Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4241-4241
Author(s):  
Stefan H. Faderl ◽  
Quin Van ◽  
Patricia E. Koch ◽  
David M. Harris ◽  
Inbal Hallevi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Western Immunoblotting ◽  
Gm Csf ◽  
Dose Dependent

Abstract Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.

scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

Abstract The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

Advanced Glycation End Products (AGEs)-Mediated Cell Growth of Human Acute Myelogenous Leukemia Via Mitogen-Activated Protein (MAP) Kinase Pathways.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2762-2762
Author(s):  
Ju Young Kim ◽  
Hyun Ki Park ◽  
Jin Sun Yoon ◽  
Eun Shil Kim ◽  
Kwang Sung Ahn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Glycation End Products ◽  
Mitogen Activated Protein ◽  
Acute Myelogenous ◽  
Dose Dependent

Abstract Advanced glycation end products (AGEs) are products of non-enzymatic glycation/oxidation of proteins/lipids that accumulate slowly during natural aging and at a much accelerated rate in a variety of disorders such as diabetes, renal failure, and Alzheimer’s disease. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signaling, activation of transcription factors and subsequent gene expression in vitro and in vivo. Most of the biologic activities associated with AGEs have been transduced by receptor for AGE (RAGE). Recently, AGEs are known to be in association with diverse cancers in terms of cellular proliferation and metastasis. However, little is known about the role of AGEs in acute myelogenous leukemia (AML). Here we examined the effects of the AGEs-RAGE interaction on the cell proliferation and intracellular signaling of AGEs in human leukemia cell lines. Expression of RAGE was observed in 8 AML cell lines examined, and up-regulated by treatment of AGE. AGE induced the proliferation of AML cell lines, HL60 and HEL, in a dose-dependent manner. Treatment with 5 μM of antisense S-ODN for RAGE did effectively inhibit cell growth of HEL cells. Exposure of HL60 and HEL with AGE induced a significant increase in the numbers of cells in S phase of cell cycle in a dose-dependent manner. AGE enhanced the expression of cell cycle regulatory proteins such as cyclin-dependent kinase (CDK) 2/4/6, cyclin D1/E/B in a dose- and a time-dependent manner. In addition, the protein levels of the cyclin-dependent kinase inhibitor (CDKI), p21 and p27, were decreased by 24 hr exposure of AGE from 10 to 200 μg/ml in HEL. Furthermore, treatment of HEL with 200 μg/ml of AGE triggered activation of mitogen-activated protein (MAP) kinases, Erk, Akt, and p38, pathways and in nuclear translocation of transcription factors NF-kB. These results indicated that AGE induced the cell growth of human AML cells, HL60 and HEL, via augmentation of cell cycle and activation of MAPK kinase pathways. Up-regulation of RAGE by exposure of AGE suggested that cellular proliferation of AML cells might be mediated in autocrine fashion.

The Synthetic Hest Shock Protein (Hsp) 90 Inhibitor EC141 Induces Degradation of the Bcr-Abl p190 Protein and Apoptosis of Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2784-2784
Author(s):  
Alessandra Ferrajoli ◽  
Yongtao Wang ◽  
Susan M. O’Brien ◽  
Stefan H. Faderl ◽  
David M. Harris ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Protein Kinase Inhibitors ◽  
High Dose ◽  
Dependent Manner ◽  
Protein Levels ◽  
Dose Dependent

