ALBERT

Description: MOZ-TIF2 is expressed as a consequence of the chromosomal inversion inv(8)(p11q13) and is associated with AML FAB subtypes M4 and M5. Mice transplanted with MOZ-TIF2 succumb to monoclonal or oligoclonal leukemias with a long median disease latency, indicating that cooperating mutations are required for MOZ-TIF2 mediated leukemogenesis. The presence of a FLT3-ITD mutation in a patient with inv(8)(p11q13) has previously been reported, suggesting FLT3-ITD as a candidate cooperating mutation. Here we report that FLT3-ITD functionally cooperates with MOZ-TIF2 in co-transduction experiments in both a serial replating and murine bone marrow transplantation assay to induce AML that is transplantable to secondary recipients. At limit dilution, both MOZ-TIF2 and FLT3-ITD retroviruses are present, demonstrating cooperative effect. Moreover, tyrosine to phenylalanine mutations of FLT3-ITD residues 589 and 591, which we have previously reported to abrogate Stat5 signaling, also abolish the ability of FLT3-ITD to cooperate with MOZ-TIF2 both in vivo and in vitro. Furthermore, a constitutively active Stat5 mutant supports factor independent serial replating activity of MOZ-TIF2 in vitro. These data suggest a multi-step transformation model in which constitutive downstream Stat5 signaling by FLT3-ITD cooperates with MOZ-TIF2 in AML induction, and indicate that FLT3-ITD potentiates the properties of self-renewal in hematopoietic progenitors through activation of STAT5.

Description: It has been determined that within the heterogeneous leukemic population, there is a rare subset of leukemia stem cells that are responsible for continued maintenance and propagation of the tumor. Recent evidence suggests that during disease pathogenesis, more mature progenitor cells are capable of acquiring stem cell-like characteristics including properties of self-renewal and the capacity to differentiate into progeny that lack self-renewing potential. Acute promyelocytic leukemia (APL) is associated with the t(15;17)(q22;q12) chromosomal translocation that produces the PML-RARa fusion protein, and is unusual among the various subtypes of AML in that it is characterized by hyperproliferation of differentiated promyelocyte progenitors. In a mouse model in which PML-RARa is expressed under the control of the endogenous murine cathepsin G promoter, we observed that whole bone marrow expressing PML-RARa derived from pre-leukemic mice has certain properties of self-renewal, such as serial replating potential in methylcellulose cultures, and serial transplantation of disease into recipient animals. Using multiparameter flow cytometry, we demonstrated that preleukemic PML-RARa animals have an expansion of mature Gr-1+c-kit-CD34− granulocytes that are not capable of self-renewal. However, in the leukemic state, the expanded population shifted to a more immature population with an immunophenotype of Gr-1+c-kit+CD34+. This population was capable of transplanting disease to secondary recipients that recapitulated the disease of the primary donor, and thus contained leukemia-initiating activity. Morphologically, these cells resembled promyelocytes, and their promyelocytic identity was confirmed by quantitative PCR detection of the expression of azurophilic primary granule components associated with the promyelocyte stage of development, including myeloperoxidase, neutrophil elastase, and cathepsin G. However, these cells lacked expression of the secondary granule protein gelatinase B associated with the more mature myelocyte stage of granulocytic differentiation. Additionally, these leukemia-initiating promyelocytes responded to differentiation and cell death signals mediated by administration of all-trans retinoic acid (ATRA). These data indicate that in APL, a highly differentiated promyelocyte compartment does in fact possess properties of leukemia stem cells. This suggests that an important step in disease pathogenesis mediated by PML-RARa is the acquisition of properties of self-renewal in a terminally differentiating progenitor that normally lacks this potential.

Description: The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase– polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor βR (PDGFβR) fusion transcript that incorporated 5′ sequences from H4 fused in frame to 3′PDGFβR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13+ and CD14+ cells but was not observed in CD3+ or CD19+ cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFβR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFβR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFβR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFβR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFβR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFβR, for efficient induction of factor-independent growth of Ba/F3 cells.

Description: The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.