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Journal of Experimental & Clinical... May 2019Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK...
BACKGROUND
Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure.
METHODS
Thiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs.
RESULTS
Here we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with β-catenin enables β-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone.
CONCLUSIONS
Our conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML.
Topics: Aurora Kinase A; Benzamides; Cell Cycle Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Forkhead Box Protein M1; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Pteridines; Pyrazoles; Signal Transduction; Thiostrepton; Up-Regulation; Polo-Like Kinase 1
PubMed: 31122263
DOI: 10.1186/s13046-019-1197-9 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Dec 2018To investigate the effect of danusertib (Danu), an inhibitor of Aurora kinase, on the proliferation, cell cycle, apoptosis, and autophagy of hepatocellular carcinoma...
OBJECTIVE
To investigate the effect of danusertib (Danu), an inhibitor of Aurora kinase, on the proliferation, cell cycle, apoptosis, and autophagy of hepatocellular carcinoma HepG2 cells and explore the underlying mechanisms.
METHODS
MTT assay was used to examine the effect of Danu on the viability of HepG2 cells to determine the IC50 of Danu. The effect of Danu on cell cycle distribution, apoptosis and autophagy were determined using flow cytometry. Western blotting was used to detect the expressions of the proteins related to cell cycle, apoptosis and autophagy. Chloroquine was used to suppress Danuinduced autophagy to test the apoptosis-inducing effect of Danu.
RESULTS
Danu significantly inhibited the proliferation of HepG2 cells with IC of 39.4 μmol and 14.4 μmol at 24 h and 48 h, respectively. Danu caused cell cycle arrest in G/M phase in HepG2 cells and led to polyploidy accumulation via up-regulating the expressions of p53 and p21 and down-regulating the expressions of cyclin B1 and DC2. Danu also caused apoptosis of HepG2 cells through up-regulating the expressions of Bax, Puma, cleaved caspase-3, cleaved caspase-9, cleaved PARP and cytochrome C and down-regulating the expressions of Bcl-xl and Bcl-2. Danu induced autophagy via activating AMPK signaling and inhibiting PI3K/PTEN/AKT/mTOR axis, and inhibition of Danu-induced autophagy with chloroquine enhanced the pro-apoptotic effect of Danu.
CONCLUSIONS
Danu inhibits cell proliferation and induces cell cycle arrest in G/M phase, apoptosis and cytoprotective autophagy in HepG2 cells.
Topics: Apoptosis; Autophagy; Benzamides; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Proteins; Protein Kinase Inhibitors; Pyrazoles
PubMed: 30613017
DOI: 10.12122/j.issn.1673-4254.2018.12.13 -
Antimicrobial Agents and Chemotherapy Feb 2019Signaling pathways play a key role in HIV-1 latency. In this study, we used the 24ST1NLESG cell line of HIV-1 latency to screen a library of structurally diverse,...
Signaling pathways play a key role in HIV-1 latency. In this study, we used the 24ST1NLESG cell line of HIV-1 latency to screen a library of structurally diverse, medicinally active, cell permeable kinase inhibitors, which target a wide range of signaling pathways, to identify inhibitors of HIV-1 latency reversal. The screen was carried out in the absence or presence of three mechanistically distinct latency-reversing agents (LRAs), namely, prostratin, panobinostat, and JQ-1. We identified inhibitors that only blocked the activity of a specific LRA, as well as inhibitors that blocked the activity of all LRAs. For example, we identified 12 inhibitors targeted toward protein kinase C or downstream kinases that blocked the activity of prostratin. We also identified 12 kinase inhibitors that blocked the reversal of HIV-1 latency irrespective of the LRA used in the screen. Of these, danusertib, an Aurora kinase inhibitor, and PF-3758309, a PAK4 inhibitor, were the most potent. The 50% inhibitory concentrations in the 24ST1NLESG cells ranged from 40 to 147 nM for danusertib (selectivity indices, >150) and from 0.1 to 1 nM for PF-3758309 (selectivity indices, >3,300). Both danusertib and PF-3758309 inhibited latency reversal in CD4 T cells isolated from HIV-1-infected donors. Collectively, our study describes a chemical approach that can be applied to elucidate the role of signaling pathways involved in LRA activity or the maintenance of HIV-1 latency and also identifies inhibitors of latent HIV-1 reactivation that could be used with antiretroviral therapy to reduce residual viremia.
