Dana E. Cullen

FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress

To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential.

Efficacy of TG101348, a Selective JAK2 Inhibitor, in Treatment of a Murine Model of JAK2V617F-Induced Polycythemia Vera

We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

Hedgehog Signaling Is Dispensable for Adult Murine Hematopoietic Stem Cell Function and Hematopoiesis

We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no apparent effect on adult hematopoiesis, including peripheral blood count, number or cell-cycle status of stem or progenitor cells, hematopoietic colony-forming potential, long-term repopulating activity in competitive repopulation assays, or stress response to serial 5-fluorouracil treatment. Furthermore, pharmacologic inhibition of Hh signaling with a potent and selective small molecule antagonist has no substantive effect on hematopoiesis in the mouse. In addition, Hh signaling is not required for the development of MLL-AF9-mediated acute myeloid leukemia (AML). Taken together, these data demonstrate that Hh signaling is dispensable for normal hematopoietic development and hematopoietic stem cell function, indicating that targeting of Hh signaling in solid tumors is not likely to result in hematopoietic toxicity. Furthermore, the Hh pathway may not be a compelling target in certain hematopoietic malignancies.

The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model

Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two-megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.

Ott1(Rbm15) has pleiotropic roles in hematopoietic development

OTT1(RBM15) was originally described as a 5′ translocation partner of the MAL(MKL1) gene in t(1,22)(p13;q13) infant acute mega karyocytic leukemia. OTT1 has no established physiological function, but it shares homology with the spen/Mint/SHARP family of proteins defined by three amino-terminal RNA recognition motifs and a carboxyl-terminal SPOC (Spen paralog and ortholog carboxyl-terminal) domain believed to act as a transcriptional repressor. To define the role of OTT1 in hematopoiesis and help elucidate the mechanism of t(1,22) acute megakaryocytic leukemia pathogenesis, a conditional allele of Ott1 was generated in mice. Deletion of Ott1 in adult mice caused a loss of peripheral B cells due to a block in pro/pre-B differentiation. There is myeloid and megakaryocytic expansion in spleen and bone marrow, an increase in the Lin−Sca-1+c-Kit+ compartment that includes hematopoietic stem cells, and a shift in progenitor fate toward granulocyte differentiation. These data show a requirement for Ott1 in B lymphopoiesis, and inhibitory roles in the myeloid, megakaryocytic, and progenitor compartments. The ability of Ott1 to affect hematopoietic cell fate and expansion in multiple lineages is a novel attribute for a spen family member and delineates Ott1 from other known effectors of hematopoietic development. It is plausible that dysregulation of Ott1-dependent hematopoietic developmental pathways, in particular those affecting the megakaryocyte lineage, may contribute to OTT1-MAL-mediated leukemogenesis.

Ott1 (Rbm15) Is Essential for Placental Vascular Branching Morphogenesis and Embryonic Development of the Heart and Spleen

ABSTRACT The infant leukemia-associated gene Ott1 (Rbm15) has broad regulatory effects within murine hematopoiesis. However, germ line Ott1 deletion results in fetal demise prior to embryonic day 10.5, indicating additional developmental requirements for Ott1. The spen gene family, to which Ott1 belongs, has a transcriptional activation/repression domain and RNA recognition motifs and has a significant role in the development of the head and thorax in Drosophila melanogaster. Early Ott1-deficient embryos show growth retardation and incomplete closure of the notochord. Further analysis demonstrated placental defects in the spongiotrophoblast and syncytiotrophoblast layers, resulting in an arrest of vascular branching morphogenesis. The rescue of the placental defect using a conditional allele with a trophoblast-sparing cre transgene allowed embryos to form a normal placenta and survive gestation. This outcome showed that the process of vascular branching morphogenesis in Ott1-deficient animals was regulated by the trophoblast compartment rather than the fetal vasculature. Mice surviving to term manifested hyposplenia and abnormal cardiac development. Analysis of global gene expression of Ott1-deficient embryonic hearts showed an enrichment of hypoxia-related genes and a significant alteration of several candidate genes critical for cardiac development. Thus, Ott1-dependent pathways, in addition to being implicated in leukemogenesis, may also be important for the pathogenesis of placental insufficiency and cardiac malformations.

Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress

Aging degrades hematopoietic stem cell (HSC) functions, including stress response; however, the involved molecular pathways are incompletely defined. Murine BM conditionally deleted for One-Twenty-Two-1 (Ott1), is able to maintain lifelong hematopoiesis and has preserved numbers of long-term HSCs, yet cannot repopulate nor sustain itself after transplantation against a competitor even when Ott1 is excised after engraftment. We show, specifically under replicative stress, that Ott1-deleted HSCs have a significant reduction of the G(0) cell-cycle fraction associated with self-renewal and undergo early failure. Therefore, Ott1 is required to preserve HSC quiescence during stress but not steady-state hematopoiesis. Reduced tolerance of replicative stress, increased myeloid potential, and greater absolute numbers are mutual characteristics of both Ott1-deleted and aged HSCs, and comparison of their gene expression profiles reveals a shared signature. Ott1-deleted HSCs share multiple aging-associated physiologic changes, including increases in NF-κB activation and DNA damage. Loss of Ott1 causes increased reactive oxygen species; however, antioxidant treatment does not rescue the competitive defect, indicating the existence of additional essential Ott1-dependent HSC pathways. In conclusion, our data establish a requirement for Ott1 in stress hematopoiesis and suggest that Ott1-dependent processes may converge with those affected by aging.

Maternal and zygotic Zfp57 modulate NOTCH signaling in cardiac development

Significance Abnormal heart development is a common birth defect. Genomic imprinting is absolutely essential for mammalian embryonic development. We found that loss of ZFP57, a master regulator of genomic imprinting, causes a number of heart morphogenetic defects. These cardiac defects are reminiscent of mutant phenotypes observed in the NOTCH signaling pathway, one of the most important pathways in development. Indeed, we demonstrate that NOTCH signaling is diminished without ZFP57. Furthermore, the maternal function of Zfp57 contributes to NOTCH signaling and embryonic heart development. Maternal and zygotic Zfp57 play redundant roles in genomic imprinting, NOTCH signaling, and heart development. Thus, our results provide mechanistic links among maternal effect, genomic imprinting, NOTCH signaling, and cardiac development. Zfp57 is a maternal–zygotic effect gene that maintains genomic imprinting. Here we report that Zfp57 mutants exhibited a variety of cardiac defects including atrial septal defect (ASD), ventricular septal defect (VSD), thin myocardium, and reduced trabeculation. Zfp57 maternal-zygotic mutant embryos displayed more severe phenotypes with higher penetrance than the zygotic ones. Cardiac progenitor cells exhibited proliferation and differentiation defects in Zfp57 mutants. ZFP57 is a master regulator of genomic imprinting, so the DNA methylation imprint was lost in embryonic heart without ZFP57. Interestingly, the presence of imprinted DLK1, a target of ZFP57, correlated with NOTCH1 activation in cardiac cells. These results suggest that ZFP57 may modulate NOTCH signaling during cardiac development. Indeed, loss of ZFP57 caused loss of NOTCH1 activation in embryonic heart with more severe loss observed in the maternal-zygotic mutant. Maternal and zygotic functions of Zfp57 appear to play redundant roles in NOTCH1 activation and cardiomyocyte differentiation. This serves as an example of a maternal effect that can influence mammalian organ development. It also links genomic imprinting to NOTCH signaling and particular developmental functions.