Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice

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 granulocyte-macrophage colony-stimulating factor (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. Yellow fluorescent protein-positive 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

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data

Hematopoietic-restricted Ptpn11E76K reveals indolent MPN progression in mice

Juvenile Myelomonocytic Leukemia (JMML) is a pediatric myeloproliferative neoplasm (MPN) that has a poor prognosis. Somatic mutations in Ptpn11 are the most frequent cause of JMML and they commonly occur in utero. Animal models of mutant Ptpn11 have probed the signaling pathways that contribute to JMML. However, existing models may inappropriately exacerbate MPN features by relying on non-hematopoietic-restricted Cre-loxP strains or transplantations into irradiated recipients. In this study we generate hematopoietic-restricted models of Ptpn11E76K-mediated disease using Csf1r-MCM and Flt3Cre. We show that these animals have indolent MPN progression despite robust GM-CSF hypersensitivity and Ras-Erk hyperactivation. Rather, the dominant pathology is pronounced thrombocytopenia with expanded extramedullary hematopoiesis. Furthermore, we demonstrate that the timing of tamoxifen administration in Csf1r-MCM mice can specifically induce recombinase activity in either fetal or adult hematopoietic progenitors. We take advantage of this technique to show more rapid monocytosis following Ptpn11E76K expression in fetal progenitors compared with adult progenitors. Finally, we demonstrate that Ptpn11E76K results in the progressive reduction of T cells, most notably of CD4+ and naïve T cells. This corresponds to an increased frequency of T cell progenitors in the thymus and may help explain the occasional emergence of T-cell leukemias in JMML patients. Overall, our study is the first to describe the consequences of hematopoietic-restricted Ptpn11E76K expression in the absence of irradiation. Our techniques can be readily adapted by other researchers studying somatically-acquired blood disorders

Hemogenic Endothelial Cells Can Transition to Hematopoietic Stem Cells through a B-1 Lymphocyte-Biased State during Maturation in the Mouse Embryo

Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45-VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate

Mice expressing KrasG12D in hematopoietic multipotent progenitor cells develop neonatal myeloid leukemia

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that bears distinct characteristics associated with abnormal fetal development. JMML has been extensively modeled in mice expressing the oncogenic KrasG12D mutation. However, these models have struggled to recapitulate the defining features of JMML due to in utero lethality, nonhematopoietic expression, and the pervasive emergence of T cell acute lymphoblastic leukemia. Here, we have developed a model of JMML using mice that express KrasG12D in multipotent progenitor cells (Flt3Cre+ KrasG12D mice). These mice express KrasG12D in utero, are born at normal Mendelian ratios, develop hepatosplenomegaly, anemia, and thrombocytopenia, and succumb to a rapidly progressing and fully penetrant neonatal myeloid disease. Mutant mice have altered hematopoietic stem and progenitor cell populations in the BM and spleen that are hypersensitive to granulocyte macrophage-CSF due to hyperactive RAS/ERK signaling. Biased differentiation in these progenitors results in an expansion of neutrophils and DCs and a concomitant decrease in T lymphocytes. Flt3Cre+ KrasG12D fetal liver hematopoietic progenitors give rise to a myeloid disease upon transplantation. In summary, we describe a KrasG12D mouse model that reproducibly develops JMML-like disease. This model will prove useful for preclinical drug studies and for elucidating the developmental origins of pediatric neoplasms

Genomic landscape of TP53-mutated myeloid malignancies

TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional clinical techniques. We performed whole-genome sequencing (WGS) of 42 acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) cases with paired normal tissue to better characterize the genomic landscape of TP53-mutated AML/MDS. WGS accurately determines TP53 allele status, a key prognostic factor, resulting in the reclassification of 12% of cases from monoallelic to multihit. Although aneuploidy and chromothripsis are shared with most TP53-mutated cancers, the specific chromosome abnormalities are distinct to each cancer type, suggesting a dependence on the tissue of origin. ETV6 expression is reduced in nearly all cases of TP53-mutated AML/MDS, either through gene deletion or presumed epigenetic silencing. Within the AML cohort, mutations of NF1 are highly enriched, with deletions of 1 copy of NF1 present in 45% of cases and biallelic mutations in 17%. Telomere content is increased in TP53-mutated AMLs compared with other AML subtypes, and abnormal telomeric sequences were detected in the interstitial regions of chromosomes. These data highlight the unique features of TP53-mutated myeloid malignancies, including the high frequency of chromothripsis and structural variation, the frequent involvement of unique genes (including NF1 and ETV6) as cooperating events, and evidence for altered telomere maintenance

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export

Mycophenolic acid induces senescence of vascular precursor cells

<div><p>Objective</p><p>Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function.</p><p>Methods</p><p>We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects.</p><p>Results</p><p>Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated β-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA.</p><p>Conclusion</p><p>Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells <i>in vitro</i>, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies <i>in vivo</i> to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.</p></div

MPA inhibits cell proliferation in a dose dependent manner.

<p>(A) Cell growth curve was evaluated using trypan blue staining to measure viable cells at 24, 48 and 72 hours. 1–5 μM concentration of MPA inhibited cell proliferation (n = 3). (B) Percentage of single cord blood-derived ECFC undergoing at least one cell division 14 days after MPA treatment (n = 3). (C) Percentage of cell clusters, LPP-ECFCs and HPP-ECFC 14 days after MPA treatment using single cell analysis (n = 3). (D) Proliferation of ECFC in the absence or presence of MPA (0.1, 0.5 and 1 μM) measured after 1, 3, 5 and 7 days by a FACS-based CFSE dilution assay. While 0.1 μM MPA did not impact ECFC division, MPA at higher concentrations significantly diminished ECFC division (n = 4). Results represent the mean ± SD. *P <0.05, **P<0.005, *** P<0.001 compared to vehicle. Abbreviations: ECFC = endothelial colony forming cell, EGM = endothelial growth medium, MPA = mycophenolic acid, LPP-ECFC = low proliferative potential—endothelial colony forming cells, HPP-ECFC = high proliferative potential-endothelial colony forming cells, CFSE = carboxyfluorescein succinimidyl ester.</p

Diminished ECFC vasculogenic function after MPA treatment.

<p>Left panel shows representative photomicrographs (magnification, 5x) of TdTomato ECFC following no treatment (vehicle) or treatment with 2.5 μM MPA in 2D assay (A) and 3D assay (D). 2D Matrigel assay showed that there is decreased average total cord length (C) and cord area (B) after MPA treatment. 3D collagen assay showed decreased total vessel area (E) after MPA treatment. *P <0.05, **P <0.005 compared to vehicle (n = 3). Abbreviations: ECFC = endothelial colony forming cells, 2D = two-dimensional, 3D = three-dimensional, EGM = endothelial growth medium, MPA = mycophenolic acid, mm = millimeter.</p