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Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

Neuroinflammation, where inflammatory cytokines are produced in excess, contributes to the pathogenesis of delirium. Microglial cells play a central role in neuroinflammation by producing and releasing inflammatory cytokines in response to infection, tissue damage and neurodegeneration. Dexmedetomidine (DEX) is a sedative, which reduces the incidence of delirium. Thus, we hypothesized that DEX may alleviate delirium by exhibiting anti-inflammatory action on microglia. In the present study, we investigated the anti-inflammatory action of DEX on human microglial HMC3 cells. The results indicated that DEX partially suppressed the IL-6 and IL-8 production by lipopolysaccharide (LPS)-stimulated HMC3 cells as well as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. Furthermore, DEX substantially suppressed IL-6 and IL-8 production by unstimulated HMC3 cells as wells as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. These observations suggest that DEX exhibits anti-inflammatory action on not only LPS-stimulated but also unstimulated microglial cells via the suppression of inflammatory signaling and cytokine production.

Context: Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. Objective: This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). Methods: We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p< 0.05. Results: Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. Conclusion: Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations-two distinct mutations on each chromosome-without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

Background It is widely accepted that Point-of Care Test (PoCT) devices are useful in the detection of coagulopathies in situations of massive bleeding such as major cardiac surgery. These devices contribute to the reduction of blood transfusion. However, their implementation remains limited in Japan because of their cost and lack of health insurance support. Study design and methods Conventional coagulation tests and thromboelastography (TEG)/Sonoclot values were measured in 50 consecutive cardiac surgery cases. Clinical background information such as operative procedures was obtained from electronic medical records, and the theoretical perioperative total blood loss was calculated by measuring the hemoglobin content and total red blood cell transfusion volume. The correlation between perioperative total blood loss and the measured laboratory values or clinical parameters was evaluated by a multivariate linear regression analysis. The risk factors of the total amount of platelet transfusion and postoperative drain bleeding volume were similarly evaluated. Results No significant association between the estimated perioperative total blood loss (eTBL) and the laboratory measurements including conventional coagulation tests, TEG and Sonoclot was observed. On the other hand, postoperative drain bleeding volume was significantly associated with postoperative Sonoclot CR (p = 0.039) as well as preoperative use of oral anticoagulants and cell saver treated blood volume. Platelet transfusion amount was significantly associated with post-CBP PF and time to peak value of Sonoclot (p = 0.014 and 0.001, respectively). Conclusion Sonoclot measurements may be useful to estimate the risks of postoperative bleeding and platelet transfusion in cardiac surgeries in Japan.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin− cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin− HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin− HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Summary Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells., Highlights • We established a model of CML stem cells derived from CML-iPSCs • ADAM8 is identified as an antigen of TKI-resistant CML cells • The inhibition of ADAM8 restored TKI sensitivity in primary samples • ADAM8 is a marker of residual CML cells in patients with optimal TKI response, The paucity and heterogeneity of CML stem cells are obstacles for analyses. In our study, a model of CML stem cells derived from CML-iPSCs identified ADAM8 as an antigen of TKI-resistant cells. In CML patients, ADAM8+ cells showed TKI resistance and residual CML cells after TKIs-treatment were concentrated in ADAM8+ population, suggesting that ADAM8 is a marker of TKI-resistant CML cells.

Background Hypertrophic obstructive cardiomyopathy (HOCM) is a type of hypertrophic cardiomyopathy associated with left ventricular outflow tract stenosis. The increased pressure gradients across the left ventricular outflow tract in patients with HOCM could lead to circulatory collapse. We describe our experience with perioperative management under femoral nerve block (FNB), lateral femoral cutaneous nerve block (LFCNB), and transthoracic echocardiography (TTE) monitoring during open reduction and internal fixation of a femoral neck fracture in a patient with severe HOCM. Case presentation A 72-year-old man, who was indicated to undergo open reduction and internal fixation of an intracapsular femoral neck fracture, had a history of treatment for hypertension and HOCM. He had heart failure for 4 years and was hospitalized several times. He was resuscitated after ventricular fibrillation and received an implantable cardioverter-defibrillator at that time. He also had severe physical limitations (New York Heart Association class III). We selected FNB and LFCNB as the methods for anesthesia and injected 0.25% levobupivacaine (20 mL) around the femoral nerve and 0.25% levobupivacaine (10 mL) into the lateral femoral nerve region. He underwent TTE during the perioperative period, which enabled us to perform hemodynamic and morphological evaluations of the heart. The intraoperative TTE findings remained stable from before the induction of anesthesia to the patient’s exit from the operating room. Postoperatively, his hemodynamic parameters continued to remain stable. Conclusions In this case, FNB and LFCNB contributed to hemodynamic stability during non-cardiac surgery. Additionally, TTE was useful for the perioperative evaluation of cardiac hemodynamics and morphology in our patient with severe HOCM.

