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Chk1 Inhibition as a novel therapeutic strategy for treating triple-negative breast and ovarian cancers
© Bryant et al.; licensee BioMed Central Ltd. 2014
Received: 1 May 2014
Accepted: 28 July 2014
Published: 7 August 2014
Chk1 inhibitors are currently in clinical trials as putative potentiators of cytotoxic chemotherapy drugs. Chk1 inhibitors may exhibit single agent anti-tumor activity in cancers with underlying DNA repair, DNA damage response or DNA replication defects.
Here we describe the cellular effects of the pharmacological inhibition of the checkpoint kinase Chk1 by the novel inhibitor V158411 in triple-negative breast cancer and ovarian cancer. Cytotoxicity, the effect on DNA damage response and cell cycle along with the ability to potentiate gemcitabine and cisplatin cytotoxicity in cultured cells was investigated. Western blotting of proteins involved in DNA repair, checkpoint activation, cell cycle and apoptosis was used to identify potential predictive biomarkers of Chk1 inhibitor sensitivity.
The Chk1 inhibitors V158411, PF-477736 and AZD7762 potently inhibited the proliferation of triple-negative breast cancer cells as well as ovarian cancer cells, and these cell lines were sensitive compared to ER positive breast and other solid cancer cells lines. Inhibition of Chk1 in these sensitive cell lines induced DNA damage and caspase-3/7 dependent apoptosis. Western blot profiling identified pChk1 (S296) as a predictive biomarker of Chk1 inhibitor sensitivity in ovarian and triple-negative breast cancer and pH2AX (S139) in luminal breast cancer.
This finding suggests that Chk1 inhibitors either as single agents or in combination chemotherapy represents a viable therapeutic option for the treatment of triple-negative breast cancer. pChk1 (S296) tumor expression levels could serve as a useful biomarker to stratify patients who might benefit from Chk1 inhibitor therapy.
KeywordsChk1 Breast cancer Ovarian cancer V158411 DNA repair Triple-negative
Breast cancer is the most common cancer affecting women and the second most common cause of death due to cancer . This highly heterogeneous disease has been classified into various subgroups (namely luminal, HER2 positive, basal-like and normal breast) based on gene expression profiles and phenotypic characteristics . Triple-negative breast cancer (TNBC) is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR) and ErbB-2/human epidermal growth factor receptor 2 (HER2) [3, 4]. TNBC shares many of the gene expression profiles and phenotypical features of basal-like breast cancer [5, 6]. The prognosis for breast cancer patients with basal-like disease, including TNBC, is generally much poorer than those of other sub-groups due to the aggressive nature of this sub-group. Whilst basal-like and triple-negative breast cancers have been demonstrated to be chemo-sensitive to neoadjuvant therapy, the relapse rates are more aggressive resulting in a worse overall survival [7, 8]. Unlike ER, PR or HER2 positive tumors where clear molecular targets for therapeutic intervention exist, identifying specific molecular targets for TNBC has proved more elusive.
Breast tumors arising from hereditary BRCA mutation carriers are mostly triple-negative and share a striking resemblance to sporadic basal-like and triple-negative breast cancers suggesting that TNBC might bear defects in DNA repair thereby endowing a degree of “BRCAness” on these cancers . BRCA mutant cell lines have been shown to be sensitive to DNA damaging agents such as cisplatin  as well as inhibitors of the repair protein poly (ADP-ribose) polymerase (PARP), a key component of the base excision DNA repair pathway [11, 12]. Recent studies have shown that basal-like TNBC cell lines are also sensitive to PARP inhibitors and cytotoxic chemotherapy drugs such as gemcitabine and cisplatin [13, 14]. Inhibition of PARP in cancer cells with defects in DNA double strand break (DSB) repair pathways (such as BRCA mutant breast cancer) results in an accumulation of single strand breaks (SSBs) leading to replication fork collapse, DSB generation and cell death. This reliance on alternative DNA repair pathways may make cancers such as TNBC sensitive to other agents that target DNA damage response pathways.
