Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.

Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

Nuphar alkaloids induce very rapid apoptosis through a novel caspase-dependent but BAX/BAK-independent pathway.

Nuphar alkaloids, originally isolated from water lilies, induce apoptosis in mammalian cells in less than 1 h, making them possibly the fastest known inducers. However, the mechanism by which this rapid apoptosis occurs remains unknown. We have investigated canonical aspects of apoptosis to determine how the nuphar alkaloid, (+)-6-hydroxythiobinupharidine (6HTBN), induces apoptosis. 6HTBN induced rapid apoptosis in various leukemia, lymphoma, and carcinoma cell lines, suggesting that its mechanism is cell-type independent. It also circumvented resistance of patient-derived chronic lymphocytic leukemia cells generated by co-culture on survival-promoting stroma. Intriguingly, 6HTBN failed to induce apoptosis in platelets. The mechanism of apoptosis involves activation of caspase 9 and caspase 3, but not caspase 8 as previously reported. The release of cytochrome c from mitochondria occurred even in the absence of BAX/BAK and in cells that retained mitochondrial membrane potential. These results suggest a novel mechanism of apoptosis that has previously not been reported. The molecular target of the nuphar alkaloids remains to be determined.

Microtubule destabilising agents: far more than just antimitotic anticancer drugs.

Vinca alkaloids have been approved as anticancer drugs for more than 50 years. They have been classified as cytotoxic chemotherapy drugs that act during cellular mitosis, enabling them to target fast growing cancer cells. With the evolution of cancer drug development there has been a shift towards new "targeted" therapies to avoid the side effects and general toxicities of "cytotoxic chemotherapies" such as the vinca alkaloids. Due to their original classification, many have overlooked the fact that vinca alkaloids, taxanes and related drugs do have a specific molecular target: tubulin. They continue to be some of the most effective anticancer drugs, perhaps because their actions upon the microtubule network extend far beyond the ability to halt cells in mitosis, and include the induction of apoptosis at all phases of the cell cycle. In this review, we highlight the numerous cellular consequences of disrupting microtubule dynamics, expanding the textbook knowledge of microtubule destabilising agents and providing novel opportunities for their use in cancer therapy.

Stereoselective Synthesis and Biological Evaluation of C1-Epimeric and Desmethyl Monomeric Nuphar Analogues.

A class of monomeric nuphar analogues that are either epimeric at C1 and C1' or lack the naturally occurring methyl group at those positions were synthesized and evaluated for biological activity. The syntheses feature enantioselective vinylogous Mukaiyama-Mannich (vM-Mannich) reactions catalyzed by chiral phosphoric acids that proceed with excellent diastereoselectivity. Biological assays reveal that both the desmethyl and C1-epimeric monomeric nuphar analogous are able to induce rapid apoptosis.

Enantioselective Formal Syntheses of 11 Nuphar Alkaloids and Discovery of Potent Apoptotic Monomeric Analogues.

Concise, scalable, and enantioselective formal syntheses of eight dimeric and three monomeric nuphar alkaloids were achieved, along with the construction of a stereochemically diverse collection of the first known monomeric analogues having apoptotic activity. The syntheses involved the development of highly enantioselective Brønsted acid catalyzed vinylogous Mukaiyama-Mannich reactions, which feature the unprecedented use of a supersilyl group to control the regio-, enantio- and diastereoselectivity. Biological studies reveal that several of these novel nuphar analogues are even more potent than their dimeric natural product counterparts.

Gossypol increases expression of the pro-apoptotic BH3-only protein NOXA through a novel mechanism involving phospholipase A2, cytoplasmic calcium, and endoplasmic reticulum stress.

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.

Monitoring oxygen levels in orthotopic human glioma xenograft following carbogen inhalation and chemotherapy by implantable resonator-based oximetry.

