ALBERT

Description: CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis, including the stem cell compartment. In the present study, we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp+/− mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead, it stimulates myeloid differentiation, which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased, the bone formation rate was similar to that found in wild-type mice. At the molecular level, we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly, potentially important regulatory proteins, endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5), were increased on Crebbp+/− endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation.

Description: Key Points With a few exceptions, the histologic and cytologic characteristics of myelodysplasia are similar in humans and mice. As in humans, MDS and MDS/MPN are distinct diseases in mice; mouse models of these diseases can serve as useful research tools.

Description: Abstract 1705 Myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) are myeloid malignancies that display features of both MDS and MPN, but cannot be properly assigned to either MDS or MPN. It is currently not known whether it originates from the hematopoietic stem cell (HSC) compartment (like MDS), from a more committed myeloid progenitor population, or a combination thereof. Fifteen to 40% of MDS/MPN patients develop acute myeloid leukemia (AML); whether the transformation occurs in a particular cell population is also unknown. We previously demonstrated that mice heterozygous for the CREB binding protein gene (Crebbp) develop MDS/MPN at 9–12 months of age and ∼40% of them progress to develop a hematologic malignancy. Thus, Crebbp+/− mice are an excellent model to address the before mentioned questions, which is important for the development of better strategies to treat MDS/MPN. For this purpose, we harvested and combined bone marrow from 1.5-year old Crebbp+/− mice (10 donors per experiment, thereby ensuring that the marrow of ∼4 donors harbored malignant hematopoietic cells) and transplanted it into lethally irradiated, wild-type recipients. Groups of mice either received unfractionated whole bone marrow (WBM) or populations purified by fluorescence-activated cell sorting. Naive Crebbp+/− mice had demonstrated functional and/or quantitative abnormalities in long-term and short-term HSCs, common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) and we therefore focused on these populations. All transplant recipients also received unfractionated wild-type “helper cells” to increase survival. Mice were closely monitored and those suspected of having developed a hematopoietic disease were sacrificed and their hematopoietic system analyzed. Four independent experiments were performed and data were combined for analysis. Among the 18 recipients who received Crebbp+/− WBM, 8 recipients (44%) developed an early-onset AML with myelofibrosis, 2–7 months after the transplant, which was not preceded by MDS. The other 10 recipients (56%) developed MDS/MPN, 12–18 months after the transplant. These mice displayed ineffective hematopoiesis, evidenced by a normocellular bone marrow, significant leukopenia, and trilineage dysplasia. One of these 10 Crebbp+/− WBM recipients that developed MDS/MPN subsequently progressed to AML. In contrast, none of the 15 recipients of Crebbp+/− HSCs (defined as Lin−Sca-1+c-Kit++ (LSK) cells, including long-term and short-term progenitors, as well as lymphoid-restricted progenitors) developed early-onset AML. Instead, 1 developed MDS/MPN while the remainder developed MDS by 11–17 months after the transplant, with one of them progressing to a disease resembling human mature T-cell leukemia. Transplantation of Crebbp+/− CMPs and GMPs also failed to cause early-onset AML and, as expected, gave rise to extremely low long-term reconstitution. Thus, these mice were mostly reconstituted by the co-transplanted wild-type “helper cells”. However, unexpectedly, 9 out of 24 (38%) showed 10% dysplastic cells and 2 (8%) developed MPD or AML with myelofibrosis. Control mice, i.e., recipients of wild-type BM cells remained healthy for the duration of the experiments. The results of these transplantation experiments show that in this mouse model, MDS/MPN is transplantable. However, it requires transplantation of WBM, since the transplantation of LSK cells resulted in MDS, suggesting that the microenvironment may play a crucial role in the etiology of MDS/MPN. This notion is in concordance with our previous study, demonstrating that Crebbp+/− mice transplanted with wild-type cells developed MPD that originated from the transplanted wild-type cells. This notion is further supported by the outcome of the CMP and GMP transplantation experiments, suggesting that abnormal myeloid progenitors are also important factors for MDS/MPN disease development. Moreover, malignant transformation seems to occur in a non-LSK cell that is more differentiated than CMPs and GMPs. Alternatively, malignant transformation requires all hematopoietic and non-hematopoietic cells to be present, again suggesting that MDS/MPN is a complex disease where both the hematopoietic compartment and its bone marrow microenvironment are affected. Disclosures: No relevant conflicts of interest to declare.

