FLT3-ITD Confers Resistance to Bortezomib By Protecting the mTOR/4EBP1 Pathway through Activation of STAT5 and Induction of Pim-1 Expression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1270-1270
Author(s):  
Ayako Nogami ◽  
Keigo Okada ◽  
Gaku Oshikawa ◽  
Shinya Ishida ◽  
Hiroki Akiyama ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Apoptotic Pathway ◽  
Expression Levels ◽  
Combined Use ◽  
Significant Difference ◽  
Induced Apoptosis ◽  
Novel Therapeutic Strategies

Abstract FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in AML, with the former strongly associated with a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in AML. The proteasomal inhibitor bortezomib (BZM) has recently been reported as a promising agent for treatment of AML. We examine here the molecular mechanisms involved in induction of apoptosis by BZM in hematopoietic cells, including AML cells, and evaluate the effects of FLT3-ITD and TKD on these mechanisms and on the sensitivity of cells to BZM-induced apoptosis. We first comparatively examined the effect of BZM on survival of hematopoietic 32D cells and human leukemic UT7 cells driven by FLT3-ITD (32D/ITD and UT7/ITD) or FLT3-D835Y (32D/TKD and UT7/TKD). BZM induced activation of Bax, decline in mitochondrial membrane potential, and activation of caspase-9, thus leading to apoptosis, more conspicuously in cells driven by FLT3-ITD than FLT3-TKD. These results indicate that FLT3-ITD, as compared with FLT3-TKD, conferred resistance to apoptosis induced by BZM through the intrinsic pathway in these cells. To elucidate the molecular mechanisms involved in BZM-induced apoptosis in these cells, we next examined the effect of BZM on expression levels of FLT3 in these cells as well as in human leukemic MV4-11 cells expressing FLT3-ITD. Intriguingly, treatment of these cells with BZM conspicuously reduced expression levels of FLT3. It was further reveled that BZM further facilitated the decline in FLT3-ITD expression in MV4-11 cells treated with actinomycin D to shut down the transcriptional activity. Furthermore, BZM significantly retarded the recovery of FLT3-ITD expression in MV4-11 cells washed out from the translation inhibitor cycloheximide. These results suggest that BZM may downregulate the FLT3-ITD expression mainly at the translational level. However, we did not observe any significant difference in extent of the BZM-induced decline in expression levels between FLT3-ITD and FLT3-TKD. We next examined the effect of BZM on the mTOR/4EBP1 pathway, which we have shown to play important roles in regulation of apoptosis downstream of FLT3-ITD. It was found that BZM downregulated this pathway more significantly in 32D/TKD cells than in 32D/ITD cells. Because we have also previously found that STAT5 activated robustly by FLT3-ITD plays an important role in modulation of the mTOR/4EBP1 pathway, we examined the effects of BZM on 32D/TKD cells forced to express the constitutively activated STAT5 mutant, STAT5A1*6. As expected, STAT5A1*6 conferred resistance to BZM-induced downregulation of the mTOR/4EBP1 pathway as well as apoptosis in 32D/TKD cells. Consistent with this, the STAT5 inhibitor pimozide, clinically in use for neuropsychiatric disorders, abrogated the resistance of 32D/ITD, UT7/ITD, and MV4-11 cells to BZM-induced inhibition of the mTOR/4EBP1 pathway and apoptosis. We finally examined the possible involvement of the STAT5 target gene product Pim-1 in acquisition of resistance to BZM by cells expressing FLT3-ITD. We fist confirmed that Pim-1 was expressed at a higher level in 32D/ITD cells than in 32D/TKD cells and that STAT5A1*6 increased the expression level of Pim-1 in 32D/TKD cells. We then examined the effects of a specific Pim kinase inhibitor, AZD-1208, and found that it synergistically downregulated the mTOR/4EBP1 pathway and induced apoptosis with BZM in 32D/ITD cells as well as 32D/TKD cells expressing STAT5A1*6. We also examined the effects of a BET bromodomain inhibitor, JQ1, which has recently been shown to inhibit the STAT5 activity and to reduce specifically the expression level of Pim-1 as well as c-Myc in MV4-11 cells. As expected, pretreatment of 32D/ITD or MV4-11 cells with JQ1 conspicuously sensitized these cells to BZM-induced apoptosis. These results suggest that BZM downregulates FLT3 expression and the mTOR/4EBP1 pathway to activate the intrinsic apoptotic pathway and that robust STAT5 activation by FLT3-ITD confers resistance to BZM on AML cells through protection of the mTOR/4EBP1 pathway at least partly by inducing Pim-1 expression. The present study may contribute to development of novel therapeutic strategies against FLT3-ITD-positive AML by combined use of BZM and the STAT5/Pim-1 pathway inhibitors. Disclosures No relevant conflicts of interest to declare.

Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4297-4297
Author(s):  
Jing Liu ◽  
Shu-Ling Wang ◽  
Lin Fang ◽  
Mao Ye ◽  
Zhi-Wei Sun ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Alternative Pathways ◽  
Cyclin Dependent Kinase Inhibitor ◽  
M Phase ◽  
Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Expression of the Co-Inhibitory Molecule PD-1 and Its Ligand PD-L1 in Patients with MDS

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5212-5212
Author(s):  
Suxia Geng ◽  
Jianyu Weng ◽  
Xin Huang ◽  
Ping Wu ◽  
Chengxin Deng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Hematologic Malignancies ◽  
Expression Level ◽  
Activated T Cells ◽  
Expression Levels ◽  
Significant Difference ◽  
Normal Controls

Abstract Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule and expresses on activated T cells, B cells and myeloid cells. PD-L1, a primary ligand PD-1, is mainly expressed on T cells and primary B cell surfaces and plays a role in the differentiation and apoptosis of these cells, induces a coinhibitory signal in activated T cells, promotes T cells apoptosis, incompetence and functional exhaustion. The expression of PD-1 is high in patients with hematologic malignancies and a high expression of PD-L1 is found on hematologic malignancies cells. To further know the characteristics of PD-1 and PD-L1 in patients with MDS, we detected theexpression of PD-1 and PD-L1 in peripheral blood (PB) and bone marrow (BM) samples from 25 RAEB and 10 RARS patients using Real-time PCR. The PD1 and PD-L1 expression levels of 13 PB and 8 BM samples from normal individuals were as controls. The PD-1 levels of PB samples from 25 RAEB patients [42.40(4.5-173.96)%] were significantly higher than that from normal controls [32.32(19.45-41.38)%, P=0.026]. While the level of PD-1 in 10 RARS patients was comparable to that of normal controls and RAEB patients (P=0.401 and P=0.352). Compared to normal controls [23.72(3.23-39.2)%], the median PD-1 level of BM from 10 RAEB patients[36.81(12.14-151.52)%] showed an increasing tendency, but the difference was not statistically significant (P=0.062). PD-L1 expression levels of PB samples from RARS patients were significantly lower than that of normal controls (P=0.009). There were no significant difference between RAEB patients and normal controls about the PD-L1 expression level in PB and BM samples (P=0.248 and P=0.181) and between RAEB and RARS patients about PD-L1 expression level in PB samples (P=0.243). PD-1 level in BM samples from 11 RAEB patients with remission was (31.32±15.75)% and it was lower significantly than that before treatment [(59.94±47.44)%, P=0.034]. After progression or transformation, the expression level of PD1 (54.72±37.27)% increased again and was higher than that in remission (P=0.028).There were also no significant difference on PD-L1 expression among before treatment, bone marrow remission and progression or transformation (P>0.05). In 8 RAEB patients transformed to AML after treatment, PD-1 level has a decreasing tendency(P =0.05)and the change of PD-L1 has no significant difference (P>0.05). In conclusion, PD1 mRNA level increased significantly in patients with RAEB and the changes of PD-1 was associated with the evolution of the disease after treatment with demethylating agents. The level of PD1 may be used as an indicator to determine the efficacy, but the changes of PD-L1 was not found regularity. Disclosures No relevant conflicts of interest to declare.