Topics: Anti-HIV Agents; Benzamides; CD4-Positive T-Lymphocytes; Cell Line; HIV Infections; HIV-1; Humans; Pyrazoles; Pyrroles; Signal Transduction; Virus Activation
PubMed: 30455231
DOI: 10.1128/AAC.01744-18 -
Cancer Cell Nov 2018The fate of disseminated tumor cells is largely determined by microenvironment (ME) niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis...
The fate of disseminated tumor cells is largely determined by microenvironment (ME) niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis progression. We investigated the underlying mechanisms using pre-clinical models and analyses of clinical data. We discovered that the osteogenic niche serves as a calcium (Ca) reservoir for cancer cells through gap junctions. Cancer cells cannot efficiently absorb Ca from ME, but depend on osteogenic cells to increase intracellular Ca concentration. The Ca signaling, together with previously identified mammalian target of rapamycin signaling, promotes bone metastasis progression. Interestingly, effective inhibition of these pathways can be achieved by danusertib, or a combination of everolimus and arsenic trioxide, which provide possibilities of eliminating bone micrometastases using clinically established drugs.
Topics: 3T3 Cells; Animals; Antineoplastic Agents; Arsenic Trioxide; Benzamides; Bone Neoplasms; Breast Neoplasms; Calcium; Calcium Signaling; Cell Line, Tumor; Cell Proliferation; Connexin 43; Everolimus; Female; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Osteogenesis; Protein Kinase Inhibitors; Pyrazoles; RAW 264.7 Cells; Tumor Microenvironment; U937 Cells
PubMed: 30423299
DOI: 10.1016/j.ccell.2018.10.002 -
Journal of Cancer 2018Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, but the overall prognosis remains disappointing especially in the advanced-stage patients....
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, but the overall prognosis remains disappointing especially in the advanced-stage patients. Aberration expression of Aurora kinases is tumorigenic and thus it has attracted interests as therapeutic targets in cancer treatment. Here, we investigated the proteomic response of HCC Hep3B cells to danusertib (Danu), a pan-Aurora kinase inhibitor, and then validated the proteomic results based on stable-isotope labeling by amino acids in cell culture (SILAC). The proteomic data identified that Danu modulated the expression of 542 protein molecules (279 up-regulated; 260 down-regulated; 3 stable). Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 107 and 24 signaling pathways were regulated by Danu, respectively. IPA analysis showed cellular growth and proliferation, and cell death and survival were among the top five molecular and cellular functions regulated by Danu. The verification experiments showed that Danu inhibited the proliferation of Hep3B cells with a 24-hr IC value of 22.03 µM. Danu treatment also arrested Hep3B cells in G/M phase via regulating the expression of key cell cycle regulators and induced apoptosis via mitochondria-dependent pathway in a dose-dependent manner. Besides, Danu induced a marked autophagy, and inhibition of autophagy enhanced the anticancer effects of Danu, indicating a cyto-protective role of Danu-induced autophagy. Our proteomic data and Western blotting assays showed the PI3K/Akt/mTOR signaling pathway was involved in the inducing effect of Danu on apoptosis and autophagy. Collectively, our findings have demonstrated that the Aurora kinases inhibition with danusertib results in global proteomic response and exerts anticancer effects in Hep3B cells involving regulation of cell cycle, apoptosis and autophagy and associated signaling pathways.
