Noriko Sugino

T memory stem cells are the hierarchical apex of adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a peripheral CD4(+) T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Despite several investigations using human specimens and mice models, the exact origin of ATL cells remains unclear. Here we provide a new insight into the hierarchical architecture of ATL cells. HTLV-1-infected cells and dominant ATL clones are successfully traced back to CD45RA(+) T memory stem (TSCM) cells, which were recently identified as a unique population with stemlike properties, despite the fact that the majority of ATL cells are CD45RA(-)CD45RO(+) conventional memory T cells. TSCM cells from ATL patients are capable of both sustaining themselves in less proliferative mode and differentiating into other memory T-cell populations in the rapidly propagating phase. In a xenograft model, a low number of TSCM cells efficiently repopulate identical ATL clones and replenish downstream CD45RO(+) memory T cells, whereas other populations have no such capacities. Taken together, these findings demonstrate the phenotypic and functional heterogeneity and the hierarchy of ATL cells. TSCM cells are identified as the hierarchical apex capable of reconstituting identical ATL clones. Thus, this is the first report to demonstrate the association of a T-cell malignancy with TSCM cells.

A novel LSD1 inhibitor NCD38 ameliorates MDS-related leukemia with complex karyotype by attenuating leukemia programs via activating super-enhancers

Lysine-specific demethylase 1 (LSD1) regulates gene expression by affecting histone modifications and is a promising target for acute myeloid leukemia (AML) with specific genetic abnormalities. Novel LSD1 inhibitors, NCD25 and NCD38, inhibited growth of MLL-AF9 leukemia as well as erythroleukemia, megakaryoblastic leukemia and myelodysplastic syndromes (MDSs) overt leukemia cells in the concentration range that normal hematopoiesis was spared. NCD25 and NCD38 invoked the myeloid development programs, hindered the MDS and AML oncogenic programs, and commonly upregulated 62 genes in several leukemia cells. NCD38 elevated H3K27ac level on enhancers of these LSD1 signature genes and newly activated ~500 super-enhancers. Upregulated genes with super-enhancer activation in erythroleukemia cells were enriched in leukocyte differentiation. Eleven genes including GFI1 and ERG, but not CEBPA, were identified as the LSD1 signature with super-enhancer activation. Super-enhancers of these genes were activated prior to induction of the transcripts and myeloid differentiation. Depletion of GFI1 attenuated myeloid differentiation by NCD38. Finally, a single administration of NCD38 causes the in vivo eradication of primary MDS-related leukemia cells with a complex karyotype. Together, NCD38 derepresses super-enhancers of hematopoietic regulators that are silenced abnormally by LSD1, attenuates leukemogenic programs and consequently exerts anti-leukemic effect against MDS-related leukemia with adverse outcome.

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34(+) hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation.

A case of minor BCR-ABL1 positive acute lymphoblastic leukemia following essential thrombocythemia and originating from a clone distinct from that harboring the JAK2-V617F mutation

Here we report on a case of Philadelphia chromosome positive B lymphoblastic leukemia (Ph+ALL), which developed following a long duration of essential thrombocythemia (ET). A mutational analysis of Janus Kinase 2 (JAK2) revealed that the V617F mutation was present in granulocytes and in hematopoietic stem and progenitor cells (HSPCs), but not in the CD34+CD19+ population that mostly consists of Ph+ALL cells, indicating that this Ph+ALL clone did not originate from the ET clone carrying the JAK2-V617F mutation. The minor BCR-ABL1 fusion was detected not only in the CD34+CD19+ population but also in HSPCs and granulocytes, indicating that the Philadelphia chromosome was acquired in an early hematopoietic stage at least prior to the commitment to B cell development. Upon dasatinib treatment, the minor BCR-ABL1 transcript rapidly disappeared in HSPCs but persisted in the CD34+CD19+ population. A relapse of Ph+ALL occurred nine months later without the disappearance of the minor BCR-ABL1 transcript in the bone marrow cells during the treatment course, suggesting that a resistant Ph+ALL clone may have arisen or been selected in the committed B cells rather than in HSPCs. This case report may partly contribute to filling the gap between previous data acquired from mice experiments and the phenomenon in real patients.

