Loss of TET2 Function in Myelodysplastic Syndrome Results in Intragenic Hypermethylation and Alterations in mRNA Splicing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 775-775 ◽  
Author(s):  
Allegra M Lord ◽  
Kendell Clement ◽  
Rebekka K. Schneider ◽  
McConkey Marie ◽  
Michelle C. Chen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Rna Sequencing ◽  
Murine Model ◽  
Splice Variant ◽  
Transcript Abundance ◽  
Differential Methylation ◽  
Mrna Splicing ◽  
Hematopoietic Differentiation

Abstract One of the most commonly mutated genes in myelodysplastic syndrome (MDS) is TET2, which actively demethylates DNA through a 5-hydroxymethylcytosine (hmC) intermediate. MDS is a disease of impaired hematopoietic differentiation, and murine models of TET2 loss display an expansion of the hematopoietic stem and myeloid progenitor pool. Previous studies examining the effect of TET2 loss on DNA methylation in myeloid malignancies have reached conflicting conclusions. We therefore aimed to better characterize the effects of TET2 loss on methylation within a well-defined set of MDS patients. We compared the DNA methylation status of bone marrow aspirates from TET2 mutant or WT cases (n=74) from a large, well-characterized cohort of MDS patients by reduced-representation bisulfite sequencing (RRBS). In order to focus specifically on the effects of TET2, each TET2 mutant patient sample was matched with a TET2 WT sample for all other factors known to affect DNA methylation, including age, sex, and disease subtype. Importantly, samples were also matched for presence of other somatic mutations, including known oncogenes and tumor suppressors. We found that global methylation was significantly increased in the TET2 mutant group. In patients with the highest TET2 mutant allele burden, the differential methylation was even greater. We next examined specific sites of differential methylation. Interestingly, though promoter regions are enriched for hmC and Tet family binding in murine ES cells, we found no difference in methylation between groups at promoters (TSS +/- 1kb). We found no difference in methylation between TET2 mutant and WT patient samples within enhancers. When we examined intragenic regions, however, we found a significant increase in methylation at intron-exon boundaries (+/- 500bp, excluding promoters) in the TET2 mutant group, which accounted for the majority of global differences in methylation. We further examined our findings in a conditional murine model of Tet2 loss. RRBS of Tet2+/+, Tet2+/- and Tet2-/- Mx1Cre+ HSPC (lin- c-Kit+ Sca-1+; HSPC) showed analagous patterns of methylation to TET2 mutant MDS samples: hypermethylation was seen globally and locally at intron-exon boundaries, but not at promoters or enhancers, in Tet2+/- and Tet2-/- vs Tet2+/+ cells. We performed hmC-DNA immunoprecipitation followed by semi-quantitative RT-PCR for a subset of the regions hypermethylated in Tet2-/- HSPCs, and found enrichment of hmC at these sites in Tet2+/+ versus Tet2-/- DNA, demonstrating that the observed hypermethylation is due to loss of hmC conversion following loss of TET2. DNA methylation at intron-exon boundaries affects mRNA splicing through interactions with RNA polymerase II binding partners. RNA sequencing of bone marrow aspirates from 2 TET2 mutant and matched WT patient pairs revealed few changes in absolute transcript abundance. We defined a gene set comprised of intragenic regions hypermethylated in TET2 mutant patients that also involved known splice variants. Using this approach, we observed shifts in splice variant transcript abundance within individual genes, specifically involving regions of aberrant hypermethylation in TET2 mutant patients. We expanded on these results with RNA sequencing of Tet2+/+ and Tet2-/- HSPCs and observed similar shifts in splice variant abundance among genes with differential intragenic methylation. In aggregate, our data show that loss of TET2 in MDS patients and in a Tet2 knockout murine model results in a global increase in DNA methylation and that hypermethylation due specifically to loss of TET2 is localized to intron-exon boundaries. Furthermore, we find that increased methylation at sites of alternate splicing correlates with shifts in splice variant ratios across a broad subset of genes. We hypothesize that this alteration in splice variant abundance may affect hematopoietic differentiation and promote the development of an MDS phenotype. Disclosures Bejar: Genoptix: Consultancy, Honoraria, Licensed IP Other; Celgene: Consultancy, Honoraria.

