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Cancer has been linked to the uncontrolled proliferation of cells and the overexpression of cell-cycle genes. The cell cycle machinery plays a crucial role in the regulation of the apoptosis to mitosis to growth phase progression. The mechanisms of the cell cycle also play an important role in preventing DNA damage. There are multiple members of the protein kinase family that are involved in the activities of the cell cycle. Essential cyclins effectively regulate cyclin-dependent kinases (CDKs), which are themselves adversely regulated by naturally occurring CDK inhibitors. Despite the fact that various compounds can effectively block the cell cycle kinases and being investigated for their potential to fight cancer. This chapter explains the detail of cell cycle and checkpoint regulators, that are crucial to the malignant cellular process. The known CDKs inhibitors and their mechanism of action in various cancers have also been addressed as a step toward the development of a possibly novel technique for the design of new drugs against cell cycle kinase proteins.

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The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.

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The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2-UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

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With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein ß and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein ß and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein ß and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.

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Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.

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Human osteosarcoma (bone cancer) is a highly malignant and the most prevalent bone tumor affecting children. Despite recent advances in the understanding of the molecular mechanism by which anticancer drugs kill osteosarcoma or block its growth, however, the mortality rate has declined only modestly. Thus, a new therapeutic approach is needed to be established. 7-hydroxystaurosporine, UCN-01, abrogates the G2 checkpoint thus enhancing the cytotoxicity of chemotherapeutic agents. In addition, it has been evaluated in clinical trials as a single antineoplastic agent in treating several cancers. However, the effects of UCN-01 on treating bone cancer has never been tested. In this study, we found that UCN-01 induced cell cycle arrest and apoptosis in the human osteosarcoma, U2-OS cells. In addition, the migration ability was also reduced, suggesting UCN-01 inhibited cell growth and migration. When U2-OS cells were treated with UCN-01, DNA damage response was triggered. The ataxia telangiectasia mutated (ATM) and the non-canonical downstream effector, ERK, was activated by UCN-01. In addition, depletion of ATM or inhibition of ERK deteriorated the cell viability in UCN-01-treated U2-OS cells. Furthermore, UCN-01 induced autophagy activation for protecting cells from apoptosis. Thus, UCN-01 might function as a single antineoplastic agent in treating human osteosarcoma.

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Enhanced rRNA synthesis is a downstream effect of many of the signaling pathways that are aberrantly activated in cancer, such as the PI3K/mTOR and MAP kinase pathways. Recently, two new rRNA synthesis inhibitors have demonstrated therapeutic effects on cancer cells while sparing normal cells. One of them, CX-5461, is currently in phase 1 clinical trials for hematological malignancies. Here, we investigate the effectiveness of transient treatment with this drug on acute lymphoblastic leukemia cells. Our results show that short exposure to CX-5461 followed by drug washout is sufficient to induce persistent G2 cell-cycle arrest and irreversible commitment to cell death, in spite of rRNA synthesis returning to normal within 24 hours of drug washout. The magnitude of cell death after transient exposure is similar to continuous exposure, but the time to cell death is relatively delayed with transient exposure. In this report, we also investigate rational drug combinations that can potentiate the effect of continuous CX-5461 treatment. We show that the checkpoint abrogator UCN-01 can relieve CX-5461-induced G2 arrest and potentiate the cytotoxic effects of CX-5461. Finally, we show that ERK1/2 is activated upon CX-5461 treatment, and that pharmacological inhibition of MEK1/2 leads to enhanced cell death in combination with CX-5461. In summary, our results provide evidence for the effectiveness of CX-5461 pulse treatment, which may minimize drug related toxicity, and evidence for enhanced effectiveness of CX-5461 in combination with other targeted agents.