Abstract High dose chemotherapy of Ph+ ALL is rarely curative and clinical responses to protein kinase inhibitors have been transient. Although new regimens combining chemotherapy with Bcr-Abl kinase inhibitors improve survival, the long-term prognosis of patients with Ph+ ALL remains guarded. Thus, novel therapeutic strategies are needed. Hsp90 is a ubiquitous molecular chaperone protein required for the folding, activation and assembly of mediators of signal transduction, cell cycle control, and transcription regulation. The Hsp90 inhibitor EC141 (Biogen Idec, Inc.) blocks the chaperone activity of Hsp90 and induces proteasomal degradation of it’s client proteins. Because Hsp90 is a chaperone of Bcr-Abl we investigated the activity of EC141 against the Ph+ ALL B-cell lines Z-119, Z-181 and Z-33 (Estrov et al. J Cell Physiol166: 618, 1996; Leukemia10:1534, 1996). First we studied the effect of EC141 on Hsp levels in Ph+ ALL cells. EC141 (50 nM) down-regulated the protein levels of Hsp90 and upregulated those of Hsp70. Then, the effect of EC141 on the proliferation of Ph+ ALL cells was evaluated using the MTT assay. EC141 inhibited the growth and metabolic activity of Z-119, Z-181 and Z-33 Ph+ ALL cells in a dose-dependent manner at concentrations ranging from 1 to 100 nM. Similar results were obtained with primary bone marrow cells from patients with Ph+ ALL. Using the ALL blast colony culture assay we found that EC141 inhibited the proliferation of marrow-derived ALL colony-forming cells in a dose-dependent fashion. To explore the mechanism of action Z-181 were incubated cells with increasing concentrations of EC141; immunoprecipitation and Western immunoblotting were used to detect changes in cellular protein levels. EC141 degraded the Bcr-Abl p190 protein and inhibited the phosphorylation of CrkL in a dose-dependent manner. Furthermore, exposure of Z-181 cells to EC141 resulted in a time- and dose-dependent activation of procaspase 3, cleavage of poly (adenosine diphosphate-ribose) polymerase and apoptotic cell death as assessed by Annexin V. Taken together, our data suggest that EC141 degrades the Bcr-Abl p190 protein, inhibits proliferation, and induces apoptosis of Ph+ ALL cells. Additional studies aimed at investigating the in vivo activity of EC141 in Ph+ ALL are warranted.

Cellular and Molecular Effects of NVP-BKM120 in Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2241-2241
Author(s):  
João Kleber Novais Pereira ◽  
João Agostinho Machado-Neto ◽  
Matheus Rodrigues Lopes ◽  
Fabiola Traina ◽  
Fernando Ferreira Costa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cyclin B1 ◽  
Pyrimidine Derivative ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Daudi Cell ◽  
Induction Of Apoptosis ◽  
Orally Bioavailable ◽  
Dose Dependent

Abstract Background: PI3K/AKT/mTOR signaling controls most hallmarks of cancer. Constitutive activation of PIK3 pathway in T cells acute lymphoblastic leukemia (T-ALL) has been reported; in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. NVP-BKM120 is an orally bioavailable 2,6-dimorpholine pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. In preclinical studies, it has shown efficacy in a variety of malignancies and is currently being investigated in phase I/II/III clinical testing, mainly for advanced solid tumors (clinicaltrials.gov). Aims: Here, we described the effects of the pan-PI3K/AKT/mTOR inhibitor NVP-BKM120 on T-ALL and BL cell lines. Methods: T-ALLcell lines, Jurkat and MOLT-4, and BL cell lines, NAMALWA and Daudi, were obtained from ATCC. NVP-BKM120 was kindly provided by Novartis, and was prepared as a 10mM stock solution in DMSO. Different concentrations of the drug were used as indicated, where cells treated only with DMSO served as control. Cell viability was measured by MTT. Colony formation was carried out in semisolid methyl cellulose medium. The induction of apoptosis was assessed by annexin-V-APC/PI and by caspase cleavage. The cell cycle was analyzed by a PI-staining method. Western blot analysis was performed by standard methods. Vital staining and flow cytometry analysis with acridine orange was performed for the detection and quantification of acidic vesicular organelles (AVOs). Comparisons between the two groups were performed by the t test. Pvalue <0.05 was considered statistically significant. Results: Cell viability decreased in a concentration-dependent manner, with an IC50 range of 7-8 μM and the clonogenic growth was significantly decreased at the concentration of 1μM and at 10µM the colony formation was completely inhibited in all cells tested. After 6 hours of NVP-BKM120 treatment, we observed an increase in apoptotic cells, as well as an increase in the cleavage of procaspase 3, 8 and 9 in Jurkat, MOLT-4 and NAMALWA cells. Compared with DMSO control, NVP-BKM120 does not have any effect during apoptosis induction in the Daudi cell line. NVP-BKM120 treatment also resulted in G2/M arrest, associated with a decrease in the G1population and a decrease in Cyclin B1 protein levels. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, P70SK6 and 4EBP1, with stable total proteins levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, followed by an increase in BAX:BCL2 ratio. Quantification of AVOs showed a dose-dependent increase of AVOs in all cells tested, after NVP-BKM120 treatment. Conclusions: NVP-BKM120 induced apoptosis in a dose-dependent manner in Jurkat, MOLT-4 and NAMALWA cells, while effects of the drug in the Daudi cell line were mainly cytostatic. In those cells, the induction of apoptosis suggested that the death receptor and mitochondrial pathways were activated after drug treatment. In our study, we found that NVP-BKM120 decreased the phosphorylation levels of BAD, which is linked to a pro-apoptotic activity, and up-regulated the BAX:BCL2 ratio. These results are consistent with the activation of caspase-9 and 3, related to the mitochondrial apoptosis. The accumulation of leukemia cells in the G2/M phase of the cell cycle has been associated with enhanced apoptosis. Our results suggest that decreased Cyclin B1 protein expression might be the molecular mechanism through which NVP-BKM120 induces G2/M arrest. The effects of NVP-BKM120 on the PI3K pathway indicate that NVP-BKM120 treatment may overcome rapamycin-induced AKT activation. P70SK6 and 4EBP1 are the two best-characterized substrates of mTOR1. Hence, the decrease in the phosphorylation levels of P70SK6 and 4EBP1 results in impaired oncogenic protein synthesis. Moreover, P70SK6 is one of the kinases whose phosphorylation by mTOR1 results in opposing autophagy. Accordingly, NVP-BKM120 resulted in increased AVOs, which is a characteristic feature of cells engaged in autophagy. In summary, our present study establishes that NVP-BKM120 effectively presents an antitumor activity against T-ALL and BL cell lines. The reduction of proliferation is possibly by down-regulation of Cyclin B1 and the increased BAX:BCL2 ratio is one of the mechanisms involved in the induction of apoptosis. Disclosures Off Label Use: NVP-BKM120 is an orally bioavailable 2,6-dimorpholino pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. .

