Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression

Abstract

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

FIG. 1

Summary of the phenotypes of wild-type Hoxa9, PIM mutant panel, and DNA-binding mutant. Hoxa9 PIM consists of the amino acid sequence ANWL. Amino acid sequences of PIM mutants are indicated. Tag Hoxa9 is an N-terminal EE-tagged Hoxa9 with amino acid sequence EEYMPEA. Hoxa9-N51S is a DNA-binding mutant. DNA binding studies were performed with EE-tagged versions of wild-type Hoxa9, PIM, and N51S mutants due to the greater abundance of protein production in coupled transcription-translation; + or − refers to positive or negative scoring in an assay. For assay details and quantitative comparison of wild-type versus mutant performance, refer to the text and the following figures. n/d, not determined; n/a, not applicable.

FIG. 2

Hoxa9-immortalized cells differentiate morphologically into neutrophils or macrophages in response to G-CSF, M-CSF, or ATRA. Myeloid progenitors immortalized by Hoxa9 (clones HF1 and HF2) were cultured in GM-CSF (A and E), G-CSF for 72 h (B and F), M-CSF for 120 h (C and G), or GM-CSF plus ATRA for 5 days (D and H) and stained with Wright-Giemsa stain. Cells immortalized by wild-type Hoxa9 (clone HF1) were cultured in GM-CSF (I) or in G-CSF for 72 h. (J) and stained for NADPH oxidase activity. Cells immortalized by PIM mutant Hoxa9-Atet were cultured in GM-CSF (K) or in G-CSF for 48 h (L) and stained for NADPH oxidase activity. Myeloid progenitors immortalized by PIM mutant Hoxa9-Atet were cultured in GM-CSF (M), G-CSF for 72 h (N), or M-CSF for 120 h (O) and stained with Wright-Giemsa stain.

FIG. 3

Progenitors having biphenotypic differentiation potential show strong expression of Hoxa9 and are clonal. (A) Identification of wild-type and mutant Hoxa9 proteins in immortalized myeloblasts from primary marrow. The abundance of Hoxa9 in total cell lysate was quantitated by immunoblot analysis using an antiserum raised against the HD C terminus of murine Hoxa9. The mobility of recombinant Hoxa9 equaled that of Hoxa9 expressed in myeloblast cell lines and is designated at the left as HoxA9. The higher mobility of the N-terminal EE-tagged Hoxa9 is designated at left as N-tag HoxA9. Lanes: 1, recombinant Hoxa9 produced by coupled transcription-translation; 2, M1AML cells containing coactivation of Hoxa9 and Meis1; 3 to 17, myeloid progenitors immortalized by Hoxb8 (lane 3), E2a-Pbx1 (lane 4); Hoxa9 (lanes 5 to 7), EE-tagged Hoxa9 (lanes 8 to 10), Hoxa9-WF (lanes 11 to 15), Hoxa9-Gtet (lane 16), and Hoxa9-Atet (lane 17). (B) Hoxa9-immortalized progenitors are clonal and thus exhibit biphenotypic potential. Shown is Southern blot analysis of DNA derived from Hoxa9-immortalized populations HF1 (lanes 1 and 5), HF1.1 (lanes 2 and 6), HF1.2 (lanes 3 and 7), and HF2 (lanes 4 and 8). DNA was cleaved with NheI and probed with Hoxa9 cDNA (lanes 1 to 4) or cleaved with BamHI and probed with neo sequences (lanes 5 to 8). The location of the genomic and proviral fragments containing Hoxa9 are indicated at the left. A background band present in all samples probed with neo sequences (lanes 5 to 8) is indicated at left as NS. The relative ratio of genomic to proviral Hoxa9 signal in lanes 1 to 3 of panel A was 1:1, confirming the presence of two proviral integrations, and was 2:1 in the sample of DNA in lane 4, confirming a single integration.

FIG. 4

M-CSF induces expression of the macrophage scavenger receptor in Hoxa9-immortalized promyelocytes. Shown is Western blot detection of the macrophage-specific scavenger receptor in promyelocytes immortalized by Hoxa9 (clone HF1 [lanes 2 to 4], subclone HF1.1 [lanes 5 to 7], and subclone HF1.2 [lanes 8 to 10]), Hoxa9-WF (clone 1; lanes 11 to 13), Hoxa9-Atet (lanes 14 to 16), and Hoxa9-Gtet (lanes 17 to 19), each grown in GM-CSF (lanes 2, 5, 8, 11, 14, and 17), G-CSF for 72 h lanes 3, 6, 9, 12, 15, and 18), or M-CSF for 120 h (lanes 4, 7, 10, 13, 16, and 19). Lane 1, mouse peritoneal macrophages (as a positive control).

