Ko Sasaki

Functional regulation of TEL by p38-induced phosphorylation

TEL is a nuclear phosphoprotein that belongs to a member of the ETS family transcription factors. TEL acts as a tumor suppressor and is essential for establishing hematopoiesis in neonatal bone marrow. Because TEL possesses multiple putative mitogen-activated protein (MAP) kinase phosphorylation sites, we here investigated functional regulation of TEL via stress signaling pathways. We showed that TEL becomes phosphorylated in vivo by activated p38 but not by JNK1. The constitutive and inducible phosphorylation sites were found to be Ser(22) and Ser(257), respectively. TEL bound to p38 and was directly phosphorylated in vitro by p38. In vivo p38-dependent phosphorylation reduced trans-repressional abilities of TEL through ETS-binding consensus site. These data indicate that TELs functions are potentially regulated by p38 which is activated by various kinds of stresses. TEL could be a constituent downstream of the specific MAP kinase in the signal transduction system.

TEL/ETV6 accelerates erythroid differentiation and inhibits megakaryocytic maturation in a human leukemia cell line UT-7/GM

TEL/ETV6 accelerates erythroid differentiation in the murine erythroleukemia cell line. To clarify the effects of TEL on megakaryocytic maturation as well as erythroid differentiation, we chose the human leukemia cell line UT‐7/GM that differentiates into the erythroid and megakaryocytic lineages by treatment with erythropoietin and thrombopoietin, respectively. Upon erythropoietin exposure, overexpressed TEL stimulated hemoglobin synthesis and accumulation of the erythroid differentiation‐specific transcripts such as γ‐globin, δ‐aminolevulinic acid synthase‐erythroid, and erythropoietin receptor. Moreover, the glycophorin A(+)/glycoprotein IIb(–) fraction appeared more rapidly in the TEL‐overexpressing cells. Interestingly, overexpression of TEL was associated with lower levels of the megakaryocytic maturation‐specific glycoprotein IIb and platelet factor 4 transcripts under the treatment with thrombopoietin. Consistently, the glycophorin A(–)/glycoprotein IIb(+) fraction increased more slowly in the TEL‐overexpressing cells. Finally, expression of endogenous TEL proteins in UT‐7/GM cells was down‐regulated following erythropoietin and thrombopoietin exposure. All these data suggest that TEL may decide the fate of human erythrocyte/megakaryocyte common progenitors to differentiate towards the erythroid lineage and against the megakaryocytic lineage. (Cancer Sci 2005; 96: 340–348)

Clinical evaluation of WT1 mRNA expression levels in peripheral blood and bone marrow in patients with myelodysplastic syndromes.

Abstract A study to evaluate WT1 mRNA expression levels in peripheral blood (PB) and bone marrow aspirate (BM) was conducted in 172 patients, including 115 with myelodysplastic syndromes (MDS), in Japan. The level of WT1 mRNA expression was evaluated according to the French–American–British (FAB) and World Health Organization (WHO) classifications (2001, 2008) and using the International Prognostic Scoring System and the WHO Prognostic Scoring System scales. WT1 mRNA expression levels in PB and BM were well correlated (r = 0.85), and they tended to increase with disease stage progression and in those at higher risk of leukemic transformation. WT1 mRNA expression can be a useful marker for the diagnosis and risk evaluation of MDS.

