Zhan Xiao

Selective Chk1 inhibitors differentially sensitize p53-deficient cancer cells to cancer therapeutics

The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA‐damaging agents. We have previously shown that siRNA‐mediated Chk1 knockdown abrogates DNA damage‐induced checkpoints and potentiates the cytotoxicity of several DNA‐damaging agents in p53‐deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A‐690002 and A‐641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and γ‐radiation in p53‐deficient but not in p53‐proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.

Differential roles of checkpoint kinase 1, checkpoint kinase 2, and mitogen-activated protein kinase–activated protein kinase 2 in mediating DNA damage–induced cell cycle arrest: implications for cancer therapy

Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus preserving their genomic integrity. The protein kinases checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and mitogen-activated protein kinase–activated protein kinase 2 (MK2) have all been shown to be involved in cell cycle checkpoint control. Recently, cell cycle checkpoint abrogation has been proposed as one way to sensitize cancer cells to DNA-damaging agents due to the expected induction of mitotic catastrophe. Due to their overlapping substrate spectra and redundant functions, it is still not clear which kinase is mainly responsible for the cell cycle arrests conferred by clinically relevant chemotherapeutics. Thus, the issue remains about which kinase is the most therapeutically relevant target and, more importantly, whether multiple kinases might need to be targeted to achieve the best efficacy in light of recent studies showing superior efficacy for pan-receptor tyrosine kinase inhibitors. To clarify this issue, we investigated the roles of the three kinases in response to different genotoxic stresses through small interfering RNA–mediated specific target knockdowns. Our result showed that only the down-regulation of Chk1, but not of Chk2 or MK2, abrogated camptothecin- or 5-fluorouracil–induced S-phase arrest or doxorubicin-induced G2-phase arrest. This was followed by mitotic catastrophe and apoptosis. Moreover, double inhibition of Chk1 and Chk2 failed to achieve better efficacy than Chk1 inhibition alone; surprisingly, inhibition of MK2, in addition to Chk1 suppression, partially reversed the checkpoint abrogation and negated mitotic catastrophe. We further showed that this is due to the fact that in MK2-deficient cells, Cdc25A protein, which is critically required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other checkpoint kinases in addition to Chk1 would be nonproductive. [Mol Cancer Ther 2006;5(8):1935–43]

A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation.

Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30–60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.

Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics

Paclitaxel (Taxol) is the most‐prescribed anti‐mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell‐cycle checkpoint kinase mediating S‐ and G2‐arrests in response to various DNA‐damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA‐damaging agents through abrogation of cell‐cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel‐treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel‐induced M‐phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M‐phase marker phospho‐histone H3. We show that Chk1 elimination attenuates the paclitaxel‐induced activation of the anti‐apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well‐established role as an enforcer of S and G2‐checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell‐death. Therefore Chk1 downregulation can not only potentiate DNA‐damaging agents, but also enhance the toxicity of anti‐microtubule agents, which significantly broadens its therapeutic applications.

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Abstract Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.

Investigation of novel 7,8-disubstituted-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones as potent Chk1 inhibitors

The synthesis and structure-activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded compounds with improved enzymatic and cellular potency.

Chk1 and Chk2 as Checkpoint Targets

DNA damage checkpoint response is initiated to arrest cell cycle progression so that DNA repair can take place and, thereby, prevents accumulation and propagation of DNA damage. In checkpoint response, Chk1 and Chk2 have major roles, primarily in arresting cells in S and G2 phase of the cell cycle. However, Chk1 appears to be the more critical player, with Chk2 playing an accessory role. Since S and G2 arrest are pro-survival events, a major effort has been devoted to the development of Chk1 inhibitors, some of which have entered clinical trials as chemo- and radio-sensitizers. On the other hand, Chk2 potentiates p53-dependent apoptosis, and inhibitors targeting this kinase may afford protection against normal tissue injury during cancer therapy.