[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect of SP600125 on phosphorylation of S6K1 in cell lines at the different differentiation stages.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Megakaryocyte pathology and bone marrow fibrosis: the lysyl oxidase connection.

Megakaryocytes (MKs), the platelet precursors, are capable of accumulating DNA greater than a diploid content as part of their cell cycle. MKs have been recognized as mediating fibrosis in a subset of hematologic malignancies, including acute megakaryoblastic leukemia and a subset of myeloproliferative neoplasms. The mechanisms responsible for fibrosis remain only partially understood. Past studies highlighted the role of growth factors in such pathologies, and recently, the protein lysyl oxidase (LOX) has been implicated in proliferation of MKs, ploidy and deposition of fibers. LOX was initially characterized as a protein responsible for the intermolecular cross-linking of elastin and collagen, and in recent years it has been identified as regulator of various pathologies, such as cancer and inflammation. Here, we review recent advances in the understanding of the contribution of MKs to the progression of myelofibrosis, highlighting the newly identified role of LOX.

Myeloperoxidase-positive acute megakaryoblastic leukemia in a dog.

A 16-month-old female spayed Labrador Retriever was referred to the University of Edinburgh for exercise intolerance, inappetence, and severe anemia. A CBC showed severe nonregenerative anemia and moderate numbers of atypical cells with morphologic features most consistent with megakaryoblastic origin. Similar cells were identified in a bone marrow aspirate and accounted for 23% of all nucleated cells. Atypical promegakaryocytes and megakaryocytes were also noted. Myelodysplastic syndrome affecting the megakaryocytic lineage was suspected. Cytologic examination of a fine-needle aspirate of the spleen revealed rare megakaryoblasts similar to those in blood and bone marrow. At necropsy, the bone marrow consisted of atypical megakaryoblasts and megakaryocytes that were also infiltrating spleen, liver, lymph nodes, renal perihilar tissue, and visceral adipose tissue, consistent with acute megakaryoblastic leukemia. Immunohistochemical analysis of splenic sections confirmed megakaryoblastic origin (immunoreactive for CD61 and von Willebrand factor). Some leukemic cells were also immunoreactive for myeloperoxidase (MPO). This aberrant immunophenotype suggested both megakaryocytic and granulocytic/monocytic differentiation of the leukemic cells. To our knowledge, this is the first report of MPO-positive acute megakaryoblastic leukemia in a dog.

Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis.

BACKGROUND: Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro. CONCLUSIONS/SIGNIFICANCE: Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.

RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.

RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the delta catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)-kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

GATA1 mutations in patients with down syndrome and acute megakaryoblastic leukaemia do not always confer a good prognosis.

Children with Down syndrome and acute megakaryoblastic leukemia (DS-AMKL) have been shown to have increased sensitivity to cytarabine based chemotherapy. The excellent prognosis in patients with DS-AMKL may be due to mutations in the GATA1 gene leading to reduced expression of the enzyme cytidine deaminase. This leads to a decreased ability to convert cytarabine into its inactive metabolite, resulting in high intracellular concentration of this cytotoxic agent. We report two cases of DS-AMKL with GATA1 mutations who had poor outcome. These patients had high expression levels of cytidine deaminase mRNA transcripts. We speculate that other factors can affect overall outcome in patients with DS-AMKL irrespective of the presence of GATA1 mutations.

JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However, there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL, based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation, and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy, we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model, JAK2T875N induced a myeloproliferative disease characterized by features of AMKL, including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL.

GATA1, cytidine deaminase, and the high cure rate of Down syndrome children with acute megakaryocytic leukemia.

Down syndrome children with acute megakaryocytic leukemia (AMkL) have higher cure rates than non-Down syndrome acute myeloid leukemia (AML) patients treated with cytosine arabinoside (ara-C). Megakaryoblasts from Down syndrome AML patients are more sensitive in vitro to ara-C than cells from non-Down syndrome AML patients. Somatic mutations in the GATA1 transcription factor have been detected exclusively and almost uniformly in Down syndrome AMkL patients, suggesting a potential linkage to the chemotherapy sensitivity of Down syndrome megakaryoblasts. Stable transfection of wild-type GATA1 cDNA into the Down syndrome AMkL cell line CMK resulted in decreased (8- to 17-fold) ara-C sensitivity and a threefold-lower generation of the active ara-C metabolite ara-CTP compared with that for mock-transfected CMK cells. High intracellular levels of uridine arabinoside (ara-U) (an inactive ara-C catabolite generated by cytidine deaminase) and cytidine deaminase transcripts were detected in GATA1-transfected CMK sublines, whereas no ara-U was detected in mock-transfected cells. Cytidine deaminase transcripts were a median 5.1-fold (P = .002) lower in Down syndrome megakaryoblasts (n = 16) than in blast cells from non-Down syndrome patients (n = 56). These results suggest that GATA1 transcriptionally upregulates cytidine deaminase and that the presence or absence of GATA1 mutations in AML blasts likely confers differences in ara-C sensitivities due to effects on cytidine deaminase gene expression, which, in turn, contributes to the high cure rate of Down syndrome AMkL patients.