Histone deacetylase inhibitors trichostatin A and valproic acid circumvent apoptosis in human leukemic cells expressing the RUNX1 chimera

Disturbance of the normal functions of wild‐type RUNX1 resulting from chromosomal translocations or gene mutations is one of the major molecular mechanisms in human leukemogenesis. RUNX1‐related chimeras generated by the chromosomal translocations repress transcriptional activity of wild‐type RUNX1 by recruiting the co‐repressor/histone deacetylase complex. Thus, histone deacetylase inhibitors are expected to restore normal functions of wild‐type RUNX1 and thereby affect the growth and differentiation ability of leukemic cells expressing the chimera. We investigated the in vitro effects of histone deacetylase inhibitors, trichostatin A and valproic acid, on human leukemic cell lines such as SKNO‐1 and Kasumi‐1 expressing RUNX1/ETO, Reh expressing TEL/RUNX1 and SKH‐1 co‐expressing RUNX1/EVI1 and BCR/ABL. We also employed K562 cells expressing BCR/ABL without such a chimera as a control. Treatment with each inhibitor increased acetylated histone 4 in all of these cell lines. Interestingly, proliferation of SKNO‐1, Kasumi‐1, SKH‐1 and Reh cells was significantly suppressed after 3‐day culture with trichostatin A or valproic acid, when compared to that of K562 cells. We observed cell cycle arrest and apoptotic induction in the RUNX1 chimera‐expressing cells by the propidium iodide staining. Up‐ and downregulation of cell cycle regulator genes appeared to be the molecular basis for the former, and activation of both extrinsic and intrinsic apoptotic caspases for the latter. We propose histone deacetylase inhibitors to be an attractive choice in the molecular targeting therapy of RUNX1‐related leukemia. (Cancer Sci 2008; 99: 414–422)

Aberrant expression of MIR9 indicates poor prognosis in acute myeloid leukaemia.

microRNAs (miRNA) play vital roles in each step of normal haematopoiesis starting at the level of haematopoietic stem cell and continuing during the differentiation process of both myeloid and lymphoid lineages (Bhagavathi & Czader, 2010; Havelange & Garzon, 2010). On the other hand, dysregulated expression of miRNA results in the development of haematological malignancies including leukaemia, lymphoma and myeloma. Considering that inactivation of RUNX1 is one of the major roles in the development of acute leukaemia, we screened expression levels of several RUNX1-inhibiting miRNAs in leukaemic bone marrow samples for preliminary study. Among them, only the amount of mature MIR9, transcribed from either MIR9-1, MIR9-2 or MIR9-3 genes, was extraordinarily increased in a certain fraction of patients (data not shown). We thus conducted a further study on mature MIR9 expression by real-time polymerase chain reaction (PCR) methods with a total of 124 bone marrow samples from 101 adult acute myeloid leukaemia (AML) patients at diagnosis and 23 controls, all of whom gave written informed consent for this study. Expression of MIR9 was normalized to the mean expression of three control small nuclear RNAs (SNORD7, RNU6-2 and SNORA74A). The patient cohort consisted of 59 men and 42 women with a median age of 62 years (range; 20–85 years). Eight, 11, 35, 17, 10, 13, 6 and 1 patients were diagnosed as AML-M0, 1, 2, 3, 4, 5, 6 and 7, respectively. Thirty-eight had normal karyotype. Twelve patients were classified as core binding factor (CBF) leukaemia including eight with t (8;21) and four with inv(16)/t(16;16). All of the 17 AMLM3 (acute promyelocytic leukaemia; APL) patients carried t (15;17). Notably, MIR9 expression was hardly detected in normal bone marrow cells. In contrast, some fraction of bone marrow samples from AML patients showed significantly increased levels of MIR9. Thus, we set a cut-off value at the highest level of MIR9 observed in normal bone marrow cells, and divided the AML patients into two groups; MIR9 (+) and MIR9 ( ) groups. Nineteen patients (19%) belonged to the positive group and 82 (81%) to the negative group. Demographic and laboratory characteristics were not significantly different between these two groups. On the other hand, the distribution of French-AmericanBritish subtypes (P = 0·008) and chromosomal findings (P = 0·017) was statistically different between the two groups. No patients in the MIR9 (+) group were diagnosed as AML-M3/t(15;17) or CBF leukaemia/t(8;21) or inv(16)/t (16;16), while three of them had 11q23 abnormalities. We observed FLT3, NPM1 and CEBPA mutations at a frequency of 19%, 8% and 33% in the entire patient cohort. There was no association with aberrant expression of MIR9 and FLT3, NPM1 or CEBPA mutations (P = 0·353, P = 0·641 or P = 0·082). Although patients received various kinds of induction chemo and/or all-trans retinoic acid (ATRA) therapy depending on the subtype of leukaemia and their age, differences in treatment regimens between the MIR9 (+) and ( ) groups in the entire and non-APL cohort were not statistically significant (P = 0·173 and P = 0·699, respectively). Patients with aberrant MIR9 expression had a lower rate of complete remission (44%) compared with those without the expression (59%), but this difference was not statistically significant (P = 0·246). We then compared overall survival (OS) and relapse-free survival (RFS) between the MIR9 (+) and MIR9 ( ) groups by Kaplan–Meier method and LogRank test. The median observation period was 513 d. The MIR9 (+) group was associated with significantly inferior OS (P = 0·005: Fig 1A) and RFS (P < 0·001: Fig 1B) compared with the MIR9 ( ) group. Even when excluding those APL patients who showed a better prognosis with ATRA therapy, the presence of MIR9 expression was significantly associated with unfavourable OS (P = 0·028:Fig 1C) and RFS (P = 0·009: Fig 1D). We performed univariate Cox regression analyses to evaluate the status of aberrant MIR9 expression as well as known prognostic factors as predictors of OS and RFS. As a result, age over 65 years (hazard ratio [HR]=1·992, P=0·035), white blood cell count greater than 50 9 10/l (HR = 2·434, P = 0·011), lactate dehydrogenase (LDH) greater than 600 iu/l (HR = 2·606, P = 0·004), and the presence of aberrant MIR9 expression (HR = 2·602, P = 0·006) were found to be significant predictors for poor OS (data not shown). On the other hand, the presence of aberrant MIR9 expression was the sole predictor of increased risk of relapse in the same group (HR = 3·560, P = 0·001). When the analyses were restricted to non-APL patients, LDH greater than 600 iu/l (HR = 2·088, P = 0·034), the presence of FLT3-internal tandem duplication mutation (HR = 3·397, P = 0·003) and MIR9 expression (HR = 2·153, P = 0·032)

