Age-related epigenetic drift in the pathogenesis of MDS and AML.
ABSTRACT: The myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS, and 331 (9.6%) genes hypermethylated in RUNX1 MDS. Thirty-two percent of genes in common between the two models (2/3 NHD13 mice and 2/3 RUNX1 mice) were also hypermethylated in at least two of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressive drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.
Project description:Genomic DNA is isolated from spleen (RUNX1 model and normal control) and bone marrow (NHD13 model and normal control). We used MCAM to identify genes that have methylation changes. Two-condition experiment, MDS model mouse vs. normal control.
Project description:BACKGROUND: Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown. METHODS: We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors. RESULTS: We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML. CONCLUSIONS: Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.
Project description:Aberrant DNA methylation often silences transcription of tumor-suppressor genes and is considered a hallmark of myeloid neoplasms. Similarly, histone deacetylation represses transcription of genes responsible for cell differentiation/death. A previous clinical study suggested potential pharmacodynamic antagonism between histone deacetylase inhibitors (HDACi) and DNA hypomethylating agents (HMA). Herein, to determine such antagonism, we used MDS/AML lines and NHD13 transgenic mice, and demonstrated that treatment with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) significantly decreased TET2 expression and global 5-hydroxymethylcytosine (5hmC) levels. Mechanistically, our RNAi screen revealed that HDAC4 was responsible for maintaining TET2 levels. Accordingly, HDAC4 knockout reduced expression levels of MTSS1, a known TET2 target, an event associated with decreased 5hmC enrichment on the MTSS1 enhancer. Retrospective analysis of GEO datasets demonstrated that lower HDAC4 levels predict worse prognosis for AML patients. In an MDS-L xenografted immunodeficient mouse model, vitamin C co-treatment prevented TET2 loss of activity seen following SAHA treatment. Accordingly, vitamin C co-treatment further reduced MDS-L cell engraftment relative to SAHA alone. In summary, our findings suggest that co-administration of a TET2 agonist with pan-HDACi treatment could effectively counter potential diminution in TET2 activity resulting from pan-HDACi treatment alone, providing a rationale for evaluating such combinations against high-risk MDS/AML.
Project description:The myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it has been suggested that additional mutations lead to progression of MDS to AML, the causative agent(s) for such mutations remains unclear. Oxidative stress is a potential cause, therefore, we evaluated levels of reactive oxygen species (ROS) in NUP98-HOXD13 (NHD13) transgenic mice, a murine model for MDS. Increased levels of ROS were detected in bone marrow nucleated cells (BMNC) that express CD71, a marker for cell proliferation, as well as immature, lineage negative bone marrow nucleated cells from NHD13 mice. In addition to the increase in ROS, increased DNA double strand breaks and activation of a G2/M phase cell cycle checkpoint were noted in NHD13 BMNC. Finally, using an in vivo assay for mutation frequency, we detected an increased mutation frequency in NHD13 BMNC. These results suggest that oxidative stress may contribute to disease progression of MDS to AML through ineffective repair of DNA damage and acquisition of oncogenic mutations.
Project description:Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with resultant cytopenias. Increased apoptosis and aberrantly functioning progenitors are thought to contribute to this phenotype. As is the case for other malignancies, overcoming apoptosis is believed to be important in progression toward acute myeloid leukemia (AML). Using the NUP98-HOXD13 (NHD13) transgenic mouse model of MDS, we previously reported that overexpression of the anti-apoptotic protein BCL2, blocked apoptosis and improved cytopenias, paradoxically, delaying leukemic progression. To further understand this surprising result, we examined the role of p53 and its pro-apoptotic effectors, PUMA and NOXA in NHD13 mice. The absence of p53 or PUMA but not NOXA reduced apoptosis and expanded the numbers of MDS-repopulating cells. Despite a similar effect on apoptosis and cell numbers, the absence of p53 and PUMA had diametrically opposed effects on progression to AML: absence of p53 accelerated leukemic progression, while absence of PUMA significantly delayed progression. This may be explained in part by differences in cellular responses to DNA damage. The absence of p53 led to higher levels of ?-H2AX (indicative of persistent DNA lesions) while PUMA-deficient NHD13 progenitors resolved DNA lesions in a manner comparable to wild-type cells. These results suggest that targeting PUMA may improve the cytopenias of MDS without a detrimental effect on leukemic progression thus warranting further investigation.