Transcriptional regulation of the cystathionine-beta -synthase gene in Down syndrome and non-Down syndrome megakaryocytic leukemia cell lines.

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21-localized gene, cystathionine-beta-synthase (CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5'-cDNA ends (5'-RACE) analysis demonstrated exclusive use of the CBS -1b promoter in the cell lines, and transient transfections with the full-length CBS -1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS -1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f- or GT-d-binding site resulted in an approximately 90% decrease of the CBS -1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to the CBS -1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns of CBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.

Telomerase and the benign and malignant megakaryoblastic leukemias of Down syndrome.

The most common form of leukemia in Down syndrome patients is megakaryoblastic leukemia. There are two forms of the disease. Transient leukemia (TL) is a form of megakaryoblastic leukemia that occurs in newborns with Down syndrome and usually disappears spontaneously within the first 3 months of life. Acute megakaryoblastic leukemia (AMKL) occurs in Down syndrome children within the first 4 years of life and is fatal without treatment. The megakaryoblasts of TL and AMKL are indistinguishable by light and electron microscopy; yet, TL is benign and AMKL is malignant. One of the hallmarks of many malignancies is the expression of telomerase. It is therefore hypothesized that the transient, benign form of megakaryoblastic leukemia (TL) would not contain telomerase activity, whereas telomerase would be demonstrable in the malignant form of the disease. Telomerase activity was determined in the blood and/or bone marrow aspirates in 29 cases of AMKL and 34 cases of TL. The authors found telomerase activity in 15 of 29 (52%) cases, of AMKL and in only 4 of 34 (12%) cases of TL (P < 0.001). Furthermore, three of the four telomerase-positive TL cases were particularly severe, of which two were fatal. Telomerase activity is found frequently in the leukemic cells of the malignant form of megakaryoblastic leukemia but rarely in the benign form of the disease (TL). Observations provide evidence that telomerase may be a critical factor for the malignant conversion of leukemic cells.

Expression of the Gb3/CD77 synthase gene in megakaryoblastic leukemia cells: implication in the sensitivity to verotoxins.

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the alpha1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.

ATPase inhibitors suppress actinomycin D-induced apoptosis in leukemia cells.

BACKGROUND: Apoptosis is mediated by many kinds of enzymes such as caspases, DNase and protein kinases. Recently, ATPase has been shown to be involved in the apoptotic system, but its role is not fully understood. MATERIALS AND METHODS: We investigated the effect of 8 species of ATPase inhibitors on actinomycin D plus colcemid-induced apoptosis in human megakaryoblastic leukemia CMK-7 cells by monitoring caspase-3 activation and DNA cleavage. RESULTS: 2,3-Butanedione monoxime (BDM), lansoprazole, cyclopiazonic acid, geldanamycin and radicicol suppressed the apoptosis. Among these compounds, geldanamycin was the strongest suppressor of both caspase-3 activation and DNA cleavage. Furthermore, Western blotting showed that radicicol suppressed the proteolytic cleavage of procaspase-9 more strongly than BDM, lansoprazole or cyclopiazonic acid. CONCLUSION: Since geldanamycin and radicicol are specific inhibitors of the ATPase in HSP90, the present result implies that ATPase activity in HSP90 plays some role in this apoptosis.

Interaction between protein kinase C and actin in megakaryocyte polyploidization.

