Mutant Fetal Liver Cells Research Articles

3026 - Bi-Allelic CEBPA Mutation Combined with a Novel GATA2 Mutation Develops an Acute Erythroid Leukemia Through a Bi-Potent Leukemic Initiating Population

Acute erythroid leukemia (AEL) is a subtype of acute myeloid leukemia (AML) representing <5% of AML cases. There are two subtypes of AEL: pure erythroid leukemia and erythroleukemia (erythroid/myeloid leukemia). Sequencing studies has comprehensively characterized the mutational landscape of AML identifying mutations that are significantly associated such as the bi-allelic CEBPA and GATA2 mutations (Papaemmanuil et al.; 2016). Recently, sequencing AEL patients also found a high frequency of bi-allelic CEBPA and GATA2 co-mutated (Ping et al.; 2016). As there are currently no models of AEL we combined three knock-in mouse strains, a novel Gata2 point mutation in the N-terminal zinc finger crossed with a knock-in of the N-terminal Cebpa mutation, and a C-terminal Cebpa mutation to generate CebpaN/C;Gata2+/+ (NC genotype) and CebpaN/C;Gata2G320D/ + (NCG genotype). To bypass Cebpa mutation perinatal lethality we performed competitive transplantations using mutant fetal liver cells and wild type bone marrow (BM) competitor cells. By analyzing myeloid reconstitution over time in the peripheral blood we found the myeloid differentiation block caused by the N-terminal mutation in NC mice was prevented when co-expressed with Gata2G320D. This correlated with a more aggressive disease in NCG compared to NC transplanted mice. 100% of NC mice developed an AML while 50% of NCG mice developed an AML, and the remaining 50% developed an AEL of the erythroleukemia subtype, identified by the presence of both myeloblasts and erythroblasts in the BM, spleen and peripheral blood. NCG AEL mice had a significantly shorter overall survival than NCG AML mice. Secondary transplant of different sorted BM populations from NCG AML or NCG AEL mice showed that Lin-cKit+FcR+ GMP-like cells could propagate and fully replicate the two diseases, identifying the putative leukemic initiating cell (LIC) population. Together our study provides a novel model of the erythroleukemia subtype of AEL where both the myeloblasts and erythroblasts develop through a bi-potent LIC population.

Differential Transcriptional Regulation of meis1 by Gfi1b and Its Co-Factors LSD1 and CoREST

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b−/− fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.

OR.2. Adoptive Transfer of Hypomorphic Rag2 Mutant Fetal Liver Cells in Reconstituted Thymic Architecture Can Prevent Peripheral Immunopathology

OR.2. Adoptive Transfer of Hypomorphic Rag2 Mutant Fetal Liver Cells in Reconstituted Thymic Architecture Can Prevent Peripheral Immunopathology

Stat5 Is Essential for Early B Cell Development but Not for B Cell Maturation and Function

The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.

Increased Myeloid Cell Responses to M-CSF and RANKL Cause Bone Loss and Inflammation in SH3BP2 “Cherubism” Mice

While studies of the adaptor SH3BP2 have implicated a role in receptor-mediated signaling in mast cells and lymphocytes, they have failed to identify its function or explain why SH3BP2 missense mutations cause bone loss and inflammation in patients with cherubism. We demonstrate that Sh3bp2 "cherubism" mice exhibit trabecular bone loss, TNF-alpha-dependent systemic inflammation, and cortical bone erosion. The mutant phenotype is lymphocyte independent and can be transferred to mice carrying wild-type Sh3bp2 alleles through mutant fetal liver cells. Mutant myeloid cells show increased responses to M-CSF and RANKL stimulation, and, through mechanisms of increased ERK 1/2 and SYK phosphorylation/activation, they form macrophages that express high levels of TNF-alpha and osteoclasts that are unusually large. M-CSF and RANKL stimulation of myeloid cells that overexpress wild-type SH3BP2 results in similar large osteoclasts. This indicates that the mutant phenotype reflects gain of SH3BP2 function and suggests that SH3BP2 is a critical regulator of myeloid cell responses to M-CSF and RANKL stimulation.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit+ fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9 (-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-Kit+ Hoxb1-b9(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4,-a11, and -c4.Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.