Hypoxia is a critical hallmark of glioma, and significantly compromises treatment efficacy. Unfortunately, techniques for monitoring glioma pO2 to facilitate translational research are lacking. Furthermore, poor prognosis of patients with malignant glioma, in particular glioblastoma multiforme, warrant effective strategies that can inhibit hypoxia and improve treatment outcome. EPR oximetry using implantable resonators was implemented for monitoring pO2 in normal cerebral tissue and U251 glioma in mice. Breathing carbogen (95% O2 + 5% CO2 ) was tested for hyperoxia in the normal brain and glioma xenografts. A new strategy to inhibit glioma growth by rationally combining gemcitabine and MK-8776, a cell cycle checkpoint inhibitor, was also investigated. The mean pO2 of left and right hemisphere were ∼56-69 mmHg in the normal cerebral tissue of mice. The mean baseline pO2 of U251 glioma on the first and fifth day of measurement was 21.9 ± 3.7 and 14.1 ± 2.4 mmHg, respectively. The mean brain pO2 including glioma increased by at least 100% on carbogen inhalation, although the response varied between the animals over days. Treatment with gemcitabine + MK-8776 significantly increased pO2 and inhibited glioma growth assessed by MRI. In conclusion, EPR oximetry with implantable resonators can be used to monitor the efficacy of carbogen inhalation and chemotherapy on orthotopic glioma in mice. The increase in glioma pO2 of mice breathing carbogen can be used to improve treatment outcome. The treatment with gemcitabine + MK-8776 is a promising strategy that warrants further investigation.

Vincristine activates c-Jun N-terminal kinase in chronic lymphocytic leukaemia in vivo.

AIMS: The authors' aim was to conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukaemia (CLL) cells in patients receiving a vincristine infusion. METHODS: Patients with CLL received 2 mg vincristine by a 5-min intravenous infusion. Blood samples were collected at baseline and up to 6 h after the vincristine infusion, and assayed for JNK activation, Noxa induction and vincristine plasma concentrations. RESULTS: Ex vivo treated peripheral CLL cells activated JNK in response to 10-100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4-6 h after the vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion. CONCLUSIONS: This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation.

Total Syntheses and Biological Evaluation of Both Enantiomers of Several Hydroxylated Dimeric Nuphar Alkaloids.

Herein, we describe the first total syntheses of five members of the dimeric nuphar alkaloids: (+)-6,6'-dihydroxythiobinupharidine (+)-1 a, (+)-6-hydroxythiobinupharidine (+)-1 b, (-)-6,6'-dihydroxythionuphlutine (-)-2 a, (-)-6,6'-dihydroxyneothiobinupharidine (-)-3 a, and (+)-6,6'-dihydroxyneothionuphlutine (+)-4 a. The latter two have not been found in nature. We have also made each of their enantiomers (-)-1 a-b, (+)-2 a, (+)-3 a, and (-)-4 a. The key step in these syntheses was the dimerization of an α-aminonitrile (a hydrolytically stable surrogate for its corresponding hemiaminal) with chiral Lewis acid complexes. We have also reassigned the literature structures of (+)-1 a-1 b­for those instances in which the NMR spectra were obtained in CD3OD­to their corresponding CD3O-adducts. Our efforts provide for the first time apoptosis data for (-)-3 a, (+)-4 a, and all five non-natural enantiomers prepared. The data indicate high apoptotic activity regardless of the enantiomer or relative stereochemical configuration at C7 and C7'.

Chromosomal instability portends superior response of rectal adenocarcinoma to chemoradiation therapy.

