Chk1 Inhibitor MK-8776 Research Articles

A subset of cancer cell lines is acutely sensitive to the Chk1 inhibitor MK-8776 as monotherapy due to CDK2 activation in S phase.

DNA damage activates Checkpoint kinase 1 (Chk1) to halt cell cycle progression thereby preventing further DNA replication and mitosis until the damage has been repaired. Consequently, Chk1 inhibitors have emerged as promising anticancer therapeutics in combination with DNA damaging drugs, but their single agent activity also provides a novel approach that may be particularly effective in a subset of patients. From analysis of a large panel of cell lines, we demonstrate that 15% are very sensitive to the Chk1 inhibitor MK-8776. Upon inhibition of Chk1, sensitive cells rapidly accumulate DNA double-strand breaks in S phase in a CDK2- and cyclin A-dependent manner. In contrast, resistant cells can continue to grow for at least 7 days despite continued inhibition of Chk1. Resistance can be circumvented by inhibiting Wee1 kinase and thereby directly activating CDK2. Hence, sensitivity to Chk1 inhibition is regulated upstream of CDK2 and correlates with accumulation of CDC25A. We conclude that cells poorly tolerate CDK2 activity in S phase and that a major function of Chk1 is to ensure it remains inactive. Indeed, inhibitors of CDK1 and CDK2 arrest cells in G1 or G2, respectively, but do not prevent progression through S phase demonstrating that neither kinase is required for S phase progression. Inappropriate activation of CDK2 in S phase underlies the sensitivity of a subset of cell lines to Chk1 inhibitors, and this may provide a novel therapeutic opportunity for appropriately stratified patients.

Abstract 943: The critical role of CDK2 activation in determining the differential sensitivity of cell lines to Chk1 and Wee1 inhibitors

Abstract Anticancer DNA damaging agents activate checkpoints that halt the cell cycle to prevent further DNA replication and mitosis until the damage has been repaired. One of the important regulatory proteins activated in this pathway is Checkpoint kinase 1 (Chk1). Hence, Chk1 inhibitors have emerged as promising novel therapeutics. In clinical trials, Chk1 inhibitors are primarily being evaluated in combination with various DNA damaging agents. However, Chk1 inhibitors also show single agent activity in a subset of cell lines, and this provides a unique therapeutic opportunity to target these tumors. The goal of this study is to define the mechanism of this sensitivity, and identify biomarkers that would permit selection of appropriate patients for clinical trials. From screening a large panel of cancer cell lines, we observed that 15% were sensitive to the Chk1 inhibitor MK-8776, with rapid (<6 h) induction of DNA double-strand breaks (DSB) in S phase cells. Most of the resistant cell lines were sensitive to the Wee1 inhibitor, MK-1775 (AZD1775), while all the cell lines were sensitive to the Wee1/Myt1 dual inhibitor, PD-166285. Wee1 suppresses activation of CDK1 and CDK2, suggesting that MK-1775-mediated activation of one or other (or both) of these kinases leads to DSB. MK-8776 did not activate CDK1, so we hypothesized that activation of CDK2 was critical. To differentiate between the role of CDK1 and CDK2 in cell lines sensitive or resistant to MK-8776, we used inhibitors to CDK1/2. We found that CVT-313 was a selective inhibitor of CDK2 at low concentrations, but that Ro-3306 failed to discriminate between these two kinases in cells. CVT-313 prevented DSB and the associated phosphorylation of histone-H2AX induced by MK-8776 and MK-1775. Cyclin E is a direct target of CDK2; phosphorylation of cyclin E leads to its degradation which was only observed in sensitive cells and prior to the formation of DSB. The acute cytotoxicity was dependent on cyclin A rather than cyclin E, as siRNA-mediated knockdown of cyclin A but not cyclin E, prevented DSB. We conclude that activation of cyclin A/CDK2 determines the sensitivity of cells to MK-8776. Resistance to MK-8776 results from failure to activate CDK2, presumably through failure to activate the upstream CDC25A phosphatase. MK-1775 can circumvent this resistance by directly activating CDK2. The untimely and abnormal activation of CDK2 is then cytotoxic to cancer cells. Citation Format: Nandini Sakurikar, Ruth Thompson, Ryan Montano, Alan Eastman. The critical role of CDK2 activation in determining the differential sensitivity of cell lines to Chk1 and Wee1 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 943. doi:10.1158/1538-7445.AM2015-943

Will Targeting Chk1 Have a Role in the Future of Cancer Therapy?