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.

Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. Methods: Chromosome 7 encodes multiple genes regulating histone methylation. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7 and non-monosomy 7. Results: We found that the siRNA-mediated knockdown of EED, or BRD4 significantly decreased cell viability in AML cells with monosomy 7 compared to the control leukemia cell lines. Stable knockdown of EED markedly attenuated proliferation as well as increased cell apoptosis specifically in leukemia cells with monosomy 7. Interestingly, PRC1 rather than PRC2 activity of EED had a predominant role in the survival of monosomy 7 leukemia cells. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to EED inhibition. We found that knockdown of GTF2I increased the sensitivity to EED inhibition via increased cell apoptosis. Because both GTF2I and EED suppressed expression levels of BCL2, it is possible that EED inhibition additively abrogate BCL2 activity in AML with monosomy 7. In addition to EED, we identified BRD4 as another essential survival factor in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. Mechanistically, we found that knockout of MLL3 or MLL5, both encoded in the chromosome 7, resulted in increased vulnerability to JQ-1 treatment. In monosomy 7, functional loss of MLL3 and MLL5 showed reduced expression levels of c-MYC and CCND1. JQ-1 treatment additively impaired the functions of these genes, and strongly inhibited cell proliferation of AML cells with monosomy 7. Conclusion: Our synthetic-lethality based approach identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Citation Format: Kensuke Matsuda, Yuki Kagoya, Hideaki Mizuno, Sho Yamazaki, Masashi Miyauchi, Mineo Kurokawa. EED or BRD4 inhibition as a novel therapeutic strategy in acute myeloid leukemia with monosomy 7 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3652.

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient’s age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient’s derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

OBJECTIVE Levofloxacin is widely used as antimicrobial prophylaxis against high-risk chemotherapy-induced neutropenia. Garenoxacin, a fluoroquinolone developed in Japan, shows a stronger in vitro antimicrobial activity against Gram-positive bacteria than levofloxacin. METHODS We retrospectively analyzed high-risk patients with acute myeloid leukemia who were administered garenoxacin (n = 36) or levofloxacin (n = 120) during chemotherapy. We compared the profiles of infections between these fluoroquinolones. RESULTS Febrile events occurred in 31 (86%) and 93 (78%) cases in the garenoxacin and levofloxacin group, respectively (P = 0.35). Bloodstream infections by Gram-positive bacteria were recorded in one (3%) case in the garenoxacin group and 25 (21%) cases in the levofloxacin group (P < 0.01). In contrast, bloodstream infections by Gram-negative microorganisms were identified in five (4%) cases in the levofloxacin group and eight (22%) cases in the garenoxacin group (P < 0.01). CONCLUSIONS These results indicate that there may be substantial differences in the antimicrobial spectrum between different fluoroquinolones. Although there are several biases due to rather small sample size and the retrospective nature, we should take the differences into consideration when we administer a prophylactic fluoroquinolone to a patient with hematological disease.

