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. 2001 Nov 6;98(23):13120-5.
doi: 10.1073/pnas.231115398. Epub 2001 Oct 30.

Meis1a Suppresses Differentiation by G-CSF and Promotes Proliferation by SCF: Potential Mechanisms of Cooperativity With Hoxa9 in Myeloid Leukemia

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Free PMC article

Meis1a Suppresses Differentiation by G-CSF and Promotes Proliferation by SCF: Potential Mechanisms of Cooperativity With Hoxa9 in Myeloid Leukemia

K R Calvo et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Hoxa9 and Meis1a are homeodomain transcription factors that heterodimerize on DNA and are down-regulated during normal myeloid differentiation. Hoxa9 and Meis1a cooperate to induce acute myeloid leukemia (AML) in mice, and are coexpressed in human AML. Despite their cooperativity in leukemogenesis, we demonstrated previously that retroviral expression of Hoxa9 alone--in the absence of coexpressed retroviral Meis1 or of expression of endogenous Meis genes--blocks neutrophil and macrophage differentiation of primary myeloid progenitors cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of Meis1 alone did not immortalize any factor-dependent marrow progenitor. Because HoxA9-immortalized progenitors still execute granulocytic differentiation in response to granulocyte CSF (G-CSF) and monocyte differentiation in response to macrophage CSF (M-CSF), we tested the possibility that Meis1a cooperates with Hoxa9 by blocking viable differentiation pathways unaffected by Hoxa9 alone. Here we report that Meis1a suppresses G-CSF-induced granulocytic differentiation of Hoxa9-immortalized progenitors, permitting indefinite self-renewal in G-CSF. Meis1a also reprograms Hoxa9-immortalized progenitors to proliferate, rather than die, in response to stem cell factor (SCF) alone. We propose that Meis1a and Hoxa9 are part of a molecular switch that regulates progenitor abundance by suppressing differentiation and maintaining self-renewal in response to different subsets of cytokines during myelopoiesis. The independent differentiation pathways targeted by Hoxa9 and Meis1a prompt a "cooperative differentiation arrest" hypothesis for a subset of leukemia, in which cooperating transcription factor oncoproteins block complementary subsets of differentiation pathways, establishing a more complete differentiation block in vivo.

Figures

Figure 1
Figure 1
Meis1a suppresses G-CSF-induced differentiation of Hoxa9-immortalized cells, increases Pbx2 abundance 3-fold, and does not alter the subcellular location of either Hoxa9 or Pbx2. Wright–Giemsa stains of Hoxa9HF1 and Hoxa9HF2 cells proliferating in GM-CSF (A and D) or 3 days after replacing GM-CSF with G-CSF (B and E). Hoxa9HF1 and Hoxa9HF2 cells expressing Meis1a 10 days after replacing GM-CSF with G-CSF (C and F, respectively). (G and H) Meis proteins permit immortalized proliferation of Hoxa9HF1 cells (G) and EE-tag-Hoxa9 cells (H) when shifted from GM-CSF to G-CSF. In G, tracing designations are uninfected cells (□), and cells infected with Meis1a virus (■), FLAG-Meis1a virus (♦), or Meis3 virus (⋄). In H, tracing designations are uninfected cells (□), and cells infected with control MSCV (♦) or Meis1a virus (■). (I) Western blot analysis demonstrating that expression of Meis1a increases Pbx2 abundance but does not alter the subcellular location of either Hoxa9 or Pbx2. Arrowheads indicate the positional of proteolytic fragments of Hoxa9 in lanes 12–14, and indicate a posttranslationally modified form of Pbx2 in lanes 16–24. The antiserum used and both the cell type and cell fraction examined are indicated across the top. (J) Northern blot analysis of total RNA from Hoxa9HF1 cells that do or do not coexpress Meis1a, and are proliferating in GM-CSF or shifted into medium with G-CSF for 11 days (indicated by + signs above lanes). The identity of each gene analyzed for expression is indicated at left.
Figure 2
Figure 2
Meis1a does not alter Hoxa9 nuclear localization in Hoxa9-immortalized myeloid progenitors. Localization of Hoxa9 in Hoxa9HF1 cells (AC) or Hoxa9HF1 cells expressing Meis1a (D–F), by using deconvolution microscopy and anti-Hoxa9 sera (green) (A, C, D, and F) and Hoescht 33258 (blue) as a nuclear stain (B, C, E, and F). (C and F) Merged images of anti-Hoxa9 plus Hoescht dye staining. (Inset) Hoxa9-immortalized cells stained with control rabbit Ig and Hoescht dye, demonstrating that cytosolic speckles arise from nonspecific staining.
Figure 3
Figure 3
Coexpression of Meis1a with Hoxa9 promotes self-renewal in response to G-CSF, self-renewal in response to SCF, and synergistic self-renewal in response to G-CSF plus SCF. Myeloid progenitors immortalized by Hoxa9 exhibit a progenitor morphology when cultured in GM-CSF (A), exhibit granulocytic differentiation when cultured in G-CSF (B), and exhibit granulocytic differentiation when cultured in G-CSF plus SCF (C) for 4 days. Myeloid progenitors immortalized by coexpression of Hoxa9 and FLAG-Meis1a exhibit a progenitor morphology when cultured in GM-CSF (D), significantly reduced granulocytic differentiation when cultured in G-CSF (E), and no granulocytic differentiation when cultured in G-CSF plus SCF (F) for 4 days. (G) Immunoblot analysis of Hoxa9 and Pbx2 in GM-CSF-dependent cell lines immortalized by Hoxa9 retrovirus (lanes 1–5) or by a bicistronic vector coexpressing Hoxa9 plus Meis1a (lanes 6–10). (H) Anti-Meis1 immunoblot of nuclear extracts derived from cultures 7–10 from G (lanes 3–6), from uninfected Hoxa9HF1 cells (lane 1), and from Hoxa9HF1 cells converted to G-CSF-dependent proliferation by expression of Meis1a (lane 2). (I and J) Abundance of five independent populations of myeloid progenitors immortalized from primary marrow by infection with Hoxa9 plus Neo retrovirus (first set of 5) or by Hoxa9 plus Meis1a retrovirus (second set of 5) 11 days after culturing 10,000 cells in G-CSF (I) or SCF (J). An asterisk signifies that the cells continued to proliferate as G-CSF- or SCF-dependent cell lines.
Figure 4
Figure 4
Meis1 induces rapid conversion of Neo-Hoxa9 cells to a phenotype that proliferates in response to G-CSF, SCF, or G-CSF plus SCF. The presence or absence of coexpressed Meis1 is designated adjacent to each tracing. Cytokines included in growth medium are indicated above each graph.
Figure 5
Figure 5
Model for the function of Meis1a in AML.

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