Tissue methylation and demethylation influence translesion synthesis DNA polymerases (TLS) contributing to the genesis of chromosomal abnormalities in myelodysplastic syndrome

2020 ◽  
pp. jclinpath-2020-207131
Author(s):  
Gabrielle Melo Cavalcante ◽  
Daniela Paula Borges ◽  
Roberta Taiane Germano de Oliveira ◽  
Cristiana Libardi Miranda Furtado ◽  
Ana Paula Negreiros Nunes Alves ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Chromosomal Abnormalities ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Normal Karyotype ◽  
Dna Demethylation ◽  
Repair System ◽  
Low Expression

AimsDNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of genes related to DNA repair system, chronic immune response and cell cycle.MethodsWe evaluated the tissue DNA methylation/hydroxymethylation in bone marrow trephine biopsies of 73 patients with MDS, trying to correlate with the mRNA expression of 21 genes (POLH, POLL, REV3L, POLN, POLQ, POLI, POLK, IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF6, IRF-7, IRF-8,IRF-9, MAD2, CDC20, AURKA, AURKB and TPX2).ResultsThe M-score (5mC) was significantly higher in patients with chromosomal abnormalities than patients with normal karyotype (95% CI –27.127779 to –2.368020; p=0.022). We observed a higher 5mC/5hmC ratio in patients classified as high-risk subtypes compared with low-risk subtypes (95% CI –72.922115 to –1.855662; p=0.040) as well as patients with hypercellular bone marrow compared with patients with normocellular/hypocellular bone marrow (95% CI –69.189259 to –0.511828; p=0.047) and with the presence of dyserythropoiesis (95% CI 17.077703 to 51.331388; p=0.001). DNA pols with translesion activity are significantly influenced by methylation. As 5mC immunoexpression increases, the expressions of POLH (r=−0.816; r2 =0.665; p=0.000), POLQ (r=−0.790; r2=0.624; p=0.001), PCNA (r=−0.635; r2=0.403; p=0.020), POLK (r=−0.633; r2=0.400; p=0.036 and REV1 (r=−0.578; r2=0.334; p=0.049) decrease.ConclusionsOur results confirm that there is an imbalance in the DNA methylation in MDS, influencing the development of chromosomal abnormalities which may be associated with the low expression of DNA polymerases with translesion synthesis polymerases activity.

Detection of Differential Mitotic Cell Ages in Bone Marrow CD34+ Cells Selected from Patients with Myelodysplastic Syndrome and Acute Leukemia by Pyrosequencing Analysis of An Epigenetic Molecular Clock DNA Signature

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2693-2693
Author(s):  
Maximilian Mossner ◽  
Olaf Hopfer ◽  
Claudia D Baldus ◽  
Uwe Neumann ◽  
Anke Kmetsch ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Molecular Clock ◽  
Mitotic Cell ◽  
Healthy Individuals ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
B Lineage