Actin stress fibers (SFs) generate intercellular tension and play important roles in cellular mechanotransduction processes and the regulation of various cellular functions. We recently found, in vascular smooth muscle cells (SMCs) cultured on a substrate, that the apical SFs running across the top surface of the nucleus have a mechanical connection with the cell nucleus and that their internal tension is transmitted directly to the nucleus. However, the effects of the connecting conditions and binding forces between SFs and the nucleus on force transmission processes are unclear at this stage. Here, we estimated the mechanical connection between apical SFs and the nucleus in SMCs, taking into account differences in the contractility of individual SFs, using experimental and numerical approaches. First, we classified apical SFs in SMCs according to their morphological characteristics: one subset appeared pressed onto the apical surface of the nucleus (pressed SFs), and the other appeared to be smoothly attached to the nuclear surface (attached SFs). We then dissected these SFs by laser irradiation to release the pretension, observed the dynamic behavior of the dissected SFs and the nucleus, and estimated the pretension of the SFs and the connection strength between the SFs and the nucleus by using a simple viscoelastic model. We found that pressed SFs generated greater contractile force and were more firmly connected to the nuclear surface than were attached SFs. We also observed line-like concentration of the nuclear membrane protein nesprin 1 and perinuclear DNA that was significantly located along the pressed SFs. These results indicate that the internal tension of pressed SFs is transmitted to the nucleus more efficiently than that of attached SFs, and that pressed SFs have significant roles in the regulation of the nuclear morphology and rearrangement of intranuclear DNA.

Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to resistance to conventional chemotherapy and poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. In this study, we aimed to find a novel therapeutic target in AML with monosomy 7. Methods and Results: Chromosome 7 encodes multiple genes regulating histone methylation such as EZH2, KTM2C and KTM2E. Although loss of these genes is considered to contribute to the development of AML with monosomy 7, it may also be required for some leukemia cell functions. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7: KG-1 and F-36P cells. We found that the siRNA-mediated knockdown of EED, BRD4 or BRDT significantly decreased cell viability in AML cells with monosomy 7 compared to K562 cells as the control without monosomy 7. Stable knockdown of EED expression by retroviral transduction of shRNA markedly attenuated proliferation as well as increased apoptosis specifically in leukemia cells with monosomy 7. Surprisingly, pharmacologic inhibition of EED, which blocks the binding of EED to the polycomb repressive complex 2 (PRC2), was only marginally effective for the monosomy 7 leukemia cells. These results suggest that EED supports survival of the monosomy 7 leukemia cells in a PRC2-independent manner. Recent studies showed that EED is not only a component of PRC2 but also interacts with the polycomb repressive complex 1 (PRC1). In contrast to modest effects of PRC2 inhibition, inhibition of PRC1 activity showed marked efficacy specifically in leukemia cells with monosomy 7. Co-inhibition of PRC1 and PRC2 synergistically suppressed proliferation of the monosomy 7-AML cells. These results collectively suggest that while both PRC1 and PRC2 cooperatively function, the PRC1 rather than PRC2 has a predominant role in the survival of monosomy 7 leukemia cells. In addition to EED, we identified BRD4 as an essential survival factors in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to BET bromodomain protein inhibition. K562 cells were individually treated with the siRNAs against 22 genes in the presence of a BET bromodomain inhibitor JQ1. We found that knockdown of MLL3 or MLL5 resulted in increased apoptosis of the JQ1-treated K562 cells. We also confirmed that pharmacological inhibition of histone methyltransferase activity of MLL3 and MLL5 showed synergistic effects with JQ1 treatment in the leukemia cells. Conclusion: We identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Disclosures Kagoya: Kyowa Kirin: Speakers Bureau; Nihon Shinyaku: Research Funding. Miyauchi:kyowa Kirin: Research Funding. Kurokawa:Novartis Pharma K.K.: Research Funding; Celgene K.K.: Consultancy, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding; Boehringer Ingelheim: Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding; Shionogi & Co., Ltd: Consultancy, Honoraria; Bioverativ Japan ltd.: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Shire Japan K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau.

Fludarabine-based therapy is widely approved as a first-line treatment for chronic lymphocytic leukemia (CLL). This treatment is occasionally associated with prolonged myelosuppression. We herein describe the cases of CLL who underwent fludarabine, cyclophosphamide and rituximab (FCR) therapy. Bone marrow examinations performed during periods of prolonged myelosuppression revealed definite myelodysplastic changes in the myeloid and erythroid lineages. G-banded karyotyping analyses revealed cytogenetic abnormalities. The patients were diagnosed with therapy-related myelodysplastic syndrome (t-MDS). Further administration of cytotoxic therapy was aborted, and no progression of t-MDS was recorded throughout the follow-up period in either case. In these cases, the t-MDS was characterized by a short latency interval and a benign clinical course. Because typical t-MDS with aggressive outcomes also occurs during prolonged myelosuppression, the transition of the clinical course in this setting should therefore be carefully watched.