Project description:The NUP98-HOXD13 (NHD13) fusion gene occurs in patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). We reported that transgenic mice expressing NHD13 develop MDS, and that more than half of these mice eventually progress to acute leukemia. The latency period suggests a requirement for at least 1 complementary event before leukemic transformation. We conducted a candidate gene search for complementary events focused on genes that are frequently mutated in human myeloid leukemia. We investigated 22 ANLL samples and found a high frequency of Nras and Kras mutations, an absence of Npm1, p53, Runx1, Kit and Flt3 mutations, and a single Cbl mutation. Our findings support a working hypothesis that predicts that ANLL cases have one mutation which inhibits differentiation, and a complementary mutation which enhances proliferation or inhibit apoptosis. In addition, we provide the first evidence for spontaneous collaborating mutations in a genetically engineered mouse model of ANLL.
Project description:Programmed cell death or apoptosis is a prominent feature of low-risk myelodysplastic syndromes (MDS), although the underlying mechanism remains controversial. High-risk MDS have less apoptosis associated with increased expression of the prosurvival BCL2-related proteins. To address the mechanism and pathogenic role of apoptosis and BCL2 expression in MDS, we used a mouse model resembling human MDS, in which the fusion protein NUP98-HOXD13 (NHD13) of the chromosomal translocation t(2;11)(q31;p15) is expressed in hematopoietic cells. Hematopoietic stem and progenitor cells from 3-month-old mice had increased rates of apoptosis associated with increased cell cycling and DNA damage. Gene expression profiling of these MDS progenitors revealed a specific reduction in Bcl2. Restoration of Bcl2 expression by a BCL2 transgene blocked apoptosis of the MDS progenitors, which corrected the macrocytic anemia. Blocking apoptosis also restored cell-cycle quiescence and reduced DNA damage in the MDS progenitors. We expected that preventing apoptosis would accelerate malignant transformation to acute myeloid leukemia (AML). However, contrary to expectations, preventing apoptosis of premalignant cells abrogated transformation to AML. In contrast to the current dogma that overcoming apoptosis is an important step toward cancer, this work demonstrates that gaining a survival advantage of premalignant cells may delay or prevent leukemic progression.
Project description:The myelodysplastic syndromes (MDS) comprise a group of premalignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it is well established that many malignancies can be transplanted, there is little evidence to demonstrate that a premalignant disease entity, such as MDS or colonic polyps, can be transplanted and subsequently undergo malignant transformation in vivo. Using mice that express a NUP98-HOXD13 (NHD13) transgene in hematopoietic tissues, we show that a MDS can be transplanted to WT recipients. Recipients of the MDS bone marrow displayed all of the critical features of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML. Even when transplanted with a 10-fold excess of WT cells, the NHD13 cells outcompeted the WT cells over a 38-week period. Limiting-dilution experiments demonstrated that the frequency of the cell that could transmit the disease was approximately 1/6,000-1/16,000 and that the MDS was also transferable to secondary recipients as a premalignant condition. Transformation to AML in primary transplant recipients was generally delayed (46-49 weeks after transplant); however, 6 of 10 secondary transplant recipients developed AML. These findings demonstrate that MDS originates in a transplantable, premalignant, long-term repopulating, MDS-initiating cell.
Project description:SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.
Project description:Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic diseases which have a high risk of progressing to acute myeloid leukemia. MDS patients have immunologic deficiency, including T and B cells dysfunction. Follicular T helper cells (Tfh, CD4+CXCR5+) are an important subset of helper T cells which help to the formation of germinal centers and B cells differentiation. In this study, we investigated the proportion and function of Tfh using NUP98-HOXD13 transgenic (NHD13) mice model with MDS phenotype. The proportion of Tfh from bone marrow and spleen of NHD13 mice decreased compared with wild type (WT) mice tested by flow cytometry. In NHD13 mice spleens, there were decreased CXCR5+ cells and increased PD-1+ cells using immunohistochemistry. The active markers (ICOS, CD40L and OX40) expressed on Tfh of NHD13 mice were decreased. In contrast, PD-1 expression on Tfh of NHD13 mice was higher than that of WT mice. After coculture with Tfh from NHD13 mice, IgG and IgM production of B cells were decreased. In conclusion, the proportion and function of Tfh in the MDS mice model were altered. The dysfunction and reduction of Tfh may inhibit B cells differentiation and antibody production. Abnormal Tfh might contribute to the immune tolerance promoting the progression of MDS.