Polymorphonuclear Leukocyte Apoptosis Is Inhibited by Platelet-released Mediators, Role of TGFβ-1

2000 ◽  
Vol 84 (09) ◽  
pp. 478-483 ◽  
Author(s):  
Nicola Martelli ◽  
Stefano Manarini ◽  
Nicola Mascetra ◽  
Piero Musiani ◽  
Chiara Cerletti ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Polymorphonuclear Leukocyte ◽  
Active Form ◽  
Dependent Manner ◽  
Gm Csf ◽  
Apoptotic Effect ◽  
Apoptotic Activity ◽  
Dose Dependent ◽  
Tgf Β1

SummaryPlatelets regulate several polymorphonuclear leukocyte (PMN) functions. We have found that thrombin-stimulated platelets potently inhibited PMN apoptosis. Cell-free supernatant from increasing concentrations of stimulated platelets inhibited PMN apoptosis in a dosedependent manner, with an effect similar to that of corresponding concentrations of platelets. At the plateau, platelet supernatant inhibited PMN apoptosis by 54.6 ± 6.8%, the anti-apoptotic activity being higher than that of GM-CSF and comparable to that of LPS. Neither IL-1ra nor a combination of anti-IL1 α + β mAb affected the activity of platelet supernatant. In contrast a mAb recognizing the active form of TGF-β1 significantly decreased this activity. Moreover, exogenous TGF-β1 inhibited PMN apoptosis in a dose-dependent manner. The active form of this cytokine was indeed present in the supernatant of stimulated platelets at a concentration able to elicit an anti-apoptotic effect. The p38 MAPK inhibitor SB203580 prevented the anti-apoptotic effect of TGF-β1 in a dose-dependent manner. Interestingly, it also prevented the anti-apoptotic effect of IL-1α, but not that of GM-CSF, LPS and dexamethasone. In conclusion, we report for the first time that PMN apoptosis is potently inhibited by platelet-released mediators, that TGF-β1 mediates an important part of this effect, and that p38 MAPK is involved in the TGF-β1 signaling leading to its anti-apoptotic effect. These results provide novel evidence to support the central role of platelets in inflammation.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia

Blood ◽  
2021 ◽  
Author(s):  
Yunchao Chang ◽  
Jaeki Min ◽  
Jamie Jarusiewicz ◽  
Marisa Actis ◽  
Shanshan Yu-Chen Bradford ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Rational Design ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Janus Kinase ◽  
Tyrosine Kinase Domain ◽  
Type I ◽  
Jak Inhibitors ◽  
Janus Kinases

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) comprises over half of Philadelphia chromosome-like (Ph-like) ALL, is associated with poor outcome in children and adults. Overexpression of CRLF2 results in activation of JAK-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of Janus kinases show variable and limited efficacy. Here we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against Janus kinases. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of multiple series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded Janus kinases and potently killed CRLF2­-rearranged cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1, and suppressed proliferation of CRLF2-rearranged ALL in vivo. While dual JAK/GSPT1-degrading PROTACs were most potent, development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading GSPT1-sparing PROTAC that demonstrated efficacy in the majority of the kinase-driven xenografts which were otherwise unresponsive to type I JAK inhibitors. Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL, and highlight the interplay of Janus kinase and GSPT1 degradation activity in this context.

scholarly journals JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome–negative CML, and megakaryocytic leukemia

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3370-3373 ◽  
Author(s):  
Jaroslav Jelinek ◽  
Yasuhiro Oki ◽  
Vazganush Gharibyan ◽  
Carlos Bueso-Ramos ◽  
Josef T. Prchal ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Myeloproliferative Disorders ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Hematologic Neoplasms ◽  
Jak2 Mutation ◽  
Acute Leukemias ◽  
Myelomonocytic Leukemia

AbstractAn activating 1849G&gt;T mutation of JAK2 (Janus kinase 2) tyrosine kinase was recently described in chronic myeloproliferative disorders (MPDs). Its role in other hematologic neoplasms is unclear. We developed a quantitative pyrosequencing assay and analyzed 374 samples of hematologic neoplasms. The mutation was frequent in polycythemia vera (PV) (86%) and myelofibrosis (95%) but less prevalent in acute myeloid leukemia (AML) with an antecedent PV or myelofibrosis (5 [36%] of 14 patients). JAK2 mutation was also detected in 3 (19%) of 16 patients with Philadelphia-chromosome (Ph)–negative chronic myelogenous leukemia (CML), 2 (18%) of 11 patients with megakaryocytic AML, 7 (13%) of 52 patients with chronic myelomonocytic leukemia, and 1 (1%) of 68 patients with myelodysplastic syndromes. No mutation was found in Ph+CML (99 patients), AML M0-M6 (28 patients), or acute lymphoblastic leukemia (20 patients). We conclude that the JAK2 1849G&gt;T mutation is common in Ph– MPD but not critical for transformation to the acute phase of these diseases and that it is generally rare in aggressive leukemias.

Simultaneous Administration of AMN107 and Imatinib in the Treatment of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 694-694 ◽  
Author(s):  
James D. Griffin ◽  
Ellen L. Weisberg
Keyword(s):  
Cell Lines ◽  
Synergistic Effects ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Imatinib Resistant

