gene alterations can confer selective sensitivity to DNA damaging agents such as cisplatin and carboplatin, topotecan, etoposide, doxorubicin, and gemcitabine. Originally presumed to inhibit DNA repair, PARP inhibitors that have recently been approved by the FDA for the treatment of advanced ovarian cancer also act as DNA damaging agents by inducing PARP-DNA complexes. These DNA damaging agents induce different types of DNA lesions that require various DNA repair genes for the repair, but commonly induce replication fork slowing or stalling, also referred to as replication stress. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. Hence, targeting DNA repair genes or DNA repair checkpoint genes augments the anti-tumor activity of DNA damaging agents. This review describes the rational basis for using DNA repair and DNA repair checkpoint inhibitors as single agents. The review also presents the strategies combining these inhibitors with DNA damaging agents for ovarian cancer therapy based on specific gene alterations.

Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.

A DNA double-strand break (DSB) is one of the most cytotoxic DNA lesions because unrepaired DSBs cause chromosomal aberrations and cell death. Although many physiological DSBs occur at DNA replication sites, the molecular mechanisms underlying this remain poorly understood. There was therefore a need to develop a highly specific method to detect DSB fragments containing DNA replication sites. Here we investigated whether pulsed-field gel electrophoresis (PFGE) combined with visualization of DNA replication sites by immunoblotting using halogenized deoxyuridines, such as BrdU and IdU, was sufficient for this detection. Our methodology enabled us to reproduce previously reported data. In addition, this methodology was also applied to the detection of bacterial infection-induced DSBs on human chromosomal DNA. Based on our findings, we propose that this strategy combining PFGE with immunoblot analysis will be applicable to studies analyzing the mechanistic details of DNA repair, the DNA damage response and the activity of DNA-damaging agents.

O6.4 - VX-970, selective inhibitor of ataxia telangiectasia and Rad3-related (ATR) protein

ABSTRACT Background: ATR is a critical regulator of the homologous recombination DNA repair pathway and the cellular response to replication stress. Inhibition of ATR is predicted to selectively enhance the sensitivity of tumor cells, in which DNA repair is often compromised, to standard of care DNA damaging agents, while relatively sparing non-tumor cells which have DNA repair mechanisms to compensate for the loss of ATR. VX-970 is a potent and selective inhibitor of ATR with a Ki Results: VX-970 substantially increases the cytotoxic activity of multiple DNA damaging agents in a broad panel of tumor cell lines, with minimal increase in non-tumor cell cytotoxicity. VX-970 markedly synergizes with cytotoxic agents in mouse xenograft models of several tumor types. At concentrations of VX-970 which inhibit tumor growth, phosphorylation of the downstream biomarker, Chk1, is observed, as is up-modulation of markers of unrepaired double strand DNA breaks, including gamma-H2AX. VX-970 is currently in two phase 1 clinical trials which employ standard 3 + 3 dose escalations in subjects with advanced solid tumors to determine the tolerability of escalating doses of VX-970, administered intravenously, in combination with gemcitabine, cisplatin or carboplatin. Conclusions: VX-970 is an inhibitor of ATR that enhances the activity of DNA damaging agents in pre-clinical models, and is in phase 1 clinical development.

