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.

scholarly journals Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 406
Author(s):  
Hiroki Akiyama ◽  
Yoshihiro Umezawa ◽  
Daisuke Watanabe ◽  
Keigo Okada ◽  
Shinya Ishida ◽  
...  
Keyword(s):  
Map Kinases ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Leukemic Cells ◽  
Bh3 Mimetics ◽  
Downstream Signaling ◽  
Hel Cells ◽  
Induced Apoptosis ◽  
Novel Therapeutic Strategies

JAK2-V617F plays a key role in the pathogenesis of myeloproliferative neoplasm. However, its inhibitor ruxolitinib has shown limited clinical efficacies because of the ruxolitinib-persistent proliferation of JAK2-V617F-positive cells. We here demonstrate that the USP9X inhibitor WP1130 or EOAI3402143 (G9) inhibited proliferation and induced apoptosis more efficiently in cells dependent on JAK2-V617F than on cytokine-activated JAK2. WP1130 preferentially downregulated activated and autophosphorylated JAK2-V617F by enhancing its K63-linked polyubiquitination and inducing its aggresomal translocation to block downstream signaling. Furthermore, JAK2-V617F associated physically with USP9X in leukemic HEL cells. Induction of apoptosis by inhibition of USP9X was mediated through the intrinsic mitochondria-mediated pathway, synergistically enhanced by BH3 mimetics, prevented by overexpression of Bcl-xL, and required oxidative stress to activate stress-related MAP kinases p38 and JNK as well as DNA damage responses in HEL cells. Although autophosphorylated JAK2-V617F was resistant to WP1130 in the ruxolitinib-persistent HEL-R cells, these cells expressed Bcl-2 and Bcl-xL at lower levels and showed an increased sensitivity to WP1130 as well as BH3 mimetics as compared with ruxolitinib-naive HEL cells. Thus, USP9X represents a promising target along with anti-apoptotic Bcl-2 family members for novel therapeutic strategies against JAK2-V617F-positive myeloproliferative neoplasms, particularly under the ruxolitinib persistence conditions.

scholarly journals JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F–positive essential thrombocythemia

Blood ◽  
2010 ◽  
Vol 116 (9) ◽  
pp. 1528-1538 ◽  
Author(s):  
Juan Li ◽  
Dominik Spensberger ◽  
Jong Sook Ahn ◽  
Shubha Anand ◽  
Philip A. Beer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Essential Thrombocythemia ◽  
Cell Function ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Transgenic Models ◽  
Disease Phenotype ◽  
Hematopoietic Stem

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F–positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2V617F mice develop reduced numbers of lineage−Sca-1+c-Kit+ cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2V617F mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Reoxygenation Induces Premature Senescence of FA Hematopoietic Progenitor/Stem Cells through a Pathway Involving ATM, P53, and P21CIP1/WAF1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 34-34
Author(s):  
Xiaoling Zhang ◽  
June Li ◽  
Daniel P. Sejas ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Kinase Inhibitor ◽  
Bone Marrow Failure ◽  
Premature Senescence ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Atm Kinase ◽  
Flow Cytometric Method