FIG. 5

Hoxa9-immortalized progenitors exhibit a promyelocytic gene expression profile and upregulate expression of terminal differentiation genes in response to G-CSF and M-CSF. (A) Northern blot analysis of genes expressed in promyelocytes immortalized by wild-type Hoxa9 (clone HF1; lanes 5, 7, and 9) or by Hoxa9-WF (clone 1; lanes 6, 8, and 10) compared with gene expression in GM-CSF-responsive progenitors undergoing terminal differentiation to neutrophils and macrophages in the presence of GM-CSF (lanes 1 to 3) and with those expressed in NIH 3T3 fibroblasts (lane 4). By comparison to lane 1, lanes 2 and 3 contain RNA from the same cells cultured for an additional 24 and 48 h, respectively, in medium plus GM-CSF. The identity of each probe is designated at the left, and the origin of the RNA samples is identified above each lane. (B) Transcriptional responses to G-CSF is the same in cells immortalized by wild-type Hoxa9, Hoxa9-Atet, or Hoxa9-Gtet. The identity of the RNA sample is indicated above each lane, and the identity of the transcript is given to the left of each panel. rec, receptor.

FIG. 6

Mutations within the PIM disrupt cooperative DNA binding with Pbx proteins but not with Meis1. (A) Gel shift analysis of cooperativity between Pbx and Meis proteins and both wild-type EE-tagged Hoxa9 (see Materials and Methods) and EE-tagged mutants of Hoxa9 within the PIM or HD, using the DNA element TGATTTAT. N-terminal EE-tagged versions of Hoxa9 and Hoxa9 mutants were used only for gel shifts assaying proteins derived via in vitro coupled transcription-translation because they produced significantly larger quantities of protein than the untagged versions. Additions to each binding reaction are indicated above the lanes. Monomeric Hoxa9 proteins bound to DNA are denoted Hoxa9 proteins; the background band is designated Bkg; heterodimers of Hoxa9 with Pbx1 or VP16-Pbx1 comigrated and are collectively denoted Pbx:HoxA9; heterodimers of E2a-Pbx1 plus Hoxa9 are designated E2a-Pbx1:Hoxa9; Hoxa9-Meis1c heterodimers are designated Meis1c:HoxA9 at the right. (B) Hoxa9-WF derived from nuclear extracts of immortalized myeloid progenitors also fails to heterodimerize with endogenous Pbx proteins. Nuclear extract from myeloid progenitors immortalized by Hoxa9 (lanes 1 to 3), Hoxa9-WF (lanes 4 to 6), or Hoxa9 treated with G-CSF for 3 days (lanes 7 to 9) was subjected to gel shift analysis using the probe TGATTTAT. Monomeric binding by Hoxa9 is indicated at the left, showing three discrete monomeric Hoxa9 bands. Western blot analysis confirmed that the Hoxa9 proteins in nuclear extracts were degraded into three smaller discrete species (data not shown).

FIG. 7

PIM mutants of Hoxa9 exhibit the same ability as wild-type Hoxa9 to reestablish differentiation arrest CM3neo cells but differ from wild-type Hoxa9 in reestablishing differentiation arrest in CM1puro cells. CM3neo (A) or CM1puro (C) cells were infected with retrovirus expressing wild-type Hoxa9 or mutants of Hoxa9 in the PIM or HD. At 48 h after infection of CM3neo cells, estrogen was removed, and the number of live nonadherent cells in the cultures was quantitated over 10 days. Populations whose differentiation arrest was reestablished by Hoxa9 proteins were analyzed for the level of Hoxa9 protein expression by immunoblotting using anti-Hoxa9 sera (B). At 48 h after infection of CM1puro cells, cells were selected for retroviral gene expression by growth in G418, and the abundance of Hoxa9 proteins was quantitated using anti-Hoxa9 immunoblotting before estrogen was removed (D). Once estrogen was removed, the abundance of live nonadherent cells was quantitated over 26 days. The identity of the Hoxa9 protein encoded by retrovirus used to infect each population is designated adjacent to the growth profiles in both panels A and C and above each lane in immunoblots B and D.