Composite lymphoma is defined as two or more distinct types of lymphoma in a single anatomical site. Among various combinations, composite B-cell and T-cell non-Hodgkin's lymphomas (CBTL) are very infrequent. Herein we describe a 66-year-old female with CBTL presenting with lymphadenopathy, multiple bone lesions and an epidural tumor. Light microscopic examination of a biopsied cervical node revealed a dual population of lymphoid cells: sheets of large cells admixed with medium-sized cells. The large cells expressed B-cell markers and showed immunoglobulin light chain restriction, consistent with diffuse large B-cell lymphoma (DLBCL). The medium-sized cells were positive for CD20 as well as T-cell markers. Because polymerase chain reaction amplification showed monoclonal rearrangement of the T-cell receptor β chain gene, this population was compatible with peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). We therefore made a diagnosis of composite DLBCL and CD20-positive PTCL-NOS. Complete remission was achieved after six cycles of R-CHOP regimen (rituximab, doxorubicin, vincristine, cyclophosphamide and prednisolone). This is the first report of CD20-positive PTCL-NOS associated with composite lymphoma. Moreover, a literature review of composite DLBCL and PTCL-NOS indicates that this rare clinical entity may be featured by efficacy of systemic chemotherapy in spite of prevalent extranodal lesions.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by megakaryocytic atypia, fibrosis in bone marrow, and extramedullary hematopoiesis. Although mutational analyses reveal that about 90% of patients with primary MF harbor JAK2, CALR or MPL mutation that constitutively activates JAK-STAT pathway, the outcome of JAK2 inhibitor treatment for MF patients still remains to be improved. However, only a limited number of humanized disease-models are currently available to develop a novel therapeutic strategy for MF. We have previously reported that induced pluripotent stem cells (iPSCs) established from MF patients (MF-iPSCs) by using retroviral vectors recapitulated the disease phenotype (Hosoi et al. 2014), indicating that MF-iPSCs are one of the useful humanized disease-models of MF. We established iPSCs from three MF patients harboring a JAK2 V617F, CALR type 1, or CALR type 2 mutations by using integration-free episomal vectors and confirmed that established MF-iPSCs harbored the identical mutations to parental cells. After hematopoietic differentiation, we obtained hematopoietic progenitor cells (HPCs) immunophenotypically defined as CD34+/CD43+ cells. Viability assays revealed that JAK2 inhibitors (Ruxolitinib and HSP90) impaired cell survival of MF-HPCs compared to normal-HPCs, suggesting that MF-HPCs also reproduce drug-sensitivity. To investigate therapeutic targets of MF, we optimized culture condition of HPCs to perform compound screening. By the use of small chemical library containing 192 compounds and two MF-iPSCs harboring a JAK2 or CALR mutation, we identified KN93, calcium/calmodulin dependent protein kinase (CAMK) 2 inhibitor, as a compound which inhibited the viability of MF-HPCs from all three patients in dose-dependent manner, compared to normal-HPCs. For validation, we used Trifluoperazine (TFP), another CAMK inhibitor and TFP had similar inhibitory effects on MF-HPCs. In Ba/F3 mouse cell line ectopically expressing MPL W515L mutant (Ba/F3 MPLmu ;IC50 of ruxolitinib = 112 nM) as other models mimicking MF than that using JAK2 mutant and CALR mutant, KN93 and TFP decreased cell growth, induced apoptosis, and suppressed the phosphorylation of Stat5, a major signaling event in MF, indicating that CAMK2 is a candidate of therapeutic target for MF. To determine the critical subtype of CAMK2 in MF, we assessed the expression of CAMK2 subtypes. In Ba/F3 MPLmu, Camk2 gamma subtype (Camk2g) was detectable among all Camk2 subtypes in mRNA levels and phosphorylation of CAMK2G protein was enhanced at the absence of interluekin-3. Knock down of CAMK2G by short-hairpin RNA decreased cell growth of Ba/F3 MPLmu. Moreover, berbamine, a CAMK2G inhibitor, also inhibited the viability of Ba/F3 MPLmu and colony forming capacity of c-kit+ primary mouse bone marrow ectopically expressing MPLW515L compared to that of expressing wild type MPL at dose-dependent manner, suggesting that CAMK2G is a critical subtype of CAMK2 in MF. To address whether CAMK2 inhibition overcome the JAK2 inhibitor-resistance, one of the reason why JAK2 inhibitor is not efficient in clinical setting, we established ruxolitinib-resistant Ba/F3_MPLmu (Ba/F3_MPLmu_R; IC50 of ruxolitinib = 642 nM) cells through one-month exposure to ruxolitinib. KN93 and TFP decreased cell growth, induced apoptosis, and suppressed the phosphorylation of Stat5 in Ba/F3_MPLmu_R and combination of CAMK2 inhibitors and ruxolitinib exhibited inhibitory effects more efficiently than single agent, suggesting that CAMK2 inhibitors show a cooperative effect with ruxolitinib against MF cells and have the potential to overcome JAK2 inhibitor-resistance. To address the effectiveness of CAMK2 inhibition in primary samples, we used CD34+ cells isolated from peripheral blood mononuclear cells of MF patients. Although either KN93 or ruxolitinib inhibited the colony forming capacity of primary MF cells with similar efficiency, combination of KN93 with ruxolitinib showed cooperative inhibition. Taken together, these findings indicate that CAMK2G would be a therapeutic target of MF patients. In conclusion, MF-iPSCs enable comparable compound screening assay and identify CAMK2G as a potential therapeutic target of MF. Our results revealed that CAMK2G inhibition could lead the novel therapeutic strategies to combine with JAK2 inhibitors and to overcome the resistance against JAK2 inhibitors. Disclosures Arai: Novartis: Research Funding. Kurokawa:Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Eizai: Research Funding; Pfizer: Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; MSD: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Teijin Pharma: Research Funding.

