scholarly journals Impact of Tet2 Deficiency, and of TET2 Mutations in Clonal Hematopoiesis, on Neutrophil/Granulocyte Immune Function

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2159-2159
Author(s):  
Elina K Cook ◽  
Michael Luo ◽  
Jeffrey Mewburn ◽  
Kimberly J Dunham-Snary ◽  
Charles Hindmarch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Immune Function ◽  
Mouse Serum ◽  
Rna Seq ◽  
Granulocytic Differentiation ◽  
Multiple Testing Correction ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Increased Risk

Abstract BACKGROUND: Neutrophils, the most abundant leukocytes and granulocytes, are important regulators of cardiovascular, inflammatory and infectious diseases, yet their role in the pathophysiology of clonal hematopoiesis of indeterminate potential (CHIP) has not been adequately addressed. The effects of inactivating CHIP-driver mutations in the epigenetic regulator TET2 in neutrophils especially, are broadly unknown. HYPOTHESIS: Tet2 inactivation in murine neutrophils, and TET2 mutations in CHIP in humans (CHIP TET2), perturb granulocyte immune effector functions. METHODS: Neutrophils were obtained (EasySep™, StemCell) from the bone marrow of 2- to 4-months-old, sex-matched, control Tet2 f/f;Vav1-icre - (Tet2 f/f) and hematopoietic knockout Tet2 f/f;Vav1-icre + (Tet2 -/-) mice. Neutrophils were cultured (RPMI+10% mouse serum/FBS) and: i) stained with Mitotracker Deep Red/Nuc Blue, co-cultured and imaged (Leica SP8-X) for 30min with GFP-labeled Staphylococcus aureus (10:1 ratio) and analyzed in FIJI; ii) cultured for 3h with vehicle or 10μg/mL of S. aureus lipotechoic acid (LTA). RNA-Seq was generated (Illumina QuantSeq 3' mRNA, single-end 75bp read lengths, 5 million reads/sample), trimmed, aligned to GRCm39 using STAR. CHIP participant DNA and RNA were sequenced previously from whole blood (Cook et al., Bld Adv 2019; Cook et al., ASH 2018, with a 48-gene panel on Ion Proton, and ribo-depleted bulk RNA on Illumina, respectively). New CHIP TET2 vs. no CHIP, and murine RNA-Seq analyses were carried out in DESeq2. Human serum granule protein levels were quantified by ELISA (VersaMax). Mann-Whitney U tests were carried out in Prism. P<0.05 was considered statistically significant, and Benjamini-Hochberg multiple testing correction was applied as needed. RESULTS: Tet2 -/- mice had 1.34-fold more bone marrow CD11b +Ly6G + neutrophils than control Tet2 f/f mice (p=0.03), consistent with myeloid expansion. Compared to Tet2 f/f, Tet2 -/- neutrophils phagocytosed fewer S. aureus (Fig1A) and moved more slowly (Fig1B). Preliminary data suggest that Tet2 -/- neutrophil extracellular trap (NET) formation in response to S. aureus was also impaired, showing fewer and less extensive NETs (Fig1C). LTA-stimulated gene expression profiles were similar between Tet2 -/- and Tet2 f/f, suggesting pre-existing differences at baseline. Unexpectedly, the most significant GO term enrichment related to upregulated viral response pathways, including interferon-stimulated genes, (e.g. Ifitm1). The cause is unknown, but this is reminiscent of the constitutive interferon response seen in myelodysplastic syndrome (MDS) patients and TET2-mutant hematopoietic stem cells, where epigenetic dysregulation of endogenous retrotransposable elements leads to a viral mimicry response. Tet2 -/- neutrophils also overexpressed Asprv1, a regulator of inflammation ostensibly acquired from a retrotransposon. Interestingly, Ccdc80, which has been linked to Tet2 and Jak2 functions, was most significantly downregulated in Tet2 -/-, along with the Pnpla1 lipid phosphatase. Finally, Tesc, a promoter of granulocytic differentiation, was upregulated in Tet2 -/-, and there were perturbations of genes encoding neutrophil granule contents. Similarly, human RNA-Seq revealed that several leukocyte (de)granulation-related genes (e.g. lactoferrin LTF, myeloperoxidase MPO) were upregulated in CHIP TET2 subjects to those without CHIP, and these corresponded with higher LTF and MPO serum titers in an expanded cohort (Fig1D,E). Finally, there were striking decreases of gene expression associated with cytotoxic (T/NK) human lymphocytes (i.e. GZMM, TRGV8, etc.). Neutrophil, lymphocyte and monocyte counts were not significantly different between the groups. CONCLUSIONS: Tet2-deficient murine neutrophils have compromised immune function, possibly due to differences in pre-stimulus state. TET2-mutation carrying neutrophils in CHIP may exhibit similar abnormalities, as has been previously noted in neutrophils isolated from MDS patients. Indeed, CHIP is now known to associate with increased risk of bacterial and viral infections, and infection risk has also previously been noted for MDS. People with CHIP have elevated peripheral blood serum MPO and LTF levels, suggesting a difference in leukocyte granule biology, likely related to neutrophils. These data aid in understanding how CHIP alters immunity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of PARP Inhibitor Therapy on p53-Deficient Hematopoietic Stem and Progenitor Cell Fitness