The DNA damage signaling response pathway (DDR) is activated by DNA breaks induced by a variety of endogenous and external insults including therapies currently used for the treatment of cancer such as ionizing radiation and cytotoxic chemotherapeutic agents such as gemcitabine, irinotecan and cisplatin. Activation of the DDR results in a number of cellular responses including checkpoint activation and cell cycle arrest, initiation of DNA repair, regulation of transcription and apoptosis [15, 16]. The serine-threonine checkpoint kinases Chk1 and Chk2 are activated by the ATR and ATM kinases in response to DNA breaks and form the key link between the sensing kinases ATR/ATM and the cell cycle machinery . Phosphorylation of Chk1 leads to checkpoint activation and cell cycle arrest at the G1/S, intra S or G2/M phases. Despite their similarity in name, Chk1 and Chk2 differ substantially in the structure of their kinase pocket [18, 19] and in their cellular function with Chk1 suggested to be the major component responsible for responses to DNA damage. Inhibiting Chk1 following genotoxic stress (such as that induced by cytotoxic chemotherapy) results in checkpoint abrogation, inhibition of DNA repair and induction of cell death in cells with a defective p53 response [20, 21]. Small molecule inhibitors of predominantly the Chk1 kinase have been readily sought as a mechanism through which the anti-tumor activity of cytotoxic chemotherapeutics may be increased whilst sparing normal cells [22, 23]. This approach is currently being tested in the clinic with a variety of agents including LY2603618 , GDC-0425, GDC-0575, MK-8776 , PF-477736  and AZD7762  in combination with a range of standard of care chemotherapy drugs.
It has also been postulated that DNA damage response checkpoints and especially Chk1 kinase activity may be critical for the normal replication of cancer cell lines with specific underlying defects in DNA repair or DNA damage response pathways, or DNA replication defects. For example, the Fanconi Anaemia (FA) repair pathway is responsible for repairing crosslinked DNA and maintaining chromosomal stability . FA deficient cell lines were found to be sensitive to Chk1 silencing by siRNA compared to FA proficient cells . We hypothesized that, given their likeness to DNA repair compromised hereditary BRCA mutated breast cancers, triple-negative breast cancer may be sensitive to cell killing by Chk1 inhibitors.
V158411 is a novel, potent, selective inhibitor of recombinant Chk1 and Chk2 kinases in vitro with IC50s of 3.5 and 2.5nM respectively . In p53 defective HT29 cells, V158411 inhibited the etoposide induced auto-phosphorylation of Chk1 on Ser296 with an IC50 of 48 nM and Chk2 on Ser516 with an IC50 of 904 nM indicating a 19-fold cellular selectivity for Chk1 over Chk2. V158411 potentiated cytotoxic chemotherapy in p53 defective cancer cells in vitro and in vivo. We therefore evaluated the single agent cytotoxic potential of V158411 against a panel of solid cancer cell lines including those derived from breast and ovarian cancer. We further profiled the panel of cell lines to understand and identify potential biomarkers predictive of response to Chk1 inhibition. The data provides a preclinical rationale to support the clinical testing of Chk1 inhibitors as single agents and in combination with cytotoxic chemotherapy in patients with triple-negative breast cancer.
Cell culture and cytotoxicity assay
All cells were obtained from the American Type Culture Collection and cultured in DMEM, RPMI or McCoys 5a containing 10% FCS (Invitrogen). The cytotoxicity of V158411 was determined following exposure of cells in 96 well plates to a 10-point titration for 72 hours. Cell proliferation was determined using sulphorhodamine B (Sigma) staining following protein precipitation with 10% TCA. For cell counts, cells were seeded in 6 well plates and counted following trypsinisation after 72 hours using a haemocytometer with trypan blue staining.
V158411 was synthesized according to the method described in  and prepared as a 20 mM DMSO stock in DMSO. Solid stocks were purchased from the indicated suppliers and prepared as concentrated stock solutions in the appropriate solvent: gemcitabine (Apin Chemicals Inc), 20 mM in H2O; cisplatin (Selleckchem), 3.33 mM in 1% NaCl in H2O; oxaliplatin (Tocris), 5 mM in H2O; carboplatin (Tocris), 25 mM in H2O; PF-477736 (Selleckchem), 20 mM in DMSO and AZD7762 (Axon Medchem), 20 mM in DMSO.
Determination of caspase-3/7 dependent apoptosis
Cells were seeded in 96 well plates and treated with 10-times the GI50 of V158411 for 24 or 48 hours. Caspase-3/7 activity was determined using a homogenous caspase-3/7 luminescence kit (Promega).
Antibodies and western blotting
Anti-pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), pChk2, pChk2 (T68), pCdc25c (S216), 53BP1, Cdc2, pCdc2 (Y15), Cyclin B1, D1 and E, PARP, pERK1/2, ERK 1/2, AKT, pAKT (S473), Bcl-XL, GAPDH and pH2AX (S139) from Cell Signaling Technologies; pChk1 (S296), FANCF and FANCD2 from Abcam, and Bcl-2 and Mcl-1 from Santa Cruz. Treated and untreated cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.
