As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.

• Keywords
• MU380, castration-resistant prostate cancer, checkpoint kinase 1, docetaxel resistance, gemcitabine, mitotic catastrophe,
• MeSH
• Cell Death drug effects   MeSH
• Checkpoint Kinase 1 antagonists & inhibitors   metabolism   MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Deoxycytidine analogs & derivatives   pharmacology   MeSH
• Docetaxel pharmacology   MeSH
• Gemcitabine MeSH
• Humans MeSH
• Mitosis * drug effects   MeSH
• Mice, SCID MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms pathology   MeSH
• Piperidines chemistry   pharmacology   MeSH
• Cell Proliferation drug effects   MeSH
• Pyrazoles chemistry   pharmacology   MeSH
• Pyrimidines chemistry   pharmacology   MeSH
• S Phase drug effects   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Deoxycytidine MeSH
• Docetaxel MeSH
• Gemcitabine MeSH
• MU380 MeSH Browser
• Piperidines MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

• Keywords
• SCH900776, TP53, checkpoint kinase 1/Chk1, chronic lymphocytic leukemia, nucleoside analogs,
• MeSH
• Apoptosis MeSH
• B-Lymphocytes cytology   MeSH
• Cell Cycle MeSH
• Checkpoint Kinase 1 antagonists & inhibitors   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   genetics   metabolism   MeSH
• Cytarabine administration & dosage   MeSH
• Deoxycytidine administration & dosage   analogs & derivatives   MeSH
• Gemcitabine MeSH
• Humans MeSH
• Mitosis MeSH
• Mutation MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Nucleosides genetics   MeSH
• Cell Proliferation MeSH
• Pyrazoles pharmacology   MeSH
• Pyrimidines pharmacology   MeSH
• Drug Screening Assays, Antitumor MeSH
• Signal Transduction MeSH
• Cell Survival MeSH
• Vidarabine administration & dosage   analogs & derivatives   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Cytarabine MeSH
• Deoxycytidine MeSH
• fludarabine MeSH Browser
• Gemcitabine MeSH
• MK-8776 MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Nucleosides MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH
• TP53 protein, human MeSH Browser
• Vidarabine MeSH

BACKGROUND: Many pathways seem to be involved in the regulation of the intra-S-phase checkpoint after exposure to ionizing radiation, but the role of p53 has proven to be rather elusive. Here we have a closer look at the progression of irradiated cells through S-phase in dependence of their p53 status. MATERIALS AND METHODS: Three pairs of tumour cell lines were used, each consisting of one p53 functional and one p53 non-functional line. Cells were labelled with bromodeoxyuridine(BrdU) immediately after irradiation, they were then incubated in label-free medium, and at different times afterwards their position within the S-phase was determined by means of flow cytometry. RESULTS: While in the p53 deficient cells progression through S-phase was slowed significantly over at least a few hours, it was halted for just about an hour in the p53 proficient cells and then proceeded without further delay or even at a slightly accelerated pace. CONCLUSIONS: It is clear from the experiments presented here that p53 does play a role for the progress of cells through the S-phase after X-ray exposure, but the exact mechanisms by which replicon initiation and elongation is controlled in irradiated cells remain to be elucidated.

• Keywords
• flow cytometry, relative movement, intra-S-phase checkpoint, x-rays,
• Publication type
• Journal Article MeSH

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

• MeSH
• Leukemia, Promyelocytic, Acute pathology   MeSH
• Apoptosis drug effects   MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• HL-60 Cells MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• G2 Phase Cell Cycle Checkpoints drug effects   MeSH
• Humans MeSH
• Morpholines pharmacology   MeSH
• DNA Repair drug effects   MeSH
• Pyrazines pharmacology   MeSH
• Pyrones pharmacology   MeSH
• Sulfones pharmacology   MeSH
• Radiation Tolerance drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one MeSH Browser
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH
• Morpholines MeSH
• Pyrazines MeSH
• Pyrones MeSH
• Sulfones MeSH