Inhibition Of Upstream Kinases Research Articles

A current understanding of drug-induced QT prolongation and its implications for anticancer therapy.

The QT interval, a global index of ventricular repolarization, varies among individuals and is influenced by diverse physiologic and pathophysiologic stimuli such as gender, age, heart rate, electrolyte concentrations, concomitant cardiac disease, and other diseases such as diabetes. Many drugs produce a small but reproducible effect on QT interval but in rare instances this is exaggerated and marked QT prolongation can provoke the polymorphic ventricular tachycardia 'torsades de pointes', which can cause syncope or sudden cardiac death. The generally accepted common mechanism whereby drugs prolong QT is block of a key repolarizing potassium current in heart, IKr, generated by expression of KCNH2, also known as HERG. Thus, evaluation of the potential that a new drug entity may cause torsades de pointes has relied on exposure of normal volunteers or patients to drug at usual and high concentrations, and on assessment of IKr block in vitro. More recent work, focusing on anticancer drugs with QT prolonging liability, is defining new pathways whereby drugs can prolong QT. Notably, the in vitro effects of some tyrosine kinase inhibitors to prolong cardiac action potentials (the cellular correlate of QT) can be rescued by intracellular phosphatidylinositol 3,4,5-trisphosphate, the downstream effector of phosphoinositide 3-kinase. This finding supports a role for inhibition of this enzyme, either directly or by inhibition of upstream kinases, to prolong QT through mechanisms that are being worked out, but include enhanced inward 'late' sodium current during the plateau of the action potential. The definition of non-IKr-dependent pathways to QT prolongation will be important for assessing risk, not only with anticancer therapies but also with other QT prolonging drugs and for generating a refined understanding how variable activity of intracellular signalling systems can modulate QT and associated arrhythmia risk.

Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade.

Because of the essential role of signal transducer and activator of transcription 3 (STAT3) in proliferation, anti-apoptosis, and chemoresistance of multiple myeloma (MM), we investigated whether icariin, a prenylated flavonol glycoside, inhibits both constitutive and inducible STAT3 activation in human myeloma cell lines. We noted that icariin could block constitutive STAT3 phosphorylation as well as its nuclear translocation and DNA binding ability in U266 cells. Icariin also suppressed IL-6-induced STAT3 activation through the inhibition of upstream kinases (Janus activated kinase-1 and -2, and c-Src). We found that icariin downregulated the protein expression of STAT3 downstream target gene products such as Bcl-2, Bcl-xl, survivin, IAP-1/2, COX-2, VEGF, and matrix metallopeptidase 9 (MMP-9) in a concentration-dependent manner. Moreover, this flavonoid also exhibited the capacity to significantly induce apoptosis and suppress proliferation of MM cells. Interestingly, this agent also significantly potentiated the apoptotic effects of bortezomib through the suppression of STAT3 activation in MM cells. Altogether, our data indicates that the potential application of icariin as a STAT3 blocker in myeloma therapy.

Abstract 4788: Discovery of potent and selective inhibitors of ERK1/2 with a unique mechanism of action

Abstract Activation of the RAS/MAPK pathway occurs via a cascade of protein phosphorylation events which culminate in the phosphorylation & activation of ERK 1/2. Aberrant activation of this pathway has been demonstrated in several human tumor types. Inhibitors of this pathway in clinical development target upstream kinases, BRAF or MEK. Only a few ERK 1/2 has been reported. The goal of the ERK program was to develop a targeted therapy that is guided by a clear patient selection / responder identification plan based on BRAF and K/NRAS mutations. Utilizing an affinity based high-throughput screening strategy (ALIS), we discover a novel class of small molecule ERK 1/2 inhibitors. Optimization of this chemical series led to the discovery of SCH 772984, which is highly selective ERK1/2 inhibitor, inhibiting cell proliferation selectively in tumor cell lines with an activated MAPK pathway and causes significant tumor regression in vivo in BRAF & RAS mutant xenograft models. Based on insights gained through crystallographic analysis, SCH 772984 and related compounds have a unique ERK binding mode which results in a novel dual mechanism of inhibition, blocking ERK phosphorylation by MEK as well as inhibiting ERK kinase activity. This novel mechanism enables the use of ERK phosphorylation as a target engagement biomarker. Biological differences between pathway inhibition at the level or ERK vs. inhibition of upstream kinases are being examined. The SAR and crystal structures of this series ERK inhibitors will be reported. Citation Format: Yongqi Deng, Ahmed Samatar, Gerald Shipps, Alan Cooper, Brian Long, Donnar Carr, Li Xiao, Alan Hruza, Tong Wang, Liang Zhu, Yang Nan, Mehul Patel, Kiran Muppalla, Hugh Zhu, Babu Sobhana Boga, Xiaolei Gao. Discovery of potent and selective inhibitors of ERK1/2 with a unique mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4788.

