Hereditary thrombocythemia due to splicing donor site mutation of THPO in a Japanese family.

Thrombopoietin (THPO) is an essential factor for platelet production. Hereditary thrombocythemia (HT) is caused by a germline mutation of THPO, MPL, or JAK2 and is inherited in an autosomal-dominant manner. We identified a Japanese family with HT due to a point mutation of the splicing donor site of the THPO gene (THPO c.13 + 1G > A). Bone marrow biopsy showed increased megakaryocytes mimicking essential thrombocythemia. One affected family member developed chronic myeloid leukemia. We cloned the mutation and developed mutated and wild type THPO expression vectors. Molecular analysis showed that the mutation causes an exon 3 skipping transcript of THPO that abrogates a suppressive untranslated upstream open reading frame. Although the transcript levels of THPO mRNA were comparable, mutated transcripts were more efficiently translated and THPO protein expression was significantly higher than that of the wild type.

Outcomes of allogeneic hematopoietic cell transplantation under letermovir prophylaxis for cytomegalovirus infection.

Cytomegalovirus (CMV) infection is a major infectious complication following allogeneic hematopoietic cell transplantation (allo-HCT). Although letermovir (LMV) prophylaxis dramatically reduces the incidence of early clinically significant CMV (csCMV) infection, it remains unclear whether it has a beneficial effect on nonrelapse mortality (NRM) and overall survival (OS). Herein, we evaluated the impact of LMV prophylaxis on posttransplant outcomes using the registry database of the Japanese Society for Transplantation and Cellular Therapy. Adult patients who underwent allo-HCT between 2017 and 2019 were analyzed (n = 6004). LMV prophylaxis was administered to 1640 patients (LMV group) and it significantly reduced the incidence of csCMV infection compared with those not administered LMV prophylaxis (15.4% vs 54.1%; p < 0.01). However, it did not improve the 1-year NRM (hazard ratio [HR], 0.93; p = 0.40) and OS (HR, 0.96; p = 0.49). In the LMV group, 74 patients had breakthrough csCMV infection and showed inferior NRM (HR, 3.44; p < 0.01) and OS (HR, 1.93; p = 0.02) compared with those without infection. After completing LMV prophylaxis, 252 patients had late csCMV infection and showed inferior NRM (HR, 1.83; p < 0.01) and OS (HR, 1.58; p < 0.01). Our findings suggest that managing breakthrough and late csCMV infections is important for improving long-term outcomes.

Subclinical minute FLT3-ITD clone can be detected in clinically FLT3-ITD-negative acute myeloid leukaemia at diagnosis.

Recent advances in next-generation sequencing (NGS) have enabled the detection of subclinical minute FLT3-ITD. We selected 74 newly diagnosed, cytogenetically normal acute myeloid leukaemia (AML) samples in which FLT3-ITD was not detected by gel electrophoresis. We sequenced them using NGS and found minute FLT3-ITDs in 19 cases. We compared cases with clinically relevant FLT3-ITD (n = 37), cases with minute FLT3-ITD (n = 19) and cases without detectable FLT3-ITD (n = 55). Molecular characteristics (location and length) of minute FLT3-ITD were similar to those of clinically relevant FLT3-ITD. Survival of cases with minute FLT3-ITD was similar to that of cases without detectable FLT3-ITD, whereas the relapse rate within 1 year after onset was significantly higher in cases with minute FLT3-ITD. We followed 18 relapsed samples of cases with clinically FLT3-ITD-negative at diagnosis. Two of 3 cases with minute FLT3-ITD relapsed with progression to clinically relevant FLT3-ITD. Two of 15 cases in which FLT3-ITD was not detected by NGS relapsed with the emergence of minute FLT3-ITD, and one of them showed progression to clinically relevant FLT3-ITD at the second relapse. We revealed the clonal dynamics of subclinical minute FLT3-ITD in clinically FLT3-ITD-negative AML. Minute FLT3-ITD at the initial AML can expand to become a dominant clone at relapse.

Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context.

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes.

Dominant-negative type of IKZF1 deletion showed a favorable prognosis in adult B-cell acute lymphoblastic leukemia.

