Miyuki Tsutsui

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

JAK2V617F mutation status and allele burden in classical Ph-negative myeloproliferative neoplasms in Japan

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, is frequently detected in classical myeloproliferative neoplasms (MPNs). In the present study, we determined the JAK2V617F allele burden in Japanese MPN patients using alternately binding probe competitive-polymerase chain reaction, a highly quantitative method recently developed by our group. Although we observed strong similarities in terms of epidemiological parameters associated with the JAK2V617F allele burden between our cohort and others, we found a higher JAK2V617F allele burden in Japanese polycythemia vera (PV) patients and lower frequencies of thrombosis in Japanese MPN patients compared with previous reports. In addition, despite the presence of high red blood cell counts, some patients bearing the JAK2V617F mutation were not diagnosed as PV, as their hemoglobin values were lower than the WHO PV criterion. In these patients, the JAK2V617F allele burden was strikingly similar to that in PV patients fulfilling the 2008 WHO criteria, suggesting that these patients can be classified as PV. Although isotopic measurement of red cell mass (RCM) is required for definitive diagnosis of PV, our data suggest that precise measurement of the JAK2V617F allele burden may improve the diagnosis of PV when RCM has not been determined.

Frequent STAT3 activation is associated with Mcl-1 expression in nasal NK-cell lymphoma.

Nasal natural killer (NK)‐cell lymphoma was resistant to various antitumor agents. Although high expression of p‐glycoprotein has been reported, other molecular mechanism of the chemo‐resistance is largely unknown. Activation of STAT3 and expression of major apoptosis‐related proteins Bcl‐2, Bcl‐x, and Mcl‐1 were analyzed by immunohistochemistry. Effects of STAT3 inhibitor AG490 on NK‐YS cell line were analyzed by Western blotting and flow cytometric apoptosis assay. STAT3 was activated in six of the nine nasal NK‐cell lymphomas (67%). In contrast, STAT3 activation was detected in 35% of diffuse large B‐cell lymphoma (DLBCL) and in 10% of follicular lymphoma (FL). Frequent activation of STAT3 was significantly correlated with Mcl‐1 expression in nasal NK‐cell lymphoma, i.e., Mcl‐1 was positive in five of six STAT3‐active cases and negative in all three STAT3‐inactive ones. In DLBCL, not only six out of seven STAT3‐active cases (86%) but also eight out of thirteen STAT3‐inactive cases (62%) were positive for Mcl‐1 expression. Latent membrane protein‐1 was positive in four nasal NK‐cell lymphomas, among which three cases showed intermediate STAT3 activation. Inhibition of STAT3 activation by JAK inhibitor AG490 decreased Mcl‐1 expression and induced apoptosis in STAT3‐active NK‐YS cells. Serum starvation rather increased the Mcl‐1 level in NK‐YS cells, and this effect was also canceled by AG490. These results suggest that activation of STAT3‐Mcl‐1 axis may play a role in the chemotherapy resistance of nasal NK‐cell lymphoma. The pathway may be one of the future therapeutic targets of this intractable disease.

Thalidomide may induce interstitial pneumonia preferentially in Japanese patients

To the Editor: A 58-year-old Japanese man with multiple myeloma (immunoglobulin D-j) was admitted to our hospital due to general fatigue, renal dysfunction and an increased level of serum M-protein on June 2006. He had no history of drug allergy. The patient was treated with seven courses of VAD (vincristine, doxorubicin and dexamethasone) regimen following tandem autologous peripheral blood stem cell transplantation (auto-PBSCT). Before auto-PBSCT, the serum M-protein became undetectable and IgD level decreased to the normal range. After obtaining the informed consent of the patient and the approbation of the Institutional Review Board of our institute, we started daily 100 mg of thalidomide (Sauramide; Penn Pharmaceuticals, South Wales, UK) on March 2008 as a maintenance therapy (1, 2). On day 50 of thalidomide administration, night sweating began, and general fatigue and low-grade fever followed on day 80. Chest and abdominal X-rays showed no significant abnormalities at this point. Administration of levofloxacin and clarithromycin for 1 wk was ineffective. The symptoms gradually worsened and C-reactive protein (CRP) level elevated from 9.7 mg ⁄dL to 17.8 mg ⁄dL in a week. A computed tomography (CT) scan revealed diffuse, ground-glass opacities in both lungs on day 87 (Fig. 1A, B), although the patient showed no respiratory symptoms such as cough, sputum, and dyspnea. The pulse oximetry at room air was 97%, and blood gas analysis showed a slight decrease in bicarbonate which maybe due to slight hyperventilation (PO2 99.8 mmHg, PCO2 30.7 mmHg at room air). We suspected that thalidomide might cause the interstitial change of the lung. The agent was immediately discontinued. The persistent fever spontaneously improved in a few days, and the infiltrative shadow in the lungs improved in a week (Fig. 1C). The CRP level decreased to the normal range, and sweating and fatigue diminished. Because the patient’s condition improved rather rapidly, we failed to

Suppression of eIF4E expression by L-Asparaginase.