Abstract Introduction: Disturbed proliferation and differentiation are the most crucial oncogeneic factors leading to malignant turnover of hematopoiesis in myeloid malignancies. Therefore, estimating the lifetime proliferation status of malignant hematopoietic cells is critical. Recently the hypothesis of an epigenetic molecular clock has been corroborated. Depending on the accumulation of CpG methylation errors throughout life after each cell division it is possible to measure an increased DNA methylation of formerly unmethylated CpG islands and subsequently relate it to the mitotic cell age. In order to elucidate the importance of disturbed proliferation in hematologic diseases we initiated a novel approach for profiling mitotic ages of hematopoietic cells in myelodysplastic syndrome and acute leukemia. Patients & Methods: Bone marrow (BM) cells of patients with myelodysplastic syndrome (MDS, IPSS-low/int-1-risk n=23, IPSS-int-2/high-risk n=27), acute myeloid leukemia (AML, n=55), acute lymphoblastic leukemia (ALL, T-lineage n=40, B-lineage n=8), and of age matched healthy individuals (n=24) were analyzed. In addition, selection of CD34+ cells was performed in MDS (n=43), in AML (n=10) as well as in healthy BM samples (n=31). CD19+ peripheral blood cells from healthy donors (n=13) served as an additional control. Genomic DNA was isolated and bisulfite converted using standard TRIZOL technique (Invitrogen, Carlsbad/CA, USA) followed by EpiTect-Bisulfite-Kit conversion (Qiagen, Hilden, Germany). PCR amplification of a CpG rich 3′ site of the Cardiac Specific Homeobox gene (CSX), considered as an epigenetic molecular clock locus, was performed as previously reported. DNA methylation was quantitative measured using the PyroMark ID Pyrosequencing system (Biotage, Uppsala, Sweden). Quantitative DNA methylation data are presented with mean ± S.E.M. Results: In MDS int-2/high-risk specific DNA methylation of BM (26.6 ± 1.8 %) and CD34+ (28.6 ± 2.7 %) was significant higher compared to low/int-1-risk MDS (BM: 19.2 ± 1.6 %, p=0.0047, CD34+: 18.7 ± 2.4 %, p=0.0093) and healthy donors (BM: 17.8 ± 0.5 %, CD34+: 17.0 ± 0.4 %, p<0.0001). Furthermore, AML BM samples showed significant higher methylation of 34.2 ± 1.7 % compared to MDS BM int-2/high-risk samples (p=0.0081). Interestingly we could detect significant higher differences in CSX methylation between paired BM/CD34+ samples in MDS low/int-1-risk, but not in MDS int-2/high-risk or AML compared to age matched healthy individuals (p=0.0063). Notably, T-lineage ALL samples did show a remarkable high mean methylation of 61.7 ± 3.1 %. However, B-lineage ALL analysis revealed a similar methylation pattern in comparison to healthy CD19+ cells (26.1 ± 1.4 % and 25.1 ± 1.4% respectively). Discussion: The significant higher CSX methylation in AML compared to int-2/high-risk and in int-2/high-risk compared to low/int-1-risk MDS or healthy individuals could possibly be considered as a disease stage related molecular marker. The intra-individual similarity of CSX methylation levels between BM and CD34+ cells in int-2/high-risk MDS patients supports the theory of a stem cell origin of this disease subgroup, whereas low/int-1-risk MDS samples reveal higher differences possibly pointing to an origin in a more differentiated progenitor cell. However, the observation of higher mitotic ages in T-lineage but not B-lineage ALL raises questions about the role of cell proliferation in distinct lymphoblastic leukemias. In summary, the determination of mitotic cell ages by quantitative DNA methylation analysis could contribute to the molecular classification of hematological malignancies and may further be used for riskassessment in patients with MDS.

Recurrent DNMT3A Mutations In Patients with Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 608-608
Author(s):  
Matthew J. Walter ◽  
Dong Shen ◽  
Jin Shao ◽  
Li Ding ◽  
Marcus Grillot ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Sample Size ◽  
De Novo ◽  
Small Sample Size ◽  
Dna Methyltransferases ◽  
Small Sample ◽  
Mutation Status ◽  
Cd34 Cells