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3275-3275
Author(s):  
Jeremy T Baeten ◽  
Irenaeus C.C. Chan ◽  
Daniel C. Link ◽  
Kelly L. Bolton
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Prior Exposure ◽  
Clinical Samples ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Fitness Advantage ◽  
Increased Risk ◽  
Bone Marrow Chimeras ◽  
The Impact

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors are an important new class of anti-cancer therapies. Therapy-related myeloid neoplasia (tMN) has been reported following PARPi therapy and is associated with adverse outcomes. We have previously shown, in retrospective data, that prior chemotherapy increases the incidence of clonal hematopoiesis (CH), especially in DNA damage response (DDR) pathway genes including TP53, PPM1D, and CHEK2 and is associated with progression to tMN. In particular, patients who receive PARPi therapy are more likely to have CH compared to other therapies or untreated patients. In the IMPACT study of CH in 10,156 cancer patients, exposure to PARPi were more likely to have CH (33%) compared to untreated patients (16%). This was particularly pronounced for DDR gene mutations, with 25% of PARPi treated patients with DDR CH compared to 2% of untreated patients. In multivariate analysis accounting for demographics and exposure to other chemotherapy or radiation therapy, exposure to PARPi conferred an increased risk of DDR CH (OR = 3.6, 95% CI 1.5-8.5, p = 0.004). From these data, we hypothesize that mutations in DDR pathway genes provide a fitness advantage to hematopoietic stem/progenitor cells (HSPCs) following PARPi treatment, leading to clonal hematopoiesis. A major limitation, however of our previous work in retrospective clinical samples, is the inability to completely adjust for the confounding effect of prior exposure to cytotoxic therapy (in particular platinum therapies) and germline BRCA1/2 mutations; both which have been shown or hypothesized to increase the risk of tMN. To test whether PARPi exposure might provide a fitness advantage to HSPCs independent of prior exposure to other therapies, we first examined the response of CRISPR-gene edited TP53-/- MOLM13 cells to the PARPi Olaparib and, as a control, Cisplatin. As expected, TP53-/- cells had increased resistance to both agents, though the response was much more pronounced in Cisplatin-treated cells (Figure 1A,B). Next, we implemented a mouse model of TP53-mutant clonal hematopoiesis, by generating mixed bone marrow chimeras transplanted with a 1:9 ratio of wildtype (CD45.1) to TP53 R172H+/- (CD45.2) cells. The "baseline" contribution of TP53 R172H+/- (CD45.2) cells to peripheral blood leukocytes 8 weeks after transplantation was determined by flow cytometry. Mice were then randomized into the following three cohorts: 1) Cisplatin (6mg/kg on days 1, 8, and 15); 2) Olaparib (50mg/kg daily for 3 weeks); and 3) vehicle alone. Peripheral blood chimerism was assessed 3, 9, and 12 weeks after initiating treatment. In addition, the contribution of TP53 R172H+/- to lineage -Sca1 +Kit + (LSK) cells in the bone marrow was determined. Cisplatin treatment resulted in a significant increase in the contribution of TP53 R172H+ to peripheral blood total leukocytes, granulocytes, and bone marrow LSK cells (Figure 1C-E). In contrast, Olaparib treated mice showed no change in CD45 chimerism. From these results we conclude that p53-deficiency does not confer a strong fitness advantage to mouse HSPCs in response to PARPi treatment. This suggests that the strong association observed between prior PARPi therapy, CH and tMN in clinical cohorts may in part be due to the confounding effects of prior (often heavy) exposure to platinum-based therapy. However, the majority of patients receiving PARPi have germline heterozygous BRCA1/2 mutations that could be contributing to their hematopoietic response to PARPi therapy. Experiments are underway to test this possibility by analyzing mixed bone marrow chimeras carrying heterozygous mutations of both Brca1 and Trp53. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rpl22 Deficiency Predisposes Hematopoietic Stem and Progenitor Cells to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 899-899 ◽  
Author(s):  
Bryan Harris ◽  
Jaqueline Perrigoue ◽  
Rachel M. Kessel ◽  
Shawn Fahl ◽  
Stephen Matthew Sykes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ribosomal Protein ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) was the most significantly reduced ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value <0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency causes a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. Because human MDS frequently progresses to AML, we examined the potential for Rpl22-deficient HSC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22-deficient HSPC exhibited an increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. To determine how Rpl22-deficiency increased the transformation potential of HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, four expression signatures were observed that were consistent with the altered behavior exhibited by Rpl22-/- HSC. Rpl22-deficient HSC exhibited increased expression of: 1) genes associated with stem cell function, consistent with the basal expansion and effective engraftment of Rpl22-/- HSC upon adoptive transfer; 2) markers of the myeloid lineage, providing a potential explanation for the myeloid bias exhibited by Rpl22-/- HSC; 3) cell cycle regulators, consistent with the increased proliferation exhibited by Rpl22-/- HSC; and 4) components of the mitochondrial respiratory chain, a metabolic program on which leukemic stem cell function depends. Together, these data suggest that Rpl22 controls a program of gene expression that regulates the predisposition of HSPC to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Simultaneous Detection ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals RNA-Seq Analysis of Clonal Hematopoiesis (CHIP) Blood Leukocytes Shows Dysregulation of Neutrophil / Innate Immunity-Related Genes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3843-3843
Author(s):  
Elina K Cook ◽  
Richard N Armstrong ◽  
Eshita Sharma ◽  
Brooke Snetsinger ◽  
Jacqueline Boultwood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiovascular Disease ◽  
Innate Immunity ◽  
Expression Profiles ◽  
Rna Seq ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Neutrophil Degranulation ◽  
Chip Chip