CHK1 plays a critical role in the anti-leukemic activity of the wee1 inhibitor MK-1775 in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) remains a difficult disease to treat and requires new therapies to improve treatment outcome. Wee1 inhibitors have been used to prevent activation of the G2 cell cycle checkpoint, thus enhancing the antitumor activity of DNA damaging agents. In this study, we investigated MK-1775 in AML cell lines and diagnostic blast samples to identify sensitive subtypes as well as possible mechanisms of resistance.MethodsIn vitro MK-1775 cytotoxicities of AML cell lines and diagnostic blasts were measured using MTT assays. The effects of MK-1775 on cell cycle progression and related proteins were determined by propidium iodide (PI) staining and flow cytometry analysis and Western blotting. Drug-induced apoptosis was determined using annexin V/PI staining and flow cytometry analysis.ResultsWe found that newly diagnosed and relapsed patient samples were equally sensitive to MK-1775. In addition, patient samples harboring t(15;17) translocation were significantly more sensitive to MK-1775 than non-t(15;17) samples. MK-1775 induced apoptosis in both AML cell lines and diagnostic blast samples, accompanied by decreased phosphorylation of CDK1 and CDK2 on Tyr-15 and increased DNA double-strand breaks (DSBs). Time-course experiments, using AML cell lines, revealed a time-dependent increase in DNA DSBs, activation of CHK1 and subsequent apoptosis following MK-1775 treatment, which could be attenuated by a CDK1/2 inhibitor, Roscovitine. Simultaneous inhibition of CHK1 and Wee1 resulted in synergistic anti-leukemic activity in both AML cell lines and primary patient samples ex vivo.ConclusionsOur study provides compelling evidence that CHK1 plays a critical role in the anti-leukemic activity of MK-1775 and highlights a possible mechanism of resistance to MK-1775. In addition, our study strongly supports the use of MK-1775 to treat both newly diagnosed and relapsed AML, especially cases with t(15;17) translocation, and supports the development of combination therapies with CHK1 inhibitors.

Detection of genotoxic and non-genotoxic carcinogens in Xpc−/−p53+/− mice

An accurate assessment of the carcinogenic potential of chemicals and pharmaceutical drugs is essential to protect humans and the environment. Therefore, substances are extensively tested before they are marketed to the public. Currently, the rodent two-year bioassay is still routinely used to assess the carcinogenic potential of substances. However, over time it has become clear that this assay yields false positive results and also has several economic and ethical drawbacks including the use of large numbers of animals, the long duration, and the high cost. The need for a suitable alternative assay is therefore high. Previously, we have proposed the Xpa*p53 mouse model as a very suitable alternative to the two-year bioassay. We now show that the Xpc*p53 mouse model preserves all the beneficial traits of the Xpa*p53 model for sub-chronic carcinogen identification and can identify both genotoxic and non-genotoxic carcinogens. Moreover, Xpc*p53 mice appear to be more responsive than Xpa*p53 mice towards several genotoxic and non-genotoxic carcinogens. Furthermore, Xpc*p53 mice are far less sensitive than Xpa*p53 mice for the toxic activity of DNA damaging agents and as such clearly respond in a similar way as wild type mice do. These advantageous traits of the Xpc*p53 model make it a better alternative for in vivo carcinogen testing than Xpa*p53. This pilot study suggests that Xpc*p53 mice are suited for routine sub-chronic testing of both genotoxic and non-genotoxic carcinogens and as such represent a suitable alternative to possibly replace the murine life time cancer bioassay.

428P - Phase 1 Study of Veliparib With Concurrent Whole Brain Radiation Therapy (WBRT) in Patients (PTS) With Brain Metastases (METS)