Prednisone versus high-dose dexamethasone for untreated primary immune thrombocytopenia. A retrospective study of the Japan Hematology & Oncology Clinical Study Group

High-dose dexamethasone (HDD) has been shown to be an effective initial treatment for immune thrombocytopenia (ITP), but it is not clear whether HDD offers any advantages over conventional-dose prednisone (PSL). We retrospectively compared the efficacy and toxicity of HDD and PSL for newly diagnosed ITP. The response was evaluated according to the International Working Group (IWG) criteria. We analyzed data from 31 and 69 patients in the HDD and PSL groups, respectively. There were no significant differences in patient characteristics between the two groups except for the incidence of the eradication of Helicobacter pylori. The response rate was better in the HDD group (42.7 vs. 28.4xa0%), and this difference was statistically significant when adjusted for other factors including the eradication of H. pylori. In the HDD group, a response was achieved earlier (28 vs. 152xa0days in median) and steroids were more frequently discontinued at 6xa0months (64.5 vs. 37.7xa0%). Among patients who achieved a response, there was no significant difference in the incidence of loss of response. There were no significant differences in the rate of adverse events, transition to chronic ITP, and splenectomy. In conclusion, HDD might enable the early cessation of steroids without a loss of response.

Presentation of familial Mediterranean fever in a heterozygous MEFV mutation triggered by immunosuppressive therapy for myelodysplastic syndrome

Familial Mediterranean fever (FMF) is a recessively inherited disease characterized by recurrent episodes of systemic inflammation. The cause of this disease is the mutations affecting both the alleles of MEFV gene. We describe here a case in a heterozygous MEFV mutation complicated with myelodysplastic syndrome (MDS). Clinical symptoms and the effectiveness of colchicines in this patient are typical for FMF. The first attack of FMF in this patient was observed during immunosuppressive therapy for MDS. This case suggests the possibility that certain immunosuppressants may trigger FMF attack in asymptomatic cases carrying MEFV heterozygous mutation.