IKZF1 deletion is a recurrent genomic alteration in B-cell acute lymphoblastic leukemia (B-ALL) and is divided into dominant-negative (DN) and loss of function (LOF) deletions. The prognostic impact of each deletion has not been fully elucidated. We retrospectively analyzed 117 patients with adult B-ALL including 60 patients with BCR::ABL1-positive B-ALL and 57 patients with BCR::ABL1-negative B-ALL by the fluorescence in situ hybridization (FISH) method for IKZF1 deletion and multiplex PCR for the 4 most common IKZF1 deletions (∆4-7, ∆2-7, ∆2-8, and ∆4-8). Samples, in which IKZF1 deletion was detected by FISH but a specific type of deletion was not identified by the PCR, were categorized as "other." Patients were classified into a DN group that had at least 1 allele of ∆4-7 (n = 23), LOF and other group (n = 40), and wildtype group (n = 54). DN type IKZF1 deletions were found in 33.3% of BCR::ABL1-positive cases and 5.2% of BCR::ABL1-negative cases. LOF and other type IKZF1 deletions were found in 43.4% of BCR::ABL1-positive cases and 24.6% of BCR::ABL1-negative cases. Patients with the DN group showed significantly higher overall survival (OS) than that of the LOF and other and WT groups (P = 0.011). Multivariate analysis including age, WBC counts, complex karyotype, and DN type IKZF1 deletion showed that the DN type of IKZF1 deletion (HR = 0.22, P = 0.013) had a positive impact and age ≥ 65 (HR = 1.92, P = 0.029) had a negative impact on OS. The prognostic impact of IKZF1 deletion depends on the type of deletion and DN type of IKZF1 deletion showed better prognosis in adult B-ALL patients.Clinical trial registration This study was part of a prospective observational study (Hokkaido Leukemia Net, UMIN000048611). It was conducted in compliance with ethical principles based on the Helsinki Declaration and was approved by the institutional review board of Hokkaido University Hospital (#015-0344).

Clinical implications of NUP98::NSD1 fusion at diagnosis in adult FLT3-ITD positive AML.

OBJECTIVES: The cryptic fusion oncogene NUP98::NSD1 is known to be associated with FLT3-ITD mutation in acute myeloid leukemia (AML), and an independent poor prognostic factor in pediatric AML. However, there are little data regarding the clinical significance of NUP98::NSD1 in adult cohort. METHODS: We conducted a multicenter retrospective study to investigate the prevalence, clinical characteristics, and prognostic impact of NUP98::NSD1 in adult FLT3-ITD-positive AML patients. RESULTS: In a total of 97 FLT3-ITD-positive AML patients, six cases (6.2%) were found to harbor the NUP98::NSD1 fusion transcript. NUP98::NSD1 positive cases had significantly higher platelet counts and a higher frequency of FAB-M4 morphology than NUP98::NSD1 negative cases. NUP98::NSD1 was found to be mutually exclusive with NPM1 mutation, and was accompanied by the WT1 mutation in three of the six cases. The presence of NUP98::NSD1 fusion at the time of diagnosis predicted poor response to cytarabine-anthracycline-based intensive induction chemotherapy (induction failure rate: 83% vs. 36%, p = .038). Five of the six cases with NUP98::NSD1 underwent allogeneic hematopoietic stem cell transplantation (HSCT). Two of the five cases have successfully maintained remission, with one of them being rescued through a second HSCT. CONCLUSIONS: Detecting NUP98::NSD1 in adult FLT3-ITD-positive AML is crucial to recognizing chemotherapy-resistant group.

HLA loci predisposing to immune TTP in Japanese: potential role of the shared ADAMTS13 peptide bound to different HLA-DR.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.

Clinical and molecular consequences of fusion genes in myeloid malignancies.

Leukemias are heterogeneous diseases characterized by aberrant hematopoietic stem and progenitor cells (HSPCs). Oncogenic fusion genes and proteins, produced via gross chromosomal rearrangements, such as chromosomal translocation, insertion, and inversion, play important roles in hematologic malignancies. These oncoproteins alter fundamental cellular properties, such as self-renewal, differentiation, and proliferation, and confer leukemogenic potential to HSPCs. In addition to providing fundamental insights into the process of leukemic transformation, these fusion genes provide targets for treatment and monitoring of myeloid leukemias. Furthermore, new technologies such as next-generation sequencing have allowed additional insights into the nature of leukemic fusion genes. In this review, we discuss the history, biologic effect, and clinical impact of fusion genes in the field of myeloid leukemias.

The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide.