ABSTRACT Background Veliparib, an oral PARP-1 and -2 inhibitor, potentiates the antitumor activity of DNA damaging agents including radiation therapy in vivo, and is shown to cross the blood-brain barrier. Updated data on safety and antitumor activity from an ongoing phase 1, dose-escalation study of veliparib with concurrent WBRT in pts with brain mets are presented here. Methods Pts with brain mets (including leptomeningeal) from primary non-CNS metastatic solid tumors (except germ cell cancer), adequate organ function, and Karnofsky performanace status (KPS) ≥70 were treated with WBRT (37.5 Gy in 15 fractions or 30 Gy in 10 fractions daily) and veliparib BID in escalating doses of 10–300 mg; the final WBRT fraction was followed by 1 extra day of veliparib. Safety and exploratory efficacy were assessed. Safety was evaluated in pts who received at least 1 dose of veliparib. Best brain tumor response (complete or partial per RECIST) rates were calculated for all pts who had at least 1 measureable lesion at baseline. Median survival time was estimated using Kaplan-Meier methodology for all dosed pts. Results To date, 66 pts (M/F, 23/43; median age 58 y) have been treated. Baseline KPS was 70, 80, 90, and 100 in 7.6, 31.8, 37.9, and 22.7% pts, respectively; primary tumor types were breast (n = 21), NSCLC (n = 23), melanoma (n = 10), colorectal (n = 3), renal (n = 1), and others (n = 8); 74.2% pts had multiple lesions, and 16.7% had prior brain stereotactic radiosurgery. Treatment-emergent dose-limiting toxicities were grade 3 hypokalemia and hyponatremia in 1 pt each at the 150 mg dose. Grade 3/4 treatment-emergent adverse events (≥4%) were fatigue (6.1%), dehydration (6.1%), anemia (4.5%), thrombocytopenia (4.5%), hyperglycemia (4.5%), and hyponatremia (4.5%). Best tumor response rate and median survival time were 36.8% and 6.6 months (m) for NSCLC, and 50% and 8.3 m for breast cancer. Conclusion Addition of veliparib up to 200 mg BID was well tolerated with concurrent standard WBRT and dose escalation is ongoing. These encouraging safety and preliminary efficacy data suggest that veliparib requires further evaluation as a radiosensitizer with WBRT in pts with NSCLC and brain mets in a randomized trial. Disclosure M.P. Mehta: Consultant: Abbott, BMS, Elekta, Merck, Novartis, Novocure, Tomotherapy, Vertex; Stock Options: Accuray, Pharmacyclics; DSMB: Apogenix; Protocol Data Review: Adnexus; Board of Directors: Pharmacyclics. W.J. Curran: Dr. Curran had served on the advisory board of Lilly, BMS, and Plexxikon. There are no conflicts. D. Wang: As a clinical investigator, and the principle investigator at Henry Ford Health System, I have conducted this clinical trial that is financially supported by Abbott Oncology. I have no other conflict of interest to be disclosed, otherwise. L. Kleinberg: I have received research funding from Abbott. J. Qian: Full-time Abbott employee and stockholder. T. Leahy: Full-time Abbott employee and stockholder. B. Desai: Full-time Abbott employee and stockholder. V.L. Giranda: Full-time Abbott employee and stockholder. All other authors have declared no conflicts of interest.

Phase I safety and pharmacokinetic (PK) study of veliparib in combination with whole brain radiation therapy (WBRT) in patients (pts) with brain metastases.

2013 Background: Veliparib is an oral PARP-1 and -2 inhibitor that enhances the antitumor activity of DNA damaging agents including radiation therapy in vivo. In pre-clinical models, veliparib crosses the blood-brain barrier. This ongoing phase I dose-escalation study evaluates the safety, PK, and provides preliminary antitumor activity of veliparib in combination with WBRT in pts with brain metastases. Methods: Pts with brain metastases from non-CNS primary solid malignancy, adequate organ function, RPA Class 2, and KPS ≥70 were treated with WBRT (37.5 Gy in 15 fractions or 30 Gy in 10 fractions) QD with veliparib BID with every fraction of WBRT in escalating doses of 10, 20, 30, 50, 100, 150, and 200 mg; the final WBRT fraction was followed by 1 extra day of veliparib. Safety, PK, and tumor response by RECIST were assessed. Results: At the time of reporting 59 pts (M/F, 21/38; median age 57 y) had been treated. Baseline KPS was 70, 80, 90, and 100 in 6.8, 32.2, 40.7, and 20.3% pts, respectively; primary tumor types were breast (n=20), NSCLC (n=20), melanoma (n=9), colorectal (n=2), and others (n=8); 71.2% pts had multiple lesions; and 18.6% had prior brain SRS. Grade 3/4 treatment-emergent adverse events (TEAEs; ≥5%) were fatigue (6.8%), anemia (5.1%), hyponatraemia (5.1%), and thrombocytopenia (5.1%); other TEAEs (≥20%) were fatigue (57.6%), headache (42.4%), nausea (40.7%), alopecia (28.8%), vomiting (22%), radiation skin reactions (22%), and decreased appetite (22%). PK of veliparib were approximately dose-proportional, with oral clearance of 21.6 ± 14.2 L/h (mean ± SD, n=45), minimal drug accumulation at day 15, and no significant effect of food on bioavailability. Tumor response was evaluable in 48 pts. Best tumor response and median survival were 37.5% and 10 months (m) for NSCLC, and 52.9% and 12.5 m for breast cancer (excluding pts with leptomeningeal disease). Conclusions: Addition of veliparib up to 200 mg BID was well tolerated with concurrent WBRT and dose escalation ongoing. The PK of veliparib was dose proportional with no food effect. Preliminary antitumor activity is encouraging and informative for the design of more definitive trials.