Abstract Hematopoietic cells are often exposed to transient hypoxia as they develop and migrate between blood and tissues. We sought to test the role of continuous cycles of hypoxia and reoxygenation in the pathogenesis of bone marrow failure in Fanconi anemia (FA). We isolated Lin− BM cells from wild-type (WT) and Fancc−/− mice, and subjected them to two cycles of hypoxic (1% O2) then reoxygenation (20% O2). Expansion of Fancc−/− Lin− BM cells was significantly decreased in response to the oxidative stress of reoxygenation, as compared with WT Lin− BM cells (1.3 vs. 4.9-fold by day 6, respectively). This inhibition was attributable to a marked decrease of Fancc−/− progenitor (Lin− ScaI− c-kit+) cells, as well as a slower expansion of Fancc−/− stem (Lin− ScaI+ c-kit+) cells than the WT stem cells following reoxygenation. Fancc−/− Lin− BM cells transduced with FANCC retroviruses exhibited a significant growth advantage to untransduced cells, particularly in hypoxia and reoxygenation conditions where the predominance of FANCC/GFP+ progenitors was observed in both the short-term liquid culture and clonogenic assays. There was no evidence of increased apoptotic death in these reoxygenated Fancc−/− BM progenitor and stem cells compared to their WT counterparts, as assessed by a flow cytometric method for caspase 3 activation (early apoptosis). Interestingly, evaluation of the apoptotic profile and cell cycle of stroma-supported long-term BM culture (LTBMC; two weeks after second reoxygenation) revealed that reoxygenated Fancc−/− LTBMC cells had reduced apoptosis compared to reoxygenated short-term culture. However, a vast majority (70.6%) of reoxygenated Fancc−/− LTBMC cells was residing in the G0 + G1 phases compared with 55.8% in WT LTBMC cells. Fancc−/− LTBMC cells stained intensely for SA-b-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21CIP1/WAF1 and to a lesser extent, p16INK4A. Moreover, reoxygenation-induced phosphorylation of p53ser15 was dependent on the ATM kinase but not ATR, as inhibition of ATM signaling by the kinase inhibitor 2-aminopurine or siRNA knockdown of ATM decreased p53ser15 phosphorylation and reduced cell senescence. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc−/− BM hematopoietic progenitor and stem cells by signaling through the ATM to p53, upregulating p21 and causing senescent cell cycle arrest. In addition, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic stem cell depletion and BM failure in FA. <B style=%

The Jak2V617F Oncogene Associated with Polycythemia Vera Regulates G1/S-Phase Transition.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3510-3510
Author(s):  
Martin Sattler ◽  
Christoph Walz ◽  
Brian J. Crowley ◽  
Jessica L. Gramlich ◽  
Kendra L. King ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Polycythemia Vera ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Dependent Manner ◽  
Hel Cells

Abstract The V617F activating point mutation in Jak2 has recently been detected in a high proportion of patients with the myeloproliferative disorders polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. Using the Jak2V617F-mutant erythroid leukemia cell line HEL as a model, potential mechanisms that contribute to transformation were investigated. Inhibition of Jak2V617F with a small molecule kinase inhibitor reduced cell growth of HEL cells in a dose dependent manner with an IC50 of 300 nM. This inhibition of growth was associated with a G1 cell cycle arrest, with minimal or delayed apoptosis. The major Jak2 target in normal hematopoietic cells, STAT5, was found to be activated by Jak2V617F. Treatment of the cells with either a Jak2 kinase inhibitor, or with a Jak2-targeted siRNA, decreased STAT5 activation, and also resulted in decreased expression of cyclin D2 and increased expression of p27Kip. Of interest, we found that Jak2V617F induced high levels of reactive oxygen species (ROS), an activity associated with several other tyrosine kinase oncogenes. Expression of a constitutively active form of STAT5 by itself was capable reducing expression of p27Kip and increasing production of ROS, suggesting that each of these signaling events are downstream of STAT5. Additionally, treatment of HEL cells with the anti-oxidant N-acetylcystein increased expression of p27Kip, suggesting that Jak2V617F regulates cell cycle progression at least in part through STAT5 activation of ROS, and ROS regulation of p27Kip. Cell growth of HEL cells was found to be blocked by anti-oxidants. Overall, our results suggest that constitutive activation of Jak2 contributes to a transforming phenotype and therefore hints at novel targets for drug development that may aid traditional therapy.

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.

ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4316-4316
Author(s):  
Hendrik Folkerts ◽  
Maria Catalina Gomez Puerto ◽  
Albertus T.J. Wierenga ◽  
Koen Schepers ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

scholarly journals Silica Nanoparticles Sensitize Human Multiple Myeloma Cells to Snake (Walterinnesia aegyptia) Venom-Induced Apoptosis and Growth Arrest

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Douaa Sayed ◽  
Mohamed K. Al-Sadoon ◽  
Gamal Badr
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Silica Nanoparticles ◽  
Biological Effects ◽  
Growth Arrest ◽  
Hematopoietic Stem ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis ◽  
The Impact ◽  
Effective Treatments