Megakaryocytes undergo a peculiar and irreversible program by which they become polyploid through repeated cycles of DNA synthesis without concomitant cell division. In order to study the possible concomitant role of protein kinase C and actin in megakaryocyte polyploidization, three cell lines (DAMI, HEL and K562), expressing some properties of the megakaryocytic lineage and known to differentiate into the megakaryocytic pathway in the presence of phorbol esters, were cultivated in the presence of phorbol myristate acetate alone (PMA, 5 x 10(-9) M, activator of protein kinase C, PKC) or concomitantly with cytochalasin B (2 micrograms/ml, inhibitor of actin polymerization). We have previously shown that DAMI, HEL and K562 cells in which actin polymerization was inhibited by cytochalasin B, acquired megakaryocytic properties in the way that they became polyploid, acquired a megakaryocytic phenotype and arrested proliferation (4). After four days of culture in the presence of PMA and cytochalasin B, the number of polyploid cells (estimated by flow cytometry) increased in comparison with control or PMA-treated cells. However, it was lower than in cytochalasin B-treated cells. Indeed, control cells predominantly diploid (2N) became polyploid with the appearance of 8N, 16N and 32N cells after addition of PMA, cytochalasin B or PMA + cytochalasin B. The endomitotic index (EI, as described in 5) which corresponds to the mean of (¿log2 DNA content expressed in N¿-1) was 0.5 +/- 0.1, 0.7 +/- 0.1 and 0.3 +/- 0.1 in control DAMI, HEL and K562 cells, respectively. The EI increased to 0.9 +/- 0.2; 1.0 +/- 0.2 and 0.4 + 0.1 in cells treated with PMA and to 1.6 +/- 0.3; 1.4 +/- 0, and 0.9 +/- 0.2 when PMA was added concomitantly to cytochalasin B. Total DNA estimated from the cell content and the percentage of cells present at each ploidy stage did not change in cytochalasin B-treated cells in comparison to control conditions. However, treatment of DAMI, HEL and K562 cells with PMA alone or concomitantly with cytochalasin B revealed that the total DNA content significantly decreased in these conditions. At last, treatment of the three cell lines with PMA alone or concomitantly with cytochalasin B for 4 days caused a complete inhibition of proliferation. In conclusion, the concomitant addition of PMA and cytochalasin B to the three cell lines lead to an augmentation of cell ploidy and to a cessation of proliferation. However, we did not observe any synergistic effect of the two compounds. The possible interaction between actin and protein kinase C is discussed in the paper.

dl-alpha-tocopherol, a potent inhibitor of phorbol ester induced shape change of erythro- and megakaryoblastic leukemia cells.

Synthetic vitamin E, dl-alpha-tocopherol, added to a human erythroleukemia HEL and a megakaryoblastic leukemia, Meg-01, cell culture produced potent dose-dependent inhibition of phorbol ester-induced adhesion and of the morphologic changes accompanying it. The inhibition was reversible by withdrawal of supplemental vitamin E from the medium. dl-alpha-Tocopherol also inhibited protein kinase C activity both at baseline and after phorbol ester stimulation. Arachidonic acid stimulated protein kinase C activity of erythroleukemia cells and promoted their adhesion, an effect that was also inhibited by dl-alpha-tocopherol. Introduction of a protein kinase C-neutralizing antibody or a protein kinase C-inhibitor substrate into permeabilized HEL cells inhibited phorbol ester-induced adhesion and shape change. dl-alpha-Tocopherol also affected the cellular distribution of protein kinase C, shifting the major portion of the enzyme to the cytosol fraction and reducing phorbol ester-induced membrane association of the enzyme. Thus, protein kinase C appears to mediate shape change and adhesion, both of which are strongly inhibited by dl-alpha-tocopherol.

Modulation of the megakaryoblastic Dami cell line differentiation by phosphodiesterase inhibitors and imidazo[1,2-a]pyrazine derivatives.

Phosphodiesterase inhibitors have been shown to modulate cell differentiation. We have previously shown that a series of imidazo[1,2-a]pyrazine derivatives displayed inhibitory effects on phosphodiesterase isoenzymes types III. IV and V isolated from Dami cells and on Dami cell growth. In the present study we have investigated the effect of these derivatives on the expression of two differentiation markers, glycoproteins Ib and IIb/IIIa of the human megakaryoblastic leukaemic Dami cell line in comparison to those elicited by 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors of types 1 (8-methoxymetyl-1-methyl-3-(2-methylpropyl) xanthine), III (Milrinone), IV (RO-201724) and V (Zaprinast). Imidazo[1,2-a]pyrazine derivatives, 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors, except 8-methoxymethyl-1-methyl-3-(2-methylpropyl) xanthine, decreased glycoprotein Ib expression. SCA40, SCA41, SCA44 and 3-isobutyl-1-methylxanthine-but not the other compounds affected the expression of glycoprotein IIb/IIIa in a positive manner. The effects of imidazo[1,2-a]pyrazine derivatives on glycoprotein expression appeared to be related to their phosphodiesterase inhibitory potency.