Cells were seeded in 6 well plates and subsequently treated with the indicated concentrations of V158411 for 24 or 48 hours. All cells were harvested, fixed in 70% ethanol and stained with propidium iodide/RNase A. Cell cycle profiles were examined by flow cytometry using a FACSArray cytometer (BD) and FACSDiva software (BD).
5x103 cells per well were seeded in 96 well plates and incubated overnight. Cells were treated with a 10-point titration of gemcitabine or cisplatin in the presence of a fixed concentration of V158411 for 72 hours. The effect on cell proliferation was determined using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega).
None of the research in this manuscript involved human subjects, human material, or human data, or used regulated vertebrates or invertebrates.
Pharmacological abrogation of Chk1 activity inhibits cell proliferation and induces caspase activation in human triple-negative breast and ovarian cancer cell lines
Sporadic basal-like or triple-negative breast cancers strongly resemble breast cancers originating from hereditary BRCA defective patients and may harbor BRCA-mimetic defects in DNA repair pathways. To test the hypothesis that triple-negative breast cancers are sensitive to Chk1 inhibitors in the absence of cytotoxic chemotherapy agents, a panel of sporadic triple-negative and luminal breast cancer cell lines  as well as several ovarian cancer cell lines were tested for their sensitivity to V158411 and compared against a panel of cell lines derived from cancers of the lung, colon or prostate.
Breast and ovarian cell lines used in this study and their sensitivity to V158411
2.6 ± 1.3
0.11 ± 0.01
2.0 ± 0.5
0.25 ± 0.02
0.18 ± 0.02
0.019 ± 0.005
0.31 ± 0.02
0.022 ± 0.003
0.39 ± 0.05
3.1 ± 1.2
0.06 ± 0.02
Chk1 inhibition induces Chk1 degradation and H2AX phosphorylation
Chk1 inhibition induces cell cycle arrest and DNA fragmentation
Western blot profiling of breast and ovarian cell lines identified Chk1 Ser296 phosphorylation as a predictive biomarker of sensitivity
V158411 potentiates cytotoxic chemotherapy in TNBC and ovarian cancer cell lines
Multiple Chk1 inhibitors are currently undergoing clinical testing in combination with a variety of cytotoxic chemotherapeutic agents for their ability to potentiate the anti-tumor efficacy of the chemotherapy drugs whilst not increasing the systemic toxicity of these drugs. Recent work using loss-of-function siRNA screens or small molecule Chk1 inhibitors have begun to identify tumor types where Chk1 is critical for cancer cell proliferation and/or viability in the absence of a DNA damaging chemotherapeutic agent. To date, neuroblastoma , melanoma , Myc driven lymphomas [35, 36], leukemia and lymphoma cell lines [37, 38] and Fanconi’s Anemia cells  have been identified to be Chk1 kinase dependent. This suggests that there may be subsets of cancers for which a Chk1 inhibitor, administered as a single agent, could be a useful therapeutic option.
We postulated that cancers with underlying defects in DNA repair, DNA damage response or DNA replication may be suitable candidates for single agent Chk1 inhibitor therapy. Spontaneous triple-negative breast cancer shares many of the characteristics of tumors derived from patients carrying mutations in the BRCA gene. BRCA is known to be involved in a variety of DNA repair pathways such as homologous recombination and base excision repair and are exquisitely sensitive to inhibitors of poly (ADP-ribose) polymerase. Recent reports have demonstrated the sensitivity of TNBC to PARP inhibitors as well as DNA damaging cytotoxic agents such as gemcitabine and cisplatin . TNBCs have been shown to have reduced expression of DNA repair genes involved in base excision repair, nucleotide excision repair and the Fanconi’s Anemia repair pathways . This suggests that like cancers that arise in BRCA mutation carriers, spontaneous TNBCs may harbor underlying defects in DNA repair and we hypothesized that this cancer sub-type may be a suitable candidate for single agent Chk1 inhibitor therapy.
This hypothesis was substantiated in a screen of 26 solid cancer cell lines where TNBC cancer cell lines were among the most sensitive to growth inhibition by the novel, selective Chk1 inhibitor V158411. Even though V158411 is an extremely selective inhibitor of Chk1, it is difficult to ascertain the absolute selectivity of any small molecule kinase inhibitor. To confirm this observation, two structurally unrelated Chk1 kinase inhibitors PF-477736  and AZD7762  potently inhibited the proliferation of TNBC breast cancer cell lines compared to two ER-positive cell lines suggesting that the anti-proliferative effects observed were due to Chk1 inhibition and not the inhibition of an off target kinase. However, the sensitivity to the Chk1 inhibitors was not just limited to the TNBC cell lines as the HER2-postive, ER-negative SKBr3 breast cancer cell line and the SKOV-3 ovarian cancer cell line (HER2-positve, ER-positive but estrogen insensitive ) were among the most sensitive cell lines to Chk1 inhibitor induced cell death. These results have recently been confirmed by another study demonstrating that four TNBC cell lines (including HCC1937, MDA-MB-157 and MDA-MB-468 used in this study) had reduced viability following Chk1 knockdown with a specific siRNA . Additional studies have demonstrated that the Chk1 inhibitor AZD7762 synergistically combined with numerous PARP1 inhibitors (including olaparib, rucaparib or ABT888) to inhibit the growth of mammary carcinoma cells in vitro and in vivo[42, 43]. In these studies, AZD7762 demonstrated little single agent activity in the breast cancer cell lines at the concentration tested. Of the cell lines tested in these papers, only two overlapped with our study.