BACKGROUND: Persistent chromosome segregation errors represent a conspicuous feature of human neoplasms. It is widely accepted that this chromosomal instability is associated with poor prognosis; however, its effect on therapeutic response is a matter of conjecture. METHODS: Here, the role of chromosome segregation errors in the response of patients with rectal adenocarcinoma to chemoradiation therapy (CRT) was examined. Pretreatment samples from 62 patients were surveyed for evidence of chromosome mis-segregation and mis-segregation frequency was correlated to the pathological response to CRT as determined by the tumor regression grade after surgical resection of irradiated tumors. RESULTS: Surprisingly, it was found that errors in chromosome segregation predicted enhanced pathological response of rectal adenocarcinoma to CRT (odds ratio, 3.9; P = .02). Furthermore, tumor response inversely correlated with the frequency of cells that exhibited segregation errors during anaphase (correlation coefficient, 0.94; P < .05). Strikingly, elevated chromosome mis-segregation combined with decreased levels of the DNA damage repair protein Mre11 portended a markedly enhanced response (odds ratio, 54.0; P = .008). CONCLUSIONS: The results of the current study demonstrate that chromosomal instability is a favorable predictor of response to CRT in patients with locally invasive rectal adenocarcinoma. Therefore, the authors propose that downstream structural damage to chromosomes resulting from segregation errors potentiates the effect of DNA-damaging therapies and synergizes with deficiencies in the DNA repair machinery. This work identifies a novel mechanistic marker that foretells treatment response to CRT and suggests that concomitant targeting of whole-chromosome segregation and DNA repair may constitute an effective therapeutic strategy.

The putative BH3 mimetic S1 sensitizes leukemia to ABT-737 by increasing reactive oxygen species, inducing endoplasmic reticulum stress, and upregulating the BH3-only protein NOXA.

S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.

Skeletal muscle and glioma oxygenation by carbogen inhalation in rats: a longitudinal study by EPR oximetry using single-probe implantable oxygen sensors.

The feasibility of EPR oximetry using a single-probe implantable oxygen sensor (ImOS) was tested for repeated measurement of pO2 in skeletal muscle and ectopic 9L tumors in rats. The ImOS (50 mm length) were constructed using nickel-chromium alloy wires, with lithium phthalocyanine (LiPc, oximetry probe) crystals loaded in the sensor loop and coated with AF 2400(®) Teflon. These ImOS were implanted into the skeletal muscle in the thigh and subcutaneous 9L tumors. Dynamic changes in tissue pO2 were assessed by EPR oximetry at baseline, during tumor growth, and repeated hyperoxygenation with carbogen breathing. The mean skeletal muscle pO2 of normal rats was stable and significantly increased during carbogen inhalation in experiments repeated for 12 weeks. The 9L tumors were hypoxic with a tissue pO2 of 12.8 ± 6.4 mmHg on day 1; however, the response to carbogen inhalation varied among the animals. A significant increase in the glioma pO2 was observed during carbogen inhalation on day 9 and day 14 only. In summary, EPR oximetry with ImOS allowed direct and longitudinal oxygen measurements in deep muscle tissue and tumors. The heterogeneity of 9L tumors in response to carbogen highlights the need to repeatedly monitor pO2 to confirm tumor oxygenation so that such changes can be taken into account in planning therapies and interpreting results.

Recurrent low-dose chemotherapy to inhibit and oxygenate head and neck tumors.

A lack of strategy to counteract hypoxia (pO2 < 10-15 mmHg) and technique to repeatedly measure tumor pO2 has restricted therapeutic optimization. We report the results obtained with an innovative anti-angiogenic strategy of recurrent low-dose (metronomic) chemotherapy to modulate hypoxia and growth of the Head and Neck tumor xenografts.The FaDu tumors were established in the flank of immune deficient mice and EPR oximetry with lithium phthalocyanine crystals was used to follow the temporal changes in tumor pO2 on treatment with gemcitabine including controls for three weeks. The FaDu tumors were hypoxic with a baseline (pre-treatment) pO2 of 2-8 mmHg. A transient increase in the tumor pO2 was evident on day 3 on treatment with a conventional schedule of gemcitabine (150 mg/kg, d1, d8, d15). No significant change in the tumor pO2 on treatment with metronomic gemcitabine (25 mg/kg on d1, d3, d5 for 3 weeks) was observed. However, tumor pO2 increased significantly on d15-d18 during treatment with a metronomic schedule of 15 mg/kg gemcitabine (d1, d3, d5 for 3 weeks). A modest decrease in the tumor growth was evident on treatment with conventional gemcitabine. Notably, tumor growth was significantly inhibited by metronomic (25 and 15 mg/kg) gemcitabine treatment. The immunohistochemistry (IHC) analyses of the tumor samples indicate a decrease in HIF-1α and TSP-1 on treatment with metronomic gemcitabine.In conclusion, a significant inhibition of tumor growth on treatment with metronomic gemcitabine was observed; however, the increase in pO2 was dose dependent. EPR oximetry can be used to follow the temporal changes in tumor pO2 to identify a therapeutic window on treatment with metronomic chemotherapy for potential combination with radiotherapy.