The development of checkpoint kinase 1 (Chk1) inhibitors as anticancer agents has a long history. In 1982, Arthur Pardee demonstrated that caffeine could abrogate DNA damage-induced G2 arrest and enhance cytotoxicity induced by nitrogen mustards. The G2 arrest provides time for the cell to repair DNA damage, and the addition of caffeine forces the cells to progress into mitosis before the damage is repaired. Caffeine was eventually shown to inhibit the DNA damage response proteins ATR and ATM, but the required concentrations could not be achieved in patients. The downstream targets of ATR and ATM are Chk1 and Chk2, respectively, but subsequent drug development has demonstrated a much more critical role for Chk1 than Chk2 in protecting cells from DNA damage. The first Chk1 inhibitor to enter clinical trials was 7-hydroxystaurosporine (UCN-01), but it had multiple offtarget effects as well as high plasma protein binding, which resulted in variable bioavailability and serious adverse effects when the binding capacity was exceeded. Many Chk1 inhibitors have subsequently been developed, one of which, MK-8776, is the subject of the report by Daud et al, which accompanies this article, in Journal of Clinical Oncology. The original therapeutic strategy required that the tumor cells are first damaged with an anticancer drug such as cisplatin or irinotecan. The resulting damage activates ATR which, in turn, activates Chk1, inhibits CDC25 phosphatases, and prevents activation of CDK1/2, the consequence of which is arrest in the S and G2 phases of the cell cycle (Fig 1). Inhibition of Chk1 reactivates CDK1/2 to abrogate this arrest before the cells can complete repair, driving the damaged S phase cells into G2 and G2 cells through an aberrant mitosis (often called mitotic catastrophe). However, subsequent experiments found variable, if any, increase in cytotoxicity with this strategy, possibly because the concentrations of drug that caused arrest in vitro were themselves cytotoxic. We concluded that inhibition of Chk1 could accelerate the rate of cell death but had limited impact on the overall extent of cell death. However, it is important to note a phase I clinical trial of the Chk1 inhibitor AZD7762 in combination with irinotecan in which one patient showed a dramatic and durable response. This outlier response was tracked to a mutation in the RAD50 gene that impeded its normal participation in irinotecan-induced arrest and repair. Consequently, there may be small subsets of patients who would truly benefit from this drug combination. It is now recognized that Chk1 inhibitors are far more effective at sensitizing cells to antimetabolites such as gemcitabine, hydroxyurea, and cytarabine. For example, we demonstrated up to a 100-fold increase in sensitivity to hydroxyurea on addition of the Chk1 inhibitor MK-8776. The sensitization observed with gemcitabine in vitro was not as pronounced (2to 10-fold), but this has become the preferred combination to test in clinical trials because gemcitabine is already approved for the treatment of many types of tumors. Gemcitabine is a prodrug which, once metabolized, can be incorporated into DNA, but perhaps more relevant here, it irreversibly CHK1 CHK1

Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

BackgroundChk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.MethodsGrowth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.ResultsMK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.ConclusionsThere are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo

Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

Sensitization of Pancreatic Cancer to Chemoradiation by the Chk1 Inhibitor MK8776

The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Checkpoint kinase 1 (Chk1) inhibitor MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models. We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)-proficient and -deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776. We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not -deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose-limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer. Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.

ATR Inhibition Broadly Sensitizes Ovarian Cancer Cells to Chemotherapy Independent of BRCA Status

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

The Novel Chk1 Inhibitor MK-8776 Sensitizes Human Leukemia Cells to HDAC Inhibitors by Targeting the Intra-S Checkpoint and DNA Replication and Repair