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

Validation and comparison of prognostic values of GNRI, PNI, and CONUT in newly diagnosed diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin lymphoma. Emerging evidence indicates that poor nutritional status determined with nutritional indices such as geriatric nutritional risk index (GNRI), prognostic nutritional index (PNI), and controlling nutritional status score (CONUT) was associated with poor prognosis of DLBCL. We conducted this multicenter retrospective study to validate and compare prognostic values of the three indices in 615 newly diagnosed DLBCL patients. The overall survival (OS) in patients with poor nutritional status determined with each of these nutritional indices were significantly inferior compared with that in those without nutritional risks (5-year OS in patients with GNRI < 95.7 and GNRI ≥ 95.7 were 56.4% and 83.5%, P < 0.001; PNI < 42.4 and PNI ≥ 42.4 were 56.1% and 81.0%, P < 0.001; CONUT > 4 and CONUT ≤ 4 were 53.1% and 77.1%, P < 0.001). GNRI and CONUT were independent prognostic predictors for OS (GNRI < 95.7, hazard ratio [HR] 1.83, 95% confidence interval [CI] 1.22-2.74, P = 0.0032; CONUT > 4, HR 1.53, 95% CI 1.05-2.23, P = 0.028) after multivariate analyses. Nutritional status determined with GNRI affected OS more strongly in the patients with nongerminal center B cell-like (nonGCB) DLBCL compared with that in those with GCB-type DLBCL. In conclusion, baseline poor nutritional status determined based on GNRI or CONUT was an independent risk factor of newly diagnosed DLBCL, and GNRI was also useful as an independent prognostic factor for patients with nonGCB-type DLBCL.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

[Aplastic anemia following drug-induced liver injury].

Aplastic anemia (AA), a hematopoietic disorder characterized by hypocellular bone marrow, is caused by immunologically-mediated hematopoietic stem cell injury. Viral infection is hypothesized as the underlying cause of hepatitis-associated AA, although its mechanism is still unclear. This report describes a case of AA following suspected drug-induced liver injury (DILI). An 18-year-old man developed severe liver dysfunction after taking oral over-the-counter drugs. The patient was diagnosed with suspected DILI based on drug-induced lymphocyte stimulation test and liver biopsy results. Although liver dysfunction improved after a course of steroid pulse therapy and liver supporting therapy, the man gradually developed pancytopenia within 3 months of DILI diagnosis, prompting the diagnosis of AA following DILI. Paroxysmal nocturnal hemoglobinuria-type cells were detected by high-sensitivity flow cytometry. Immunosuppressive therapy with antithymocyte globulin and cyclosporin was administered, with pancytopenia improvement. To the best of our knowledge, this is the first report in the literature with a case of AA following DILI, and we believe it is important for evaluating the pathogenesis of drug-induced and hepatitis-associated AA.

Severe adverse events by tyrosine kinase inhibitors decrease survival rates in patients with newly diagnosed chronic-phase chronic myeloid leukemia.

OBJECTIVE: This multicenter cooperative study aimed to analyze the adverse events (AEs) associated with tyrosine kinase inhibitors (TKIs) used as initial treatment for chronic-phase chronic myeloid leukemia (CML-CP) and their impact on outcome. METHODS: We retrospectively evaluated 450 patients with CML-CP who received TKIs between 2004 and 2014. RESULTS: The 5-year overall survival (OS) and event-free survival (EFS) rates were 95.1% and 89.0%, respectively. Patients with comorbidities (46.4%) and aged ≥60 years (50.4%) at diagnosis had significantly inferior OS to those without comorbidities and aged <60. Patients achieved higher rates of major molecular response (MMR) at 6 and 12 months after initial treatment with dasatinib or nilotinib compared to imatinib, but final MMR rates were almost the same. Sixty-six percent of patients required treatment modifications from first-line TKI therapy; the main reasons were AEs (48.4%) and failure (18%). Grade III-IV AEs in first-line TKI therapy were significantly correlated to inferior OS/EFS compared to grade 0-II AEs. CONCLUSION: Although long-term outcomes were similar in CML-CP patients treated with each TKI regardless of first-line TKI selection, severe AEs in first-line TKI therapy decreased their survival rates. Early change in TKIs is recommended, when faced with severe AEs of specific TKIs.

[Recurrent invasive pneumococcal disease in a patient with IgG-κ smoldering multiple myeloma].

