Goichi Yoshimoto

Self-Renewing Hematopoietic Stem Cell Is the Primary Target in Pathogenesis of Human Chronic Lymphocytic Leukemia

We report here that in chronic lymphocytic leukemia (CLL), the propensity to generate clonal B cells has been acquired already at the hematopoietic stem cell (HSC) stage. HSCs purified from patients with CLL displayed lymphoid-lineage gene priming and produced a high number of polyclonal B cell progenitors. Strikingly, their maturation into B cells was restricted always to mono- or oligo-clones with CLL-like phenotype in xenogeneic recipients. These B cell clones were independent of the original CLL clones because they had their own immunoglobulin VDJ genes. Furthermore, they used preferentially VH genes frequently used in human CLL, presumably reflecting the role of B cell receptor signaling in clonal selection. These data suggest that HSCs can be involved in leukemogenesis even in mature lymphoid tumors.

FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD–specific STAT5 activation

Myeloid cell leukemia-1 (MCL-1) is an essential survival factor for hematopoiesis. In humans, hematopoietic stem cells (HSCs) express MCL-1 at the highest level in response to FMS-like tyrosine kinase-3 (FLT3) signaling. We here show that this FLT3-dependent stem cell maintenance system also plays a critical role in survival of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). The CD34(+)CD38(-) LSC fraction expresses high levels of FLT3 as well as MCL-1, even compared with normal HSCs. Treatment with FLT3 ligand induced further MCL-1 up-regulation in LSCs in all AML cases tested. Interestingly, the group of samples expressing the highest levels of MCL-1 constituted AML with FLT3-internal tandem duplications (ITD). In FLT3-ITD AML cell lines, cells expressed a high level of MCL-1, and an inhibition of MCL-1 induced their apoptotic cell death. A tyrosine kinase inhibitor suppressed MCL-1 expression, and induced apoptosis that was reversed by the enforced MCL-1 expression. Finally, transduction of FLT3-ITD into HSCs strongly activated MCL-1 expression through its signal transducer and activator of transcription 5 (STAT5)-docking domains. This effect was completely abrogated when STAT5 activation was blocked. Thus, the acquisition of FLT3-ITD ensures LSC survival by up-regulating MCL-1 via constitutive STAT5 activation that is independent of wild-type FLT3 signaling.

Human Flt3 Is Expressed at the Hematopoietic Stem Cell and the Granulocyte/Macrophage Progenitor Stages to Maintain Cell Survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.

Identification of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the human common myeloid progenitor

To establish effective therapeutic strategies for eosinophil-related disorders, it is critical to understand the developmental pathway of human eosinophils. In mouse hematopoiesis, eosinophils originate from the eosinophil lineage-committed progenitor (EoP) that has been purified downstream of the granulocyte/macrophage progenitor (GMP). We show that the EoP is also isolatable in human adult bone marrow. The previously defined human common myeloid progenitor (hCMP) population (Manz, M.G., T. Miyamoto, K. Akashi, and I.L. Weissman. 2002. Proc. Natl. Acad. Sci. USA. 99:11872–11877) was composed of the interleukin 5 receptor α chain+ (IL-5Rα+) and IL-5Rα− fractions, and the former was the hEoP. The IL-5Rα+CD34+CD38+IL-3Rα+CD45RA− hEoPs gave rise exclusively to pure eosinophil colonies but never differentiated into basophils or neutrophils. The IL-5Rα− hCMP generated the hEoP together with the hGMP or the human megakaryocyte/erythrocyte progenitor (hMEP), whereas hGMPs or hMEPs never differentiated into eosinophils. Importantly, the number of hEoPs increased up to 20% of the conventional hCMP population in the bone marrow of patients with eosinophilia, suggesting that the hEoP stage is involved in eosinophil differentiation and expansion in vivo. Accordingly, the phenotypic definition of hCMP should be revised to exclude the hEoP; an “IL-5Rα–negative” criterion should be added to define more homogenous hCMP. The newly identified hEoP is a powerful tool in studying pathogenesis of eosinophilia and could be a therapeutic target for a variety of eosinophil-related disorders.

T Cell Leukemia/Lymphoma 1 and Galectin-1 Regulate Survival/Cell Death Pathways in Human Naive and IgM+ Memory B Cells through Altering Balances in Bcl-2 Family Proteins

BCR signaling plays a critical role in purging the self-reactive repertoire, or in rendering it anergic to establish self-tolerance in the periphery. Differences in self-reactivity between human naive and IgM+ memory B cells may reflect distinct mechanisms by which BCR signaling dictates their survival and death. Here we demonstrate that BCR stimulation protected naive B cells from apoptosis with induction of prosurvival Bcl-2 family proteins, Bcl-xL and Mcl-1, whereas it rather accelerated apoptosis of IgM+ memory B cells by inducing proapoptotic BH3-only protein Bim. We found that BCR-mediated PI3K activation induced the expression of Mcl-1, whereas it inhibited Bim expression in B cells. Phosphorylation of Akt, a downstream molecule of PI3K, was more sustained in naive than IgM+ memory B cells. Abundant expression of T cell leukemia/lymphoma 1 (Tcl1), an Akt coactivator, was found in naive B cells, and enforced expression of Tcl1 induced a high level of Mcl-1 expression, resulting in prolonged B cell survival. In contrast, Galectin-1 (Gal-1) was abundantly expressed in IgM+ memory B cells, and inhibited Akt phosphorylation, leading to Bim up-regulation. Enforced expression of Gal-1 induced accelerated apoptosis in B cells. These results suggest that a unique set of molecules, Tcl1 and Gal-1, defines distinct BCR signaling cascades, dictating survival and death of human naive and IgM+ memory B cells.