In light of these results, we attempted to understand the mechanism by which single agent Chk1 inhibitors induced TNBC and ovarian cancer cell death. The clearest marker of response to Chk1 inhibition in all the sensitive cell lines was the time dependent increase in phosphorylation of H2AX on serine 139. Phosphorylation on this site is generally associated with an increase in DNA double strand breaks . Coupled with Chk1 inhibition reducing cell viability and inducing caspase-3/7 dependent apoptosis and DNA fragmentation, cell death following Chk1 inhibition appears to be most likely via increased DNA double strand breaks. The mechanism for the generation of these breaks is not completely clear. However, since Chk1 inhibition caused a dramatic decrease in the fraction of cells in G1 that were unable to complete S-phase and accumulate in mitosis suggests that replication fork collapse and the subsequent formation of DSBs by the DNA endonuclease Mus81/Eme1  is responsible.
In all breast and ovarian cancer cell lines, including those relatively resistant to V158411 single agent cytotoxicity, reduction in total Chk1 protein levels following V158411 treatment was evident. This was especially notable at the higher concentrations of V158411 and appeared to correlate with an increase in pH2AX (S139) in the V158411 sensitive cell lines. V158411 treatment of leukemia and lymphoma cell lines induced Chk1 degradation that was proteasome dependent  whilst degradation of Chk1 was observed in HT29 cells following treatment with four structurally distinct Chk1 inhibitors in combination with gemcitabine: V158411, LY2603618, MK-8776 and GNE-900 . Chk1 is deactivated through protein degradation in response to replication and genotoxic stress. Phosphorylation of Chk1 at serine 317 and 345 by ATR promotes Chk1 activation but also induces the ubiquitin-proteasome dependent degradation of Chk1 [47–49]. Given that V158411 induces phosphorylation of Chk1 at serine 317 and 345 in breast and ovarian cancer cells, this degradation of Chk1 reflects the normal homeostatic mechanism of checkpoint resetting.
The precise mechanism for the sensitivity of the TNBC and ovarian cancer cell lines compared to other solid cancer cell lines remains to be fully understood. The resistance of the two ER-positive breast cancer cell lines BT474 and MCF-7 could not be overcome with 4-hydroxytamoxfien suggesting that estrogen receptor signaling did not contribute to the relative sensitivities in the breast cancer cell lines. Chk1 expression has been demonstrated to be elevated in histological grade 3 TNBC primary tumors compared to other grade 3 breast cancers . Sensitivity of the TNBC and ovarian cancer cell lines did not correlate with total Chk1 protein expression levels but did correlate closely with the levels of phosphorylation of Chk1 on serine 296 and to a lesser extent serine 317 but not with serine 345. This observation matched that of Cole et al. who identified neuroblastoma as a potential therapeutic target for Chk1 inhibition and that sensitivity to Chk1 inhibition by either siRNA or small molecules correlated with Chk1 S296 phosphorylation. The HER2 positive breast cancer cell line SKBr3 was extremely sensitive to growth inhibition by all three Chk1 inhibitors tested. Whilst this cell line had low levels of Chk1 phosphorylation at serine 296, it was the only cell line with high expression of pH2AX (S139) potentially indicative of a reasonable level of DNA breakage in proliferating cells. Shibata et al.,  identified elevated expression levels of pChk1 (S317) and to a lesser extent pH2AX (S139) as being predictive of the sensitivity of breast cancer cell lines to the Chk1 inhibitor PF-477736. Sensitivity to V158411 appeared independent of both p53 and kRas mutational status, both of which have previously been implicated in Chk1’s mechanism of action . The outlier in this analysis was the ovarian cancer cell line ES-2. This cell line had high expression levels of pChk1 (S296) but was relatively resistant to growth inhibition by all three Chk1 inhibitors. Further work is needed to understand the relative resistance of this cell line to Chk1 inhibition.
