Yoshitaka Sunami

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

JAK2 , CALR , and MPL mutation spectrum in Japanese patients with myeloproliferative neoplasms

Recurrent somatic mutations in the JAK2 , MPL , and CALR genes have been described in patients diagnosed with Philadelphia-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These mutations are generally

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.

Detection of MPLW515L/K Mutations and Determination of Allele Frequencies with a Single-Tube PCR Assay

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells

Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

Autoimmune hemolytic anemia in a patient with TAFRO syndrome

TAFRO syndrome is a systemic inflammatory disorder characterized by low platelet counts, anasarca, fever, reticulin fibrosis, renal dysfunction, and organomegaly. Patients with TAFRO syndrome occasionally have courses complicated by immunological diseases. Herein, we describe a case of TAFRO syndrome associated with autoimmune hemolytic anemia (AIHA). The patient was admitted because of menorrhagia. She had thrombocytopenia, pleural effusion and ascites, hepatomegaly, and multiple lymphadenopathies. Her symptoms worsened, especially fever, pleural effusion and ascites, and she developed AIHA. Steroid pulse therapy followed by 45 mg of prednisolone (PSL) improved not only the symptoms of TAFRO syndrome but also those of AIHA. There have been no reports, to our knowledge, of AIHA associated with TAFRO syndrome, and detailed studies on this syndrome are needed.

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.

Homomultimerization of mutant calreticulin is a prerequisite for MPL binding and activation

Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.

Various Neurological Symptoms by Neurolymphomatosis as the Initial Presentation of Primary Testicular Lymphoma

Neurological symptoms induced by the infiltration of malignant lymphoma into the nervous systems are subsumed under the term neurolymphomatosis (NL). Here, we report the case of a 30-year-old Japanese man with primary testicular lymphoma complicated, as seen in various neurological findings, by secondary NL prior to testicular swelling. Painless right scrotal enlargement was noticed more than 1 month after the appearance of neurological complications such as right upper extremity numbness, dysarthria, facial palsy, and diplopia. Proactive investigation and biopsies of extranodal sites at high risk of central nervous system infiltration of malignant lymphoma, such as the testes, should be considered when secondary NL is suspected based on imaging findings.