Mitogen- and stress-activated protein kinase 1 activates osteoclastogenesis in vitro and affects bone destruction in vivo

Mitogen- and stress-activated protein kinase (MSK) 1 is an important regulator of immune response and mitogenic signaling. In this study, we report for the first time that MSK1 was activated by the osteoclast differentiation factor receptor activator of nuclear factor kappa B ligand (RANKL) in osteoclast precursor cells. Inhibition of upstream kinases ERK1/2 and p38, but not JNK, suppressed MSK activation upon RANKL stimulation. An MSK1 inhibitor efficiently prevented the induction of c-Fos and NFATc1 and CREB phosphorylation by RANKL. Inhibition of MSK1 also successfully blocked RANKL-induced osteoclastogenesis. MSK knockdown with small interfering RNA significantly inhibited osteoclast differentiation and bone resorption. MSK1 did not affect osteoclast survival. The induction of c-Fos and NFATc1 and the phosphorylation of CREB and ATF2 were also inhibited by MSK1 knockdown. Moreover, knockdown of MSK1 significantly blocked recruitment of c-Fos to the NFATc1 promoter upon RANKL stimulation. Therefore, NFATc1-inducible osteoclast-specific genes were downregulated by MSK1 blockade. NFATc1 retrovirus transduction almost completely rescued the differentiation defect of MSK1-silenced cells. In vivo knockdown of MSK1 reduced RANKL-induced bone resorption as well as osteoclast formation. Thus, our results suggested that MSK1 is an important novel molecule involved in RANKL signaling and osteoclast differentiation.

A Novel STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition in a Wide Range of Hematopoietic Malignancies without Growth Suppression of Normal Hematopoietic Cells

Abstract 577Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like JAK family kinases and Src family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials.Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases and Src family kinases. In HEL92.1.7 cells with constitutively active mutation of JAK2, OPB-31121 treatment inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). Src-dependent constitutive phosphorylation of STAT3 was also inhibited by OPB-31121 without inhibition of Src in H1650 cells that had active mutantation of EGF receptor. In addition, STAT3 immunoprecipitated from OPB-31121-treated cells was neither phosphorylated by JAK2 nor Lyn, a Src family kinase, in vitro without decrease in auto phosphorylation of upstream kinases,OPB-31121 demonstrated strong growth inhibitory effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including myeloma, AML with JAK2 mutation and CML. It is revealed that STAT3 is constitutively activated by oncogenic autocrine of IL-6 pathway or tyrosine kinase signal from oncoprotein in these cell lines. We also demonstrated growth inhibition or reduction of cell lines including HEL92.1.7 (AML with JAK2 mutation, T/C: 16%), KU812 (CML, T/C: 2%), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation, T/C: 5.9%) in NOD/SCID mice. For further analyses, we used human leukemia model mice where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth reduction of leukemia cells of BCR-ABL-positive ALL (T/C: 4%, Figure B), CML-BC with T315I mutation in BCR-ABL (T/C: 15.3%), and AML (T/C: 15.9%). Notably, OPB-31121-induced growth reduction was extremely selective for leukemia cells. Normal hematopoietic cells of mice were hardly affected by OPB-31121, whereas, cytarabine showed non-specific growth suppression of both leukemia cells and normal hematopoietic cells (Figure C). The safety of OPB-31121 on normal hematopoietic cells was also confirmed by colony formation assay, where OPB-31121 hardly affected colony formation of human cord blood cells at 100 nM. For further analyses, we transplanted human cord blood cells into NOG mice and investigated the growth inhibitory effect of OPB-31121 on normal hematopoietic cells in vivo. No significant growth suppression of human normal hematopoietic cells was observed in OPB-31121 treated mice (T/C: 99.9%, Figure D).Taken together, we conclude that OPB-31121 holds promise as a non-myelosuppressive therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase I/II trial in Japan. [Display omitted] Disclosures:Hayakawa:Otsuka Pharmaceutical Co. Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co. Ltd.: Employment. Sumida:otsuka Pharmaceutical Co. Ltd.: Employment. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

A Novel Direct STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition In a Wide Range of Hematopoietic Malignancies and Effectively Suppresses the Chemotherapy Resistant Quiescent Cells In Vivo