The underlying mechanism for the sensitivity of these cancer cell types to single Chk1 inhibitor therapy is not yet clear and the phosphorylation events identified as potential predictive markers of sensitivity (pChk1 (S296), (S317) and pH2AX (S139)) are most likely symptomatic rather than the cause of the underlying sensitivity. This observation suggests that the molecular defects in these cell lines occur in pathways for which Chk1 can mutually compensate to protect genomic integrity and therefore Chk1 inhibition is lethal. An example of this so far discovered is the Fanconi’s Anemia (FA) DNA repair pathway. Cells defective in FA were sensitive to Chk1 siRNA and the small molecule Go6976 due to an accumulation of unrepairable DNA double strand breaks . The basal like breast cancer cell line HCC9137 harbors a homozygous truncation mutation in the DNA repair gene BRCA1 and this reduced capacity to repair DNA breaks may underlie this cell lines sensitivity. Underlying defects in DNA repair would be predicted to confer increased sensitivity to DNA damaging cytotoxic drugs such as cisplatin. The correlation between sensitivity to cisplatin and V158411 was cell line dependent and not consistent across the panel of breast and ovarian lines studied. For example, the BRCA defective cell line HCC1937 was highly sensitive to cisplatin and V158411 whilst the ovarian cell line SKOV-3 was equally sensitive to V158411 but 8-fold more resistant to cisplatin than the HCC1937 cell line. This suggests that different mechanisms may account for the V158411 sensitivity in different cell lines.
Cells under replicative stress due to oncogene amplification or activation of oncogenic signaling pathways are addicted to Chk1 kinase activity for the completion of a normal S-phase [35, 36]. The sensitivity of neuroblastoma and melanoma cell lines has been suggested to be likely related to oncogenic replicative stress. In the case of neuroblastoma, this has been linked to members of the Myc family of oncogenes [33, 34]. Therefore the sensitivity of the breast and ovarian cell lines in this study could be attributed to underlying defects in DNA repair or DNA damage signaling pathways, oncogene induced replicative stress or a combination of the two. Chk1 has been demonstrated to be important for replication origin firing, high rates of replication fork progression and replication fork stabilization [34, 54, 55]]. Inhibition of Chk1 may result in increased replication origin firing and reduced fork progression leading to increased regions of RPA bound ssDNA. This in turn leads to ATR activation and tumor cell apoptosis.
Triple-negative status was not a sufficient prognostic marker for response to Chk1 inhibition as other cell line types such as the HER2 positive SKBr3 breast cancer cell line and SKOV-3 ovarian cancer cell line were among the most sensitive cell lines to growth inhibition by V158411. For this treatment to be better focused in the clinic to those patients most likely to benefit then additional biomarkers prognostic of sensitivity would be extremely beneficial. High expression of pChk1 (S296) in untreated tumors and the induction of pH2AX (S139) post Chk1 inhibitor therapy appear to be the most sensitive and predictive markers for response to Chk1 inhibitor therapy. However, phospho-protein profiling of tumor biopsies by immunohistochemistry or western blotting is not without its technical challenges. Alternative methods of identifying potential clinical responders, such as gene signature profiles, would aid the use of Chk1 inhibitors as monotherapy.
From this study and others, it is becoming clear that Chk1 inhibitors may have clinical utility as a single agent as well as in combination with cytotoxic chemotherapy agents in a variety of human cancer types. Chk1 inhibitors either as single agents or in combination with cytotoxic chemotherapy are a potentially viable therapeutic option for the treatment of triple-negative breast cancer in the clinic. High tumor expression of pChk1 (S296) could serve as a useful biomarker to select those patients who would most likely benefit from Chk1 inhibitor therapy.
Clinical testing of Chk1 inhibitors is currently focused on their ability to potentiate the anti-tumor efficacy of cytotoxic chemotherapy drugs and anti-metabolite therapies. Recent studies have demonstrated single agent activity of Chk1 inhibitors in cancer cells harboring defects in DNA damage repair or response pathways or high levels of replicative stress. V158411 either as a single agent or in combination with cytotoxic chemotherapy are potentially viable therapeutic options for the treatment of triple-negative breast cancer in the clinic. High tumor expression of pChk1 (S296) could serve as a useful biomarker to select those patients who would most likely benefit from Chk1 inhibitor therapy. The BRCAness of triple-negative breast cancer may underlie the sensitivity of this cancer type to Chk1 inhibition.
We thank Dr Mark Christie (Akranim Ltd) for critically reading and revising the manuscript for intellectual content. This work was funded by Vernalis R&D Ltd who approved the manuscript for publication.
