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NIPBL/NPMc+ interplay in myeloid differentiation
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Figure 5. The impairment of myeloid cell differentiation in nipblb-MO and NPMc+ injected embryos is caused by hyper-activation of canonical Wnt signaling. (A- G) Whole mount in situ hybridization analyses of spi1b. The expression of spi1b was increased in nipblb-MO (B) and NPMc+-injected embryos (E) in comparison to that in controls (A). The myeloid phenotype was rescued by injection of Wnt inhibitor dkk1b mRNA (C, F) and treatment with indomethacin (D, G). Quantification of the observed phenotypes in (H) and (I). Images were processed as described in the Online Supplementary Methods. The scale bar represents 100 μm. *P<0.05 and ***P<0.001. ctrl: control; MO: morpholino.
humans, heterozygous loss-of-function mutations of NIPBL cause CdLS.37 Nevertheless, CdLS patients with mutations in NIPBL or in other cohesin genes are not pre- disposed to myeloid neoplasms. Rare cases of leukemia have been reported in patients with CdLS, but were prob- ably caused by other inherent aspects of this latter pathol- ogy.38–40 The mutations in cohesin genes responsible for CdLS and leukemia insurgence are considered different for their pathophysiological output: cohesin mutations in tumors occur in somatic adult cells while germline cohesin mutations in CdLS patients occur in embryonic tissue. Moreover, cohesin mutations in cancer might not trigger, but contribute to tumorigenesis only with other mutations such as TET2,41 NPM1,9,42 DNMT3A43 or FLT3-ITD.44 Indeed, we speculate that the decrease in NIPBL expres- sion observed in AML patients might not be due to specif- ic somatic mutations occurring at the gene level, but rather to a secondary effect caused by genetic lesions that have arisen elsewhere (i.e., NPM1).
In nipblb-loss-of-function embryos we observed defects in myeloid differentiation with an increased number of myeloid progenitors and a decrease of mature myeloid cells. These hematopoietic defects share similarities with the myeloproliferative phenotype of NPMc+ embryos.12,13 Indeed, we also confirmed that the forced expression of NPMc+ generated a phenotype with expansion of HSC and myeloid precursors. However, the main difference in the hematopoietic phenotype generated by nipblb knock- down and NPMc+ forced expression was in the HSC pop- ulation. In this regard, while HSC numbers were signifi-
cantly increased in NPMc+ embryos, in nipblb-MO inject- ed embryos they were almost comparable to those in con- trols. This finding is not surprising as we demonstrated that nipblb acts downstream of, and is regulated by, NPMc+. It is therefore conceivable that, also in the hematopoietic cascade that drives myeloid differentiation, the effects of nipblb are exerted on a population derived from HSC, such as myeloid progenitors.
We further observed that in nipblb knockdown and NPMc+ overexpressed zebrafish embryos, the Wnt/β- catenin pathway was hyper-activated. In the process of hematopoiesis, many signaling pathways are critical dur- ing the different developmental stages of HSC and for the maturation of these cells into differentiated lineages. In particular, the canonical Wnt pathway exerts its action on specific cell populations, being fine-tuned in a dosage- dependent fashion.45 We demonstrated that Nipbl plays an active role in this modulation. Indeed, in a previous study, we showed that the downregulation of nipblb in zebrafish embryos at 24 hpf led to decreased activation of the canonical Wnt pathway.30 In this work we demonstrated that, from 48 hpf, Nipbl acts in an opposite way by hyper- activating the Wnt pathway in the whole embryo. It is conceivable that, given its double effects on the Wnt path- way, Nipblb might be considered a new factor in canoni- cal Wnt pathway modulation.
Recently several Wnt inhibitors have been identified as potential drugs to reduce tumor cell viability in lymphoma and myeloma cell lines in vitro and in vivo.46 Thus, Wnt/β- catenin inhibitors, such as indomethacin that has already
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