Asako Takeuchi

Recombinant human soluble thrombomodulin safely and effectively rescues acute promyelocytic leukemia patients from disseminated intravascular coagulation

We treated individuals for disseminated intravascular coagulation (DIC) caused by acute promyelocytic leukemia (APL) (n=9) using human soluble thrombomodulin (rTM) in combination with all-trans retinoic acid (ATRA) and chemotherapy, and compared the clinical outcomes with historical control patients (n=8) treated with ATRA and/or chemotherapy. Two control patients developed intracranial vascular incidents. On the other hand, no bleeding related mortality was noted in rTM-treated patients. Notably, treatment with rTM rescued patients from DIC earlier than historical controls (log rank test, p=0.019). These results suggest that administration of rTM should be considered for the treatment of individuals with DIC associated with APL.

Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+ /CD38- cells and sensitizes them to antileukemia agents.

To verify molecular mechanisms by which leukemia stem cells (LSCs) maintain a dormant state, we explored the activity of the major prosurvival signal pathways in CD34+/CD38− compartment, supposed to contain LSCs, and CD34+/CD38+ counterparts from patients with acute myelogenous leukemia (AML, n = 11) by fluorescence‐activated cell sorting (FACS). CD34+/CD38− cells expressed a greater amount of p‐janus kinase 2 (JAK2) and p‐signal transducer and activator of transcription 5 (STAT5) than CD34+/CD38+ counterparts in all patients except for one case. In addition, we found that CD34+/CD38− cells were relatively resistant to cytarabine‐ and the inhibitor of the fms‐like tyrosine kinase 3 (FLT3)‐mediated growth inhibition, as measured by the clonogenic assay. Interestingly, blockade of JAK2/STAT5 signaling by the specific JAK2 inhibitor AZ960 stimulated cell cycling in CD34+/CD38− cells in conjunction with downregulation of cyclin‐dependent kinase inhibitor p21waf1 and sensitized these cells to the growth inhibition mediated by cytarabine and the FLT3 kinase inhibitor. Moreover, exposure of CD34+/CD38− cells to AZ960 potently induced apoptosis in parallel with downregulation of antiapoptotic protein Bcl‐xL, as measured by Western blot analysis. Taken together, JAK2/STAT5 signaling may be a promising molecular target to eradicate CD34+/CD38− leukemia cells in individuals with AML.

Expression of p-JAK2 predicts clinical outcome and is a potential molecular target of acute myelogenous leukemia.

Our study determined if Janus kinase 2 (JAK2) was activated in acute myelogenous leukemia (AML; n = 77, excluding acute promyelocytic leukemia) by immunohistochemistry (IHC) using a phosphor‐specific antibody against JAK2. p‐JAK2 was detectable in all cases, although its levels varied between patient samples (high levels, n = 31; low levels, n = 46). The quantification of levels of p‐JAK2 by IHC was well correlated with that assessed by Western blot analyses and fluorescence‐activated cell sorting (FACS). Levels of p‐JAK2 were directly correlated with high white blood cell count (52.3 × 103/L in patients with high p‐JAK2 vs. 28.3 × 103/L in patients with low p‐JAK2, p < 0.01) and were inversely correlated with complete remission rates (45% in patients with high p‐JAK2 vs. 78% in patients with low p‐JAK2, p < 0.003). In addition, multivariate analysis confirmed that high levels of p‐JAK2 remained a significant factor for overall survival (hazard ratio = 2.213; 95% confidence interval, 1.212–4.041, p = 0.023). Moreover, we found that AZ960, a novel and specific inhibitor of the JAK2 kinase, potently inhibited the clonogenic growth and induced apoptosis of freshly isolated AML cells from patients in association with cleavage of caspase 3 and downregulation of anti‐apoptotic Bcl‐xL proteins. Taken together, JAK2 may be a promising molecular target for treatment of AML.

Effect of recombinant human soluble thrombomodulin on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation.

From 2001 to 2012, 71 individuals with hematological diseases received HSCT in our institution. Of these, 41 developed disseminated intravascular coagulation (DIC) in association with various underlying conditions. The patients who developed DIC after 2008 (n = 23) were treated by recombinant human soluble thrombomodulin (rTM), and the others (n = 11) were treated by either heparin and/or antithrombin III concentrate. Seven patients did not receive any anticoagulant therapy. Of note, treatment for coagulopathy by rTM significantly improved clinical outcomes of patients at day 100 and dramatically prolonged their overall survival (P = 0.044). Taken together, rTM is useful to improve clinical outcomes of transplant recipients with coagulopathy.

The novel function of CD82 and its impact on BCL2L12 via AKT/STAT5 signal pathway in acute myelogenous leukemia cells.

The aim of this study was to explore the biological functions of a tetraspanin family protein CD82 expressed aberrantly in chemotherapy-resistant CD34+/CD38− acute myelogenous leukemia (AML) cells. Microarray analysis of patient-isolated CD34+/CD38− AML cells revealed that the levels of anti-apoptotic protein BCL2L12 were downregulated after CD82 depletion by specific short hairpin RNA (shRNA). Western blot analysis indicated that BCL2L12 was aberrantly expressed in patient-isolated AML cells and AML cell lines. Furthermore, CD82 blockade by a specific antibody downregulated BCL2L12 in parallel with dephosphorylation of signal transducer and activator of transcription 5 (STAT5) and AKT, whereas pharmacological inhibition of STAT5 and AKT activation decreased BCL2L12 expression in leukemia cells. In addition, shRNA-mediated downregulation of BCL2L12 increased the levels of cleaved caspase-3 and suppressed proliferation of leukemia cells, impairing their engraftment in immunodeficient mice. Taken together, our results indicate that CD82 regulated BCL2L12 expression via STAT5A and AKT signaling and stimulated proliferation and engrafting of leukemia cells, suggesting that CD82 and BCL2L12 may be promising therapeutic targets in AML.

STAT5A regulates DNMT3A in CD34+/CD38− AML cells

Signal transducer and activator of transcription 5 (STAT5) is activated in CD34(+)/CD38(-) acute myelogenous leukemia (AML) cells. Inhibition of STAT5 induced apoptosis and sensitized these cells to the growth inhibition mediated by conventional chemotherapeutic agents. The present study attempted to identify molecules that are regulated by STAT5 in CD34(+)/CD38(-) AML cells by utilizing cDNA microarrays, comparing the gene expression profiles of control and STAT5A shRNA-transduced CD34(+)/CD38(-) AML cells. Interestingly, DNA methyltransferase (DNMT) 3A was downregulated after depletion of STAT5A in CD34(+)/CD38(-) AML cells. Reporter gene assays found that an increase in activity of DNMT3A occurred in response to activation of STAT5A in leukemia cells. On the other hand, dephosphorylation of STAT5A by AZ960 decreased this transcriptional activity. Further studies utilizing a chromatin immunoprecipitation assay identified a STAT5A-binding site on the promoter region of DNMT3A gene. Forced expression of STAT5A in leukemia cells caused hypermethylation on the promoter region of the tumor suppressor gene, PTEN, and downregulated its mRNA levels, as measured by methylation-specific and real-time polymerase chain reaction, respectively. Taken together, these data suggest that STAT5A positively regulates levels of DNMT3A, resulting in inactivation of tumor suppressor genes by epigenetic mechanisms in AML cells.