- Feng Z, Xu S, Liu M, Zeng YX, Kang T: Chk1 inhibitor Go6976 enhances the sensitivity of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy in vitro and in vivo. Cancer Lett. 2010, 297: 190-197.View ArticlePubMedGoogle Scholar
- Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de RM, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein LP, Borresen-Dale AL: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001, 98: 10869-10874.View ArticlePubMedPubMed CentralGoogle Scholar
- Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N: Triple-negative breast cancer–current status and future directions. Ann Oncol. 2009, 20: 1913-1927.View ArticlePubMedGoogle Scholar
- Bosch A, Eroles P, Zaragoza R, Vina JR, Lluch A: Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research. Cancer Treat Rev. 2010, 36: 206-215.View ArticlePubMedGoogle Scholar
- Foulkes WD, Stefansson IM, Chappuis PO, Begin LR, Goffin JR, Wong N, Trudel M, Akslen LA: Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst. 2003, 95: 1482-1485.View ArticlePubMedGoogle Scholar
- Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S, Bishop T, Benitez J, Rivas C, Bignon YJ, Chang-Claude J, Hamann U, Cornelisse CJ, Devilee P, Beckmann MW, Nestle-Kramling C, Daly PA, Haites N, Varley J, Lalloo F, Evans G, Maugard C, Meijers-Heijboer H, Klijn JG, Olah E, Gusterson BA, Pilotti S, Radice P, Scherneck S, Sobol H, et al: Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res. 2005, 11: 5175-5180.View ArticlePubMedGoogle Scholar
- Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F, Ollila DW, Sartor CI, Graham ML, Perou CM: The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007, 13: 2329-2334.View ArticlePubMedGoogle Scholar
- Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M, Cristofanilli M, Hortobagyi GN, Pusztai L: Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol. 2008, 26: 1275-1281.View ArticlePubMedGoogle Scholar
- Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, Gonzalez-Angulo AM, Hortobagyi GN, Arun BK: Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol. 2008, 26: 4282-4288.View ArticlePubMedGoogle Scholar
- Bhattacharyya A, Ear US, Koller BH, Weichselbaum RR, Bishop DK: The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin. J Biol Chem. 2000, 275: 23899-23903.View ArticlePubMedGoogle Scholar
- Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005, 434: 917-921.View ArticlePubMedGoogle Scholar
- Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T: Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 2005, 434: 913-917.View ArticlePubMedGoogle Scholar
- Alli E, Sharma VB, Hartman AR, Lin PS, McPherson L, Ford JM: Enhanced sensitivity to cisplatin and gemcitabine in Brca1-deficient murine mammary epithelial cells. BMC Pharmacol. 2011, 11: 7-View ArticlePubMedPubMed CentralGoogle Scholar
- Hastak K, Alli E, Ford JM: Synergistic chemosensitivity of triple-negative breast cancer cell lines to poly(ADP-Ribose) polymerase inhibition, gemcitabine, and cisplatin. Cancer Res. 2010, 70: 7970-7980.View ArticlePubMedPubMed CentralGoogle Scholar
- Dai Y, Grant S: New insights into checkpoint kinase 1 in the DNA damage response signaling network. Clin Cancer Res. 2010, 16: 376-383.View ArticlePubMedPubMed CentralGoogle Scholar
- Smith J, Tho LM, Xu N, Gillespie DA: The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res. 2010, 108: 73-112.View ArticlePubMedGoogle Scholar
- Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ: Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 2000, 14: 1448-1459.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen P, Luo C, Deng Y, Ryan K, Register J, Margosiak S, Tempczyk-Russell A, Nguyen B, Myers P, Lundgren K, Kan CC, O’Connor PM: The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation. Cell. 2000, 100: 681-692.View ArticlePubMedGoogle Scholar
- Cai Z, Chehab NH, Pavletich NP: Structure and activation mechanism of the CHK2 DNA damage checkpoint kinase. Mol Cell. 2009, 35: 818-829.View ArticlePubMedGoogle Scholar
- Bucher N, Britten CD: G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer. Br J Cancer. 2008, 98: 523-528.View ArticlePubMedPubMed CentralGoogle Scholar