Description: Characterization of chromosomal translocations in leukemias has led to a better understanding of the causes of disease, as well as to new cures. The transcriptional co-activators p300 and CBP are not only targets of translocations, but perturbation of their normal function is central to the transforming effects of most translocation oncogenes in acute myelogenous leukemia. In our prior studies, we demonstrated that genetic inactivation of p300 or CBP function resulted in perturbed hematopoietic differentiation, and the development of hematologic malignancies. However, given the large size and many functional domains of p300/CBP, it has been difficult to determine which specific domains and activities are necessary for them to suppress hematologic tumor formation. We have now developed a novel forward-genetic mouse model that allows us to systematically evaluate p300 structure-function and determine which functional domain mediates hematologic tumor suppression in vivo. By systematically mutating functional domains within p300 and expressing the mutants in embryonic stem (ES) cells, we have been able to generate a series of chimeric mice lines, each expressing a particular mutation in p300. Our current work entails the careful examination of the peripheral blood and bone marrow for each mouse line in order to determine the importance of specific p300 domains for the suppression of hematologic malignancies that arise in the absence of p300. Moreover, this study is helping us gain insight into the mechanisms responsible for the establishment and maintenance of normal, functional stem and progenitor cell populations. This information will be important for elucidating the transcriptional programs that are likely disrupted in leukemias, thus revealing molecular mechanisms that can be used to direct the development of new cures.

Description: Retrovirus-mediated gene transfer into long-lived human pluripotent hematopoietic stem cells (HSCs) is a widely sought but elusive goal. A major problem is the quiescent nature of most HSCs, with the perceived requirement for ex vivo prestimulation in cytokines to induce stem cell cycling and allow stable gene integration. However, ex vivo culture may impair stem cell function, and could explain the disappointing clinical results in many current gene transfer trials. To address this possibility, we examined the ex vivo survival of nonobese diabetic/severe combined immune-deficient (NOD/SCID) repopulating cells (SRCs) over 3 days. After 1 day of culture, the SRC number and proliferation declined twofold, and was further reduced by day 3; self-renewal was only detectable in noncultured cells. To determine if the period of ex vivo culture could be shortened, we used a vesicular stomatitis virus G protein (VSV-G) pseudotyped retrovirus vector that was concentrated to high titer. The results showed that gene transfer rates were similar without or with 48 hours prestimulation. Thus, the use of high-titer VSV-G pseudotyped retrovirus may minimize the loss of HSCs during culture, because efficient gene transfer can be obtained without the need for extended ex vivo culture.

Description: Cyclic adenosine monophosphate response element binding (CREB)–binding protein (CBP) and p300 are multidomain transcriptional coactivators that help assemble large regulatory complexes at sites of active transcription. Nullizygosity of CBP or p300 results in pervasive defects in hematopoiesis. To systematically assess the structural domains of p300 required for normal hematopoiesis, we used recombinase-mediated cassette exchange to create an allelic series of coisogenic embryonic stem cells, each expressing a different mutant of p300 from the endogenous locus. We found that deletion of either the KIX or CH1 domain caused profound and pervasive defects in hematopoiesis, whereas the loss of most other domains had only lineage-restricted effects. When expressed from the p300 locus, an extra copy of CBP largely compensated for a lack of p300. Surprisingly, mutation of the p300 histone acetyltransferase (HAT) domain had minimal effects on hematopoiesis, and actually increased progenitor and stem cell numbers and proliferative potential. Our results suggest that, in distinct contrast to other organ systems, HAT activity does not provide a critical function for hematopoietic development and emphasizes the importance of enzyme-independent functions of p300.

Description: The results of previous studies have shown that the development of hematopoiesis during fetal life can occur in the absence of Steel factor (SF ) signaling. On the other hand, impairment of this mechanism can severely compromise the ability of cells from adult bone marrow to regenerate hematopoiesis on their transplantation into myeloablated recipients. This apparent paradox could result from changes during ontogeny in the responsiveness of hematopoietic stem cells to regulators that may substitute for SF as well as from differences in the availability of such factors during embryogenesis and in the myeloablated adult. To investigate these possibilities, we studied the effect of W41 and W42 mutations on the numbers, phenotype, and posttransplant self-renewal behavior of primitive hematopoietic cells present in the fetal liver (FL) of 14.5-day-old mouse embryos. In W41/W41 FL, day-12 spleen colony-forming units and long-term culture-initiating cells appeared both quantitatively and qualitatively similar to their counterparts in the FL of +/+ embryos. W41/W41 FL also contained near normal numbers (≈50% of controls) of transplantable lymphomyeloid stem cells with competitive reconstituting ability in myeloablated adult +/+ recipients (as assessed for up to at least 16 weeks posttransplant). Moreover, both the original phenotype of these W41/W41 competitive repopulating units (CRUs) and their clonal posttransplant output of mature progeny were normal. Similarly, when myeloablated adult +/+ mice were cotransplanted with 5 × 104 +/+ FL cells and a sevenfold to 70-fold excess of W41/W41 FL CRUs, the contribution of the +/+ FL CRUs to the circulating white blood cell count present 5 weeks later was markedly reduced as compared with that of mice that received only +/+ FL cells. However, over the next 3 months, the proportion of mature white blood cells that were derived from +/+ precursors increased significantly (P 〈 .002) in all groups (to ≥30%), indicating that the ability to sustain hematopoiesis beyond 5 weeks is more SF-dependent than the ability to initially reconstitute both lymphoid and myeloid compartments. Cells from individual FL of W42/+ matings also showed an initial ability (at 7 to 8 weeks posttransplant) to competitively repopulate both lymphoid and myeloid compartments of myeloablated +/+ adult recipients. However, in contrast to recipients of normal or W41/W41 FL cells, the repopulation obtained with the W42 mutant stem cells was transient. Secondary transplants confirmed the inability of the W42 mutant cells to regenerate or even maintain a population of transplantable stem cells. Taken together with previous results from studies of CRUs in adult W mice, these findings support the concept of changes in the way hematopoietic stem cells at different stages of development respond to the stimulatory conditions evoked in the myeloablated recipient. In addition, they provide the first definitive evidence that SF is a limiting physiological regulator of sustained hematopoietic stem cell self-renewal in vivo.

