-
Combined inhibition of aurora kinases and Bcl-xL induces apoptosis through select BH3-only proteins.The Journal of Biological Chemistry Feb 2023Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in...
Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that alisertib and danusertib, two small-molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two antiapoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of alisertib- or danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a Bcl-2 homology (BH)3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with alisertib or danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three BH3-only proteins of the Bcl-2 family, as mediators of alisertib-ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon alisertib treatment. On the other hand, we found that alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into truncated Bid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.
Topics: Humans; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Aurora Kinases; bcl-2-Associated X Protein; bcl-X Protein; Cell Line, Tumor; Colonic Neoplasms; Enzyme Activation; HCT116 Cells; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2
PubMed: 36621626
DOI: 10.1016/j.jbc.2023.102875 -
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 -
International Journal of Molecular... Jan 2013Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib...
Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358) have been indicated as possible substrates of human flavin-containing monooxygenase 3 (hFMO3). Here we report the in vitro rate of oxidation of these drugs by wild-type hFMO3 and its polymorphic variant V257M. The conversion of Tozasertib and Danusertib to their corresponding metabolites, identified by LC-MS, by the purified wild-type and V257M hFMO3 show significant differences. In the case of Tozasertib, the V257M variant shows a catalytic efficiency, expressed as k(cat)/K(m), similar to the wild-type: 0.39 ± 0.06 min-1µM-1 for V257M compared to 0.33 ± 0.04 min-1µM-1 for the wild type. On the other hand, in the case of Danusertib, V257M shows a 3.4× decrease in catalytic efficiency with k(cat)/K(m) values of 0.05 ± 0.01 min-1µM-1 for V257M and 0.17 ± 0.03 min-1µM-1 for the wild type. These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism affects the N-oxidation of relevant anticancer drugs, with important outcome in their therapeutic effects. These findings demonstrate that codon 257 is important for activity of the hFMO3 gene and the codon change V to M has an effect on the catalytic efficiency of this enzyme.
PubMed: 23358255
DOI: 10.3390/ijms14022707 -
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 -
Theranostics 2021Doxorubicin is a widely used anticancer drug. However, its major side effect, cardiotoxicity, results from cardiomyocyte loss that causes left ventricle (LV) wall...
Thyroid hormone plus dual-specificity phosphatase-5 siRNA increases the number of cardiac muscle cells and improves left ventricular contractile function in chronic doxorubicin-injured hearts.
Doxorubicin is a widely used anticancer drug. However, its major side effect, cardiotoxicity, results from cardiomyocyte loss that causes left ventricle (LV) wall thinning, chronic LV dysfunction and heart failure. Cardiomyocyte number expansion by thyroid hormone (T3) during preadolescence is suppressed by the developmental induction of an ERK1/2-specific dual specificity phosphatase 5 (DUSP5). Here, we sought to determine if a brief course of combined DUSP5 suppression plus T3 therapy replaces cardiomyocytes lost due to preexisting doxorubicin injury and reverses heart failure. We used -jetPEI to deliver DUSP5 or scrambled siRNA to ~5-week-old C57BL6 mice followed by 5 daily injections of T3 (2 ng/µg body weight). Genetic lineage tracing using -MerMer::Rosa26fs-Confetti mice and direct cardiomyocyte number counting, along with cell cycle inhibition (danusertib), was used to test if this treatment leads to cardiomyocyte generation and improves LV contractile function. Three doses of doxorubicin (20 µg/g) given at 2-weekly intervals, starting at 5-weeks of age in C57BL6 mice, caused severe heart failure, as evident by a decrease in LV ejection fraction. Mice with an ~40 percentage point decrease in LVEF post-doxorubicin injury were randomized to receive either DUSP5 siRNA plus T3, or scrambled siRNA plus vehicle for T3. Age-matched mice without doxorubicin injury served as controls. In uninjured adult mice, transient therapy with DUSP5 siRNA and T3 increases cardiomyocyte numbers, which is required for the associated increase in LV contractile function, since both are blocked by danusertib. In mice with chronic doxorubicin injury, DUSP5 siRNA plus T3 therapy rebuilds LV muscle by increasing cardiomyocyte numbers, which reverses LV dysfunction and prevents progressive chamber dilatation. RNA therapies are showing great potential. Importantly, a GMP compliant -jetPEI system for delivery of siRNA is already in use in humans, as is T3. Given these considerations, our findings provide a potentially highly translatable strategy for addressing doxorubicin cardiomyopathy, a currently untreatable condition.
Topics: Animals; Antibiotics, Antineoplastic; Benzamides; Cardiotoxicity; Cell Count; Cell Proliferation; Doxorubicin; Dual-Specificity Phosphatases; Insulin-Like Growth Factor I; Mice; Myocardial Contraction; Myocytes, Cardiac; Protein Kinase Inhibitors; Pyrazoles; RNA, Small Interfering; Triiodothyronine; Ventricular Dysfunction, Left; Ventricular Function, Left; Ventricular Remodeling
PubMed: 33754028
DOI: 10.7150/thno.57456 -
Nature Chemical Biology Nov 2012Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors...
Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABL(T315I). To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABL(T315I) CML cells on c-Myc through nonobvious off targets.
Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Fusion Proteins, bcr-abl; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Nitriles; Piperazines; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins c-myc; Pyrazoles; Pyrimidines; Quinolines; Structure-Activity Relationship; Systems Biology
PubMed: 23023260
DOI: 10.1038/nchembio.1085 -
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 -
Biochemical Pharmacology Feb 2012Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs...
Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K(m) values of 45 ± 8 μM and 25 ± 4 μM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.
Topics: Benzamides; Benzydamine; Binding Sites; Cell-Free System; Humans; Models, Molecular; Molecular Structure; Oxygenases; Piperazines; Protein Conformation; Pyrazoles; Sulindac
PubMed: 22177984
DOI: 10.1016/j.bcp.2011.11.029 -
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 -
Molecular Cancer Jun 2012Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase...
BACKGROUND
Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL.
RESULTS
We compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival.
CONCLUSIONS
PHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.
Topics: Animals; Antineoplastic Agents; Apoptosis; Aurora Kinase B; Aurora Kinases; Benzamides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Enzyme Activation; Fusion Proteins, bcr-abl; Humans; Mice; Mutation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Vincristine
PubMed: 22721004
DOI: 10.1186/1476-4598-11-42