PubMed: 29937924
DOI: 10.7150/jca.20822 -
Journal of Hematology & Oncology Jun 2018Bcr-Abl inhibitors paved the way of targeted therapy epoch. Imatinib was the first tyrosine kinase inhibitor to be discovered with high specificity for Bcr-Abl protein... (Review)
Review
Bcr-Abl inhibitors paved the way of targeted therapy epoch. Imatinib was the first tyrosine kinase inhibitor to be discovered with high specificity for Bcr-Abl protein resulting from t(9, 22)-derived Philadelphia chromosome. Although the specific targeting of that oncoprotein, several Bcr-Abl-dependent and Bcr-Abl-independent mechanisms of resistance to imatinib arose after becoming first-line therapy in chronic myelogenous leukemia (CML) treatment.Consequently, new specific drugs, namely dasatinib, nilotinib, bosutinib, and ponatinib, were rationally designed and approved for clinic to override resistances. Imatinib fine mechanisms of action had been elucidated to rationally develop those second- and third-generation inhibitors. Crystallographic and structure-activity relationship analysis, jointly to clinical data, were pivotal to shed light on this topic. More recently, preclinical evidence on bafetinib, rebastinib, tozasertib, danusertib, HG-7-85-01, GNF-2, and 1,3,4-thiadiazole derivatives lay promising foundations for better inhibitors to be approved for clinic in the near future.Notably, structural mechanisms of action and drug design exemplified by Bcr-Abl inhibitors have broad relevance to both break through resistances in CML treatment and develop inhibitors against other kinases as targeted chemotherapeutics.
Topics: Fusion Proteins, bcr-abl; Humans; Molecular Targeted Therapy; Protein Kinase Inhibitors; Structure-Activity Relationship; Treatment Outcome
PubMed: 29925402
DOI: 10.1186/s13045-018-0624-2 -
Journal of Cancer 2018: To elucidate the expression of Aurora kinases (AURK) and the anticancer effects of pan-aurora kinase inhibitor Danusertib in hepatocarcinogenesis model in C56Bl6 mice....
: To elucidate the expression of Aurora kinases (AURK) and the anticancer effects of pan-aurora kinase inhibitor Danusertib in hepatocarcinogenesis model in C56Bl6 mice. : Thirty mice C56Bl6 were randomly divided into Group A or control, Group B animals who underwent experimental hepatocarcinogenesis with diethylnitrosamine (DEN), and Group C animals with DEN-induced hepatocarcinogenenesis that treated with pan-aurora kinase inhibitor Danusertib. Primary antibodies for immunochistochemistry (IHC) included rabbit antibodies against Ki-67, DKK1, INCENP, cleaved caspase-3, NF-κB p65, c-Jun, β-catenin. Hepatocyte growth factor receptor (C-MET/HGFR) and Bcl-2 antagonist of cell death (BAD) serum levels were determined using a quantitative sandwich enzyme immunoassay technique. : Inhibition of AURK reduced the number of DEN-induced liver tumours. Apoptosis and proliferation was very low in both DEN-induced and anti- AURK groups respectively. The hepatocellular adenoma cells of DEN-treated mice uniformly had ample nuclear INCENP whereas in anti- AURK markedly decreased. Expression of β-catenin, NF-kB and c-Jun did not differ in liver tumors of both AURK -depleted and non-depleted mice. Depletion of AURK reduced the number of DEN-induced hepatic tumours. However, their size did not differ significantly between the groups.
PubMed: 29581770
DOI: 10.7150/jca.22329 -
Cell Death & Disease Feb 2018The Aurora kinase family (Aurora A, B and C) are crucial regulators of several mitotic events, including cytokinesis. Increased expression of these kinases is associated...