Characterisation of SCL DNA-Binding Functions In Vivo in Haematopoiesis.

The basic Helix-Loop-Helix (bHLH) transcription factor SCL is required for specification of haematopoietic stem cells (HSCs) and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. We challenged this concept by showing that SCL DNA-binding activity was dispensable for some of its functions using SCL−/− ES cells rescued with a DNA-binding defective SCL mutant (SCL-RER) (Porcher et al. Development,1999). We have now studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line SCL-RER mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of primitive erythroid progenitors was observed in SCL RER/RER mutant yolk sacs. At day E12.5 and E13.5, homozygote mutant SCL RER/RER embryos were smaller and paler than wild-type (wt) and heterozygote littermates but presented at the expected mendelian frequency. Lethality was first observed at day E14.5. However, 7% of homozygote mice were born from heterozygous crosses. Surviving adult mice presented with mild microcytic hypochromic anemia. Assessment of progenitor replating potential showed qualitative defects with poor haemoglobinisation of homozygote-derived fetal and adult CFU-Es compared to controls. To understand the cause of the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for either early progenitors or late normoblasts. We observed decreased or increased mRNA expression of most genes tested in mutant-derived erythroid populations when compared to controls matched for differentiation stage, thereby showing that SCL DNA-binding activities are required for both activation and repression of target genes. As examples, mRNA for red cell membrane protein Band 4.2 was dramatically decreased; in contrast, alpha-globin expression was up-regulated in early progenitors, indicating that SCL DNA-binding activity might be required for repression of alpha-globin levels in this setting. We then pursued the analysis of SCL-mediated alpha-globin gene regulation by chromatin immunoprecipitation (ChIP) analysis and tested 4 DNase I hypersensitive sites (DHS) previously shown to bind SCL. From material derived from mutant fetal liver cells, we observed slight variations of SCL binding on 3 out of the 4 cis-acting elements when compared to controls. Importantly, there was a dramatic decrease in SCL binding on the fourth DHS site (HS-12). We concluded that SCL DNA-binding activity was likely to be directly required for repression of alpha-globin levels in erythroid progenitors. Interestingly, we have recently characterised the interaction of SCL with a co-repressor complex comprising the oncoprotein ETO-2 in erythroid cells. We have now shown by ChIP that ETO-2 occupies the alpha-globin locus on HS-12 in wt erythroid progenitors, but not in more mature cells, therefore suggesting a role for the SCL/ETO-2 complex in repression of erythroid-specific genes in the early stages of erythroid maturation. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of HSCs and allows characterisation of DNA-binding requirements throughout development. Combined with studies of the dynamic of SCL-containing multiprotein complexes during erythroid maturation, this model will help define the molecular pathways involved in erythropoiesis.

Oncogenic Ras Requires SHP-2 To Induce Myeloproliferative Disease (MPD).