Abstract 1776: Antitumor activity of Chk1 inhibitor LY2606368 as a single agent in SW1990 human pancreas orthotopic tumor model

Abstract ChK1 is a key kinase in the DNA damage response signaling network, including cell cycle checkpoints, DNA repair, apoptosis and transcription, thus emerging as an attractive target in anti-cancer therapy. LY2606368, a potent and selective ATP competitive inhibitor of the Chk1 protein kinase, is currently in clinical development. LY2606368 has been reported to inhibit Chk1 auto-phosphorylation activated by DNA damaging agents, and induce phosphorylation of H2AX, a DNA damage maker in multiple cancer cell lines in vitro. In addition, LY2606368 has demonstrated potent single agent activity and potentiates the anti-tumor activity of DNA damaging agents in vivo. Pancreatic cancer is one of the least curable cancers, with an approximate 5% overall 5-year survival for all patients. In the current study, we evaluated the efficacy of LY2606368 (as its methanesulfonate salt, hydrate; LY2606368.MeSO3H.H2O = LY2940930) in pancreatic cancer cell lines in vitro and xenograft tumors (including subcutaneous and orthotopic models) in vivo. LY2606368 alone significantly inhibited the cell proliferation in a variety of pancreatic cell lines (SW1990, SU86.86, Bx-PC3, AsPC-1, CFPAC-1, Capan-2, HPAF-II) with SW1990 being the most sensitive (IC50=1.5 nM). In SW1990 subcutaneous xenograft model, LY2606368 showed substantial dose-dependent inhibition of tumor growth. In SW1990 pancreas orthotopic model, which represents the local and metastatic growth pattern seen in pancreas cancer patients, LY2606368 treatment resulted in over 92% inhibition of primary tumor growth as well as 100% inhibition of metastasis to lymph node, spleen and intestine. The anti-tumor effect of LY2606368 treatment was further demonstrated in comparing with gemcitabine (the standard of care for pancreas cancer patient) in SW1990 orthotopic model. The anti-tumor and/or anti-metastasis mechanism of LY2606368 was also investigated, including phosphorylation of H2AX, cell proliferation, cell survival, and soft agar anchorage-independent growth. Together, our findings demonstrate the therapeutic potential of LY2606368 as a single agent in the treatment of pancreatic cancer 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 1776. doi:1538-7445.AM2012-1776

DNA Damage Response Pathways and Cell Cycle Checkpoints in Colorectal Cancer: Current Concepts and Future Perspectives for Targeted Treatment

Although several drugs have been designed in the last few years to target specific key pathways and functions in colorectal cancer (CRC), the backbone of CRC treatment is still made up of compounds which rely on DNA damage to accomplish their role. DNA damage response (DDR) and checkpoint pathways are intertwined signaling networks that arrest cell cycle, recognize and repair genetic mistakes which arise during DNA replication and transcription, as well as through the exposure to chemical and physical agents that interact with nucleic acids. The good but highly variable activity of DNA damaging agents in the treatment of CRC suggests that intrinsic alterations in DDR pathways and cell cycle checkpoints may contribute differentially to the way cancer cells react to DNA damage. In the present review, our aim is to depict the recent advances in understanding the molecular basis of the activity of DNA damaging agents used for the treatment of CRC. We focus on the known and potential drug targets that are part of these complex and intertwined pathways. We describe the potential role of the checkpoints in CRC, and how their pharmacological manipulation could lead to chemopotentiation or synergism with currently used drugs. Novel therapeutic agents playing a role in DDR and checkpoint inhibition are assessed. We discuss the possible rationale for combining PARP inhibition with DNA damaging agents, and we address the link between DDR and EGFR pathways in CRC.