Abstract Abstract 608 Myelodysplastic syndrome (MDS) genomes are characterized by global DNA hypomethylation with concomitant hypermethylation of gene promoter regions compared to CD34+ cells from normal bone marrow samples. Currently, the underlying mechanism of altered DNA methylation in MDS genomes and the critical target genes affected by methylation remain largely unknown. The methylation of CpG dinucleotides in humans is mediated by DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B. DNMT3A and DNMT3B are the dominant DNA methyltransferases involved in de novo DNA methylation and act independent of replication, whereas DNMT1 acts predominantly during replication to maintain hemimethylated DNA. The function of these proteins in cancer cells is less well defined. Our group recently found that DNMT3A mutations are common in de novo acute myeloid leukemia (62/281 cases, 22%) and are associated with poor survival (Ley, et al, unpublished), providing a rationale for examining the mutation status of DNMT3A in MDS patients. MDS cases (n=150) were classified according to the French-American-British (FAB) system. The patients included refractory anemia (RA; n=67), RA with ringed sideroblasts (RARS; n=5), RA with excess blasts (RAEB; n=72), and RA with excess blasts in transformation (RAEB-T; n=6). The median International Prognostic Scoring System (IPSS) score was 1 (range 0–3), and the median myeloblast count was 4 (range 0–28%). We designed and validated 28 primer pairs covering the coding sequences and splice sites of all 23 exons for DNMT3A. Paired DNA samples were obtained from the bone marrow (tumor) and skin (normal) of each patient so that somatic mutations could be distinguished from inherited variants/polymorphisms. 17,120 reads were produced by capillary sequencing, providing at least 1X coverage for 82.6% of the target sequence (low/no coverage was obtained for 2 out of 28 amplicons). A semiautomated analysis pipeline was used to identify sequence variants and we restricted our analysis to nonsynonymous and splice site nucleotide changes. All mutations were confirmed by independent PCR and sequencing. We identified nonsynonymous DNMT3A mutations in 12/150 bone marrow samples (8% of cases). All the mutations were heterozygous (10 missense, 1 nonsense, 1 frameshift) and were computationally predicted (by SIFT and/or PolyPhen2) to have deleterious functional consequences. DNMT3A mRNA is expressed in normal CD34+ bone marrow cells and was expressed in all MDS patient samples tested (n=28), independent of mutation status. There was no difference in the expression level of total DNMT3A mRNA in CD34+ cells harvested from mutant (n=3) vs. non-mutant MDS samples (n=25). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/12 mutations. The clinical characteristics of the 12 patients with DNMT3A mutations were similar to those of the 138 patients without mutations. Specifically, DNMT3A mutations were present in all MDS FAB subtypes (excluding CMML which was not tested) and in patients with IPSS scores ranging from 0–3. Mutations were not associated with a specific karyotype. In addition, there was no correlation between mutation detection and the myeloblast count of the banked bone marrow specimen, suggesting that mutations were not missed due to the cellular heterogeneity in the samples. We compared the overall (OS) and event-free survival (EFS) of the 12 patients with DNMT3A mutations vs. 138 patients without a mutation and observed a significantly worse OS in patients with mutations (p=0.02), with a median survival of 433 and 945 days, respectively. There was a trend towards worse EFS for patients with mutations (p=0.05). A multivariate analysis for outcomes could not be performed due to the small sample size of patients with mutations, indicating that a larger cohort from a clinical trial will be needed to properly address the affect of DNMT3A mutations on outcomes. The small sample size also precluded us from addressing whether the response to the hypomethylating agents 5-azacytidine or decitabine correlated with the mutation status of DNMT3A. If validated in larger cohort studies, we propose that DNMT3A mutation status could help risk stratify de novo MDS patients for more aggressive treatment early in their disease course. Disclosures: Westervelt: Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau. DiPersio:Genzyme: Honoraria.

scholarly journals Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 827-827
Author(s):  
Cara Lunn Shirai ◽  
James N Ley ◽  
Brian S. White ◽  
Justin Tibbitts ◽  
Jin Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Transgenic Mice ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Mrna Splicing ◽  
Splice Acceptor Site ◽  
Isoform Expression

Abstract Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to U2AF1(WT) at ≥4 months post-transplant in both primary and secondary transplant recipient mice (p≤0.05, n=3-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR <5%). We observed a preference of the mutant U2AF1(S34F) to skip exons (p=1.3e-05, n=72) and alternative splice sites (p=0.014, n=45) with a T in the -3 position relative to the AG splice acceptor site of differentially-spliced genes; this effect has been previously reported in AML patient samples with U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24. We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p<0.001, n=5-6). We are currently testing these isoform changes for their functional contribution to mutant U2AF1-associated phenotypes. Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals RNA Sequencing of Bone Marrow Derived Mesenchymal Stromal Cells (MSCs) of Healthy Young, Healthy Old and Patients with Myelodysplastic Syndrome (MDS) Reveals Transcriptomic Changes upon Aging of the Niche and Onset of MDS

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4300-4300
Author(s):  
Johann-Christoph Jann ◽  
Maximilian Mossner ◽  
Florian Nolte ◽  
Tobias Boch ◽  
Verena Nowak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Myelodysplastic Syndrome ◽  
Rna Sequencing ◽  
Normal Aging ◽  
Gene Set Enrichment Analysis ◽  
Differentially Expressed ◽  
Molecular Changes ◽  
Gene Sets