A Novel Ring-Substituted Diindolylmethane 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) Methane Abrogates ERK Activation and Induces Apoptosis in Acute Myeloid Leukemia (AML).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3399-3399
Author(s):  
Rooha Contractor ◽  
Ismael J. Samudio ◽  
Zeev Estrov ◽  
David Harris ◽  
James A. McCubrey ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Erk Activation ◽  
New Class ◽  
Induced Apoptosis ◽  
Extracellular Regulated Kinase

Abstract We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.

Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4469-4469
Author(s):  
Hilmar Quentmeier ◽  
Sonja Eberth ◽  
Julia Romani ◽  
Margarete Zaborski ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

BCR/ABL-Mediated Myeloid Expansion Is Promoted by C/EBPβ, a Regulator of Emergency Granulopoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3747-3747
Author(s):  
Yoshihiro Hayashi ◽  
Hideyo Hirai ◽  
Hisayuki Yao ◽  
Satoshi Yoshioka ◽  
Sakiko Satake ◽  
...  
Keyword(s):  
Median Survival ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Expression Level ◽  
Hematopoietic Stem ◽  
Emergency Situations ◽  
Proliferation And Differentiation ◽  
Myeloid Progenitors

Abstract Abstract 3747 Chronic phase chronic myeloid leukemia (CP-CML) is characterized by the increase of myeloid cells in the peripheral blood (PB) and bone marrow (BM). We have previously shown that the C/EBPβ transcription factor is required for emergency granulopoiesis, increased proliferation and differentiation of granulocytic precursors in emergency situations such as infection (Hirai H et al., Nature Immunol. 2006). Enhanced myelopoiesis is a common feature between emergency situations and CP-CML. However, little is known about the roles of C/EBPβ in the pathogenesis of CP-CML. The aim of this study is to elucidate the regulation and function of C/EBPβ in BCR/ABL-mediated myeloid expansion. We first assessed the expression level of C/EBPβ in hematopoietic stem cells and myeloid progenitors in BM obtained from healthy donors or CP-CML patients. The transcript of C/EBPβ is expressed at significantly higher level in common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in CP-CML BM than those in normal BM. When BCR/ABL was retrovirally transduced into a mouse hematopoietic stem cell line, EML, C/EBPβ expression was significantly upregulated. Treatment of the EML-BCR/ABL cells with imatinib mesylate normalized the expression level of C/EBPβ. These data suggested that C/EBPβ was upregulated in response to the downstream signaling of BCR/ABL. In order to investigate the function of C/EBPβ in BCR/ABL-mediated myeloid expansion, BCR/ABL was retrovirally introduced into BM cells obtained from 5-FU treated C/EBPβ knockout (KO) mice and their properties were compared with those of BCR/ABL-transduced BM cells from wild type (WT) mice. When the transduced cells were cultured in cytokine-free semisolid methylcellulose medium, the number and the size of the colonies of C/EBPβ KO cells were significantly smaller. Flow cytometric analysis of the colony-forming cells revealed that the BCR/ABL-transduced C/EBPβ KO BM cells gave rise to higher frequency of c-kit+ cells and lower CD11b+ cells than BCR/ABL-transduced WT BM cells (%c-kit+ cells=8.2±3.0% vs. 11.3±3.5%, p=0.002, %CD11b+ cells=75.1±2.1% vs. 90.0±4.2%, p=0.003). In addition, BCR/ABL-transduced C/EBPβ KO BM cells revealed higher replating efficiency than BCR/ABL-transduced WT BM cells. To investigate the role of C/EBPβ in leukemogenesis, BCR/ABL-transduced BM cells from C/EBPβ KO mice or WT mice were transplanted into lethally irradiated recipient mice. In mice transplanted with BCR/ABL-transduced C/EBPβ KO cells, the increase of white blood cell count was delayed (Figure) and higher frequency of c-kit+ cells were observed in the BM at day 19 post transplantation (16.0±2.6% vs. 5.5±4.6%, p=0.01). Spleen size of mice transplanted with BCR/ABL-transduced WT cells is much larger than that of BCR/ABL-transduced C/EBPβ KO cells (Figure). The median survival of mice transplanted with BCR/ABL-transduced WT cells was 19 days. In contrast, the median survival of mice transplanted with BCR/ABL-transduced C/EBPβ KO cells was 31 days (p=0.0005). In summary, C/EBPβ is upregulated by BCR/ABL and the absence of C/EBPβ resulted in delayed proliferation and differentiation of myeloid cells both in vitro and in vivo. Our results suggest that C/EBPβ is involved in the BCR/ABL-mediated myeloid expansion in CP-CML and that C/EBPβ can be the novel molecular target for the therapy of CML. We are currently investigating the molecular mechanisms which mediate the upregulation of C/EBPβ and the direct targets of C/EBPβ in CP-CML. Disclosures: No relevant conflicts of interest to declare.

