Synergistic Activity of Combinations of JAK2 Kinase Inhibitor with PI3K/mTOR, MEK or PIM Kinase Inhibitor Against Human Myeloproliferative Neoplasm Cells Expressing JAK2V617F

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 798-798 ◽  
Author(s):  
Warren Fiskus ◽  
Rekha Rao Manepalli ◽  
Ramesh Balusu ◽  
Kapil N. Bhalla
Keyword(s):  
Progenitor Cells ◽  
Mtor Inhibitor ◽  
Kinase Inhibitor ◽  
Jak2 V617f ◽  
Protein Translation ◽  
Mek Inhibitor ◽  
Cytokine Signaling ◽  
Synergistic Activity ◽  
Cytotoxic Effects ◽  
Pim1 Kinase

Abstract Abstract 798 The mutant JAK2-V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). JAK2-V617F activates downstream signaling through the STAT, RAS/MAPK and PI3/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). We have previously reported that pan-histone deacetylase (HDAC) inhibitors e.g. panobinostat (PS) (Novartis Pharmaceuticals), depleted mRNA expression of JAK2-V617F, and disrupted the chaperone association of with hsp90 with JAK2-V617F, thereby promoting the degradation of JAK2-V617F by the proteasome. This led to attenuation of the levels and downstream transcriptional activity of STAT3 and STAT5, resulting in growth arrest and apoptosis of MPN HPCs. Additionally, co-treatment with PS and a JAK2 kinase inhibitor, TG101209, further depleted JAK/STAT signaling and synergistically induced apoptosis of JAK2-V617F expressing HEL92.1.7 and Ba/F3-JAK2V617F cells, as well as exerted greater lethality against primary CD34+CD38-Lin- MPN versus normal CD34+ HPCs. In the present studies, we determined the cytotoxic effects of inhibiting JAK2-STAT3/5 in conjunction with pharmacologic targeting of the collateral, pro-growth and pro-survival signaling through PI3K/AKT, RAF-MEK or PIM1 kinases in MPN cells. For this, the cytotoxic effects of co-treatment with TG101209 and the MEK inhibitor (AZD6244, AstraZenaca), dual PI3K/mTOR inhibitor (BEZ235, Novartis) or the PIM1 kinase inhibitor (SGI-1776, SuperGen) were evaluated in HEL92.1.7, Ba/F3-JAK2V617 and primary human MPN cells. Treatment with BEZ235 dose-dependently attenuated the levels of p-JAK2, p-STAT5, p-STAT3, p-AKT, p-ERK1/2 and p-4EBP1. Co-treatment with BEZ235 and TG101209 was synergistically lethal against the cultured MPN and primary CD34+ MF-MPN cells (combination indices < 1.0). Co-treatment with AZD6244 and TG101209 also induced synergistic apoptosis of cultured MPN cells (combination indices of < 1.0). This was associated with greater attenuation of the levels of p-AKT and p-ERK1/2. PIM1 is a cytoplasmic serine/threonine kinase that serves as a downstream effector of several cytokine signaling pathways promoting cell survival and proliferation. PIM1 collaborates in Myc-induced transformation and known to phosphorylate 4EBP1 and eIF4B, thereby promoting protein translation. Co-treatment with TG101209 and the PIM1 kinase inhibitor, SGI-1776 also induced synergistic apoptosis of HEL92.1.7 cells and Ba/F3-JAK2V617F cells (combination indices < 1.0) but not of Ba/F3-hEpoR cells. PIM kinase mediates PRAS40 phosphorylation and induces mTORC1 activity in phosphorylating 4EBP1. Consistent with this, co-treatment with SGI-1776 and TG-101209 inhibited p-PRAS40 and p-4EBP1 levels in cultured MPN but not in normal progenitor cells. These findings demonstrate for the first time that combined treatment with a MEK inhibitor, PIM1 kinase inhibitor or dual PI3K/mTOR inhibitor enhances the anti-JAK2-V617F activity of TG101209 in cultured and primary human MPN cells. Our findings support the rationale to determine the in vivo activity of TG101209 in combination with inhibitors of MEK, PIM1 or PI3K/mTOR kinase against human MPN cells. Disclosures: No relevant conflicts of interest to declare.