Abstract 1103: Linkage between endogenous Chk2 activation and p14ARF expression in p53-mutated cells revealed by proteomic and genomic analyses of the NCI60

Abstract Chk2 is a multi-substrate kinase involved in the DNA damage response and genomic stability. Chk2 inhibitors are currently being evaluated in cancer models, with the rationale of enhancing the activity of DNA damaging agents in p53-deficient tumors while protecting normal tissues. Part of the ATM-Chk2-p53 axis, Chk2 is overexpressed or activated in various malignant tumors. Here we determined Chk2 copy number, total and single-exon transcriptional levels, and total and activated (pT68) protein across the 60 cell lines of the NCI DTP screen. We correlated these parameters with p53 mutational status, miRNA expression (Agilent miRNA Microarray V2), and performed supervised transcript (Agilent Whole Human Genome Oligo Microarray) class comparison analysis between high and low pT68 Chk2 cell lines using BRB Array Tools 3.8. All the lines used for this purpose were known to be p53-mutated. Our phosphoproteomic analyses demonstrate that a number of cell lines have high endogenous Chk2 activation (ovarian OVCAR3 and OVCAR4, renal RFX-393, lung NCI-H322M, and EKVX, melanoma UACC-62 and −257, leukemia RPMI-8226 and breast BT-549) whereas the wild-type p53 tumors almost invariably show undetectable Chk2 activation. Expression of total Chk2 protein and mRNA were generally positively correlated to each other. Exon array analyses across the NCI-60 suggest exon 3 splicing in almost all Chk2 high expresser cell lines and premature truncation between exons 8 and 10 in the ovarian SKOV3 cells, which could be related to the low level of Chk2 expression in that cell line. One microRNA, hsa-miR-455-3p, showed a highly significant negative correlation with both Chk2 transcript levels and with total protein levels (Spearman's r =-0.48, p < 10-3 and r=-0.35, p < 0.01 respectively) and was predicted to hybridize to Chk2 mRNA by miRBase (www.mirbase.org). Among the 181 probes differentially expressed in cells with highly activated Chk2, 123 mapped to known gene IDs, and 58 were eligible for network analysis with Ingenuity Pathways Analysis 8.0 (Ingenuity Systems Inc.). Functions associated with the networks enriched in genes differentially expressed by high pT68 cells included: cell cycle, cell-to-cell signaling, cell growth and proliferation, cancer. Remarkably, the tumor suppressor CDKN2A (p14ARF) showed a 132-fold difference between the two groups considered (unpaired t test, two-tailed p < 10-7; false discovery rate < 10-7), being highly overexpressed in high pT68 Chk2 cell lines. Several EGFR-Ras-MAPK related genes were downregulated in high pT68 cell lines. Together, these data suggest that Chk2 is part of a multifaceted regulation network in cancer. Although traditionally considered as a low-penetrance tumor suppressor, it clearly plays more complex roles in tumor biology, possibly behaving as an activated oncogene in specific genomic contexts. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1103.

Abstract 5408: Exploiting the defective p53/MDM2/p14ARF pathway in neuroblastoma by targeting checkpoint kinase Chk1 with AZD7762