Inhibition of LSD1 Influences Multiple Mechanisms of Epigenetic Gene Regulation During Terminal Erythroid Maturation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3442-3442
Author(s):  
Michael Getman ◽  
Samantha J England ◽  
James Palis ◽  
Laurie A Steiner
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Histone Demethylase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Local Effects ◽  
Erythroid Progenitor ◽  
Significant Difference ◽  
Erythroid Maturation

Abstract Abstract 3442 The maturation of a committed erythroid progenitor to a functional red blood cell is a complex process involving significant changes in gene expression during a time of rapid cell division and nuclear condensation. LSD1 (Lysine-Specific Histone Demethylase 1) is a histone H3 lysine 4 (H3K4) and lysine 9 (H3K9) demethylase that plays pivotal role in this process. LSD1 participates in both enhancer and repressor complexes, and facilitates repression of γ-globin by participating in the Direct Repeat Erythroid Complex (Cui, MCB, 2011). LSD1 inhibitors Tranylcypromine (TCP) and Pargyline (PG) are being investigated as potential therapies for the β-globinopathies, however little is known about the broader functional or genomic consequences of LSD1 inhibition on terminal erythroid maturation. Both TCP and PG impair erythroid maturation in Extensively Self Renewing Erythroblasts (ESREs), a primary cell model of terminal erythroid maturation. ESREs are primary cells derived from fetal liver that proliferate extensively in culture, but retain the ability to appropriately mature and enucleate (England, Blood, 2011), making them ideal for functional and genomic studies of terminal erythroid maturation. In untreated or vehicle (DMSO) treated cultures >90% of cells are benzidine positive by day3 of maturation. In contrast, cultures treated with 400um PG, 1um TCP, or 2um TCP were 72, 42, and 33% benzidine positive by maturation day3, respectively. Cells in the TCP-and PG- treated cultures also had morphologic evidence of impaired maturation, with larger nuclei and more basophilic cytoplasm. In addition to its role as a histone demethylase, LSD1 stabilizes DNMT1 (DNA methyltransferase 1; Wang, Nat Genet 2009). We hypothesized that loss of DNA methylation contributes to the maturation impairment seen with LSD1 inhibitors, and that inhibition of DNMTs with decitabine would also impair terminal erythroid maturation. Consistent with this hypothesis, ESREs treated with decitabine demonstrated a dose-dependent impairment of maturation similar to that seen with PG and TCP. To elucidate the molecular mechanisms underlying the maturation impairment in TCP- and PG- treated cultures, levels of H3K4me2 and methylated DNA (5-methyl cytosine, 5-mC) were assessed both globally and at specific loci. An ELISA (Enzyme-linked Immunosorbent Assay) was used to assess global levels of H3K4me2 and 5-mC in vehicle-, PG-, and TCP-treated cultures after 24 hours of maturation. Global levels of H3K4me2 were significantly higher in PG- and TCP- treated samples than control. In maturing cells, there was no significant difference in the level of 5-mC in vehicle- and inhibitor- treated cultures. It is well established, however, that global DNA methylation decreases with erythroid maturation (Seashore, Science, 2011), and a significant decrease in 5-mC occurs in ESREs during the first 24hrs of maturation. As TCP- and vehicle- treated cultures mature differently, the effect of TCP on 5-mC levels was also assessed in self-renewing ESREs at the proerythroblast stage. Unlike maturing cells, TCP-treated proerythroblasts had a significant decrease in 5-mC levels compared to control. Chromatin immunoprecipitation (ChIP) was used to examine the local effects of LSD1 inhibition on H3K4me2 enrichment at erythroid-specific promoters. TCP-treated cultures had non-uniform changes in H3K4me2 enrichment, with levels increased at some promoters (e.g. protein 4.1,εy-globin), but unchanged at others (e.g. β-globin). To further study the relationship between LSD1 inhibition and H3K4me2 levels, ChIP-seq was used to identify LSD1 sites that co-localized with putative enhancers, defined as peaks of H3K4me2 binding > than 1kb from a transcription start site. ChIP-qPCR was used to compare the level of H3K4me2 at 5 validated enhancer-associated LSD1 sites in vehicle- and TCP-treated cells. The effect of TCP was variable, with only 2/5 enhancer-associated LSD1 sites having increased H3K4me2. Lastly, the local effects of inhibitors on 5-mC were examined using a methyl binding domain pulldown coupled with qPCR. In TCP-treated cells, 5-mC levels declined at several loci, most notably at the εy-globin promoter. Taken together, these results suggest that the impaired erythroid maturation associated with LSD1 inhibition results from the perturbation of multiple mechanisms of epigenetic regulation. Disclosures: No relevant conflicts of interest to declare.