Background Bone marrow infiltration is widely accepted as an important prognostic factor in malignant lymphomas. Although multiplex polymerase chain reaction for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements is a helpful tool for the confirmation of minimal infiltration in B cell lymphomas, it has not been elucidated that IgH clonality of the bone marrow at the initial evaluation is a valuable prognostic factor and a predicting marker for the initial therapy in the patients with B cell lymphomas who had a bone marrow biopsy with negative pathology. Methods All patients with DLBCL (n=61) and indolent B cell non-Hodgkin's lymphomas (iB-NHL) including FL, MCL and MALT lymphoma (n=51) who had a bone marrow biopsy and a bone marrow IgH clonality obtained at diagnosis in our institute it the period between March 2002 and 2016 were included in this study. The clinical information including the overall survival (OS), progression free survival (PFS), laboratory findings, backgrounds, and the response to the first line therapy were collected from the clinical charts. Result Bone marrow infiltration by pathological examination was reported in 20% (12/61) of DLBCL patients and in 24% (12/51) of iB-NHL patients (Path-pos group). In the patients with negative pathological report, 21% of the DLBCL patients (13/61) and 33% of the iB-NHL patients (17/51) were positive bone marrow involvement by PCR (Path-neg/PCR-pos group). The other 59% of the DLBCL patients (36/61) and 43% of the iB-NHL patients (22/51) were negative bone marrow involvement by both pathological examination and PCR (Path-neg/PCR-neg group). In DLBCL cohort, the 5-year OS rates in Path-pos, Path-neg/ PCR-pos, and Path-neg/ PCR-neg groups were 46.9% (95% CI, 13.2% to 75.3%), 92.3% (95% CI, 56.6% to 98.9%), and 85.5% (95% CI, 68.7% to 93.7%), respectively (p=0.0735). The 5-year PFS rates in Path-pos, Path-neg/PCR-pos, and Path-neg/PCR-neg group were 33.3% (95% CI, 10.3% to 58.8%), 76.9% (95% CI, 44.2% to 91.9%), and 71.1% (95% CI, 52.9% to 83.3%), respectively (p=0.0331). There were no significant difference between Path-neg/ PCR-pos group and Path-neg/ PCR-neg group in the 5-year OS and PFS rates (p=0.698). In DLBCL patients, the positivity of pathological examination of bone marrow was identified as a valuable prognostic factor, but the positivity of PCR was not a prognostic factor. In iB-NHL cohort, the 5-year OS rates in Path-pos, Path-neg/PCR-pos, and Path-neg/PCR-neg groups were 77.5% (95% CI, 44.8% to 92.3%), 100% (95% CI, 100% to 100%), and 94.4% (95% CI, 66.6% to 99.2%), respectively (p=0.114). The 5-year PFS rates in Path-pos, Path-neg/PCR-pos, and Path-neg/PCR-neg group were 45.7% (95% CI, 17.3% to 70.5%), 46.8% (95% CI, 19.6% to 70.2%), and 87.1% (95% CI, 57.3% to 96.6%), respectively (p=0.152). There were almost no difference between Path-pos, Path-neg/PCR-pos, and Path-neg/PCR-neg group in the 5-year OS rate, but the 5-year PFS rate in Path-pos and Path-neg/PCR-pos group tend to be lower than in Path-neg/PCR-neg group (p=0.0641 and p=0.0891, respectively). In iB-NHL cohort, in addition to the positivity of pathological examination, the positivity of PCR is possible to be a prognostic factor. Univariate analysis of PFS in pathology negative patients in iB-NHL revealed that performance status was a significant prognostic factor, and IgH clonality also seemed to be a prognostic factor. Multivariate analysis of PFS in pathology negative patients in iB-NHL showed that there was no significant independent risk factor. In the response to the first line therapy in DLBCL cohort, the rate of complete response in Path-pos, Path-neg/PCR-pos, and Path-neg/PCR-neg groups were 83.3%, 90.9%, and 80.0%, respectively (p=0.8871). In iB-NHL cohort, the rate of complete response were 60.0%, 71.4%, and 82.4%, respectively (p=0.4623). In each cohort, there were no difference between each groups in overall response rate and complete response rate. Conclusion Positive IgH clonality with negative pathology in bone marrow is a valuable prognostic factor for PFS at diagnosis in the patients with iB-NHL. Disclosures Kurokawa: Eizai: Research Funding; MSD: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Teijin Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Pfizer: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Astellas Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding.

