// Lisa Deng 1,2 , Elizabeth L. Virts 1,4 , Reuben Kapur 1,2,3,4 and Rebecca J. Chan 1,2,4 1 Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA 2 Department of Medical & Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA 3 Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA 4 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA Correspondence to: Rebecca J. Chan, email: // Keywords : JMML, Shp2, PI3K p110δ, mouse model, in vivo Abbreviations : JMML: juvenile myelomonocytic leukemia; GM-CSF: granulocyte macrophage colony-stimulating factor; GOF: gain-of-function; PI3K: phosphatidyl inositide-3 kinase; LDMNC: low-density mononuclear cell Received : July 27, 2017 Accepted : September 08, 2017 Published : October 03, 2017 Abstract Juvenile myelomonocytic leukemia is a childhood malignancy that lacks effective chemotherapies and thus has poor patient outcomes. PI3K p110δ has been found to promote hyperproliferation of cells expressing mutant Shp2. In this study, we tested the efficacy of a PI3Kδ inhibitor in mice expressing the Shp2 gain-of-function mutation, E76K. We found that in vivo treatment of mice led to significantly decreased splenomegaly, reduced frequency of bone marrow progenitor cells, and increased terminally differentiated peripheral blood myeloid cells. The survival of drug-treated mice was significantly prolonged compared to vehicle-treated controls, although mice from both groups ultimately succumbed to a similar myeloid cell expansion. PI3Kδ inhibitors are currently used to treat patients with relapsed lymphoid malignancies, such as chronic lymphocytic leukemia. The current findings provide evidence for using PI3Kδ inhibitors as a treatment strategy for JMML and potentially other myeloid diseases.
PTPN11 gain-of-function mutation is the most common mutation found in patients with juvenile myelomonocytic leukemia and DNMT3A loss occurs in over 20% of acute myeloid leukemia patients. We studied the combined effect of both Ptpn11 gain-of-function mutation (D61Y) and Dnmt3a haploinsufficiency on mouse hematopoiesis, the presence of which has been described in both juvenile myelomonocytic leukemia and acute myeloid leukemia patients. Double mutant mice rapidly become moribund relative to any of the other genotypes, which is associated with enlargement of the spleen and an increase in white blood cell counts. An increase in the mature myeloid cell compartment as reflected by the presence of Gr1+Mac1+ cells was also observed in double mutant mice relative to any other group. Consistent with these observations, a significant increase in the absolute number of granulocyte macrophage progenitors (GMPs) was seen in double mutant mice. A decrease in the lymphoid compartment including both T and B cells was noted in the double mutant mice. Another significant difference was the presence of extramedullary erythropoiesis with increased erythroid progenitors in the spleens of Dnmt3a+/-;D61Y mice relative to other groups. Taken together, our results suggest that the combined haploinsufficiency of Dnmt3a and presence of an activated Shp2 changes the composition of multiple hematopoietic lineages in mice relative to the individual heterozygosity of these genes.
Background: Accumulating evidence suggests the origin of Juvenile Myelomonocytic Leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN).
Results: E9.5 YS EMPs from VavCre+;PTPN11D61Y embryos demonstrated growth hypersensitivity to GM-CSF and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11E76K;ROSAYFP mice in which oncogene expression was restricted to EMPs. YFP+ progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN.
Conclusions: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. This article is protected by copyright. All rights reserved.
Juvenile myelomonocytic leukemia (JMML) is a commonly fatal myeloid leukemia occurring in very young children bearing germline or somatic mutations in genes within the RAS-ERK signaling pathway including NF1 , CBL, KRAS, NRAS , and PTPN11 (1). Leukemia relapse following allogeneic stem cell transplant is uncharacteristically high at 40–50% for JMML patients, and findings from the recently published manuscript by Dong et al .
Juvenile myelomonocytic leukemia (JMML) is a commonly fatal myeloid leukemia occurring in very young children bearing germline or somatic mutations in genes within the RAS-ERK signaling pathway including NF1, CBL, KRAS, NRAS, and PTPN11 (1).Leukemia relapse following allogeneic stem cell transplant is uncharacteristically high at 40-50% for JMML patients, and findings from the recently published manuscript by Dong et al. (2) provide some clues that might account for this high relapse rate.The authors found that Nestin-Cre-mediated knock-in of Ptpn11 E76K/+ , resulting in mutant Shp2 expression in bone marrow (BM) mesenchymal stem/progenitor and neural cells, induced activation of otherwise normal hematopoietic stem and progenitor cells to differentiate into myeloid cells.The resulting leukemia recapitulated the features of JMML, with splenomegaly, myelocytosis, and increased myelopoiesis in the BM.While Mx1-Creand LysM-Cremediated knock-in of Ptpn11 E76K/+ have previously been shown to cause a JMML-like myeloproliferative neoplasm (MPN) in mice (3), this is the first study demonstrating that BM niche-restricted expression of Ptpn11 E76K/+ is capable of producing disease.The elegance of the present study lies in the use of five tissue-specific Cre promoters which identified mesenchymal stem/progenitor cells and osteoprogenitors as the critical mutated BM microenvironment cell types with the capacity to promote myeloid disease, ruling out differentiated osteoblasts and endothelial cells.Relevant to human disease and hematopoietic stem cell (HSC) transplantation as the standard treatment for JMML, the authors found that lethally irradiated Ptpn11 E76K/+ Nestin-Cre + mice transplanted with wild-type (WT) BM cells develop a donor-derived MPN, demonstrating the mutant BM microenvironment's pathogenic effect on transplanted normal cells.A potential mechanism accounting for the development of the donor WT cell-derived
Abstract Endothelial cells take pivotal roles in the heart and the vascular system and their differentiation, subspecification and function is determined by gene expression. A stable, in vitro cardiac endothelial cell line could provide high cell numbers as needed for many epigenetic analyses and facilitate the understanding of molecular mechanisms involved in endothelial cell biology. To test their suitability for transcriptomic or epigenetic studies, we compared the transcriptome of cultured immortalized mouse cardiac endothelial cells (MCEC) to primary cardiac endothelial cells (pEC). Whole transcriptome comparison of MCEC and pEC showed a correlation of 0.75–0.77. Interestingly, correlation of gene expression declined in endothelial cell-typical genes. In MCEC, we found a broad downregulation of genes that are highly expressed in pEC, including well-described markers of endothelial cell differentiation. Accordingly, systematic analysis revealed a downregulation of genes associated with typical endothelial cell functions in MCEC, while genes related to mitotic cell cycle were upregulated when compared to pEC. In conclusion, the findings from this study suggest that primary cardiac endothelial cells should preferably be used for genome-wide transcriptome or epigenome studies. The suitability of in vitro cell lines for experiments investigating single genes or signaling pathways should be carefully validated before use.