Oncogenic mutations in NRAS or KRAS2 are found in 20–40% of myeloid malignancies including acute myeloid leukemia, high risk subtypes of myelodysplastic syndrome, and in juvenile myelomonocytic leukemia (JMML) and other types of MPD. Mutant Ras proteins accumulate in the active, GTP-bound state due to impaired intrinsic GTPase activity and resistance to GTPase activating proteins (GAPs). Alternative genetic mechanisms, such as BCR-ABL fusion and inactivation of the NF1 tumor suppressor, also deregulate Ras signaling in myeloid malignancies. Moreover, the association of germline mutations in NF1 with a markedly increased risk of JMML and studies of KrasG12D and Nf1 mutant mice argue strongly that hyperactive Ras can initiate MPD in vivo. Activating mutations in PTPN11, which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in JMML. As is the case for NF1, germline PTPN11 mutations impart an increased risk of developing JMML. These observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1. While NF1 encodes a GAP for Ras that directly reduces Ras-GTP levels, data from various systems have shown that SHP-2 is a positive effector of Ras activation. However, exactly how depohsophorylation of SHP-2 substrates regulates Ras output and how other SHP-2 target proteins modulate its effects are incompletely understood. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. To test the hypothesis that Ras is strictly downstream of SHP-2 in primary hematopoietic cells, we used the conditional alleles LSL-KrasG12D and Ptpn11flox/flox coupled with the interferon-inducible Mx1-Cre transgene. Juvenile mice were injected with pI:pC, resulting in expression of K-RasG12D and inactivation of Ptpn11. These mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre, LSL-KrasG12D mice (Braun et al., PNAS 101(2):597–602), although with slightly increased latency. Importantly, however, myeloid progenitors invariably retained at least one functional Ptpn11 allele despite uniform activation of the conditional KrasG12D allele. These data suggested that there was strong selective pressure to retain at least one functional Ptpn11 allele, even in the presence of oncogenic K-Ras expression. To address this hypothesis directly, we infected LSL-KrasG12D, Ptpn11flox/flox, and compound mutant fetal liver cells with a Cre-expressing retrovirus and enumerated myeloid progenitor colonies in methylcellulose medium. Remarkably, no granulocyte-macrophage colonies developed from Cre-expressing Ptpn11flox/flox fetal liver cells either in the presence or absence of KrasG12D. Infecting compound mutant cells with a Ptpn11-IRES-Cre virus fully restored the aberrant growth phenotype of KrasG12D mutant cells. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro. Our data support a model in which SHP-2 has essential Ras-independent functions in hematopoiesis (i.e. that Ras is not strictly downstream of SHP-2). SHP-2 may be required either for efficient activation of canonical Ras effector pathways and/or to regulate signaling parallel to Ras. Our data imply that SHP-2 inhibition will ablate both normal and neoplastic hematopoietic progenitors. This may have a therapeutic role in preparing patients with JMML and other high risk MPDs for hematopoietic stem cell transplantation.

Hematopoietic, angiogenic and eye defects in Meis1 mutant animals.

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.

Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver.

Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II-null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non-cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

AbstractPlatelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

Lack of the Polycomb-group gene rae28 causes maturation arrest at the early B-cell developmental stage.

The rae28 gene (rae28) is a murine homologue of the Drosophila polyhomeotic gene, which is a member of the Polycomb-group genes. In this study, we examined the role of rae28 in lymphocyte development. Because homozygous rae28-deficient (rae28-/-) mice died in the perinatal period, we examined lymphocyte development by generating chimeric mice reconstituted with green fluorescence protein-labeled mutant fetal liver cells as well as in in vitro culture systems. We further examined RAE28 expression by reverse transcriptase polymerase chain reaction assay in human leukemic cells with B-lineage acute lymphoblastic leukemia (ALL). Severe B-cell maturation arrest was observed in rae28-/- between pro- and pre-B lymphocyte stages. B-cell development was also delayed in heterozygous neonates. Furthermore, interleukin-7-dependent colony-forming ability was impaired not only in homozygous lymphocytes but also in heterozygotes. Its human homologue, RAE28, is located on chromosome 12p13, which frequently is associated with chromosomal abnormalities and loss of heterozygosity in patients with hematologic malignancies. To determine whether a link exists between RAE28 and leukemia, we examined RAE28 expression in leukemic cells from pediatric patients with B-lineage ALL. RAE28 expression was not detected in four B-cell precursor ALL cases of a total of 43 examined, although RAE28 is normally expressed constitutively during the process of B-cell maturation as assessed in isolated cell populations. rae28 plays an important role in the early B-cell developmental stage in a gene dosage-dependent manner. Furthermore, the human RAE28 locus may provide a candidate gene causing the molecular pathogenesis of childhood B-cell precursor ALL.

Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1.

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.