- Cho SH, Toouli CD, Fujii GH, Crain C, Parry D: Chk1 is essential for tumor cell viability following activation of the replication checkpoint. Cell Cycle. 2005, 4: 131-139.View ArticlePubMedGoogle Scholar
- Kawabe T: G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther. 2004, 3: 513-519.PubMedGoogle Scholar
- Garrett MD, Collins I: Anticancer therapy with checkpoint inhibitors: what, where and when?. Trends Pharmacol Sci. 2011, 32: 308-316.View ArticlePubMedGoogle Scholar
- King C, Diaz H, Barnard D, Barda D, Clawson D, Blosser W, Cox K, Guo S, Marshall M: Characterization and preclinical development of LY2603618: a selective and potent Chk1 inhibitor. Invest New Drugs. 2013, 32: 213-226.View ArticlePubMedGoogle Scholar
- Guzi TJ, Paruch K, Dwyer MP, Labroli M, Shanahan F, Davis N, Taricani L, Wiswell D, Seghezzi W, Penaflor E, Bhagwat B, Wang W, Gu D, Hsieh Y, Lee S, Liu M, Parry D: Targeting the replication checkpoint using SCH 900776, a potent and functionally selective CHK1 inhibitor identified via high content screening. Mol Cancer Ther. 2011, 10: 591-602.View ArticlePubMedGoogle Scholar
- Blasina A, Hallin J, Chen E, Arango ME, Kraynov E, Register J, Grant S, Ninkovic S, Chen P, Nichols T, O’Connor P, Anderes K: Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1. Mol Cancer Ther. 2008, 7: 2394-2404.View ArticlePubMedGoogle Scholar
- Zabludoff SD, Deng C, Grondine MR, Sheehy AM, Ashwell S, Caleb BL, Green S, Haye HR, Horn CL, Janetka JW, Liu D, Mouchet E, Ready S, Rosenthal JL, Queva C, Schwartz GK, Taylor KJ, Tse AN, Walker GE, White AM: AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008, 7: 2955-2966.View ArticlePubMedGoogle Scholar
- Wang W: Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet. 2007, 8: 735-748.View ArticlePubMedGoogle Scholar
- Chen CC, Kennedy RD, Sidi S, Look AT, D’Andrea A: CHK1 inhibition as a strategy for targeting Fanconi Anemia (FA) DNA repair pathway deficient tumors. Mol Cancer. 2009, 8: 24-View ArticlePubMedPubMed CentralGoogle Scholar
- Stokes S, Foloppe N, Fiumana A, Drysdale M, Bedford S, Webb P: Indolyl- pyridone derivatives having checkpoint kinase 1 inhibitory activity [WO/2009/093012]. World Intellectual Property Organization. 2009Google Scholar
- Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW: A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006, 10: 515-527.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang W, Soares J, Greninger P, Edelman EJ, Lightfoot H, Forbes S, Bindal N, Beare D, Smith JA, Thompson IR, Ramaswamy S, Futreal PA, Haber DA, Stratton MR, Benes C, McDermott U, Garnett MJ: Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells. Nucleic Acids Res. 2013, 41: D955-D961.View ArticlePubMedGoogle Scholar
- Cole KA, Huggins J, Laquaglia M, Hulderman CE, Russell MR, Bosse K, Diskin SJ, Attiyeh EF, Sennett R, Norris G, Laudenslager M, Wood AC, Mayes PA, Jagannathan J, Winter C, Mosse YP, Maris JM: RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma. Proc Natl Acad Sci U S A. 2011, 108: 3336-3341.View ArticlePubMedPubMed CentralGoogle Scholar
- Brooks K, Oakes V, Edwards B, Ranall M, Leo P, Pavey S, Pinder A, Beamish H, Mukhopadhyay P, Lambie D, Gabrielli B: A potent Chk1 inhibitor is selectively cytotoxic in melanomas with high levels of replicative stress. Oncogene. 2013, 32: 788-796.View ArticlePubMedGoogle Scholar
- Murga M, Campaner S, Lopez-Contreras AJ, Toledo LI, Soria R, Montana MF, D'Artista L, Schleker T, Guerra C, Garcia E, Barbacid M, Hidalgo M, Amati B, Fernandez-Capetillo O: Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nat Struct Mol Biol. 2011, 18: 1331-1335.View ArticlePubMedPubMed CentralGoogle Scholar
- Ferrao PT, Bukczynska EP, Johnstone RW, McArthur GA: Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells. Oncogene. 2012, 31: 1661-1672.View ArticlePubMedGoogle Scholar
- Davies KD, Humphries MJ, Sullivan FX, von C,I, Le HY, Mohr PJ, Wang B, Blake JF, Lyon MA, Gunawardana I, Chicarelli M, Wallace E, Gross S: Single-agent inhibition of Chk1 is antiproliferative in human cancer cell lines in vitro and inhibits tumor xenograft growth in vivo. Oncol Res. 2011, 19: 349-363.View ArticlePubMedGoogle Scholar