Methotrexate-associated lymphoplasmacytic lymphoma complicated with type 2 cryoglobulinemia

A 62-year-old female suffering from rheumatoid arthritis(RA) for 16 years was referred to our hospital with out ofbreath on slight exertion. She had been prescribed withmethotrexate (MTX; 4 mg per week) and prednisolone for10 years for the treatment of RA. Upon physical exami-nation, tender liver and spleen that extended below thexiphoid were palpable with a protuberant abdomen. Com-puted tomography (CT) showed hepatosplenomegaly,bilateral pleural effusions and ascites in the absence of lungfield abnormalities or lymphadenopathy. Bone marrowexamination was performed to demonstrate an infiltrationof clonal B cells confirmed by immunocytological analysiswith flow cytometry. She was diagnosed as having MTX-associated lymphoproliferative disorder (LPD). The effu-sions in the body cavities and hepatosplenomegaly weredramatically improved only with the withdrawal of MTX.She had been asymptomatic for 2 years until the currenthospitalization. She presented herself in the outpatientdepartmentwithacomplaintofheadache,vertigoandlossofvisionandthenshewasreadmittedbecauseofextensivefluidretention in the chest, abdomen and lower extremities. A CTscan showed recurrence of the massive effusions in the bodycavities and hepatosplenomegaly (Fig. 1a). Histologicalanalysis for the liver specimen obtained by an ultrasound-guided needle biopsy showed a typical relatively monoto-nousappearanceoflymphocytes,plasmacytoidlymphocytesand plasma cells (Fig. 1b). Proliferating cells were stronglyexpressedwithCD20(Fig. 1c).Immunocytologicalanalysiswith flow cytometry demonstrated that the atypical lym-phocytes were positive for CD19, CD20 surface moleculeswith jlight chain-restricted surface immunoglobulin (Ig)-Mand negative for CD5, CD10 and CD23 surface molecules.Bone marrow examination showed a lymphoplasmacyticinfiltrate composed of lymphocytes with basophilic cyto-plasm and plasmacytoid cells (Fig. 1d). Laboratory studieswere remarkable for a white blood cell (WBC) of 2.8 910

Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells

The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

Hypomethylation of the Treg-specific demethylated region in FOXP3 is a hallmark of the regulatory T-cell subtype in adult T-cell leukemia

A subtype of adult T-cell leukemia cells can be distinguished based on the hypomethylated state of their FOXP3 gene. These cells have Treg properties, and the patients have a poor prognosis. Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1. Because of its immunosuppressive property and resistance to treatment, patients with ATL have poor prognoses. ATL cells possess the regulatory T cell (Treg) phenotype, such as CD4 and CD25, and usually express forkhead box P3 (FOXP3). However, the mechanisms of FOXP3 expression and its association with Treg-like characteristics in ATL remain unclear. Selective demethylation of the Treg-specific demethylated region (TSDR) in the FOXP3 gene leads to stable FOXP3 expression and defines natural Tregs. Here, we focus on the functional and clinical relationship between the epigenetic pattern of the TSDR and ATL. Analysis of DNA methylation in specimens from 26 patients with ATL showed that 15 patients (58%) hypomethylated the TSDR. The FOXP3+ cells were mainly observed in the TSDR-hypomethylated cases. The TSDR-hypomethylated ATL cells exerted more suppressive function than the TSDR-methylated ATL cells. Thus, the epigenetic analysis of the FOXP3 gene identified a distinct subtype with Treg properties in heterogeneous ATL. Furthermore, we observed that the hypomethylation of TSDR was associated with poor outcomes in ATL. These results suggest that the DNA methylation status of the TSDR is an important hallmark to define this heterogeneous disease and to predict ATL patient prognosis. Cancer Immunol Res; 4(2); 136–45. ©2015 AACR.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×108) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.