Engulfment of hematopoietic stem cells caused by down-regulation of CD47 is critical in the pathogenesis of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 20% of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.

Mobilization of Human Lymphoid Progenitors after Treatment with Granulocyte Colony-Stimulating Factor

Hemopoietic stem and progenitor cells ordinarily residing within bone marrow are released into the circulation following G-CSF administration. Such mobilization has a great clinical impact on hemopoietic stem cell transplantation. Underlying mechanisms are incompletely understood, but may involve G-CSF-induced modulation of chemokines, adhesion molecules, and proteolytic enzymes. We studied G-CSF-induced mobilization of CD34+CD10+CD19−Lin− and CD34+CD10+CD19+Lin− cells (early B and pro-B cells, respectively). These mobilized lymphoid populations could differentiate only into B/NK cells or B cells equivalent to their marrow counterparts. Mobilized lymphoid progenitors expressed lymphoid- but not myeloid-related genes including the G-CSF receptor gene, and displayed the same pattern of Ig rearrangement status as their bone marrow counterparts. Decreased expression of VLA-4 and CXCR-4 on mobilized lymphoid progenitors as well as multipotent and myeloid progenitors indicated lineage-independent involvement of these molecules in G-CSF-induced mobilization. The results suggest that by acting through multiple trans-acting signals, G-CSF can mobilize not only myeloid-committed populations but a variety of resident marrow cell populations including lymphoid progenitors.

FLT3 mutations in normal karyotype acute myeloid leukemia in first complete remission treated with autologous peripheral blood stem cell transplantation.

Summary:We retrospectively analysed the significance of FLT3 mutations in patients with acute myeloid leukemia (AML) having a normal karyotype, who were treated with high-dose chemotherapy and autologous peripheral blood stem cell transplantation (auto-PBSCT). In all, 34 patients with normal karyotype AML in first complete remission receiving high-dose chemotherapy and auto-PBSCT were analysed based on the presence or absence of FLT3/ITDs and FLT3/D835. They were 16 males and 18 females and with a median age of 41.5 years. FLT3/ITDs were detected in eight of 34 patients (23.5 %), and FLT3 D835 mutations in two of 34 patients (5.9%). White blood cell count (P=0.0087), serum concentration of lactate dehydrogenase (P=0.005), and percentages of peripheral blood (P=0.0131) and bone marrow (BM) blasts (P=0.0312) were significantly higher in patients showing the FLT3 mutations. Overall survival (OS) and disease-free survival (DFS) were similar between patients with or without FLT3 mutations (5 year DFS, 67.5 vs 68.55%, P=0.819; 5 year OS, 64.81 vs 78.88%, P=0.4457, by the log-rank test). FLT3 mutations demonstrate no further prognostic impact in patients with normal karyotype AML in first CR treated with high-dose chemotherapy and auto-PBSCT. Myeloablative chemotherapy supported by auto-PBSCT may overcome any poor prognostic implications of FLT3 mutations.

Monitoring of minimal residual disease (MRD) is useful to predict prognosis of adult patients with Ph-negative ALL: results of a prospective study (ALL MRD2002 Study)

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) for patients with Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) in first complete remission (CR1) is much more intensive than multi-agent combined chemotherapy, although allogeneic HSCT is associated with increased morbidity and mortality when compared with such chemotherapy. Minimal residual disease (MRD) status has been proven to be a strong prognostic factor for adult patients with Ph-negative ALL.MethodsWe investigated whether MRD status in adult patients with ALL is useful to decide clinical indications for allogeneic HSCT. We prospectively monitored MRD after induction and consolidation therapy in adult patients with Ph-negative ALL.ResultsOf 110 adult ALL patients enrolled between July 2002 and August 2008, 101 were eligible, including 59 Ph-negative patients. MRD status was assessed in 43 patients by the detection of major rearrangements in TCR and Ig and the presence of chimeric mRNA. Thirty-nine patients achieved CR1, and their probabilities of 3-year overall survival and disease-free survival (DFS) were 74% and 56%, respectively. Patients who were MRD-negative after induction therapy (n = 26) had a significantly better 3-year DFS compared with those who were MRD-positive (n = 13; 69% vs. 31%, p = 0.004). All of 3 patients who were MRD-positive following consolidation chemotherapy and did not undergo allogeneic HSCT, relapsed and died within 3 years after CR.ConclusionsThese results indicate that MRD monitoring is useful for determining the clinical indications for allogeneic HSCT in the treatment of ALL in CR1.

Oral valganciclovir as preemptive therapy is effective for cytomegalovirus infection in allogeneic hematopoietic stem cell transplant recipients.

Between March 2007 and January 2008, the safety and efficacy of oral valganciclovir (VGC) preemptive therapy for cytomegalovirus (CMV) infection was evaluated in ten consecutive patients who received allogeneic hematopoietic stem cell transplantation (HSCT). Patients were screened once or twice per week after engraftment using CMV pp65 antigenemia assay. When more than 2 CMV antigen-positive cells per 50,000 leukocytes were detected, preemptive therapy with oral VGC was initiated at a dose of 900 mg twice daily for 3 weeks. Nine patients (90%) completed the 3-week VGC treatment except for one patient who developed febrile neutropenia. There was no other significant toxicity. CMV antigen-positive cells were rapidly decreased in all nine patients and became undetectable by the end of the VGC treatment. None of the patients developed CMV disease. CMV infection relapsed in four of the ten patients (40%) after the VGC treatment. These observations suggest that preemptive therapy with VGC is effective for preventing CMV disease in allogeneic HSCT patients. Further studies with a large number of patients will be necessary to determine the optimal initial- and maintenance-dose of VGC.