Abstract Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm. Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes. Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change > 1.5, false discovery rate, FDR < 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR < 0.05, normalized enrichment score > 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia. Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting of the bone marrow microenvironment improves outcome in a murine model of myelodysplastic syndrome

Blood ◽  
2016 ◽  
Vol 127 (5) ◽  
pp. 616-625 ◽  
Author(s):  
Sophia R. Balderman ◽  
Allison J. Li ◽  
Corey M. Hoffman ◽  
Benjamin J. Frisch ◽  
Alexandra N. Goodman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Disease Progression ◽  
Murine Model ◽  
Bone Marrow Microenvironment ◽  
Time Dependent ◽  
In Vivo Model ◽  
Key Points

Key Points An in vivo model of MDS displays time-dependent defects in HSPCs and in microenvironmental populations. Normalization of the marrow microenvironment alters disease progression and transformation and improves hematopoietic function.

Exportin 4, a Potential Amplifier of TGF-β Signaling, Is Increased in Primary Baboon Bone Marrow Erythroblasts Following Decitabine Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1582-1582
Author(s):  
Donald Lavelle ◽  
Tatiana Kouznetsova ◽  
Kestis Vaitkus ◽  
Peter Larsen ◽  
Maria Hankewych ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Splicing Factor ◽  
Hematopoietic Differentiation ◽  
Cell Disease ◽  
Human Genechip

Abstract The DNA demethylating drug decitabine increased fetal hemoglobin (HbF) to therapeutic levels and reduced the level of DNA methylation of the γ-globin gene promoter in patients with sickle cell disease and in experimental baboons. Whether decreased DNA methylation of the γ-globin gene is solely responsible for increased HbF following decitabine treatment is unknown. Increased platelet counts in patients with sickle cell disease and myelodysplastic syndrome and in experimental baboons following decitabine treatment suggest that decitabine also affects hematopoietic differentiation. To investigate to what extent the mechanism responsible for the ability of decitabine to reactivate HbF and alter hematopoietic differentiation may involve the induction of other unknown genes, the global pattern of gene expression in purified primary bone marrow erythroblasts pre- and post-decitabine treament was analyzed. Baboons were phlebotomized for ten days (Hct 20) followed by adminstration of decitabine for ten days (0.52mg/kg/d; sc). RNA was isolated from nucleated erythroblasts purified from bone marrow aspirates obtained pre- and post-decitabine treatment. Purification of erythroblasts was performed by sedimentation in Percoll gradients followed by immunomagnetic column purification using an anti-baboon RBC antibody (Pharmingen). To assess the feasibility of using human Genechip arrays to detect differences in expression of baboon transcripts, RNA isolated from purified erythroblasts of a single baboon pre- and post-decitabine was hybridized in triplicate to human Genechip Focus arrays (Affymetrix) containing over 8500 genes. The expression of 48 genes was increased >2 fold in the post-decitabine treated sample compared to the pre-treatment sample. Among the more highly induced genes were HLA-A (3 fold), HLA-B (5.7 fold), exportin 4 (3.9 fold), and splicing factor 3b1 (3.7 fold). Reverse transcriptase PCR using human primer sets was performed to analyze the expression of these genes in pre- and post-decitabine treated bone marrow erythroblasts in independent samples from three additional baboons. Induction of HLA-A, exportin 4, and splicing factor 3b1 was confirmed in all three post-decitabine treated samples. The exportin 4 gene encodes a protein involved in nuclear export of the Smad3 protein. Activated TGF-β receptors phosphorylate Smad3 and induce its nuclear import to affect gene transcription. Following dephosphorylation of Smad3 in the nucleus, transport of the protein to the cytoplasm mediated by exportin 4 has been proposed to allow the propagation of multiple rounds of activation by activated TGF-β receptors thus amplifying TGF-β signaling (Kurisaki et al; Mol Cell Biol26:1318, 2006). Because TGF-β increases HbF synthesis in cultured erythroid progenitors and also induces erythroid and megakaryocytic differentiation, we suggest that induction of exportin 4 by decitabine may play a role in the ability of this drug to increase HbF synthesis and alter hematopoietic differentiation. Our results thus confirm the feasibility of using human Genechip arrays to assess gene expression levels in baboons. Furthermore, we have indentified a gene induced by decitabine that potentially amplifies TGf-β signaling and thus may play a role in the ability of this drug to increase HbF and alter hematopoietic differentiation.