Pharmacological targeting of bone marrow mesenchymal stromal/stem cells for the treatment of hematological disorders

The therapeutic effects of mesenchymal stromal/stem cells (MSCs) are mainly based on three characteristics: immunomodulation, tissue regeneration, and hematopoietic support. Cell therapy using culture-expanded MSCs is effective in some intractable bone and hemato-immune disorders; however, its efficacy is limited. In this article, we review the previous efforts to improve the clinical outcomes of cell therapy using MSCs for such disorders. We describe pharmacological targeting of endogenous bone marrow-derived MSCs as a crucial quality-based intervention to establish more effective MSC-based therapies.

Intravascular large B‐cell lymphoma cells in the bone marrow smear preparation

Intravascular large B-cell lymphoma (IVLBCL) is a subtype of extranodal large B-cell lymphoma characterized by the growth of lymphoma cells only within the lumina of small vessels in the various organs (Swerdlow et al, 2008). Because of its rarity and its variety of the clinical presentations, the definite diagnosis of IVLBCL often requires long time with repeated applications of biopsy on the various organs. Bone marrow is demonstrated to be one of the affected organs (Masaki et al, 2009) and a diagnostic site in IVLBCL (Murase et al, 2007). However, the detection of lymphoma cells within the lumina of small vessels and/or sinuses in the bone marrow biopsy specimen is not necessarily guaranteed because, at least in part, the quantity of specimen yield by a single painful procedure is not large. We show characteristic morphology of IVLBCL cells in the May-Grünwald Giemsa (MG)-stained bone marrow smear preparation, which could be one of the useful features for the diagnosis of IVLBCL. From February 2006 through April 2010, 11 patients were clinically diagnosed as having intravascular lymphoma in our institute. In the retrospective analysis, five out of the 11 patients fulfilled the following presentations for the definite diagnosis of IVLBCL: (i) the proposed clinical diagnostic criteria for IVLBCL (Masaki et al, 2009), (ii) the characteristic accumulation of 18[F]-fluorodeoxyglucose (FDG) in the positron emission tomography (PET)/computed tomography (CT) (Miura & Tsudo, 2010) and (iii) the existence of atypical lymphoid cells within the lumina of small vessels in the biopsy specimens (Asada et al, 2007). In the examination of MG-stained bone marrow smear preparation, four out of the five patients presented with an involvement of IVLBCL cells (Table I). Among the four patients, the IVLBCL cells demonstrated similar unique morphological features in the three patients (Fig 1, Case #2, #3 and #5). The IVLBCL cells are large-sized cells with basophilic cytoplasm, vacuoles in the cytoplasm, not fine chromatin in the nuclei. Cell aggregates were found in two out of the three patients (Fig 1, Case #2 and #5). Bone marrow is one of the initial sites of histological examination for the diagnosis of lymphoma. Biopsy specimen of bone marrow is reported to be useful for the diagnosis of IVLBCL (Masaki et al, 2009). In our retrospective analysis, however, the existence of lymphoma cells within the lumina of small vessels and/or sinuses in the hematoxyline-eosin (HE)-stained bone marrow biopsy specimen was confirmed in none of the patients with definite diagnosis of IVLBCL (Table I). Regarding the discrepancy between the previous reports and our results, we would speculate that there are methodological differences in detection of lymphoma cells such as the quantity of biopsy specimen yield, the number of biopsy specimen sections reviewed and the use of immunohistochemical analysis. On the other hand, the preparation of MG-stained smear of bone marrow aspirate is a relatively uniform procedure. In our analysis, the involvement of IVLBCL cells in the bone marrow smear was observed in four out of the five patients in which multiple accumulations of FDG in the bones were confirmed in the PET/CT examination (Table I). Three out of the four patients presented with IVLBCL cells with a similar unique characteristic morphology in the MG-stained bone marrow smear preparation (Fig 1). We would like to suggest that lymphoma cells shown here are one of the patterns of IVLBCL cells and that further investigations including 18[F]-FDG-PET/CT and/or random skin biopsy should be applied actively to make an early diagnosis of IVLBCL for such patients.