Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.     

The homeodomain transcription factor Prep1 (pKnox1) is required for hematopoietic stem and progenitor cell activity

Most of the hypomorphic Prep1^i/i embryos (expressing 3-10% of the Prep1 protein), die between E17.5 and P0, with profound anemia, eye malformations and angiogenic anomalies [Ferretti, E., Villaescusa, J.C., Di Rosa, P., Fernandez-Diaz, L.-C., Longobardi, E., Mazzieri, R., Miccio, A., Micali, N., Selleri, L., Ferrari G., Blasi, F. (2006). Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype. Mol. Cell. Biol. 26, 5650-5662]. We now report on the hematopoietic phenotype of these embryos. Prep1^i/i fetal livers (FL) are hypoplastic, produce less common myeloid progenitors colonies (CFU-GEMM) in cytokine-supplemented methylcellulose and have an increased number of B-cells precursors that differentiate poorly. Prep1^i/i FL is able to protect lethally irradiated mice only at high cell doses but the few protected mice show major anomalies in all hematopoietic lineages in both bone marrow (BM) and peripheral organs. Prep1^i/i FL cells compete inefficiently with wild type bone marrow in competitive repopulation experiments, suggesting that the major defect lies in long-term repopulating hematopoietic stem cells (LTR-HSC). Indeed, wt embryonic expression of Prep1 in the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), cKit⁺Sca1⁺Lin⁻AA4.1⁺ (KSLA) cells and B-lymphocytes precursors agrees with the observed phenotype. We therefore conclude that Prep1 is required for a correct and complete hematopoiesis.     

Mapping of MN1 Sequences Necessary for Myeloid Transformation

The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.     

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients'' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34⁺ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1^−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1^−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.     

Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress

PREP1 (PKNOX1) maps in the Down syndrome (DS) critical region of chromosome 21, is overexpressed in some DS tissues and might be involved in the DS phenotype. By using fibroblasts from DS patients and by overexpressing Prep1 in F9 teratocarcinoma and Prep1^i/i MEF to single out the role of the protein, we report that excess Prep1 increases the sensitivity of cells to genotoxic stress and the extent of the apoptosis directly correlates with the level of Prep1. The apoptotic response of Prep1-overexpressing cells is mediated by the pro-apoptotic p53 protein that we show is a direct target of Prep1, as its depletion reverts the apoptotic phenotype. The induction of p53 overcomes the anti-apoptotic role of Bcl-X_L, previously shown to be also a Prep1 target, the levels of which are increased in Prep1-overexpressing cells as well. Our results provide a rationale for the involvement of PREP1 in the apoptotic phenotype of DS tissues and indicate that differences in Prep1 level can have drastic effects.     

HOXA9 Modulates Its Oncogenic Partner Meis1 To Influence Normal Hematopoiesis

While investigating the mechanism of action of the HOXA9 protein, we serendipitously identified Meis1 as a HOXA9 regulatory target. Since HOXA9 and MEIS1 play key developmental roles, are cooperating DNA binding proteins and leukemic oncoproteins, and are important for normal hematopoiesis, the regulation of Meis1 by its partner protein is of interest. Loss of Hoxa9 caused downregulation of the Meis1 mRNA and protein, while forced HOXA9 expression upregulated Meis1. Hoxa9 and Meis1 expression was correlated in hematopoietic progenitors and acute leukemias. Meis1^+/− Hoxa9^−/− deficient mice, generated to test HOXA9 regulation of endogenous Meis1, were small and had reduced bone marrow Meis1 mRNA and significant defects in fluorescence-activated cell sorting-enumerated monocytes, mature and pre/pro-B cells, and functional B-cell progenitors. These data indicate that HOXA9 modulates Meis1 during normal murine hematopoiesis. Chromatin immunoprecipitation analysis did not reveal direct binding of HOXA9 to Meis1 promoter/enhancer regions. However, Creb1 and Pknox1, whose protein products have previously been reported to induce Meis1, were shown to be direct targets of HOXA9. Loss of Hoxa9 resulted in a decrease in Creb1 and Pknox1 mRNA, and forced expression of CREB1 in Hoxa9^−/− bone marrow cells increased Meis1 mRNA almost as well as HOXA9, suggesting that CREB1 may mediate HOXA9 modulation of Meis1 expression.While the Hox homeobox genes are widely recognized as important developmental genes (26), we and others have shown that several Hox genes, and Hoxa9 in particular, are important for both normal hematopoiesis (27, 28) and leukemic transformation (25, 29). While the Hoxa9 gene plays a role in embryonic development, much of the research on this gene has focused on its role as an oncogene that is often upregulated in acute myeloid leukemias (12, 29). In an analysis of 6,817 genes, Hoxa9 was the most highly positively correlated with treatment failure in acute myeloid leukemia patients (18). Meis1 is a member of the TALE family of non-Hox homeobox genes, which was initially identified as a frequent viral integration site in myeloid leukemias arising in BXH2 mice (32). The Hoxa9 gene is also upregulated in many of the leukemias arising in the BXH2 animals (33). Forced expression of HOXA9 in murine bone marrow (BM) cells in culture results in immortalization of myeloid progenitor cells (4, 15), while transplantation of HOXA9-infected BM cells results in the eventual induction of acute myelogenous leukemia (25). In contrast, transplantation of BM cells infected with HOXA9 plus MEIS1 results in rapid development of disease (25). Both HOXA9 and MEIS1 are expressed following forced expression of the MLL oncogene (47) or in patients with MLL gene rearrangements (22).Hoxa9 is expressed in numerous tissues during development, including rib (8), limb (17), motor neuron progenitors (10), reproductive tract (9), and mammary gland (7). Hoxa9 is also expressed in normal adult BM (24, 43), and loss of Hoxa9 leads to multiple relatively mild defects in normal hematopoiesis (23, 27, 28). Retroviral expression studies have also shown that HOXA9 and MEIS1 are important for myeloid blood cell differentiation (3, 4). Despite the broad expression of Hoxa9 and other Hox genes, relatively little is known about how the HOX proteins function. An important advance was the discovery that many HOX proteins gain DNA binding specificity by forming complexes with the PBX (6, 31), MEIS1 (41), and PREP1 (2) proteins. Although HOXA9 is capable of binding DNA alone (42), it forms cooperative DNA binding complexes with MEIS1 alone (41) and in a triple complex with PBX proteins (40, 44). Despite these apparent advances, relatively few downstream targets for HOX proteins, and HOXA9 in particular (11), have been confirmed.During ongoing studies of the mechanism of action of the HOXA9 protein, we discovered that HOXA9 appeared to upregulate the Meis1 mRNA and protein. Given the numerous biological connections between HOXA9 and MEIS1, we embarked on studies to explore this pathway. Forced expression of HOXA9 in BM cells upregulated the Meis1 mRNA and protein, while loss of Hoxa9 resulted in a reduction in the Meis1 mRNA and protein. In addition, in a biological model to assess Hoxa9 modulation of Meis1, compound mutant animals that were homozygous null at the Hoxa9 locus and heterozygous at the Meis1 locus showed a significant loss of murine BM monocytes, mature B cells, and pre/pro-B-cell progenitors and an increase in orthochromatophilic erythroblasts in postnatal-day-15 mice compared to results for all controls, suggesting that HOXA9 regulates Meis1 during normal hematopoiesis. Chromatin immunoprecipitation (ChIP) analysis did not show direct binding of HOXA9 to distal or proximal Meis1 genomic regions. However, these studies, together with PCR analysis, showed that HOXA9 binds to and upregulates two genes, Creb1 and Pknox1 (the protein product is subsequently referred to as PREP1), whose protein products have previously been reported to upregulate Meis1 expression (13, 14). Addition of CREB1 to Hoxa9^−/− bone marrow cells increased Meis1 mRNA nearly as effectively as HOXA9. Taken together, our data show that HOXA9 indirectly modulates its DNA binding and oncogenic partner MEIS1 and that the DNA-binding property of HOXA9 is required for this process. We further show that Hoxa9 modulation of Mes1 is biologically important during normal hematopoiesis and that CREB1 may mediate the regulation of Meis1 by HOXA9.     