Abstract Introduction: A defective p53/MDM2/p14ARF pathway seems to be the principal mode of checkpoint dysfunction in metastatic neuroblastoma following DNA damage, which probably accounts for chemoresistance after initial treatment response. Checkpoint kinase inhibitor AZD7762 (AstraZeneca, currently in clinical trials) has the potential to enhance the action of DNA-damaging agents in some advanced solid malignancies. In this study, we assessed the potential synergy between AZD7762 and chemotherapeutic agents in the in vitro and in vivo treatment of neuroblastoma cells which harbor aberrations in the p53/MDM2/p14ARF pathway. Experimental Design: AZD7762 was tested in combination with chemotherapeutic agents both in vitro and in vivo against a representative panel of neuroblastoma cell lines. Ten were p53/MDM2/p14ARF pathway-defective (four with p53 mutations, three with MDM2 amplification, and three with p14ARF deletion) and three were wild type. Using these lines, p53/p21 functional assay, drug induced cytotoxicity, S/G2 checkpoint abrogation, and therapy of xenografts were examined. Synergism with chemotherapy was quantified using combination index (CI). CI<1 indicates synergism, CI=1 additive effects, and CI>1 antagonism. Results: Based on p53/p21 functional assays, all four p53 mutant lines failed to show endogenous p21 induction after DNA damage (defined as p53 pathway nonfunctional). Similarly, p53 was nonfunctional in two of the three MDM2 amplified and one of the three p14ARF deleted cell lines. In cytotoxicity assays, p53 nonfunctional lines were more resistant to DNA-damaging agents when compared to p53 functional lines. Synergy between AZD7762 and DNA-damaging agents (cisplatin, doxorubicin, etoposide, melphalan, SN38 and topotecan) was strong in all p53 nonfunctional lines (average CI values: 0.44 - 0.63), but not in p53 functional lines (average CI values greater than 1.0). In contrast, gemcitabine showed even stronger synergy with AZD7762 regardless of p53 functional status (CI values: 0.11 - 0.47). AZD7762 treatment resulted in the abrogation of DNA damage-induced S/G2 checkpoint arrest, and in the potentiation of antitumor activity of DNA-damaging agents in four mouse xenograft models. Conclusions: AZD7762 potentiated both in vitro and in vivo the antitumor effects of DNA-damaging agents against neuroblastoma. These results suggest that the addition of AZD7762 may reverse or prevent drug resistance, especially if a defective p53/MDM2/p14ARF pathway is the underlying mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5408.

Imaging with FLT-PET demonstrates that PF-477736, an inhibitor of CHK1 kinase, overcomes a cell cycle checkpoint induced by gemcitabine in PC-3 xenografts

3045 Background: The development of strategies to monitor the molecular and cellular response to novel agents that target the cell cycle is vital to provide proof of mechanism and biological activity of these compounds. The protein kinase CHK1 is activated following DNA damage in the S and G2-phases of the cell cycle and mediates cell cycle arrest. In vitro studies demonstrate that inhibition of CHK1 can overcome cell cycle arrest induced by DNA damage and enhance cytotoxic activity of DNA damaging agents. In vivo studies show that combining DNA damaging agents with a CHK1 inhibitor potentiates antitumor activity. We hypothesize that functional imaging with 18F-fluorine-L-thymidine (FLT), a PET-tracer where tumor uptake is maximal in the S and G2 phases of the cell cycle can be used to non-invasively monitor the induction and therapeutic inhibition of a cell cycle checkpoint in vivo. Methods: Nude mice harbouring PC-3 xenografts were treated with vehicle controls, gemcitabine, the CHK1-inhibitor PF-477736 or gemcitabine + PF-477736. FLT-PET scans were performed and tumors harvested for ex-vivo biomarkers to assess S-phase, M-phase and DNA-repair. Results: Gemcitabine induced a 8.3 ±0.8 fold increase in tumoral uptake of FLT at 21 hours that correlated with a 3.3 ±0.2-fold increase in thymidine kinase activity and S-phase arrest as demonstrated by BrdU incorporation and elevated expression of cyclin-A. Treatment with PF-477736 at 17 hours after gemcitabine abrogated the early FLT-flare at 21 hours by 82% (p<0.001). This was associated with both an increased fraction of cells in mitosis and G1-phase of the cell cycle as determined by phos-histone H3 and flow cytometry. Furthermore, the combination of gemcitabine and PF-477736 enhanced DNA damage as measured by phos-gamma-H2AX and significantly delayed tumor growth when compared to tumors treated with gemcitabine alone. Conclusion: These data clearly indicate that the CHK1-inhibitor PF-477736 can overcome the cell cycle checkpoint induced by gemcitabine and increase associated DNA damage in tumors in-vivo. The PET studies indicate that functional imaging with FLT-PET is a promising strategy to monitor responses to therapeutic agents that target cell cycle checkpoints. [Table: see text]

JNK Activation and Fas Up-Regulation Precede Proteasomal Degradation of Topoisomerase I in SN38-Mediated Cytotoxicity Against Multiple Myeloma.