- Bryant C, Scriven K, Massey AJ: Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells. Mol Cancer. 2014, 13: 147-View ArticlePubMedPubMed CentralGoogle Scholar
- Ribeiro E, Ganzinelli M, Andreis D, Bertoni R, Giardini R, Fox SB, Broggini M, Bottini A, Zanoni V, Bazzola L, Foroni C, Generali D, Damia G: Triple negative breast cancers have a reduced expression of DNA repair genes. PLoS One. 2013, 8: e66243-View ArticlePubMedPubMed CentralGoogle Scholar
- Lau KM, Mok SC, Ho SM: Expression of human estrogen receptor-alpha and -beta, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc Natl Acad Sci U S A. 1999, 96: 5722-5727.View ArticlePubMedPubMed CentralGoogle Scholar
- Albiges L, Goubar A, Scott V, Vicier C, Lefebvre C, Alsafadi S, Commo F, Saghatchian M, Lazar V, Dessen P, Delaloge S, Andre F, Quidville V: Chk1 as a new therapeutic target in triple-negative breast cancer. Breast. 2014, 23: 250-258.View ArticlePubMedGoogle Scholar
- Tang Y, Hamed HA, Poklepovic A, Dai Y, Grant S, Dent P: Poly(ADP-ribose) polymerase 1 modulates the lethality of CHK1 inhibitors in mammary tumors. Mol Pharmacol. 2012, 82: 322-332.View ArticlePubMedPubMed CentralGoogle Scholar
- Booth L, Cruickshanks N, Ridder T, Dai Y, Grant S, Dent P: PARP and CHK inhibitors interact to cause DNA damage and cell death in mammary carcinoma cells. Cancer Biol Ther. 2013, 14: 458-465.View ArticlePubMedPubMed CentralGoogle Scholar
- Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, Pommier Y: GammaH2AX and cancer. Nat Rev Cancer. 2008, 8: 957-967.View ArticlePubMedPubMed CentralGoogle Scholar
- Forment JV, Blasius M, Guerini I, Jackson SP: Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation. PLoS One. 2011, 6: e23517-View ArticlePubMedPubMed CentralGoogle Scholar
- Rawlinson R, Massey AJ: gammaH2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments. BMC Cancer. 2014, 14: 483-View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang YW, Otterness DM, Chiang GG, Xie W, Liu YC, Mercurio F, Abraham RT: Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway. Mol Cell. 2005, 19: 607-618.View ArticlePubMedGoogle Scholar
- Zhang YW, Brognard J, Coughlin C, You Z, Dolled-Filhart M, Aslanian A, Manning G, Abraham RT, Hunter T: The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress. Mol Cell. 2009, 35: 442-453.View ArticlePubMedPubMed CentralGoogle Scholar
- Leung-Pineda V, Huh J, Piwnica-Worms H: DDB1 targets Chk1 to the Cul4 E3 ligase complex in normal cycling cells and in cells experiencing replication stress. Cancer Res. 2009, 69: 2630-2637.View ArticlePubMedPubMed CentralGoogle Scholar
- Verlinden L, Vanden BI, Eelen G, Drijkoningen M, Verlinden I, Marchal K, Wolf-Peeters C, Christiaens MR, Michiels L, Bouillon R, Verstuyf A: The E2F-regulated gene Chk1 is highly expressed in triple-negative estrogen receptor /progesterone receptor /HER-2 breast carcinomas. Cancer Res. 2007, 67: 6574-6581.View ArticlePubMedGoogle Scholar
- Shibata H, Miuma S, Saldivar JC, Huebner K: Response of subtype-specific human breast cancer-derived cells to poly(ADP-ribose) polymerase and checkpoint kinase 1 inhibition. Cancer Sci. 2011, 102: 1882-1888.View ArticlePubMedPubMed CentralGoogle Scholar
- Gilad O, Nabet BY, Ragland RL, Schoppy DW, Smith KD, Durham AC, Brown EJ: Combining ATR suppression with oncogenic Ras synergistically increases genomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner. Cancer Res. 2010, 70: 9693-9702.View ArticlePubMedPubMed CentralGoogle Scholar
- Elstrodt F, Hollestelle A, Nagel JH, Gorin M, Wasielewski M, van den OA , Merajver SD, Ethier SP, Schutte M: BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res. 2006, 66: 41-45.View ArticlePubMedGoogle Scholar
- Maya-Mendoza A, Petermann E, Gillespie DA, Caldecott KW, Jackson DA: Chk1 regulates the density of active replication origins during the vertebrate S phase. EMBO J. 2007, 26: 2719-2731.View ArticlePubMedPubMed CentralGoogle Scholar
- Petermann E, Woodcock M, Helleday T: Chk1 promotes replication fork progression by controlling replication initiation. Proc Natl Acad Sci U S A. 2010, 107: 16090-16095.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/570/prepub
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