Autoimmune neutropenia (AIN), resulting from granulocyte-specific autoantibodies, is much less frequent than other autoimmune hematologic disorders including autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). These autoimmune disorders may precede, synchronize, or follow collagen disorders, viral infections, and lymphoid neoplasms. Herein we present the first case of AIN in association with Helicobacter pylori-negative mucosa-associated lymphoid tissue (MALT) lymphoma with nodal dissemination. In our case, AIN, accompanied by ITP, occurred prior to the clinical manifestation of lymphoma. AIN and ITP were well managed afterwards, but they relapsed in accordance with the recurrence of lymphoma. The administration of prednisolone at 0.5 mg/kg daily alleviated the cytopenias within a week. In general, combination chemotherapy is performed for the treatment of lymphoma-associated autoimmune hematologic disorders and indeed seems to be effective. Our case indicates that corticosteroid monotherapy may be effective for lymphoma-associated AIN especially when AIN precedes the onset of lymphoma.

This study was conducted as a retrospective, observational, exploratory cohort study with the aim of elucidating the safety profile of micafungin at doses exceeding 150 mg daily. We identified adult patients with hematological diseases who had received micafungin therapy for ≥ 7 consecutive days. Twenty-six patients administered micafungin at 300 mg daily (high-dose group) were compared with 58 patients administered micafungin at 150 mg daily (standard-dose group). The most frequent adverse events (AEs) were hepatotoxicity, hypertension and diarrhea. AEs were recorded in 42 (72%) and 19 (73%) patients in the standard-dose and high-dose groups, respectively (p = 1.00). Hepatobiliary AEs were noted in 28 (48%) and 15 (58%) patients, respectively (p = 0.48). Serious AEs and resultant treatment discontinuation were infrequent. Our results suggest that micafungin was safe and well tolerated at 300 mg daily.