The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5120-5120
Author(s):  
Hatice Demet Kiper ◽  
Burcin Tezcanli Kaymaz ◽  
Ozlem Purclutepe ◽  
Ceyda Tunakan Dalgic ◽  
Nur Selvi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Conflicts Of Interest ◽  
Time Dependent ◽  
Blue Dye ◽  
Dependent Manner ◽  
Down Regulation ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

Gene Expression Levels Of Imatinib Transporters and Molecular Response To Imatinib Therapy In Chronic Myeloid Leukemia Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4959-4959
Author(s):  
Hemant Malhotra ◽  
Pratibha Sharma ◽  
Shipra Bhargava ◽  
Bharti Malhotra ◽  
Madhu Kumar
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Conflicts Of Interest ◽  
Imatinib Therapy ◽  
Molecular Response ◽  
Transcript Expression ◽  
Analysis Study ◽  
Expression Levels ◽  
Significant Difference

Abstract Imatinib mesylate (IM) is the standard first-line treatment for most CML patients. After an initial response, approximately 30 to 40% patients develop resistance to the drug. Various mechanisms of resistance to Imatinib therapy have been identified. One of the mechanisms proposed is varying expression levels of the drug transporters. In the present study, we determined the relative expression levels of Imatinib transporter genes (hOCT1, ABCB1, ABCG2) in CML patients by quantitative real time polymerase chain reaction (qRT-PCR) and correlated these levels with molecular response. One hundred and ten CML patients were considered for gene expression analysis study for hOCT1 gene and eighty seven CML patients were considered for gene expression analysis study for ABCB1 and ABCG2 genes. CML patients who were on IM therapy for more than 2 years were divided into two groups: Responders: patients who achieve a Complete Molecular response (CMR) or a Major Molecular Response (MMR) [bcr/abl: abl ratio <1% as assessed by RQ-PCR] and Non-responders: those without CMR or MMR (bcr/abl: abl ratio =/> 1% as assessed by RQ-PCR). The relative transcript expression levels of the three genes were compared between responders and non-responders. No significant difference in the expression levels of hOCT1, ABCB1 and ABCG2 was found between the two categories - responders versus non-responders (p value > 0.05). The median transcript expression levels of hOCT1, ABCB1 and ABCG2 genes in responders were 30.63, 10.14 and 0.59 versus 40.13, 8.34 and 0.53 in non-responders, respectively. We conclude that, in our study, the mRNA expression levels of IM transporter genes did no correlate with molecular response in CML patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ethnic Differences in MicroRNA-375 Expression Level and DNA Methylation Status in Type 2 Diabetes of Han and Kazak Populations

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Xiangyun Chang ◽  
Siyuan Li ◽  
Jun Li ◽  
Liang Yin ◽  
Ting Zhou ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Methylation Status ◽  
Cpg Methylation ◽  
Expression Level ◽  
Related Factor ◽  
Expression Levels ◽  
Relative Expression ◽  
Cpg Sites ◽  
Significant Difference