A proof of principle clinical trial to determine whether conjugated linoleic acid modulates the lipogenic pathway in human breast cancer tissue.

Conjugated linoleic acid (CLA) is widely used as a "nutraceutical" for weight loss. CLA has anticancer effects in preclinical models, and we demonstrated in vitro that this can be attributed to the suppression of fatty acid (FA) synthesis. We tested the hypothesis that administration of CLA to breast cancer patients would inhibit expression of markers related to FA synthesis in tumor tissue, and that this would suppress tumor proliferation. Women with Stage I-III breast cancer were enrolled into an open label study and treated with CLA (1:1 mix of 9c,11t- and 10t,12c-CLA isomers, 7.5 g/d) for ≥ 10 days before surgery. Fasting plasma CLA concentrations measured pre- and post-CLA administration, and pre/post CLA tumor samples were examined by immunohistochemistry for Spot 14 (S14), a regulator of FA synthesis, FA synthase (FASN), an enzyme of FA synthesis, and lipoprotein lipase (LPL), the enzyme that allows FA uptake. Tumors were also analyzed for expression of Ki-67 and cleaved caspase 3. 24 women completed study treatment, and 23 tumors were evaluable for the primary endpoint. The median duration of CLA therapy was 12 days, and no significant toxicity was observed. S14 expression scores decreased (p = 0.003) after CLA administration. No significant change in FASN or LPL expression was observed. Ki-67 scores declined (p = 0.029), while cleaved caspase 3 staining was unaffected. Decrements in S14 or Ki-67 did not correlate with fasting plasma CLA concentrations at surgery. Breast tumor tissue expression of S14, but not FASN or LPL, was decreased after a short course of treatment with 7.5 g/day CLA. This was accompanied by reductions in the proliferation index. CLA consumption was well-tolerated and safe at this dose for up to 20 days. Overall, CLA may be a prototype compound to target fatty acid synthesis in breast cancers with a "lipogenic phenotype".

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.

BACKGROUND: Chk1 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. METHODS: Growth 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. RESULTS: MK-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. CONCLUSIONS: There 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.

The cancer therapeutic potential of Chk1 inhibitors: how mechanistic studies impact on clinical trial design.

Many anticancer agents damage DNA and activate cell cycle checkpoints that permit time for the cells to repair their DNA and recover. These checkpoints have undergone intense investigation as potential therapeutic targets and Chk1 inhibitors have emerged as promising novel therapeutic agents. Chk1 was initially recognized as a regulator of the G2/M checkpoint, but has since been demonstrated to have additional roles in replication fork stability, replication origin firing and homologous recombination. Inhibition of these pathways can dramatically sensitize cells to some antimetabolites. Current clinical trials with Chk1 inhibitors are primarily focusing on their combination with gemcitabine. Here, we discuss the mechanisms of, and emerging uses for Chk1 inhibitors as single agents and in combination with antimetabolites. We also discuss the pharmacodynamic issues that need to be addressed in attaining maximum efficacy in vivo. Following administration of gemcitabine to mice and humans, tumour cells accumulate in S phase for at least 24 h before recovering. In addition, stalled replication forks evolve over time to become more Chk1 dependent. We emphasize the need to assess cell cycle perturbation and Chk1 dependence of tumours in patients administered gemcitabine. These assessments will define the optimum dose and schedule for administration of these drug combinations.

Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA.

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Variations in Mre11/Rad50/Nbs1 status and DNA damage-induced S-phase arrest in the cell lines of the NCI60 panel.

BACKGROUND: The Mre11/Rad50/Nbs1 (MRN) complex is a regulator of cell cycle checkpoints and DNA repair. Defects in MRN can lead to defective S-phase arrest when cells are damaged. Such defects may elicit sensitivity to selected drugs providing a chemical synthetic lethal interaction that could be used to target therapy to tumors with these defects. The goal of this study was to identify these defects in the NCI60 panel of cell lines and identify compounds that might elicit selective cytotoxicity. METHODS: We screened the NCI60 panel in search of cell lines that express low levels of MRN proteins, or that fail to arrest in S-phase in response to the topisomerase I inhibitor SN38. The NCI COMPARE program was used to discover compounds that preferentially target cells with these phenotypes. RESULTS: HCT116 cells were initially identified as defective in MRN and S phase arrest. Transfection with Mre11 also elevated Rad50 and Nbs1, and rescued the defective S-phase arrest. Cells of the NCI60 panel exhibited a large range of protein expression but a strong correlation existed between Mre11, Rad50 and Nbs1 consistent with complex formation determining protein stability. Mre11 mRNA correlated best with protein level suggesting it was the primary determinant of the overall level of the complex. Three other cell lines failed to arrest in response to SN38, two of which also had low MRN. However, other cell lines with low MRN still arrested suggesting low MRN does not predict an inability to arrest. Many compounds, including a family of benzothiazoles, correlated with the failure to arrest in S phase. The activity of benzothiazoles has been attributed to metabolic activation and DNA alkylation, but we note several cell lines in which sensitivity does not correlate with metabolism. We propose that the checkpoint defect imposes an additional mechanism of sensitivity on cells. CONCLUSIONS: We have identified cells with possible defects in the MRN complex and S phase arrest, and a series of compounds that may preferentially target S phase-defective cells. We discuss limitations of the COMPARE program when attempting to identify compounds that selectively inhibit only a few cell lines.

Comparative Activity and Off-Target Effects in Cells of the CHK1 Inhibitors MK-8776, SRA737, and LY2606368.

DNA damage activates the checkpoint protein CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of CHK1 (CHK1i) enhance damage-induced cell death. Additionally, CHK1i elicits single agent cytotoxicity in some cell lines. We compared three CHK1i that have undergone clinical trials and exhibited different toxicities. Each CHK1i inhibits other targets at higher concentrations, and whether these contribute to the toxicity is unknown. We compared their sensitivity in a panel of cell lines, their efficacy at inhibiting CHK1 and CHK2, and their ability to induce DNA damage and abrogate damage-induced S phase arrest. Published in vitro kinase analyses were a poor predictor of selectivity and potency in cells. LY2606368 was far more potent at inhibiting CHK1 and inducing growth arrest, while all three CHK1i inhibited CHK2 at concentrations 10- (MK-8776 and SRA737) to 100- (LY2606368) fold higher. MK-8776 and SRA737 exhibited similar off-target effects: higher concentrations demonstrated transient protection from growth inhibition, circumvented DNA damage, and prevented checkpoint abrogation, possibly due to inhibition of CDK2. Acquired resistance to LY2606368 resulted in limited cross-resistance to other CHK1i. LY2606368-resistant cells still abrogated DNA damage-induced S phase arrest, which requires low CDK2 activity, whereas inappropriately high CDK2 activity is responsible for sensitivity to CHK1i alone. All three CHK1i inhibited protein synthesis in a sensitive cell line correlating with cell death, whereas resistant cells failed to inhibit protein synthesis and underwent transient cytostasis. LY2606368 appears to be the most selective CHK1i, suggesting that further clinical development of this drug is warranted.