Synergistic Activity of Co-Treatment with PIM1 Kinase Inhibitor SGI-1776 and Histone Deacetylase Inhibitor Panobinostat or Heat Shock Protein 90 Inhibitor AUY922 against Human CML and Myeloproliferative Neoplasm (MPN) Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2651-2651 ◽  
Author(s):  
Warren C. Fiskus ◽  
Kathleen M. Buckley ◽  
Rekha Rao ◽  
Yongchao Wang ◽  
Atul Joshi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Histone H3 ◽  
Myeloproliferative Neoplasm ◽  
Heat Shock Protein 90 ◽  
Jak2 V617f ◽  
Hematopoietic Stem ◽  
Pim1 Kinase ◽  
Hel Cells ◽  
Induced Apoptosis

Abstract Abstract 2651 Poster Board II-627 The oncoprotein PIM1 is a cytoplasmic serine/threonine kinase, which is overexpressed and implicated in the pathogenesis of hematologic malignancies. PIM1 activity promotes cell survival and proliferation, and is a downstream mediator of cytokine receptor signaling involving the JAK/STATs. PIM1 phosphorylates and inactivates Bad, which enhances the anti-apoptotic functions of Bcl-2. PIM1 also mediates the phosphorylation and inactivation of cell cycle inhibitors p21 and p27, thereby promoting progression through G1 and G2/M phases of the cell cycle. PIM1 phosphorylates serine 10 on histone H3, and has been shown to phosphorylate and stabilize Myc, thereby collaborating in Myc-induced transformation. Further, PIM1 is known to phosphorylate 4EBP1 on the same residues (threonine 37 and 46) as mTOR kinase. It can also phosphorylate eIF4B, thereby maintaining high levels of protein translation. PIM1 is known to be chaperoned as a heat shock protein 90 client protein. In the present studies, we demonstrate that treatment with the PIM kinase inhibitor SGI-1776 (SuperGen Inc.) dose-dependently (500 nM to 2.0 μM) depleted p-BAD (Ser112), p-p70S6K, p-4EBP1, p-Ser10 Histone H3 and c-Myc levels, while inducing p27 and p21 expression, in the cultured human CML-BC K562 and erythroleukemia HEL92.1.7 (with homozygous JAK2-V617F mutation). Treatment with SGI-1776 induced apoptosis of K562 and HEL92.1.7 (HEL) cells as well as Ba/F3-hEpoR-JAK2-V617F but not Ba/F3-hEpoR cells. Similar effects of SGI-1776 were observed in primary CML-BC and MPN (myeloproliferative neoplasm) cells. Treatment with the non-geldanamycin analogue hsp90 inhibitor AUY922 (Novartis Pharma) or with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharma) disrupted the chaperone association of PIM1 with hsp90 and increased binding to hsp70, thereby promoting the proteasomal degradation of PIM1 in K562 and HEL cells. Treatment with PS (20–100 nM) also depleted PIM1 mRNA as measured by qPCR. Co-treatment with SGI-1776 (0.5 to 2.0 μM) and clinically achievable concentrations of AUY922 (e.g., 25 nM) caused greater depletion of pBAD (Ser112), pp70S6K, p4EBP1, cyclin B1, c-Raf, c-Myc, and AKT and synergistically induced apoptosis of K562 and HEL92.1.7 cells (combination indices < 1.0). As compared to treatment with either agent alone, co-treatment with SGI-1776 and AUY922 or PS also exerted greater cytotoxicity against primary CML-BC and MF-MPN cells. Importantly, co-treatment with PS significantly enhanced SGI-1776-induced apoptosis of purified primary CD34+CD38-Lin- hematopoietic stem cells expressing JAK2-V617F derived from a patient with primary myelofibrosis. Taken together, these pre-clinical findings indicate that simultaneously depleting PIM kinase levels by AUY922 or PS and inhibiting PIM1 kinase activity with SGI-1776 induces synergistic apoptosis against cultured and primary CML-BC and MPN cells. Additionally, our findings suggest that co-treatment with PS and SGI-1776 may be an effective treatment strategy against hematopoietic stem cells expressing JAK2-V617F in patients with MF-MPN. Disclosures: Atajada: Novartis: Employment. Quadt:Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

Combined MEK and mTOR suppression is synergistic in human NSCLC and is mediated via inhibition of protein translation

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 10615-10615 ◽  
Author(s):  
H. M. McDaid ◽  
M. Legrier ◽  
C. H. Yang ◽  
H. G. Yan ◽  
L. Lopez-Barcons ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Human Lung ◽  
Lung Tumor ◽  
Mtor Inhibitors ◽  
Protein Translation ◽  
Mek Inhibitor ◽  
Human Lung Cancer ◽  
Index Method

