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NIPBL/NPMc+ interplay in myeloid differentiation
NPMc+, which functions as an oncogene in vitro8 and has a role in aberrant hematopoiesis in vivo. Indeed, murine models expressing NPMc+ in the hematopoietic lineage develop myeloproliferative disease9 and leukemia10,11 while the forced expression of human NPMc+ in zebrafish caus- es an increase in primitive early myeloid cells and defini- tive hematopoietic stem cells (HSC).12,13 It has also been demonstrated, with similar results in zebrafish and human AML blasts, that the expression of NPMc+ activates canonical Wnt signaling, providing insight into the molec- ular pathogenesis of AML bearing NPM1 mutations.12 Indeed, the canonical Wnt/β-catenin pathway has been shown to be crucial for the regulation of HSC prolifera- tion, differentiation and apoptosis.14
Recently, mutations in cohesin genes were found to be strongly correlated with NPM1 mutations although they do not seem to affect the prognosis of patients with AML.15 The cohesin complex is composed of different pro- teins that form a complex (SMC1, SMC3, RAD21, STAG1 and STAG2), and by additional regulator proteins (NIPBL, MAU2, ESCO1, ESCO2 and HDAC8). This multifunc- tional complex regulates the cohesion of sister chromatids during cell division, but also gene transcription and chro- matin architecture. Recently, the genes of the cohesin complex were found to be mutated in almost 10% of patients with myeloid malignancies, while an additional 15% of patients had reduced expression of cohesin tran- scripts, suggesting a role for the cohesin complex in the pathogenesis of AML.5 In a cohort of patients studied by Thota and colleagues,15 the most frequently mutated genes of the cohesin complex were STAG2 (5.9%), RAD21 (2%), and SMC3 (2%), whereas mutations in the other cohesins were less frequent (<1%). Somatic mutations in cohesin subunits are mutually exclusive and, being mainly non- sense and frameshift mutations, result in a predicted loss- of-function phenotype.16 It should be noted that cohesin mutations in AML, but not in other kind of tumors,17 are associated with a normal karyotype in malignant cells; therefore, the role of cohesins in tumor development is not correlated with their function in sister chromatid cohesion but rather with their role in mediating DNA accessibility to gene regulatory elements.15 Indeed, in vitro and in vivo models of cohesin haploinsufficiency show a delay in the differentiation of HSC, which are expanded in an immature state.18–21
In this work, we studied the expression of cohesin genes in a cohort of adults with AML and found a specific down- regulation of NIPBL when NPM1 was mutated. Interestingly, we also found that nipblb was downregulated in our zebrafish model for NPMc+ expression. The zebrafish (Danio rerio) is a powerful model for studying hematologic diseases as it shares several hematopoietic genes with higher vertebrates, and mutations in human leukemia-causative genes disrupt normal hematopoiesis in zebrafish, suggesting a functional conservation of these genes during evolution. Our zebrafish model with loss-of- function of nipblb showed dysregulation of myeloid cell differentiation with increased numbers of myeloid precur- sors and a decrease of mature myeloid cells. The hematopoietic phenotype presented by nipblb-loss-of-func- tion zebrafish embryos recapitulated the myeloid defects presented by embryos with NPMc+ overexpression and was due to hyper-activation of the canonical Wnt path- way. Indeed, overexpression of the dkk1b Wnt inhibitor or indomethacin treatment rescued the phenotype.
Our study provides new insights into the molecular mechanisms underlying NIPBL function, identifying the canonical Wnt pathway as one of its targets and indicating that it plays a role with NPMc+ in the development of AML. Using the well-suited zebrafish model, we estab- lished a platform to further investigate the mechanisms through which NPMc+ and NIPBL might interact and con- tribute to leukemic transformation.
Methods
Patients
Diagnostic bone marrow samples from healty subjects and 40 adult patients affected by AML were collected and characterized as described in the Online Supplementary Methods. Patients’ materi- al was collected after obtaining informed consent (protocol ASG- MA-052A approved on May 8th, 2012 by Azienda San Gerardo). The clinical features of the participants are reported in Online Supplementary Table S1. Human material and derived data were used in accordance with the Declaration of Helsinki.
Animals
Zebrafish embryos were raised and maintained according to international (European Union Directive 2010/63/EU) and national (Italian decree n. 26 of March 4th, 2014) guidelines on the protec- tion of animals used for scientific purposes, as described in the Online Supplementary Methods.
Reverse transcription and real-time quantitative polymerase chain reaction assays
RNA was extracted from human and zebrafish embryos using TRIZOL reagents (Life Technologies, Carlsbad, CA, USA), follow- ing the manufacturer’s protocol. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments on human sam- ples were performed with the Universal Probe Library system (Roche Diagnostics, Basel, Swiss), as described in the Online Supplementary Methods. Probes and primers are reported in Online Supplementary Tables S2 and S3.
Western blotting
Protein extracts were prepared, loaded and quantified as described in the Online Supplementary Methods. The antibodies used are listed in Online Supplementary Table S4. Images were acquired using an Alliance MINI HD9 AUTO Western Blot Imaging System (UVItec Limited, Cambridge, UK) and analyzed with the related software.
In situ hybridization and immunofluorescent analyses Whole mount in situ hybridization (WISH) experiments, were carried out as described by Thisse et al.22 and in the Online Supplementary Methods. Pictures were acquired with a Leica DFC480 photo camera (Leica, Wetzlar, Germany). Immunostaining was performed as described previously.23 The antibodies used are listed in Online Supplementary Table S4. Images
were acquired as described in the Online Supplementary Methods.
Sudan black staining
Sudan black staining was performed as described by Cvejic et al.24 and in the Online Supplementary Methods.
Fluorescence-activated cell sorting analyses
Embryo dissociation was performed as described previously.25 Fluorescence-activated cell sorting (FACS) analyses were per- formed as described in the Online Supplementary Methods on
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