Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9

Abstract

Transcriptional deregulation through the production of dominant-acting chimeric transcription factors derived from chromosomal translocations is a common theme in the pathogenesis of acute leukemias; however, the essential target genes for acute leukemogenesis are unknown. We demonstrate here that primary myeloid progenitors immortalized by various MLL oncoproteins exhibit a characteristic Hoxa gene cluster expression profile, which reflects that preferentially expressed in the myeloid clonogenic progenitor fraction of normal bone marrow. Continued maintenance of this MLL-dependent Hoxa gene expression profile is associated with conditional MLL-associated myeloid immortalization. Moreover, Hoxa7 and Hoxa9 were specifically required for efficient in vitro myeloid immortalization by an MLL fusion protein but not other leukemogenic fusion proteins. Finally, in a bone marrow transduction/transplantation model, Hoxa9 is essential for MLL-dependent leukemogenesis in vivo, a primary requirement detected at the earliest stages of disease initiation. Thus, a genetic reliance on Hoxa7 and Hoxa9 in MLL-mediated transformation demonstrates a gain-of-function mechanism for MLL oncoproteins as upstream constitutive activators that promote myeloid transformation via a Hox-dependent mechanism.

Figure 1.

Consistent expression of select Hox genes in murine and human cells transformed by MLL fusion genes. (A) RT-PCR analysis was conducted on RNA isolated from MPMP cell lines immortalized by the MLL fusion genes indicated at the top of the lanes. Primers employed for RT-PCR were specific for various Hox or Meis transcripts indicated to the left of the panels. (B) RT-PCR was performed on human cell lines that express MLL fusions with various partner proteins, which are indicated along with the leukemia subtypes at the top of the gel lanes. Cell lines are as follows: HB1119 (lane 1), RS411 (lane 2), MV4-11 (lane 3), ML21 (lane 4), MonoMac6 (lane 5), and THP1 (lane 6).

Figure 2.

Myeloid transformation requires sustained function of MLL-ENL and correlates with maintenance of Hox gene expression. (A) Myeloid clonogenic activity in normal bone marrow is associated with expression of Hoxa cluster genes. CFUs (per 10,000 plated cells) were determined after 7 d of culture in methylcellulose for whole bone marrow mononuclear cells (BM) or cellular fractions enriched based on the expression (Lin⁺) or absence (Lin^-) of lineage markers. RT-PCR results are shown below for the Hox transcripts indicated to the right of each panel. (B) Conditional MLL-ENL proteins are schematically illustrated and consist of ER fusions at the C terminus of transformation-competent MLL-ENL (MER) or a mutant lacking the critical transformation domain of ENL (MEC). (C) Conditional immortalization of myeloid progenitors by MLL-ENL. Results of serial myeloid replating assays are shown for myeloid progenitors transduced with the constructs indicated at the top of the panels, cultured in the presence (+) or absence (-) of 4-OHT. Results of RT-PCR assays performed on cells harvested at day 7 of each plating are shown below for the transcripts indicated on the right. (D) MLL fusion protein activity is continuously required for the maintenance of transformation. A myeloid precursor cell line (MER3) immortalized by MLL/ENL-ER was plated in methylcellulose culture in the presence (+) or absence (-) of 4-OHT, and CFUs (per 10,000 plated cells) were determined on day 7 (lanes 1,2, respectively). Serial replating was performed as illustrated below, and CFUs again determined on day 7 (lanes 3,4, respectively).

Figure 3.

Hoxa7 and Hoxa9 are independently required for efficient in vitro immortalization by MLL-ENL. (A) Representative results (CFUs/10,000 plated cells) of serial myeloid replating assays are shown for myeloid progenitors transduced with vector alone (V), MLL-ENL (ME), or E2A-HLF (EH) as indicated. Genotypes of BM cells used for transductions are indicated above the panels. (B) Morphologies for colony types observed in methylcellulose assays initiated with cells transduced by MLL-ENL (rows 1 and 2) or E2A-HLF (row 3). Genotypes of transduced cells are indicated above the panels. (C) Coexpression of Hoxa9 in combination with MLL-ENL rescues in vitro immortalization of Hoxa9^-/- BM cells. Results are shown as CFU/10,000 plated cells. Transduced constructs are indicated below, along with indications of ability to establish IL-3-dependent cell lines in liquid media.

Figure 4.

Hoxa9 is essential for the leukemogenic properties of MLL-ENL. (A) Survival curves for cohorts of animals that were transplanted with MLL-ENL transduced progenitors from Hoxa9^+/+ (n = 15) or Hoxa9^-/- (n = 13) BM donors. (B) Coexpression of Hoxa9 in combination with MLL-ENL rescues in vivo leukemogenicity of Hoxa9^-/- BM cells. Survival curves are shown for cohorts of animals that were transplanted with Hoxa9^-/- progenitors transduced with Hoxa9 alone (n = 10), Hoxa9 + MLL-ENL (n = 10), or cell lines (lines 3 and 4) immortalized by coexpressed Hoxa9 and MLL-ENL. (C) Histology for spleens and livers from mice transplanted with transduced BM cells. Transduced genes and BM genotypes are indicated at the top.

Figure 5.

Hoxa9-dependent preleukemia in mice transplanted with MLL-ENL transduced BM. BM cells were analyzed for expression of Gr-1 and Mac-1 at 5 wk posttransplant. Numbers of Gr-1⁺/Mac-1⁺ cells were increased in recipients of wild-type BM transduced with MLL-ENL but not Hoxa9^-/- BM, which showed levels of Gr-1⁺/Mac-1⁺ cells similar to those of control mice. Donor BM genotypes and transduced genes are indicated to the right. FACS data are representative of two animals in each cohort.

Figure 6.

Schematic model depicting Hoxa-dependent myeloid transformation by MLL oncoproteins. Differentiation-inducing signals from the bone marrow microenvironment promote commitment of normal pluripotential HSCs toward terminal myeloid development that is associated with the loss of 5′Hoxa gene expression. MLL oncoproteins disrupt this critical regulatory circuit, enhancing myeloid progenitor self-renewal via maintenance of 5′Hoxa gene expression, blocking differentiation events ultimately progressing to AML.