Abstract Chronic myelogenous leukemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) are caused by the Bcr-Abl tyrosine kinase oncogene. The Abl inhibitor imatinib is an effective, frontline therapy for early, chronic phase CML. However, accelerated or blast crisis phase CML and Ph+ ALL patients often relapse because of drug resistance that results from the emergence of imatinib-resistant point mutations within the Bcr-Abl kinase domain. The aminopyrimidine ATP-competitive inhibitor, AMN107, was designed to fit into the ATP-binding site of the Bcr-Abl protein in such a way as to exhibit higher efficacy against imatinib-resistant Bcr-Abl point mutants. AMN107 is active against many imatinib-resistant Bcr-Abl mutants in vitro and in vivo, and is significantly more potent than imatinib against wild-type Bcr-Abl. AMN107 is currently showing promise in phase I/II clinical trials involving CML patients who are unresponsive to imatinib, and thus could potentially be used as a single agent in selected patients resistant or intolerant to imatinib. Alternatively, the use of more than one inhibitor of Abl should effectively lower the number of residual Bcr-Abl-expressing cells having the potential to undergo mutation, and therefore could potentially suppress the emergence of drug-resistant Bcr-Abl mutations. Thus, AMN107 and imatinib could be administered together to achieve higher responsiveness in CML patients. In the current study, we investigated the combination of imatinib and AMN107 in a panel of wild-type and imatinib-resistant Bcr-Abl-expressing cell lines, including 32D.p210, K562, F486S-Ba/F3, F317L-Ba/F3, M351T-Ba/F3, and T315I-Ba/F3. We found evidence of additive to synergistic effects in several of the cell lines examined. In addition, the combination of AMN107 and imatinib was studied in vivo using a bioluminescent Bcr-Abl model of CML. Mice harboring murine 32D.p210 cells engineered to stably express firefly luciferase were treated with vehicle, AMN107 alone (15mg/kg), imatinib alone (75mg/kg), or both AMN107 and imatinib at their respective doses. Mice treated with both agents were observed to carry an overall lower tumor burden (as measured by levels of total body bioluminescence and percent spleen weights) than vehicle-treated mice and mice treated with each agent alone. These results suggest that the combination of imatinib and AMN107 may be a more effective treatment for CML than either agent alone.

WP1066: A Novel PI-3K Inhibitor with Antileukemic Activity in Philadelphia Chromosome-Positive ALL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 850-850
Author(s):  
Alessandra Ferrajoli ◽  
Stefan Faderl ◽  
Tony Wang ◽  
Waldemar Priebe ◽  
Hagop Kantarjian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tyrosine Kinases ◽  
Philadelphia Chromosome ◽  
Annexin V ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Dose Dependent

Abstract Prognosis for patients with Philadelphia chromosome (Ph) positive ALL remains dismal. Ph+ ALL is characterized by the activation of several tyrosine kinases that provide the neoplastic clone with its proliferative capacity and survival advantage. We found that WP1066, a novel derivative of the tyrphostin AG490, inhibits the JAK-STAT pathway and cytokine-dependent and independent signaling pathways. Therefore, we sought to investigate the activity of WP1066 in Ph+ ALL. To do this, we first studied the effect of WP1066 on the Ph+ ALL cell lines Z-119 and Z-181 (Estrov Z et al. J. Cell Physiol.166(3):18, 1996). Using the MTT assay we found that WP1066 inhibited the growth of both Z-119 and Z-181 cells in a concentration-dependent manner with only 8% and 4% of the cells surviving at a concentration of 4 mM, respectively. Similarly, the clonogenic growth of both Z-119 and Z-181 cells was effectively inhibited by WP1066 with more than 90% reduction in colony numbers at concentration of 4 mM. Using Western Immunoblott analysis of cell lysates, we found that 4 mM of WP1066 induced caspase-3 cleavage in a time- and dose-dependent manner in both Z-119 and Z-181 cells. In addition, WP-1066 downregulated uncleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and upregulated cleaved PARP protein levels in a time-dependent manner after 2 hours of exposure to 4 mM. We further evaluated induction of apoptosis using the annexin V-FITC assay and showed a dose dependent increase of the fraction of apoptotic cells in both Z-119 and Z-181 cells. After 24 hour of exposure to 4 mM of WP1066 the fraction of apoptotic cells increased by 23% and 43%, respectively. To elucidate the mechanisms by which WP1066 induces growth inhibition and apoptosis in Ph+ ALL cells, we investigated the effect of this agent on the phosphatidylinositol 3-kinase (PI-3K) pathway because the PI-3K pathway is constitutively activated in Ph+ leukemias. We found that WP1066 inhibited the phosphorylation of AKT in a time-dependent fashion in both cell lines and that this inhibitory effect lasted for 24 hours. In conclusion, our data suggest that exposure to WP1066 induces caspase-dependent apoptosis, is associated with PI3-K inhibition and reduces the growth of the Ph+ cell lines Z-119 and Z-181. The activity of WP1066 in Ph+ ALL should be further studied.

scholarly journals HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4385-4385
Author(s):  
Zuzana Chyra ◽  
Maria Gkotzamanidou ◽  
Masood A. Shammas ◽  
Vassilis L. Souliotis ◽  
Yan Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Large Panel ◽  
Dose Dependent

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

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