Actin stress fibers (SFs) play important roles in cellular mechanotransduction and regulation of various cellular functions. SFs generate internal tension and contribute to physical interactions between cells and extracellular matrices. It has recently been suggested that cytoskeletons have the potential to interact with the nuclei via nuclear membrane proteins. However, it remains unclear at this stage whether SFs are involved in a mechanical interaction with the nucleus, and their internal forces are transmitted directly to the nucleus and influence the intranuclear DNA.

Chronic myelomonocytic leukemia (CMML), the most frequent disease entity of myelodysplastic syndrome/myeloproliferative neoplasm is a clonal hematopoietic malignancy that is characterized by persistent monocytosis, morphologic myeloid dysplasia, and progression to acute myeloid leukemia. The pathogenesis of CMML remains entirely elusive because of the lack of suitable mouse models and the difficulties in the establishment of CMML cell lines. We have previously reported that we established induced pluripotent stem cells (iPSC) from CMML CD34 positive leukemic cells (CMML-iPSC) as a new disease model. Co-cultured with C3H10T1/2 stromal cells in the presence of vascular endothelial growth factor, CMML-iPSC generated CD34 CD43 double-positive hematopoietic progenitor cells (CMML-HPC). CMML-HPC have recapitulated important disease features of parental CMML cells in terms of genetic abnormalities, acceleration of cell proliferation, and aberrant surface markers expression. In addition, a novel human CMML xenograft mouse model has been established through secondary transplantation of human HPCs from CMML-iPSC-derived teratomas. This model produced HPCs that mimicked the properties of CMML in vivo. To identify key molecular abnormalities that contribute to the pathophysiology of CMML, we conducted comprehensive gene expression and DNA methylation profiling analyses of normal and CMML parental CD34 positive cells, iPSC, and their hematopoietic progenies, respectively. Correlation analysis revealed that gene expression and DNA methylation status between normal and CMML iPSC-derived HPC exhibited similar pattern (R2 = 0.92 and 0.96, respectively), although normal and CMML parental CD34 positive cells were quite different (R2 = 0.72 and 0.90, respectively), indicating that reprogramming followed by re-differentiation may enable to obtain more homogenous population of normal and CMML cells that reside in almost the same differentiation stage. These results allowed us to determine the difference in the genetic and epigenetic status between normal and CMML iPSC-derived HPC, which remained through reprogramming and re-differentiation, in order to find out causative genes in the pathogenesis of CMML. Using these combined omics platforms, we identified SLIT and NTRK like family member 4 (SLITRK4) as a candidate gene involving in pathogenesis of CMML, whose expression was enhanced and whose promoters were hypo-methylated in CMML-HPC. In other CMML patients' CD34 positive leukemic cells, the expression of SLITRK4 was up-regulated compared to healthy CD34 positive bone marrow cells and other leukemia cells. In addition, we revealed SLITRK4 had pro-proliferative activity as the knockdown of SLITRK4 inhibited proliferation of leukemic cell lines OCI-AML3. To elucidate whether SLITRK4 exert any biological functions in CMML, we established CMML-iPSC clones harboring hetero-knockout (wt/-) or homo-knockout (-/-) of SLITRK4 gene by CRISPR/Cas9 system. Although SLITRK4 (wt/-) and (-/-) clones did not exhibit any morphological and proliferative difference in CMML-iPSC, the production of HPC from CMML-iPSC was dramatically attenuated in SLITRK4-dependent manner. Therefore, while little has been known about the roles of SLITRK molecules in tumorigenesis, we demonstrated SLITRK4 was indispensable for generation of CMML leukemic cells and suggested the possibility of novel molecular therapy targeting SLITRK4, based on the findings obtained from our combined omics platforms. In summary, we identified SLITRK4 as a novel candidate gene responsible for the pathogenesis of CMML through our combined omics platform using patient-derived iPSC. This platform may provide a potential to trace causative genes in a variety of diseases. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria.