10615 Background: Lung cancer is a heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of RAS and AKT. These intersect at various points rendering the network highly redundant. Hence, dual suppression of both, using MEK and mTOR inhibitors may be a promising rational drug combination that can overcome the intrinsic plasticity of this signaling network. Methods: The concurrent combination of the MEK inhibitor CI-1040 and the mTOR inhibitor AP23573 was evaluated in non-small cell lung cancer (NSCLC) cell lines, using the combination index method of Chou and Talalay. The concurrent combination of Rapamycin and the MEK inhibitor PD0325901 was also evaluated in animal models of human lung cancer. Results: The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor rapamycin, or its analog AP23573, were synergistic in human lung cancer cell lines. Statistically significant enhancement of cell death by the combination of MEK and mTOR inhibitors was not observed. Rather, synergism was associated with suppression of proliferation and inhibition of protein translation. Concurrent suppression of both MEK and mTOR inhibited ribosomal biogenesis by forty percent after twenty-four hours and was associated with a block in the initiation phase of translation. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone, or PD0325901 at the maximum tolerated dose, in xenograft and human heterotransplanted lung tumor models grown in nude mice. Conclusions: These data support therapeutic strategies in lung cancer that simultaneously suppress RAS and mTOR signaling networks, and provide evidence that both pathways converge on factors that regulate protein translation initiation. No significant financial relationships to disclose.

scholarly journals Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

scholarly journals Second-generation tricyclic pyrimido-pyrrolo-oxazine mTOR inhibitor with predicted blood–brain barrier permeability

2021 ◽  
Author(s):  
Chiara Borsari ◽  
Erhan Keles ◽  
Andrea Treyer ◽  
Martina De Pascale ◽  
Paul Hebeisen ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Neurological Disorders ◽  
Second Generation ◽  
Mtor Inhibitor ◽  
Kinase Inhibitor ◽  
Brain Barrier ◽  
Barrier Permeability ◽  
Blood Brain Barrier Permeability ◽  
Blood Brain ◽  
Brain Barrier Permeability

Here we present the first pyrimido-pyrrolo-oxazine-based mTOR kinase inhibitor (11) predicted to penetrate the blood brain barrier (BBB). Thus, 11 has a potential in treatments of neurological disorders.

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

2003 ◽  
Vol 284 (6) ◽  
pp. L1020-L1026 ◽  
Author(s):  
Stephen M. Carlin ◽  
Michael Roth ◽  
Judith L. Black
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Airway Remodeling ◽  
Muscle Cells ◽  
Mek Inhibitor ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-μm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFABwere all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3′-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

scholarly journals Characterization of CD34+ hematopoietic progenitor cells in JAK2 V617F and CALR -mutated myeloproliferative neoplasms

2016 ◽  
Vol 48 ◽  
pp. 11-15 ◽  
Author(s):  
Anna Angona ◽  
Alberto Alvarez-Larrán ◽  
Beatriz Bellosillo ◽  
Raquel Longarón ◽  
Laura Camacho ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina

Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3593-3605 ◽  
Author(s):  
M.A. Dyer ◽  
C.L. Cepko
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Kinase Inhibitor ◽  
Retinal Development ◽  
Cell Types ◽  
Cell Cycle Exit ◽  
Retinal Progenitor Cells ◽  
Mouse Development ◽  
Retinal Progenitor ◽  
Cyclin Kinase Inhibitor

A precise balance between proliferation and differentiation must be maintained during retinal development to obtain the correct proportion of each of the seven cell types found in the adult tissue. Cyclin kinase inhibitors can regulate cell cycle exit coincident with induction of differentiation programs during development. We have found that the p57(Kip2) cyclin kinase inhibitor is upregulated during G(1)/G(0) in a subset of retinal progenitor cells exiting the cell cycle between embryonic day 14.5 and 16.5 of mouse development. Retroviral mediated overexpression of p57(Kip2) in embryonic retinal progenitor cells led to premature cell cycle exit. Retinae from mice lacking p57(Kip2) exhibited inappropriate S-phase entry and apoptotic nuclei were found in the region where p57(Kip2) is normally expressed. Apoptosis precisely compensated for the inappropriate proliferation in the p57(Kip2)-deficient retinae to preserve the correct proportion of the major retinal cell types. Postnatally, p57(Kip2) was found to be expressed in a novel subpopulation of amacrine interneurons. At this stage, p57(Kip2)did not regulate proliferation. However, perhaps reflecting its role during this late stage of development, animals lacking p57(Kip2) showed an alteration in amacrine subpopulations. p57(Kip2) is the first gene to be implicated as a regulator of amacrine subtype/subpopulation development. Consequently, we propose that p57(Kip2) has two roles during retinal development, acting first as a cyclin kinase inhibitor in mitotic progenitor cells, and then playing a distinct role in neuronal differentiation.

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