Han population is six times as likely as Kazak population to present with type 2 diabetes mellitus (T2DM) in China. We hypothesize that differential expression and CpG methylation of miR-375 may be an ethnic-related factor that influences the incidence of T2DM. The expression level of miR-375 was examined using real-time PCR on Kazak and Han T2DM plasma samples. Furthermore, the methylation levels of CpG sites of miR-375 promoter were determined by MassARRAY Spectrometry in these samples. The relative expression levels of plasma miR-375 in Kazak T2DM samples are 1, and the relative expression levels of plasma miR-375 in Han T2DM samples are 3. The mean level of miR-375 methylation, calculated from the methylation levels of the CpG sites, was 8.47% for the Kazak T2DM group and 10.38% for the Han T2DM group. Further, five CpG units showed a statistically significant difference between Kazak and Han T2DM samples, and, among them, four were hypomethylated and only one CpG unit showed hypermethylation in Kazak T2DM samples. These findings indicate that the expression levels of plasma miR-375 and its CpG methylation in the promoter region are ethnically different, which may contribute to the different incidence of diabetes observed in Kazak and Han populations.

scholarly journals Preclinical Analysis of Linsitinib for the Treatment of Polycythemia Vera

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2825-2825
Author(s):  
Titiksha Basu ◽  
Albert Gruender ◽  
Christine Dierks ◽  
Heike L Pahl
Keyword(s):  
Polycythemia Vera ◽  
Patient Population ◽  
Drug Targets ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Marrow Transplant ◽  
Insulin Growth Factor ◽  
Healthy Control ◽  
Induced Apoptosis

Abstract Even after the discovery of JAK2V617F in majority of Polycythemia Vera (PV) patients and the generation of JAK2 inhibitors, the treatment of PV remains to be improved. An increased understanding of aberrant signaling in PV cells may yield novel drug targets for this patient population. The formation of erythroid colonies in the absence of exogenous erythropoietin (Epo), so called Epo-independent erythroid colonies (EECs), is a pathognomonic hallmark of PV. Over 20 years ago, Axelrad and colleagues proposed that EEC formation is not due to Epo-independence. Rather, they postulated that PV cells are hypersensitive to Insulin Growth Factor-1 (IGF-1). Subsequently, increased IGF-1 receptor (IGF-1R) phosphorylation was demonstrated in PV MNCs. In addition, serum concentrations of IGF-1 binding protein (IGF-1BP) are significantly elevated in PV patients. It has been shown that IGF-1 cooperates with JAK2V617F to increase JAK/STAT signaling, elevating constitutive phosphorylation of STAT factors and thereby augmenting aberrant proliferation. We therefore hypothesized that inhibition of IGF-1 signaling would ameliorate the PV phenotype, even in the presence of JAK2V617F. The dual IGF-1R and insulin receptor (IR) kinase inhibitor linsitinib (formerly called OSI-906) was used to investigate the contribution of the IGF-1 pathway to PV pathophysiology. In methylcellulose colony assays, linsitinib significantly reduced the EEC formation from PV MNCs. In contrast, in the presence of EPO, colony formation from either PV or healthy control MNCs was not affected. These data suggest that linsitinib specifically inhibits growth of the PV clone. Linsitinib likewise significantly reduced the cytokine independent proliferation of Ba/F3 cells expressing JAK2V617F but did not affect proliferation of Ba/F3 cells expressing either JAKWT or JAK2V617F in the presence of cytokine (mIL3). These data again argue that IGF-1 signalling is required for aberrant, cytokine independent growth. In addition to reducing proliferation, Linsitinib also induced apoptosis in IL3-independent JAK2V617F transduced Ba/F3 cells. Using the JAK2V617F bone marrow transplant (BMT) model, we investigated the effect of linsitinib on the PV phenotype in vivo. Linsitinib treatment significantly reduced the elevated RBC count, hematocrit and hemoglobin in this PV mouse model. In addition, the white blood cell count was decreased. The data presented here provide a pre-clinical rationale for expanding the investigation of linsitinib as well as similar IGF-1 pathway inhibitors for the treatment of PV. The effect of combining JAK2 and IGF-1 inhibition may prove especially promising in this patient population and is currently being investigated. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document