Background. Multiple myeloma (MM), an almost incurable disease, is the second most common blood cancer. Initial chemotherapeutic treatment could be successful; however, resistance development urges the use of higher toxic doses accompanied by hematopoietic stem cell transplantation. The establishment of more effective treatments that can overcome or circumvent chemoresistance has become a priority. We recently demonstrated that venom extracted fromWalterinnesia aegyptia(WEV) either alone or in combination with silica nanoparticles (WEV+NPs) mediated the growth arrest and apoptosis of prostate cancer cells. In the present study, we evaluated the impact of WEV alone and WEV+NP on proliferation and apoptosis of MM cells.Methods. The impacts of WEV alone and WEV+NP were monitored in MM cells from 70 diagnosed patients. The influences of WEV and WEV+NP were assessed with flow cytometry analysis.Results. WEV alone and WEV+NP decreased the viability of MM cells. Using a CFSE proliferation assay, we found that WEV+NP strongly inhibited MM cell proliferation. Furthermore, analysis of the cell cycle using the propidium iodide (PI) staining method indicated that WEV+NP strongly altered the cell cycle of MM cells and enhanced the induction of apoptosis.Conclusions. Our data reveal the biological effects of WEV and WEV+NP on MM cells that enable these compounds to function as effective treatments for MM.

scholarly journals INNO-406 inhibit the growth of chronic myeloid leukemia and promote its apoptosis via targeting PTEN

2020 ◽  
Author(s):  
Jiandong Sun ◽  
Yilin Wang ◽  
Lirong Sun
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Molecular Mechanism ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Myeloproliferative Neoplasm ◽  
Lyn Kinase ◽  
Apoptotic Protein ◽  
Induced Apoptosis ◽  
Kinase Inhibitory Activity

Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm. INNO-406 is a novel tyrosine kinase inhibitor (TKI) that possess specific Lyn kinase inhibitory activity with no or limited activity against other sarcoma (Src) family member kinases. The present study aimed to confirm the anti-tumor effect of INNO-406 on CML cells, and elucidate the molecular mechanism underlying its effect. Methods The cell proliferation and apoptosis were detected by MTT, western blot and flow cytometry respectively. Results As suggested by the findings, INNO-406 significantly inhibited the proliferation and induced apoptosis of CML cells. In addition, INNO-406 promoted the expression level of PETN. Rescue experiment revealed that PTEN knockdown reversed the effect of INNO-406 which indicated the correlation between INNO-406 and PTEN. Further study determined that PTEN inhibited the phosphorylation of AKT and 4EBP1 and subsequently altered the expression of apoptotic protein expressions including bax, cytochrome c (cyto-c), cleaved caspase3 and bcl-2. In vivo study further confirmed that INNO-406 inhibited the growth of CML cells in vivo by targeting PTEN. Conclusion Based on the above findings, this work extended our understanding of INNO-406 in the chemotherapy of CML and its molecular mechanism.

scholarly journals Combination approach to diagnosis and treatment of an elderly patient with chronic Ph-negative myeloproliferative neoplasm and concomitant surgical pathology. Clinical observation

MD-Onco ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 61-65
Author(s):  
Yu. E. Ryabukhina ◽  
P. A. Zeynalova ◽  
O. I. Timofeeva ◽  
F. M. Abbasbeyli ◽  
T. V. Ponomarev ◽  
...  
Keyword(s):  
Elderly Patient ◽  
Essential Thrombocythemia ◽  
Early Stage ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Left Femur ◽  
Hematopoietic Stem ◽  
Chronic Myeloproliferative Neoplasms

Chronic myeloproliferative neoplasms (CMPN), Ph-negative, are of clonal nature, develop on the level of hematopoietic stem cell and are characterized by proliferation of one or more hematopoietic pathways. Currently, the group of Ph-negative CMPN includes essential thrombocythemia, primary myelofibrosis, polycythemia vera, myeloproliferative neoplasm unclassifiable.Identification of mutations in the Jak2 (V617F), CALR, and MPL genes extended understanding of biological features of Ph-negative CMPN and improved differential diagnosis of myeloid neoplasms. Nonetheless, clinical practice still encounters difficulties in clear separation between such disorders as primary myelofibrosis, early-stage and transformation of essential thrombocythemia into myelofibrosis with high thrombocytosis. Thrombocytosis is one of the main risk factors for thromboembolic complications, especially in elderly people.A clinical case of an elderly patient with fracture of the left femur developed in the context of Ph-negative CMPN (myelofibrosis) with high level of thrombocytosis is presented which in combination with enforced long-term immobilization and presence of additional risk created danger of thrombosis and hemorrhage during surgery and in the postoperative period.