Overexpression of MIR9 indicates poor prognosis in acute lymphoblastic leukemia

Abstract Aberrant expression of MIR9 predicts a poor prognosis in acute myelogenous leukemia. To evaluate its clinical significance in acute lymphoblastic leukemia, we analyzed expression levels of MIR9 in bone marrow samples from patients with acute lymphoblastic leukemia and compared them to those in normal bone marrow cells. Approximately 20% of them showed higher expression compared with controls. There was a tendency that patients who showed overexpression of MIR9 underwent worse clinical courses, but without statistical significance. However, when the analyses were restricted to patients who did not receive a stem cell transplant, overexpression of MIR9 was significantly associated with worse overall survival. Interestingly, exaggerated MIR9 expression and higher white blood cell count at presentation were independent unfavorable prognostic factors in all patients for overall survival by multivariate analysis. The presence of higher MIR9 expression could be a useful indicator for treatment stratification.

Acute myeloid leukemia with t(7;21)(q11.2;q22) expresses a novel, reversed-sequence RUNX1―DTX2 chimera

The RUNX1 gene is frequently rearranged in acute leukemia. We cloned a novel RUNX1 chimeric gene generated by t(7;21)(q11.2;q22) in a patient with acute myeloid leukemia. 3′-rapid amplification of cDNA ends analysis showed a tail-to-tail fusion between RUNX1 on 21q22 and DTX2 on 7q11.2, with an insertion of short complementary sequence from UPK3B adjacent to DTX2. DTX2 encodes a putative E3-ubiquitin ligase with no known biological function. There are two possible functions of RUNX1-reversed UPK3B–DTX2: one from aberrant RUNX1 chimeric protein and the other from the reversed sequence of DTX2. The predicted aberrant protein expressed under the RUNX1 promoter was highly structurally similar to RUNX1a. In a reporter assay, the aberrant protein inhibited the trans-activation function of RUNX1 in a dominant-negative manner, similar to RUNX1a. In contrast, the DTX2 reversed sequence may degrade wild-type DTX2 transcript or suppress its translation. In conclusion, we identified a novel fusion RUNX1 partner, DTX2, which chimerize in a reverse direction. This is the first example of RUNX1 chimera in an opposing direction generated by chromosomal translocation in leukemia. In addition to the aberrantly truncated RUNX1 protein, the DTX2 antisense sequence may play some role in the development of leukemia carrying the t(7;21) translocation.

Chronic myelogenous leukemia in chronic phase transforming into acute leukemia under treatment with dasatinib 4 months after diagnosis.

We report a 64-year-old woman morphologically diagnosed with chronic myelogenous leukemia in the chronic phase. Despite having achieved a complete hematological response following treatment with dasatinib, she developed lymphoblastic crisis 4xa0months later. Blastic cells were in a CD45-negative and SSC-low fraction, and positive for CD10, CD19, CD34, and HLA-DR expression and rearrangement in the immunoglobulin heavy chain gene. Chemotherapy using the HyperCVAD/MA regimen led to a complete cytogenetic response, and after cord blood transplantation, she obtained a complete molecular remission. However, the crisis recurred 6xa0months later. Another salvage therapy using L-AdVP regimen followed by nilotinib led to a complete molecular remission. Retrospective analyses using flow cytometry and polymerase chain reaction revealed a minimal blastic crisis clone present in the initial marrow in chronic phase. This case is informative as it suggests that sudden blastic crisis may occur from an undetectable blastic clone present at initial diagnosis and that leukemic stem cells may survive cytotoxic chemotherapy that eliminates most of the blastic cells.