A 68-year-old female with smoldering multiple myeloma (IgG-κ type) was admitted to the hospital owing to general fatigue, fever, and pain in the right leg. On the day following admission, she developed shock, and a blood culture revealed Streptococcus pneumoniae. She was diagnosed with septic shock and invasive pneumococcal disease (IPD). She received antibiotics and intravenous immunoglobulin and improved after several days. She had a history of recurrent IPD and had received the pneumococcal polysaccharide vaccine 23 (PPSV23) 2 years earlier. Therefore, we inquired with the National Institute of Infectious Diseases if the pneumococcal serotype isolated from her present IPD contained PPSV23. The results showed that her serotype was 19F, a serotype present in PPSV23. We administered pneumococcal conjugate vaccine 13 (PCV13) ; however, she was unable to mount high enough opsonophagocytic assay titers against some serotypes, including 19F. We think she was unable to mount effective humoral immune responses to PPSV23 or PCV13 owing to her underlying disease, smoldering myeloma. It should be considered how IPD can be effectively prevented in patients with multiple myeloma.

Risk factors of human herpesvirus 6 encephalitis/myelitis after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Human herpesvirus 6 (HHV-6) encephalitis/myelitis is now a well-known complication after allogeneic stem cell transplantation (allo-HSCT), particularly after cord blood transplantation (CBT). In this study, we evaluated the risk factors of HHV-6 encephalitis/myelitis. METHODS: We evaluated 253 patients who received allo-HSCT from 2007 to 2015 at our institute. HHV-6 encephalitis/myelitis was defined as HHV-6 DNA detection in the cerebrospinal fluid or peripheral blood by polymerase chain reaction in the presence of typical manifestations without other concurrent condition that led to the manifestations. RESULTS: HHV-6 encephalitis/myelitis occurred in 11 patients (4.5%) (9 encephalitis, 3.7%; 2 myelitis, 0.8%). Multivariate analysis showed that CBT, mycophenolate mofetil (MMF) for graft-versus-host disease prophylaxis, history of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and engraftment syndrome (ES) were significantly associated with incidence of HHV-6 encephalitis/myelitis (P=.025, P=.017, P=.017, and P=.014, respectively). CONCLUSION: Although it has been shown that CBT, ES, and history of allo-HSCT are risk factors for HHV-6 encephalitis/myelitis, our study demonstrated MMF is also a risk factor for the disease.

Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation.

OBJECTIVE: Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide-LMIR3 interaction in the development of IBD. DESIGN: The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3(-/-), mast cell-deficient Kit(W-sh/W-sh), Kit(W-sh/W-sh)LMIR3(-/-) or Kit(W-sh/W-sh) mice engrafted with WT or LMIR3(-/-) bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. RESULTS: LMIR3 deficiency exacerbated DSS-induced colitis in mice. Kit(W-sh/W-sh) mice harbouring LMIR3(-/-) mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide-LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide-LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. CONCLUSIONS: LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3(-/-) mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide-LMIR3 binding.

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells.

High levels of HES1 expression are frequently found in BCR-ABL(+) chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC-like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC-like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.

Human CD300C delivers an Fc receptor-γ-dependent activating signal in mast cells and monocytes and differs from CD300A in ligand recognition.

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.

Relative impact of THPO mutation causing hereditary thrombocythemia.

Germline mutations of THPO were reported as causes of hereditary thrombocythemia. Six previously reported distinct sites of the mutation were clustered at the 5`-untranslated region or the exon 3 splicing donor site of the THPO gene. Each mutation was identified in an independent pedigree, and the differences between the mutations were not compared. We cloned six distinct THPO mutations (THPO c.-47delG, THPO c.-31G>T, THPO c.13G>A, THPO c.13+1G>A, THPO c.13+2T>C, and THPO c.13+5G>A) and compared the molecular mechanisms that underlie the increased production of THPO protein. At the transcript level, all of the mutations except THPO c.-47delG showed an exon 3 skipping transcript, including two mutations (THPO c.-31G>T and THPO c.13+5G>A) that were distant from the splicing donor site. THPO c.-47delG showed the same full-length transcript as that of the wild-type transcript. At the protein level, all mutations resulted in a higher level of production of thrombopoietin (THPO) protein compared with wild-type THPO. There are only two distinct patterns of mechanisms for increased production of THPO: exon 3 skipping that deleted upstream suppressive open reading frame (ORF)7 and one base deletion that shifted ORF7 to connect to the initial codon of THPO in-frame. The common mechanisms of hereditary thrombocytosis due to THPO mutations are unleashed THPO translations, which are usually suppressed by upstream out-of-frame ORF7.