Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3⁻ AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.
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.
Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. SIGNIFICANCE:The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL. Cancer Discov; 4(9); 1088-1101
Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and β-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias.
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.
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.
Key Points• NOD-specific Sirpa polymorphism is the genetic determinant of highly efficient xenograft activity in NOD-based immunodeficient mouse models.Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2 nullIl2rgnull mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1 nullIl2rgnull strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology. (Blood. 2013;121(8):1316-1325) IntroductionImmunodeficient mice are widely used to reconstitute human hematopoiesis by xenotransplantation of hematopoietic stem cells (HSCs). 1,2 This "humanized" mouse model provides a powerful tool with which to evaluate the biologic properties of human HSCs and progenitors in vivo. 3,4 Such xenotransplantation systems have also been used to study human cancer stem cells. [5][6][7][8] Elimination of the lymphoid system is the first step to achieving reconstitution of human hematopoiesis. To deplete T and B cells, the scid mutation in the Prkdc gene [9][10][11] or disruption of the recombination activating gene 1 or 2 (Rag1 and Rag2) 12,13 has been introduced into various mouse strains. In addition, to deplete natural killer (NK) cells or their functions, the IL-2 receptor common ␥ chain subunit (Il2rg) [14][15][16] or beta-2-microglobulin (B2m) [17][18][19] is disrupted.However, depletion of lymphoid cells is not sufficient and it has been shown empirically that additional strain-specific factors modulate human hematopoietic engraftment in the xenotransplantation setting. For example, within the SCID strain, the SCID with the NOD background was the gold standard for the xenotransplantation assay based on its high efficiency. 11 In fact, recent studies have shown that among the lymphoid-depleted mouse strains, the NOD-scid Il2rg null (NSG/NOG) 14,15 and NOD.Rag1 null Il2rg null (NOD-RG) 20 strains are the most efficient; the BALB/c.Rag2 null Il2rg null (BALB-RG) strain is the next efficient 21,22 ; and the C57BL/6 strains with scid, 23 Rag2 null , Rag2 null B2m null , Rag2 null Prf null , 24 or Rag2 null Jak3 null25 mutations are unable to reconstitute human hematopoiesis. The NOD strain has multiple immune deficiencies, ...
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