Ibrutinib is not an effective drug in primografts of TCF3-PBX1

AimThe Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib (PCI-32765) is effective in patients with multiple myeloma, non-Hodgkin lymphoma and chronic lymphoblastic leukemia. We previously showed that primary cells of children with TCF3-PBX1 positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) express BTK and are sensitive to ibrutinib in vitro. However, preclinical studies in mice are lacking that justify clinical implementation.MethodsImmunocompromised NSG mice were engrafted with a luciferase-positive TCF3-PBX1 leukemic cell line or primary leukemic cells and treated with ibrutinib or placebo. Additionally, primary cells were exposed in vitro to 4 main induction drugs as monotherapy and in combination with ibrutinib.ResultsTreatment with ibrutinib of mice engrafted with a TCF3-PBX1 cell line, TCF3-PBX1 positive or TCF3-PBX1 negative primary leukemic cells did not result in prolonged life span compared to placebo treated mice. In vitro sensitivity to ibrutinib was unaltered in leukemic cells obtained from engrafted mice compared to the original material. However, ibrutinib treatment did not affect leukemic cell viability and tumor outgrowth, nor could lymphocytosis be detected. Ibrutinib was biologically active, since hCD19⁺ cells harvested from ibrutinib treated mice had no detectable levels of phospho-BTK at tyrosine 223 (pBTK Y223), whereas pBTK Y223 was still detectable in placebo treated cases. In combination tests, we noticed an antagonistic effect of ibrutinib on vincristine sensitivity, which was not observed for prednisolone, L-asparaginase and daunorubicin.ConclusionsWe conclude that ibrutinib is not the precision medicine of choice for TCF3-PBX1 positive BCP-ALL.     

Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b.

Hoxa9, Meis1 and Pbx1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either Pbx1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor-dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b failed to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells.     

Characteristics of preleukemia cells induced in mice.

A high proportion of irradiated C57BL/6 mice inoculated with the radiation leukemia virus D-RadLV develop overt T-cell leukemias originating in the thymus. In unirradiated hosts the incidence is much lower. As early as 10 days after injection of D-RadLV the bone marrow contains “preleukemia” cells which, although not frankly leukemic, will develop into leukemia cells if transferred into a specially pretreated recipient mouse. In the present report, certain properties of D-RadLV-induced leukemia and preleukemia cells are compared. In this model system, leukemia cells express the T-cell surface component Thy-1 (Thy-1⁺) whereas preleukemia cells do not (Thy-1^?). But preleukemia cells could be induced in vitro by thymopoietin or ubiquitin to become Thy-1⁺, suggesting that they are prothymocytes. Unlike leukemia cells, preleukemia cells injected into normal recipients immunized them against transplants of leukemias induced by the same D-RadLV virus. Evidently D-RadLV virus induces a critical change in prothymocytes which in a later (Thy-1⁺) phase of differentiation is manifest in overt leukemia transformation.