AbstractAbstract 3277Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like Janus kinase (JAK) family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis-resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials.Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases. OPB-31121 treatment of HEL92.1.7 cells that had constitutive active mutation of JAK2 inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). STAT3 phosphorylation by JAK2 in vitro was also inhibited by OPB-31121 under constant JAK2 autophosphorylation. On the other hand, it did not inhibit dimerization and nuclear translocation of STAT3 once STAT3 was phosphorylated. Also, direct association between OPB-31121 and STAT3 was suggested in vitro. These data implies that one of the mechanisms of OPB-31121 action was the direct inhibition of STAT3 phosphorylation without JAK kinase inhibition. [Display omitted] OPB-31121 demonstrated strong growth suppressive effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including acute myeloid leukemia (AML) with JAK2 mutation or fms-related tyrosine kinase 3 (FLT3) mutation, chronic myeloid leukemia (CML), and myeloma. It is revealed that STAT3 is constitutively activated by tyrosine kinase signal from oncoprotein or oncogenic autocrine of IL-6 pathway in these cell lines. Of note, OPB-31121 had little growth inhibitory effect on normal hematopoietic cells and hardly affected colony formation of human cord blood cells at 100 nM. We also demonstrated growth suppression or regression of cell lines including HEL92.1.7, KU812 (CML), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation) in NOD/SCID mice (T/C: 1.8 to 39.5 %). For further analyses, we used human leukemia model mouse where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth suppression of leukemia cells of BCR-ABL-positive acute lymphoblastic leukemia (ALL), CML-blast crisis (BC), CML-BC with T315I mutation in BCR-ABL, and AML with FLT3/ITD (T/C: 4 to 58 %, Figure B). Furthermore, treatment with cytarabine induced accumulation of quiescent cells that were thought to be relatively resistant to chemotherapy, whereas OPB-31121 did not cause such accumulation, suggesting its effectiveness on quiescent cells (Figure C). We are now investigating the effect of OPB-31121 on leukemia-initiating cells and the results will be shown at the meeting. Taken together, we conclude that OPB-31121 holds promise as a therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase 1 trial in Hong-Kong, Korea, and the USA. Disclosures:Hayakawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Matsuyama:Otsuka Pharmaceutical Co., Ltd.: Employment. Harada:Otsuka Pharmaceutical Co., Ltd.: Employment. Hashimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Ohi:Otsuka Pharmaceutical Co., Ltd.: Employment. Kodama:Otsuka Pharmaceutical Co., Ltd.: Employment. Sumida:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

Sorafenib Induces Growth Arrest and Apoptosis of Human Glioblastoma Cells through the Dephosphorylation of Signal Transducers and Activators of Transcription 3

Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953-62. (c)2010 AACR.

Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes

BackgroundDuring endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.ResultsUsing a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.ConclusionOur results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate.

Atrial Natriuretic Peptide Inhibits Mitogen-activated Protein Kinase through the Clearance Receptor: POTENTIAL ROLE IN THE INHIBITION OF ASTROCYTE PROLIFERATION

The modulation of the activity of mitogen-activated protein kinase (MAPK) by endogenous growth factors or growth inhibitors provides a potential means of regulating cell proliferation. We determined the effect of the endogenous anti-proliferative peptide, atrial natriuretic peptide (ANP), on the ability of MAPK to phosphorylate myelin basic protein. In astrocytes, MAPK activity was significantly stimulated (up to 3-fold) by three known glial mitogens, endothelin-3, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. ANP inhibited by 55-70% the ability of each of these mitogens to activate MAPK. The effects of ANP were equipotent to those caused by C-ANP 4-23, a peptide that specifically binds to the natriuretic peptide clearance receptor. Additionally, both natriuretic peptides caused a 70-80% inhibition of the sodium vanadate-stimulated MAPK activity, complete inhibition of the okadaic acid-stimulated activity, and inhibition of the mitogen-stimulated phosphorylation of MAPK. To understand the potential mechanism by which the natriuretic peptides act, we found that both ANP and C-ANP inhibited the mitogen-stimulated activity of the immediate upstream kinase in the cascade, MAPK kinase (MEK). C-ANP also strongly inhibited the endothelin-3-, platelet-derived growth factor-, and phorbol 12-myristate 13-acetate-induced stimulation of DNA synthesis in the astrocytes, while both okadaic acid and sodium vanadate significantly reversed these anti-proliferative actions. Our results identify ANP as a peptide hormone that inhibits growth factor-stimulated MAPK. These data suggest that the ability of the natriuretic peptides to inhibit MAPK may be important for their anti-growth actions. This effect likely occurs via the inhibition of upstream kinase(s), including MEK, uniquely resulting from ligand binding to the natriuretic peptide clearance receptor.