A Drosophila model identifies calpains as modulators of the human leukemogenic fusion protein AML1-ETO

Abstract

The t(8:21)(q22;q22) translocation is 1 of the most common chromosomal abnormalities linked to acute myeloid leukemia (AML). AML1-ETO, the product of this translocation, fuses the N-terminal portion of the RUNX transcription factor AML1 (also known as RUNX1), including its DNA-binding domain, to the almost entire transcriptional corepressor ETO (also known as MTG8 or RUNX1T1). This fusion protein acts primarily by interfering with endogenous AML1 function during myeloid differentiation, although relatively few genes are known that participate with AML1-ETO during leukemia progression. Here, we assessed the consequences of expressing this chimera in Drosophila blood cells. Reminiscent of what is observed in AML, AML1-ETO specifically inhibited the differentiation of the blood cell lineage whose development depends on the RUNX factor Lozenge (LZ) and induced increased numbers of LZ(+) progenitors. Using an in vivo RNAi-based screen for suppressors of AML1-ETO, we identified calpainB as required for AML1-ETO-induced blood cell disorders in Drosophila. Remarkably, calpain inhibition triggered AML1-ETO degradation and impaired the clonogenic potential of the human t(8;21) leukemic blood cell line Kasumi-1. Therefore Drosophila provides a promising genetically tractable model to investigate the conserved basis of leukemogenesis and to open avenues in AML therapy.

Fig. 1.

AML1-ETO specifically inhibits LZ-dependent blood cell differentiation. (A–D) Pan-hematopoietic expression of AML1-ETO under the control of srp-gal4 does not affect plasmatocyte development (A and B: crq) but inhibits crystal cell differentiation (E and F: PO45/CG8193). This repression is not due to a reduction in lz expression (C and D: lz) but to the competition between AML1-ETO and LZ to regulate LZ target genes (G and H: PO45/CG8193). (A–H): Lateral views of stage 11 embryos. Genotypes are indicated in the lower part of each panel. Arrows in (G and H) indicate ectopic activation of PO45 induced by LZ in the plasmatocytes and posterior endoderm.

Fig. 2.

Both moieties of the AML1-ETO fusion protein are concomitantly required to block crystal cell differentiation. lz-gal4-driven expression of AML1-ETO, but not that of its AML1 (AML1∂ETO) or ETO moiety, inhibits crystal cell differentiation (A–D and I–L: PO45; Q–T: heat-revealed crystal cells). Formation and maintenance of the LZ⁺ cells (marked by lz-gal4, UAS-gfp) is not impaired (E–H, M–P, and U–X: GFP). (A–H) Dorsal views of stage 13 embryos. (I–P) Third instar larval lymph gland. (Q–X) Dorsal views of the posterior segments of third instar larvae. Genotypes are indicated in the upper part of the figure.

Fig. 3.

AML1-ETO expression induces a preleukemic state. (A–D) lz-gal4, UAS-gfp third instar wandering larvae of the indicated genotypes were bled and the absolute number of GFP+ cells (A and C) as well as the proportion of LZ-GFP⁺ cells expressing the crystal cell differentiation marker PO45 (B and D) were determined. (A) AML1-ETO but not AML1∂ETO or ETO induces a net increase in LZ-GFP⁺ circulating blood cells as compared to control larvae. (B) In the presence of AML1-ETO, the ratio of progenitors (GFP⁺, PO45⁻) to differentiated (GFP⁺, PO45⁺) crystal cells is almost inverted. Neither AML1∂ETO nor ETO affects this ratio. (C and D) Switching off the expression of AML1-ETO at the early L3 stage does not suppress the increase in circulating LZ-GFP⁺ cells (C) at the wandering larvae stage but partially restore crystal cell differentiation (P < 0.001) (D). **, significant difference (Student's t test, P < 0.001) compared to the wild-type strain.

Fig. 4.

calpB is required for AML1-ETO function. (A) Absolute number of circulating LZ-GFP⁺ cells in third instar larvae. (B) Ratio of circulating progenitors (GFP⁺, PO45⁻) to differentiated (GFP⁺, PO45⁺) crystal cells in third instar larvae. (C–F) PO45 expression in stage 13 embryos. (G–J) PO45 expression in third instar larval lymph gland. The phenotypes induced upon expression of AML1-ETO in the LZ⁺ blood cell lineage are suppressed when AML1-ETO is coexpressed with a UAS-dsRNA against calpB or when it is expressed in a calpB^−/− mutant background (P < 0.001). Similarly, coexpressing LZ with AML1-ETO significantly suppressed AML1-ETO-induced LZ⁺ cell increase and differentiation bias (P < 0.001). **, significant difference (Student's t test, P < 0.001) compared to the wild-type strain. (K-N) GFP (green) and LZ or AML1-ETO (red) expression in circulating larval blood cells from lz-gal4, UAS-gfp (K), lz-gal4, UAS-gfp; UAS-dsCalpB (L), lz-gal4, UAS-gfp; UAS-aml1eto (M), and lz-gal4, UAS-gfp; UAS-aml1eto;UAS-dsCalpB (N) larvae. LZ expression is not affected by calpB loss of function whereas AML1-ETO levels are strongly decreased. Nuclei were stained with DAPI (blue). (K'-N′) show the red channel from panels (K–N).

Fig. 5.

Calpain inhibition reduces the clonogenicity of Kasumi-1 cells. (A) Relative viability of Kasumi-1 cells treated with increasing doses of ALLN or calpain inhibitor III (Inh III). (B) Relative colony numbers obtained upon treatment of Kasumi-1 or HL-60 cells with 10 μM ALLN or 30 μM Calpain Inhibitor III. (A and B) Significant differences between control and treated cells are indicated: *, P< 0.01; **, P < 0.001. (C) Colony forming activity and differentiation potential of primary blood cells treated with 10 μM ALLN or 30 μM calpain inhibitor III. B/CFU-E, blast/colony-forming unit erythroid; M, macrophage; G, granulocyte; GM, granulocyte-macrophage; and GEMM, granulocyte-erythrocyte-macrophage-megakaryocyte. (D) Western blots showing AML1-ETO or GAPDH expression in Kasumi-1 cells treated with 10 μM ALLN or 30 μM calpain inhibitor III. The relative levels of AML1-ETO (normalized to GAPDH) are indicated in the lower part of the panel.