scholarly journals Identification of the Epigenetic Reader BRD4 As a Novel Therapeutic Target in JAK2 V617F+ MPN Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2829-2829
Author(s):  
Alexandra Keller ◽  
Barbara Peter ◽  
Johannes Zuber ◽  
Philipp Bernhard Staber ◽  
Peter Bettelheim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Jak2 V617f ◽  
G1 Arrest ◽  
Mrna Levels ◽  
Neoplastic Cells ◽  
Hel Cells ◽  
Ic50 Values ◽  
Brd4 Inhibition

Abstract Myeloproliferative neoplasms (MPN) are characterized by clonal expansion and accumulation of erythrocytes, platelets, and myeloid cells in the bone marrow (BM) and other organs. Classical MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 V617F mutation is frequently detected in neoplastic cells in patients with MPN. Although MPN are chronic and indolent diseases in most patients, fatal progression may occur. So far, the only curative approach for these patients is hematopoietic stem cell transplantation. Therefore, current research is evaluating new therapeutic targets and the effects of various targeted drugs. The epigenetic reader bromodomain-containing protein 4 (BRD4) has recently been identified as a promising target in acute myeloid leukemia. In the present study, we investigated the potential value of BRD4 as a molecular target in MPN. We employed two JAK2 V617F+ cell lines, SET-2 and HEL, as well as BM samples obtained from 18 MPN patients (ET: n=7; PV: n=7; PMF: n=4). Three BRD4 inhibitors were applied: JQ1, BI2536, and BI6727. As assessed by qPCR, primary MPN cells as well as SET-2 cells and HEL cells were found to express BRD4 mRNA. In 3 H-thymidine uptake experiments, all three BRD4 blockers were found to suppress the proliferation of the two MPN cell lines and of primary MPN cells in 8/8 patients tested. The effects of these drugs were dose-dependent, with the following IC50 values obtained in SET-2 cells: JQ1, 50-100 nM; BI2536, 20-40 nM; BI6727, 50-75 nM; and in HEL cells: JQ1, 100-500 nM; BI2536, 20-40 nM; BI6727, 30-50 nM. In primary MPN cells, all three agents tested produced IC50 values between 500 and 1000 nM. In normal BM cells, JQ1 did not produce a reasonable IC50 value (>5000 nM). In one patient sample (PMF), we analyzed the effect of JQ1 on the percentage of putative (neoplastic) stem cells (CD34+/CD38-). In this experiment, exposure to JQ1 was followed by a decrease in the percentage of CD34+/CD38- cells compared to control medium (control: 0.16% vs JQ1: 0.045%). To confirm the role of BRD4 as a potential target in MPN cells, we performed target-knockdown experiments in SET-2 cells and HEL cells using two different BRD4 shRNAs (#602 and #1817) and a random shRNA as control. In these experiments, the shRNA-induced knockdown of BRD4 was found to block proliferation in SET2 cells and HEL cells when compared to untransfected cells or random shRNA-transduced cells. In a next step, we examined the mechanism of drug-induced growth inhibition. In cell cycle experiments, BI2536 and BI6727 were found to induce a G2/M phase arrest in both cell lines. By contrast, JQ1 induced a G1 arrest in HEL cells, but did not show a significant effect on cell cycle progression in SET-2 cells. We also asked whether BRD4 inhibition is associated with induction of apoptosis in MPN cells. All three BRD4 blockers induced apoptosis in SET-2 cells and HEL cells at relatively high concentrations after 48 hours, with ED50 values of >5 µM for JQ1 and 0.5-5.0 µM for BI2536 and BI6727. Finally, we asked whether exposure to BRD4 inhibitors is associated with modulation of BRD4 mRNA or MYC mRNA expression. As assessed by qPCR, JQ1, BI2536, and BI6727 were found to downregulate BRD4 mRNA levels as well as MYC mRNA levels in SET-2 cells and HEL cells. In conclusion, our data show that BRD4 is expressed in JAK2 V617F+ MPN cells and that BRD4 inhibition is associated with decreased proliferation and survival of neoplastic cells. The clinical value of BRD4 as a novel target in MPN cells remains to be determined. Disclosures Zuber: Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Staber:Genactis: Research Funding; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda-Millenium: Research Funding; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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