Since the emergence of tyrosine kinase inhibitors (TKIs), long-term survival of patients with chronic myelogenous leukemia (CML) has been improved. However, those TKIs have not fully succeeded in curing CML, mainly due to TKI-resistant CML stem cells. CML stem cells are often difficult to analyze because they represent an extremely minor population of CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs) from bone marrow (BM) cells of two patients with CML in chronic phase (CML-CP) and obtained CML pre-hematopoietic progenitor cells (CML-pre-HPCs), immature hematopoietic cells phenotypically defined by CD34+/CD45-/CD43+ cells. In semisolid and liquid cultures, CML-pre-HPCs recapitulated the principal features of CML stem cells, multi-potency and the resistance against imatinib. Gene expression enrichment analysis for CML-pre-HPCs demonstrated that several gene sets, including those related to the maintenance of hematopoietic stem cells were enriched. In addition, we found that a disintegrin and metalloprotease 8 (ADAM8), also known as CD156, was highly enhanced in CML-pre-HPCs and the expression level of ADAM8 was even increased after the treatment of imatinib in vitro. To address the significance of ADAM8 in CMP-CP patient, we evaluated purified ADAM8+ cells by fluorescence-activated cell sorting (FACS) in primary samples. First, FACS analysis found that ADAM8+ cells were enriched more in BM samples of patient with newly diagnosed CML-CP than normal or other types of leukemias among CD34+ fraction. ADAM8+ cells were enriched in CD34+/CD38- fraction compaered to CD34+/38+ fraction in BM of CML-CP patients, indicating that ADMA8+ cells represent immature hematopietic cells. In cell viability assays, ADAM8+/CD38+ CML cells in newly diagnosed CML-CP patient enhibited imatinib-resistance and imatinib-induced apoptosis in vitro was strongly suppressed in ADAM8+ CML cells compared to ADAM8- cells. Even in CD34+/CD38+ fraction, which was previously known as TKI-sensitive fraction, ADAM8+ cells exhibited TKI-resistance in both cell viability and apoptosis assay, indicating that ADAM8 would be a useful marker of TKI-resistant CML cells. Finally, to evaluate the significance of ADAM8 as a marker of TKI-resistant CML cells in vivo, we measured the frequency of CML cells in BM samples of CML-CP patients who had achieved major or complete molecular response (MMR; n = 2 or CMR; n = 1) after the administration of TKIs by limiting dilution analysis. In CML patients with MMR, CML cells remained in ADAM8+ cells at higher frequency in spite of steep decline of CML cells in ADAM8- cells The frequency of CML cells was as high in CD34+/CD38+/ADAM8+ fraction as in CD34+/38- CML stem cell fraction. Even in a patient with CMR, residual CML cells were detected in ADAM8+ population among CD34+/CD38+ fraction, whereas CML cells were undetectable in ADAM8- population. In conclusion, we have established a powerful platform with CML-iPSCs to investigate the pathophysiology of TKI-resistant CML stem cells. Using this platform, we have identified ADAM8 as a novel marker of TKI-resistant CML cells. CD34+/CD38+/ADAM8+ fraction, as well as CD34+/CD38- fraction, was an important population that defines residual CML cells even in CML-CP patients with deep molecular response after the treatment of TKI. ADAM8 would become an attractive candidate of novel therapeutic targets against TKI-resistant CML cells. Disclosures Kataoka: Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.

Composite lymphoma is defined as two or more distinct types of lymphoma in a single anatomical site. Among various combinations, composite B-cell and T-cell non-Hodgkin's lymphomas (CBTL) are very infrequent. Herein we describe a 66-year-old female with CBTL presenting with lymphadenopathy, multiple bone lesions and an epidural tumor. Light microscopic examination of a biopsied cervical node revealed a dual population of lymphoid cells: sheets of large cells admixed with medium-sized cells. The large cells expressed B-cell markers and showed immunoglobulin light chain restriction, consistent with diffuse large B-cell lymphoma (DLBCL). The medium-sized cells were positive for CD20 as well as T-cell markers. Because polymerase chain reaction amplification showed monoclonal rearrangement of the T-cell receptor β chain gene, this population was compatible with peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). We therefore made a diagnosis of composite DLBCL and CD20-positive PTCL-NOS. Complete remission was achieved after six cycles of R-CHOP regimen (rituximab, doxorubicin, vincristine, cyclophosphamide and prednisolone). This is the first report of CD20-positive PTCL-NOS associated with composite lymphoma. Moreover, a literature review of composite DLBCL and PTCL-NOS indicates that this rare clinical entity may be featured by efficacy of systemic chemotherapy in spite of prevalent extranodal lesions.