Topoisomerase I is an essential constitutive nuclear enzyme which is expressed in multiple myeloma (MM) cells as well as bone marrow stromal cells (BMSCs). SN38 is the highly potent active metabolite of topoisomerase I inhibitor irinotecan. Our investigations demonstrated significant differential toxicity of SN38 against MM cell lines and patient cells versus BMSCs. SN38 directly inhibited constitutive and inducible IL-6 and VEGF secretion by BMSCs, despite minimal impact upon BMSC viability, consistent with the S-phase specificity of camptothecin-induced apoptosis. In contrast to conventional therapy, adhesion of MM cells to BMSCs failed to confer tumor cell resistance to SN38.Proteasomal degradation of topoisomerase I in response to formation of topoisomerase I-cleavable complexes has been proposed as a resistance mechanism to camptothecins in solid malignancies. In our studies, proteasomal degradation of nuclear topoisomerase I was observed in MM.1S cells, but failed to protect from apoptosis. Moreover, RPMI-derived doxorubicin resistant Dox40 cells and dexamethasone-sensitive MM.1S cells were equally sensitive to SN38; in contrast to MM.1S, Dox40 displayed very little evidence of topoisomerase I degradation even at high concentrations of SN38. Furthermore, SUDHL4 lymphoma cells displayed significant resistance to SN38 despite lack of proteasomal degradation of topoisomerase I, indicating that proteasomal degradation is not a consistent mechanism of resistance to topoisomerase I inhibition in hematological malignancies.By elucidating the mechanisms of apoptosis downstream to SN38-induced DNA damage, approaches to augment the anti-tumor activity of this class of compounds may be revealed. We identified several intracellular signals translating topoisomerase I inhibition into apoptosis in MM. In MM.1S cells, SN38 at physiological concentrations induced JNK activation within 2 hours and cell surface Fas up-regulation that was maximal at 6–8 h, sustained for at least 24 h, and closely followed by cleavage of pro-caspase 8 and pro-caspase 3 independently from caspase 9. These pro-apoptotic signaling events were sustained despite proteasomal degradation of topoisomerase I, which occurred after JNK activation. Importantly, exogenous Fas activation by Fas-activator CH11 in combination with SN38 resulted in synergistic toxicity to MM cells.Downstream from caspase 8, PARP is an important DNA repair enzyme. PARP inhibitors have already shown significant activity in animal models, may augment chemotherapy, and may be potentially useful in combination with topoisomerase I inhibitors. In our experiments, PARP inhibition by NU1025 augmented SN38-induced apoptosis in a synergistic fashion, providing the rationale to target PARP in order to enhance the anti-tumor activity of DNA damaging agents in general, and irinotecan in particular.These findings have clinical significance because identification of downstream apoptotic signaling following topoisomerase I inhibition will both elucidate mechanisms of resistance and optimize future combination chemotherapy against MM. Furthermore, enhanced activity in combination with novel chemotherapeutic agents will provide the framework for future clinical trials of topoisomerase I inhibitors in MM.

Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor.

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4'-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(-) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard-phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard-DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

The interplay between mixed function oxygenases and DNA alteration under PAH pollution

Our experience in DNA alteration assessment tells us that aquatic vertebrates, in general, and also some of the invertebrates, by living in polluted areas acquire altered DNA; DNA alterations being defined either as single-strand breaks, and/or alkali sensitive or single-strand specific nuclease-cleavable sites (sse). This can be shown by several methods: (i) Alkaline filter elution; 1 (ii) fluorescence analysis of DNA unwinding; 2 (iii) alkaline sucrose density gradient centrifugation; 3 (iv) alkaline unwinding analysis with hydroxyapatite differentiation; 4 (v) nucleoid sedimentation analysis; 5 (vi) supercoiled DNA relaxation detection by electrophoresis; 6 and (vii) measurement of DNA molecular weight distributions with the electron microscope. 7 The amount of sse per DNA mass can be a function of either an increased activity of DNA damaging agents or agents that inhibit DNA repair. Among DNA damaging agents are polycyclic aromatic hydrocarbons (PAH), that can cause sse by the ‘fast effect’ 8 or after induction upon uptake, through the action of mixed function oxygenase (MFO). Since MFO induction within certain limits is a function of PAH concentration measurement of this enzyme activity enables at least partial interpretation of the DNA alteration status, which can be a two-phase process.