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Repair of DNA-targeted anticancer agents is an active area of investigation of both fundamental and clinical interest. However, most studies have focused on a small number of compounds limiting our understanding of both DNA repair and the DNA damage response. S23906 is an acronycine derivative that shows strong activity toward solid tumors in experimental models. S23906 forms bulky monofunctional DNA adducts in the minor groove which leads to destabilization of the double-stranded helix. We now report that S23906 induces formation of DNA double strand breaks that are processed through homologous recombination (HR) but not Non-Homologous End-Joining (NHEJ) repair. Interestingly, S23906 exposure was accompanied by a higher sensitivity of BRCA2-deficient cells compared to other HR deficient cell lines and by an S-phase accumulation in wild-type (wt), but not in BRCA2-deficient cells. Recently, we have shown that S23906-induced S phase arrest was mediated by the checkpoint kinase Chk1. However, its activated phosphorylated form is equally induced by S23906 in wt and BRCA2-deficient cells, likely indicating a role for BRCA2 downstream of Chk1. Accordingly, override of the S phase arrest by either 7-hydroxystaurosporine (UCN-01) or AZD7762 potentiates the cytotoxic activity of S23906 in wt, but not in BRCA2-deficient cells. Together, our findings suggest that the pronounced sensitivity of BRCA2-deficient cells to S23906 is due to both a defective S-phase arrest and the absence of HR repair. Tumors with deficiencies for proteins involved in HR, and BRCA2 in particular, may thus show increased sensitivity to S23906, thereby providing a rationale for patient selection in clinical trials.

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The ataxia telangiectasia mutated serine/threonine kinase (ATM)/checkpoint kinase 2 (CHEK2, best known as CHK2) and the ATM and Rad3-related serine/threonine kinase (ATR)/CHEK1 (best known as CHK1) cascades are the 2 major signaling pathways driving the DNA damage response (DDR), a network of processes crucial for the preservation of genomic stability that act as a barrier against tumorigenesis and tumor progression. Mutations and/or deletions of ATM and/or CHK2 are frequently found in tumors and predispose to cancer development. In contrast, the ATR-CHK1 pathway is often upregulated in neoplasms and is believed to promote tumor growth, although some evidence indicates that ATR and CHK1 may also behave as haploinsufficient oncosuppressors, at least in a specific genetic background. Inactivation of the ATM-CHK2 and ATR-CHK1 pathways efficiently sensitizes malignant cells to radiotherapy and chemotherapy. Moreover, ATR and CHK1 inhibitors selectively kill tumor cells that present high levels of replication stress, have a deficiency in p53 (or other DDR players), or upregulate the ATR-CHK1 module. Despite promising preclinical results, the clinical activity of ATM, ATR, CHK1, and CHK2 inhibitors, alone or in combination with other therapeutics, has not yet been fully demonstrated. In this Trial Watch, we give an overview of the roles of the ATM-CHK2 and ATR-CHK1 pathways in cancer initiation and progression, and summarize the results of clinical studies aimed at assessing the safety and therapeutic profile of regimens based on inhibitors of ATR and CHK1, the only 2 classes of compounds that have so far entered clinics.

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Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect.

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The Ras GTPases comprising three main isoforms H-, N- and K-Ras operate at the plasma membrane as molecular switches in essential signaling pathways. Active concentration of the minor phospholipid phosphatidylserine in the inner leaflet of the plasma membrane contributes to the electrostatic potential that is required for K-Ras anchoring to the plasma membrane. We recently observed that staurosporine and related analogs: 7-oxostaurosporine, UCN-01 and UCN-02, long known as relatively non-specific protein kinase inhibitors, block endosomal sorting and recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine to endosomes and endomembranes with concomitant mislocalization of K-Ras. Staurosporines are therefore a new tool to study phosphatidylserine trafficking. We discuss whether the mechanism of action of UCN-01, an FDA-approved staurosporine analog used as an anti-cancer therapeutic, is related to effects on phosphatidylserine subcellular distribution. Given the high prevalence of expression of constitutively active K-Ras in human cancers, we ask whether inhibitors of phosphatidylserine trafficking may have important therapeutic applications.

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Aim and Methods To investigate the effect of UCN-01(7-hydroxystaurosporine) on cell migration and invasion ability of DU-145, an invasive human prostate cancer cell line.Results It was found that UCN-01 at non-cytotoxic doses (100 nmol· L-1) significantly inhibited prostate cancer DU-145 cell invasion and migration behaviors.Moreover, this anti-invasion and migration activity of UCN-01 was associated with an up-regulation of cell adhesion molecule E-cadherin. Conclusion These results indicate for first time that UCN-01 inhibits the invasion and migration of human prostate cancer cells.Thus, clinical application of UCN-01 may contribute to the potential benefit for suppression of prostate cancer invasion and metastasis.