The Aurora kinase family (Aurora A, B and C) are crucial regulators of several mitotic events, including cytokinesis. Increased expression of these kinases is associated with tumorigenesis and several compounds targeting Aurora kinase are under evaluation in clinical trials (a.o. AT9283, AZD1152, Danusertib, MLN8054). Here, we demonstrate that the pan-Aurora kinase inhibitor Tozasertib (VX-680 and MK-0457) not only causes cytokinesis defects through Aurora kinase inhibition, but is also a potent inhibitor of necroptosis, a cell death process regulated and executed by the RIPK1, RIPK3 and MLKL signalling axis. Tozasertib's potency to inhibit RIPK1-dependent necroptosis and to block cytokinesis in cells is in the same concentration range, with an IC50 of 1.06 µM and 0.554 µM, respectively. A structure activity relationship (SAR) analysis of 67 Tozasertib analogues, modified at 4 different positions, allowed the identification of analogues that showed increased specificity for either cytokinesis inhibition or for necroptosis inhibition, reflecting more specific inhibition of Aurora kinase or RIPK1, respectively. These results also suggested that RIPK1 and Aurora kinases are functionally non-interacting targets of Tozasertib and its analogues. Indeed, more specific Aurora kinase inhibitors did not show any effect in necroptosis and Necrostatin-1s treatment did not result in cytokinesis defects, demonstrating that both cellular processes are not interrelated. Finally, Tozasertib inhibited recombinant human RIPK1, human Aurora A and human Aurora B kinase activity, but not RIPK3. The potency ranking of the newly derived Tozasertib analogues and their specificity profile, as observed in cellular assays, coincide with ADP-Glo recombinant kinase activity assays. Overall, we show that Tozasertib not only targets Aurora kinases but also RIPK1 independently, and that we could generate analogues with increased selectivity to RIPK1 or Aurora kinases, respectively.
Topics: Animals; Apoptosis; Aurora Kinases; Cell Line; Humans; Mice; Piperazines; Receptor-Interacting Protein Serine-Threonine Kinases
PubMed: 29434255
DOI: 10.1038/s41419-017-0245-7 -
Nature Communications Apr 2017The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic...
The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases.
Topics: Animals; Antineoplastic Agents; Aurora Kinases; Benzamides; Biphenyl Compounds; Bone Neoplasms; Bone and Bones; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; High-Throughput Screening Assays; Humans; Mammary Neoplasms, Experimental; Mice; Morpholines; Protein Kinase Inhibitors; Pyrazoles; Pyridones; Tissue Culture Techniques; Tumor Microenvironment
PubMed: 28429794
DOI: 10.1038/ncomms15045 -
International Journal of Molecular... Nov 2015Ovarian carcinoma (OC) is one of the most common gynecological malignancies, with a poor prognosis for patients at advanced stage. Danusertib (Danu) is a pan-inhibitor...
Danusertib Induces Apoptosis, Cell Cycle Arrest, and Autophagy but Inhibits Epithelial to Mesenchymal Transition Involving PI3K/Akt/mTOR Signaling Pathway in Human Ovarian Cancer Cells.
Ovarian carcinoma (OC) is one of the most common gynecological malignancies, with a poor prognosis for patients at advanced stage. Danusertib (Danu) is a pan-inhibitor of the Aurora kinases with unclear anticancer effect and underlying mechanisms in OC treatment. This study aimed to examine the cancer cell killing effect and explore the possible mechanisms with a focus on proliferation, cell cycle progression, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) in human OC cell lines C13 and A2780cp. The results showed that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both cell lines. Danu arrested cells in G₂/M phase and led to an accumulation of polyploidy through the regulation of the expression key cell cycle modulators. Danu induced mitochondria-dependent apoptosis and autophagy in dose and time-dependent manners. Danu suppressed PI3K/Akt/mTOR signaling pathway, evident from the marked reduction in the phosphorylation of PI3K/Akt/mTOR, contributing to the autophagy inducing effect of Danu in both cell lines. In addition, Danu inhibited EMT. In aggregate, Danu exerts potent inducing effect on cell cycle arrest, apoptosis, and autophagy, but exhibits a marked inhibitory effect on EMT. PI3K/Akt/mTOR signaling pathway contributes, partially, to the cancer cell killing effect of Danu in C13 and A2780cp cells.
Topics: Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Epithelial-Mesenchymal Transition; Female; Humans; Mitochondria; Ovarian Neoplasms; Phenotype; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 26580601
DOI: 10.3390/ijms161126018