scholarly journals Unique Metabolic Vulnerabilities of Myelodysplastic Syndrome Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1511-1511
Author(s):  
Brett M. Stevens ◽  
Krysta L. Engel ◽  
Austin E. Gillen ◽  
Rachel Culp-Hill ◽  
Angelo Dalessandro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
High Risk ◽  
Oxidative Phosphorylation ◽  
Myelodysplastic Syndrome ◽  
Single Cell ◽  
Rna Sequencing ◽  
Protein Translation ◽  
Mass Cytometry ◽  
Metabolic Properties

Abstract Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study. Methods: Bone marrow specimens from MDS patients at various disease stages, including serial samples during progression, were obtained. Single cell techniques including mass cytometry, antibody based single cell RNA sequencing (CITE-Seq) and transcriptional profiling with RNA sequencing were used to elucidate novel mechanisms of progression. Selective targeting of primitive MDS cells was tested using several agents. Results: Our previous work characterizing MDS stem cells (MDSC) showed significant similarities between MDSCs and AML LSCs (Stevens et al, Nat. Communications, 2018). However, little is known about lower risk disease. In order to understand transcriptional changes and their relationship to metabolism across pathogenesis, the transcriptome of blasts from patients with LR, intermediate (INT), or HR IPSS scores was investigated. The first major transcriptional difference identified was enrichment of glycolysis pathway at LR and INT stage. In contrast, HR MDS demonstrated enrichment of oxidative phosphorylation. Furthermore, comparison of intermediate to HR MDS showed increased RNA polymerase and Ribosome pathways at the HR stage. These changes demonstrate the progressive alteration of metabolic properties during MDS pathogenesis with cells first relying on mechanisms associated with normal stem cells (i.e. glycolysis) and later transitioning to a state associated with AML stem cells (i.e. reliance on oxidative phosphorylation). Using serial specimens of patients of who progressed from LR to HR MDS we performed CITE-Seq and mass cytometry. CITE-seq in serial specimens showed up-regulation of protein translation and oxidative phosphorylation in a subset of MDS stem and progenitor cells (CD34+ at transcript and antibody level) present at LR stage and conserved at HR stage (Fig 1A-C). MDSCs also acquired surface antigens including CD99 and CD52 upon progression from LR to HR. Analysis of the mass cytometry data showed significant overlap with CITE-Seq data including increased CD123+ and MCL1 expression in MDS stem cells upon progression. In order to understand therapeutic vulnerabilities as they relate to progression, we investigated ex vivo drug response in LR and HR specimens. MDS samples were challenged with two regimens, Ven/Aza, a regimen known to inhibit OXPHOS; and omacetaxine and azacitidine (Oma/Aza), which inhibits translation. CITE-seq showed that MDSC were selectively sensitive to these agents (Fig 1D). Importantly, addition of either drug regimen caused ablation of MDSC at LR and HR stages and these changes were most profound in cells with LSC properties. Based on preclinical findings, we are investigating MDS patients treated with Ven/Aza or Oma/Aza via CITE-seq and metabolomics for correlation of clinical response with properties of MDSC. Preliminary studies show that patients that respond to Oma/Aza present with a population of MDSC with transcriptional signatures of protein translation and LSCs (Fig. 1E). Studies are underway to investigate overlapping properties of ven/aza resistance in AML to resistance in MDS specifically investigating fatty acid metabolism in MDSC. Conclusions: Analysis of MDS patient bone marrow reveals acquisition of aberrant metabolic properties at both low and high risk stages of disease. These distinct aspects of MDSC biology create unique and targetable features. Figure 1 Figure 1. Disclosures Pollyea: Genentech: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Consultancy; Glycomimetics: Other.

Sign in / Sign up

Export Citation Format

Share Document