Description: In recent studies on acute myeloid leukemia (AML), genes involving DNA methylation (DNMT3A, IDH1/2, TET2) were identified as frequently mutated. Around 17% of AML patients were found to have a mutation in isocitrate dehydrogenase-1 or -2 (IDH1/2). In these patients, 2-hydroxylglutarate (2-HG), which is generated from a neomorphic activity of mutant IDH1/2, accumulates and inhibits DNA hydroxylmethylation mediated by TET family proteins. Interestingly, mutations in the de novo DNA methyltransferase 3A (DNMT3A) and IDH1/2 mutations co–occur in a statistically significant portion of AML patients. In current known mouse models, neither Dnmt3a knockout (KO) and IDH1/2 mutation alone initiates overt hematopoietic malignancy, leading to our hypothesis that Dnmt3a and IDH1/2 mutations may act synergistically to initiate hematopoietic malignancy. To create a Dnmt3a knockout IDH1/2 mutant double mutant mouse model, we have employed a transplantation approach to create Idh2R140Q (one of the abundant IDH2 mutation in AML patients) overexpressing stem cells on a Dnmt3a KO (Dnmt3a–/–) and wild-type (WT) background by retroviral transduction. Idh2WT overexpression was also used as a control group. With a latency of 180 days after transplantation, a hematopoietic disease with a median survival of 197 days was observed in the Dnmt3a–/––Idh2R140Q group. Anemia, thrombocytopenia and monocytosis were observed in morbid Dnmt3a–/––Idh2R140Q mice. The pathological examination of Dnmt3a–/––Idh2R140Q mice showed myelodysplasia in one or more lineages, an accumulation of less differentiated myeloid progenitors in the bone marrow and pronounced extramedullary hematopoiesis in the spleen. Moreover, approximately 20% of Dnmt3a–/––Idh2R140Q mice developed AML. Together, these features led to the diagnosis of MDS/MPN (Myelodysplastic Syndrome / Myeloproliferative Neoplasms) with high transformation rate to AML. In comparison, WT–Idh2R140Q mice also developed less severe MDS/MPN characterized by myeloid differentiation bias and extramedullary hematopoiesis without lethality in one year after bone marrow transplantation. The MDS/MPN of Dnmt3a–/––Idh2R140Q developed into MPD after secondary transplantation with Lin- cKit+ progenitors, while the same progenitors from the control genotypes did not cause hematopoietic diseases in secondary transplantation . The profiling of 2-HG with gas chromatography mass spectrometry (GC-MS) in the serum of morbid mice transplanted with Dnmt3a–/––Idh2R140Q showed an 80-fold increase compared to normal mouse serum, while the 2-HG content in mice transplanted with WT–Idh2R140Q cells was 10-fold higher than that of normal mouse serum. This suggests that Dnmt3a loss-of-function can promote the synthesis of 2-HG by the Idh2R140Q mutation. The metabolomic profiling on cKit+ bone marrow cells identifies 43 metabolites differentially present in malignant Dnmt3a–/––Idh2R140Q cells compared with groups of other genotypes. Furthermore, the unsupervised cluster analysis shows Dnmt3a–/––Idh2R140Q cells have a distinct metabolome profile compared with cells of other genotypes. This suggests a synergistic effect on metabolome between the two genetic backgrounds. The essential amino acid and glycolysis pathway metabolites– are among the most enriched differential present metabolites. In addition, the glutamine anaplerosis pathway is highly upregulated in the Dnmt3a–/––Idh2R140Q group indicated by the increase of a-ketoglutarate and glutamate. In contrast, the WT – Idh2R140Q and Dnmt3a KO groups have no alteration in glutamine anaplerosis pathway metabolites, indicating a synergy on 2-HG synthesis between Dnmt3a knockout and Idh2R140Q. Moreover, an excess of glutamine during in vitro culture significantly promotes the colony forming ability Dnmt3a–/––Idh2R140Q HSPCs, while there’s no effect in Dnmt3 Idh2WT and WT–Idh2R140Q HSPCs. In summary, our research shows for the first time, Dnmt3a loss-of-function promotes 2-HG synthesis with mutant Idh2 and strongly aggravates the phenotype induced by Idh2 mutation. Our research also shows the synergistic effect on the metabolome of two genetic backgrounds. These data likely explain the high frequency of co-mutations between DNMT3A and IDH1/2 that promotes aggressive AML in patients. Disclosures: No relevant conflicts of interest to declare.