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NIPBL/NPMc+ interplay in myeloid differentiation
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Figure 1. Expression of cohesin genes in bone marrow samples from adults with acute myeloid leukemia divided into two subgroups according to the absence or presence of NPM1 mutations and in a zebrafish model for NPM1 mutation (NPMc+). (A-D) Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses in 40 adults with acute myeloid leukemia indicated that the expression of NIPBL was decreased when NPM1 was mutated. The expression levels of the other cohesin genes analyzed did not correlate with the presence of NPM1 mutations. (E) RT-qPCR analyses of cohesin gene expression in a zebrafish model with overexpression of human NPMc+ showed that the expression of nipblb was downregulated in comparison to that of controls at 3 days post-fertilization (dpf). (F) RT- qPCR time course analyses at 24, 36 and 48 hours post-fertilization (hpf) of nipblb expression in embryos injected with NPMc+ transcript. nipblb was significantly downregulated at 48 hpf. (G) RT-qPCR analyses of nipblb expression in zebrafish embryos at 3 dpf injected with different doses of human NPMc+. The downregu- lation of nipblb was significantly dependent on the dose of NPMc+ injected. (H) Western blot analyses of Nipbl protein expression in embryos at 3 dpf injected with 100 pg/embryo of NPMc+ transcript. Vinculin marker was used for normalization. Western blot images were processed as described in the Online Supplementary Methods. *P<0.05, **P<0.01 and ***P<0.001. ns: non-significant; AML: acute myeloid leukemia; MO: morpholino; ctrl: control.
obtained with nipblb-MO injection, we used a second morpholino targeting the 5’UTR region of nipblb (5’UTRnipblb-MO), which has been previously used and validated.29 The injection of this second morpholino reduced the Nipbl protein levels, as determined by west- ern blot analyses, confirming its efficacy in blocking pro- tein production. It also recapitulated the hematopoietic defects observed with the injection of the ATG morpholi- no, showing an increased number of myeloid precursors positive for spi1b and a decreased number of mature myeloid cells. Moreover, when co-injected at subcritical doses, the two nipblb-MO cooperated to induce the myeloid phenotype (Online Supplementary Figure S4).
nipbl downregulation and NPMc+ overexpression both induce the hyper activation of the canonical Wnt pathway
Previous experimental and clinical evidence suggested a close correlation between AML development and canoni- cal Wnt pathway dysregulation, especially in patients with NPM1 mutations.12 To examine whether nipblb and NPMc+ regulate the activation status of the canonical Wnt pathway, we used the Tg(TOPdGFP) transgenic line that
bears the GFP reporter gene under the control of four enhancers and the basal promoter of lef1, a β-catenin- dependent transcription factor.32 Tg(TOPdGFP) embryos were injected with nipblb-MO or NPMc+ mRNA and the expression of gfp and axin2, one of the direct targets of activated β-catenin, were analyzed by quantitative RT- PCR at 3 dpf.33 Both genes were upregulated following nip- blb knockdown or NPMc+ overexpression, indicating a hyper-activation of the canonical Wnt pathway (Figure 3A,B). To determine whether the Wnt signaling was enhanced specifically in hematopoietic cells, confocal images of the CHT of Tg(TOPdGFP) embryos were ana- lyzed (Figure 3C). GFP+ cells were increased in both nipblb- MO- and NPMc+-injected embryos (10 increased/10 scored). Specifically, nipblb downregulation almost dou- bled the number of GFP+ cells in the CHT (n=27±5) in comparison to the number in controls (n=14±3), (Figure 3D,E, quantification in I). NPMc+ overexpression also increased the number of GFP+ cells in the CHT (n=20±4) in comparison to the number in controls (n=14±3), (Figure 3D-G, quantification in I). To further demonstrate that the increase of GFP+ cells in the CHT was due to the hyper- activation of the canonical Wnt pathway, we injected the
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