Interactions between the novel Chk1 inhibitor MK-8776 and the histone deacetylase (HDAC) inhibitor (HDACI) vorinostat were examined in human leukemia cells harboring wild-type (wt) or deficient p53. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various p53-wt or -deficient leukemia cell lines, whereas p53 knockdown by short hairpin RNA (shRNA) sensitized p53-wt cells to lethality of this regimen. Leukemia cell lines carrying FLT3-ITD were also sensitive to the MK-8776/vorinostat regimen. Synergistic interactions were associated with inhibition of Chk1 activity, interference with the intra-S-phase checkpoint, disruption of DNA replication, and downregulation of proteins involved in DNA replication (e.g., Cdt1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γ-H2A.X formation, and apoptosis. Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared with their wt counterparts and displayed downregulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary acute myelogenous leukemia (AML) blasts, particularly against the CD34(+)/CD38(-)/CD123(+) population enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34(+) cells. Together, these findings indicate that the novel Chk1 inhibitor MK-8776 markedly potentiates HDACI lethality in leukemia cells displaying various genetic backgrounds through mechanisms involving disruption of the intra-S checkpoint, DNA replication, and DNA repair. They also argue that leukemic cells, including those bearing oncogenic mutations associated with poor prognosis, for example, p53 deletion/mutation or FLT3-ITD, may also be susceptible to this strategy.

Abstract 3413: Dissecting the combination of gemcitabine and the Chk1 inhibitor MK-8776 in vitro and in vivo: cell cycle perturbation and the impact of administration schedule.

Abstract Gemcitabine inhibits ribonucleotide reductase, prevents synthesis of deoxyribonucleotides, and induces arrest of cells in S phase. Subsequent inhibition of Chk1 causes the stalled replication forks to collapse, thereby increasing cell death. We have observed up to 10-fold decrease in the growth-inhibitory concentration (IC50) of gemcitabine upon incubation with the Chk1 inhibitor MK-8776 across multiple cell lines. In contrast to most in vitro studies, we have briefly incubated cells with gemcitabine to more closely reflect the exposure to drug that occurs in cancer patients. This permits analysis of the recovery from S phase arrest as would occur clinically. Following treatment, the rate of recovery depends on the concentration of gemcitabine, with S phase arrest lasting for 24 h at low concentrations and 48 - 72 h at higher concentrations. MK-8776 (2 μM) was then added for 6 h periods reflecting the approximate exposure that occurs in human plasma at its maximum tolerated dose. The maximum decrease in IC50 occurred when MK-8776 was added 18-24 h after gemcitabine. In addition to there being more cells in S phase at this time, we observed that replication forks also evolve over time to become more Chk1 dependent. Homologous recombination of DNA as assessed by the formation of RAD51 foci does not occur at the time cells initially arrest, but begins to occur 8 - 16 h later. Inhibition of Chk1 prevents RAD51 foci formation leading to replication fork collapse. Hence there are two interacting mechanisms that contribute to the optimum schedule of this drug combination: gemcitabine-mediated accumulation of cells in S phase, and Chk1-mediated stabilization of replication forks. To assess cell cycle perturbation in vivo, tumors were stained for Ki67 which detects cells at all phases of the cell cycle except Go, and geminin which stains cells in S and G2. Results were expressed as the ratio of geminin:Ki67; i.e., the ratio of cells in cycle that are in S/G2. In untreated AsPc-1 human pancreas tumor xenografts, about 25% of the cells were in S/G2, while administration of 150 mg/kg gemcitabine (equates to 450 mg/m2 in humans) showed 80-90% of the cells in S/G2 24 h after drug administration. When 50 mg/kg MK-8776 was administered 18 h after 150 mg/kg gemcitabine, there was greater tumor growth delay than concurrent administration of the drugs. We are currently conducting a clinical trial in patients with bladder cancer to assess the cell cycle perturbation that occurs following administration of standard dose of 1000 mg/m2 gemcitabine. Initial results show a significant S/G2 arrest 24 h after drug administration. These results emphasize the importance of assessing cell cycle perturbation in patient tumors, and provide justification for a clinical trial of gemcitabine followed 1 day later by MK-8776. Citation Format: Alan Eastman, Ryan Montano, Ruth Thompson, Nadeem Khan, Huagang Hou, Lionel D. Lewis, John D. Seigne, Marc S. Ernstoff. Dissecting the combination of gemcitabine and the Chk1 inhibitor MK-8776 in vitro and in vivo: cell cycle perturbation and the impact of administration schedule. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3413. doi:10.1158/1538-7445.AM2013-3413

Abstract 3420: The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair.