L -Asparaginase ( L -ASP) is an important anti-tumor agent used in the treatment of childhood acute lymphocytic leukemia and some kinds of non-Hodgkin’s lymphoma [9] . Extranodal NK-cell lymphoma clinically shows high sensitivity to L -ASP [10–12] , and we have previously reported a specifically high anti-tumor effect of L -ASP against NK-cell tumor cell lines and samples [13] . L -ASP causes rapid depletion of asparagine and glutamine and thus suppresses growth of malignant cells [14] . However, the antitumor mechanism of L -asparaginase and the determinants of its sensitivity are not fully understood [15] . High expression of asparagine synthetase, which opposes the action of L -ASP, only partially explains the resistance to L -ASP in pediatric acute lymphocytic leukemia [16] . Iiboshi et al. [17] showed that L -ASP inhibits the rapamycin-targeted pathway, including p70 S6K and 4E-BP1 in Jurkat cells. In this study, we confirmed the result and further revealed rather specific depletion of eIF4E by L ASP. A much lower concentration of L -ASP depleted eIF4E and its down-stream effector Mcl-1 and induced apoptosis in NK-tumor cell lines. We first confirmed the effect of 1 IU/ml of L -ASP on the phosphorylation states of p70 S6K and S6 ribosomal Eukaryotic translation initiation factor 4E (eIF4E) is considered oncogenic because external expression of eIF4E in the primary fibroblast leads to deregulated proliferation and malignant transformation [1] . Over-expression of eIF4E generates lymphoma in vivo, and its cooperation with c-myc markedly accelerates B-cell lymphomagenesis [2] . Indeed, eIF4E is over-expressed in a wide variety of human tumors, including carcinomas of the colon, breast, and some non-Hodgkin lymphomas [3–5] . The protein plays a key regulatory role in cap-dependent mRNA translation by binding to the 7-methylguanosine cap structure of mRNAs and delivering them to the eIF4F translation initiation complex [6] . The translation of a subset of mRNAs with a complex 5 -UTR is more sensitive to the availability of eIF4E than most cellular RNAs with a short, unstructured UTR [7] . The eIF4E-sensitive mRNAs typically encode growth and survival factors like c-myc, cyclin D1, vascular endothelial growth factor and ornithine decarboxylase [8] . The Akt/mammalian target of rapamycin (mTOR) signal inactivates eIF4E-binding protein 1 (4E-BP1), which binds to and prevents the function of eIF4E. The pathway thus facilitates translation of eIF4E-sensitive growth-promoting mRNAs. Received: November 11, 2009 Accepted after revision: February 16, 2010 Published online: April 27, 2010

Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.

Blastic plasmacytoid dendritic cell neoplasm accompanied by autoimmune hemolytic anemia achieving complete remission with hydroxyurea and steroids.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematological malignancy previously known as blastic natural killer (NK) cell lymphoma or CD4+/CD56+ hematodermic neoplasm. BPDCN is currently believed to arise from plasmacytoid dendritic cell precursors. We report the first case of BPDCN achieving complete remission with hydroxyurea (HU) and steroid treatment. An 87-year-old man came to Juntendo University Hospital, Tokyo, Japan, due to bruise-like skin lesions on the trunk, thighs and upper arms (Fig. 1a). Whole body computed tomography scans disclosed

Accurate Flow Cytometric Gating of the Large Lymphocyte Region Is a Powerful Screening Method for Detecting Hairy Cell Leukemia Presenting with a Low Tumor Burden

Hairy cell leukemia typically presents with pancytopenia and often mimics aplastic anemia. Making an accurate diagnosis is crucial, as treatment with the purine analogues cladribine and pentostatin brings about durable complete remission in the majority of patients. Surface kappa and lambda flow cytometric analyses of peripheral blood or bone marrow are a powerful screening tool, although routine gating of the entire lymphocyte region may fail to show light chain restriction due to a low tumor burden. We herein demonstrate that accurate subgating of the large lymphocyte region is essential and recommend the application of this method in all cases of pancytopenia of unknown etiology.

Ischemic Cardiomyopathy with a Rapid Progression from Thrombotic Thrombocytopenic Purpura.

An 83-year-old woman who complained of dizziness and nausea visited our hospital. An electrocardiogram showed ST-segment elevation in multiple leads and an echocardiogram showed severe hypokinesis of the anteroseptal wall of the left ventricle. However, emergency coronary angiography showed no stenotic lesions in any coronary arteries. A laboratory examination showed thrombocytopenia, renal dysfunction, and hemolysis. We therefore diagnosed the patient with thrombotic thrombocytopenic purpura (TTP). While we were preparing to initiate plasma exchange therapy, she suddenly developed cardiopulmonary arrest. A postmortem examination revealed microthrombi in the small vessels of the myocardium. We herein report a case of ischemic cardiomyopathy with a rapid progression from TTP.

Indolent T‐lymphoblastic proliferation concomitant with acinic cell carcinoma mimicking T‐lymphoblastic lymphoma: case report and literature review

Indolent T‐lymphoblastic proliferation (iT‐LBP) is a non‐clonal benign condition showing extrathymic proliferation of T‐lymphoblasts positive for CD3, CD4, CD8, and TdT. Isolated iT‐LBP has been observed, but the majority of iT‐LBPs have been seen in conjunction with other disorders, including Castleman disease, hepatocellular carcinoma, follicular dendritic cell tumours, angioimmunoblastic T‐cell lymphoma, myasthenia gravis, and acinic cell carcinoma (ACC). The clinical course of iT‐LBP is indolent, and no therapy is usually required. A major concern is misdiagnosis as T‐lymphoblastic lymphoma, and a correct diagnosis of iT‐LBP often requires not only pathological analysis but also careful monitoring of the clinical course. The aim of this study was to broaden the knowledge of pathologists and physicians concerning this as yet not well‐recognised entity.