Abstract While histone deacetylase inhibitors (HDACIs, e.g., vorinostat) are approved for CTCL, they have limited single-agent activity in AML. Consequently, new mechanisms and targeted agents capable of improving HDACI anti-leukemic efficacy are urgently needed. Recently, new insights into HDACI mechanisms of actions have implicated interruption of DNA replication and DNA damage checkpoints, raising the possibility that agents targeting Chk1, a key modulator that monitors the DNA replication process and mediates activation of DNA damage checkpoints, may improve HDACI activity. To test this hypothesis, interactions between the novel Chk1 inhibitor MK8776 and the HDAC inhibitor (HDACI) vorinostat were examined in human AML cells harboring wild-type (wt) or deficient p53 and/or FLT3-ITD. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various AML cell lines, although lethality was significantly more pronounced in p53-deficient cells. Notably, AML cells carrying FLT3-ITD were particularly sensitive to the MK8776/vorinostat regimen, regardless of p53 status. Synergistic interactions were associated with inhibition of Chk1 activity (e.g., diminished Ser296 phosphorylation), interference with the intra-S phase checkpoint (cell cycle analysis), disruption of DNA replication (EdU incorporation), and down-regulation of proteins involved in DNA replication (e.g., CDT1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γH2A.X formation and increased Chk2 Thr68 phosphorylation, and apoptosis. Moreover, AML cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wt-counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Interestingly, MK8776 induced Ser15, but not Ser20, phosphorylation and accumulation of p53 in p53-wt cells, events that were largely blocked by HDACIs. Moreover, p53 knock down by shRNA significantly sensitized these cells to MK8776 or HDACIs, particularly in combination. Finally, the MK8776/vorinostat regimen was active in primary AML blasts, particularly against CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34+ cells. Together, these findings indicate that the novel Chk1 inhibitor MK8776 markedly potentiates HDACI lethality in AML cells displaying various genetic backgrounds through mechanisms involving disruption of DNA replication, the intra-S checkpoint, and DNA repair. They also argue that leukemic cells, and particularly those bearing oncogenic mutations associated with poor prognosis e.g., p53 deletion/mutation or FLT3-ITD, may be more susceptible than their wt-counterparts to this strategy. Citation Format: Shuang Chen, Maciej Kmieciak, Liang Zhou, Hui Lin, Xin-Yan Pei, Yun Dai, Steven Grant. The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3420. doi:10.1158/1538-7445.AM2013-3420

The Mre11 Nuclease Is Critical for the Sensitivity of Cells to Chk1 Inhibition

The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, and for stabilizing stalled replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be cytotoxic. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon incubation with the Chk1 inhibitor MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/Nbs1 complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11 nuclease activity leads, not only to a decrease in the amount of ssDNA following Chk1 inhibition, but also inhibits the formation of DSB, suggesting that DSB are a consequence of ssDNA formation. These findings were corroborated by the discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following treatment. However, once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when incubated with MK-8776. Our findings suggest that Mre11 provides the link between aberrant activation of Cdc25A/Cdk2 and Mus81. The results highlight a novel role for Mre11 in the production of DSB and may help define which tumors are more sensitive to MK-8776 alone or in combination with DNA damaging agents.

Abstract 2040: The Mre11 nuclease is critical for sensitivity of cells to Chk1 inhibition

Abstract The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, thereby providing time for cells to repair their DNA and recover. Chk1 also stabilizes stalled replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be cytotoxic. Using the novel Chk1 inhibitor MK-8776 (previously known as SCH900776), we screened a panel of cell lines and found that some are killed rapidly at low concentrations while other cell lines continue to grow in the presence of much higher concentrations. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon incubation with MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/Nbs1 (MRN) complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11 nuclease activity leads to a decrease not only in the amount of ssDNA following Chk1 inhibition, but also in the formation of DSB, suggesting that DSB arise as a consequence of ssDNA formation. These findings were corroborated by the discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following treatment, but once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when incubated with MK-8776. Inhibition of Chk1 also leads to aberrant activation of Cdc25A and Cdk2, and our findings suggest that Mre11 provides the link between Cdk2 activation and Mus81. We propose that the variable sensitivity of cell lines to Chk1 inhibition reflects defects in this pathway. These data highlight a novel role for Mre11 in the production of DSB and may help define which tumors are more sensitive to MK-8776 alone or in combination with DNA damaging agents. 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 2040. doi:1538-7445.AM2012-2040