Abstract BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) involves the peripheral blood (PB) expansion of progeny of a hematopoietic stem or progenitor cell that is somatically mutated in a hematological cancer-associated gene (most often TET2 or DNMT3A). CHIP associates with comorbid diseases of aging such as cardiovascular disease. Murine knockout (Tet2 or Dnmt3a) and engraftment models of CHIP develop exacerbated cardiovascular disease and their mutated myeloid cells are more reactive to inflammatory stimuli. However, whether blood leukocytes in human CHIP are hyper-inflammatory remains speculative. We recently found people with CHIP have higher serum levels of certain pro-inflammatory cytokines and chemokines than controls (Cook et al, ASH 2017). Thus, we hypothesized that PB effector cells in people with CHIP will be enriched for pro-inflammatory gene expression and pathways. METHODS: The presence of CHIP (variant allele frequency, VAF>0.02) was determined in the whole PB of 30 hematologically healthy adults >65 years old at Baycrest and Sunnybrook Health Sciences Centers (Toronto, Canada) using Ion Proton DNA sequencing targeting 48 commonly mutated genes in myeloid neoplasms. RNA-Seq (HISeq 4000, Illumina, 75bp paired-end sequencing reads with a depth of >50 million/sample) was performed on corresponding ribo-depleted whole PB samples (PAXgene), reads were aligned with HISAT2, gene counts quantified with featureCount, and analyzed with DESeq2. FDR<0.1 was used as a cutoff for differential gene expression analyses. Correlations with clinical and comorbidity data were tested with logistic regressions. RESULTS: People with CHIP ("CHIP+", n: males=8, females=13; TET2=12, DNMT3A=8, SF3B1=1; VAF range=0.03-0.40) compared to those without CHIP ("CHIP-", n: males=3, females=6) had six significantly downregulated genes (e.g. GZMM) and 10 upregulated genes (e.g. DEFA4, LTF, MPO, see Figure 1A). Hierarchical clustering of these top genes yielded two groups, one consisting of most of the CHIP- cases (8/9 cases, in a cluster of 11, see Figure 1A). The three CHIP+ cases that clustered with CHIP- had VAFs lower than 0.15. Of the 16 differentially regulated genes between CHIP+ and CHIP-, nine were recognized by reactome, and most overlapped (≥6 genes) with pathways involving neutrophil degranulation and innate immunity (Figure 1B). DEFA4, LTF, CRISP3, BPI and MPO specifically encode components of neutrophil granules, with various anti-microbial and homeostatic functions. However, mean neutrophil counts (4.6±1.6 vs. 4.4±1.6 10^9/L for CHIP+ vs. CHIP-) and neutrophil to lymphocyte ratios (3.2±1.4 vs. 2.8±2.1 in CHIP+ vs. CHIP-) did not significantly differ between the groups. This suggests that mutations of CHIP may affect neutrophil/immune-related function or phenotype, potentially contributing to comorbid disease. For example, greater expression of alpha-defensins (i.e. DEFA4) in CHIP may involve dysregulated granulocyte maturation and inflammatory function as seen in myelodysplasia (Droin et al, 2010 Blood), suggesting a potential dysregulation of inflammation and immunity. Higher VAFs (>0.15) associated with higher ECOG scores (poorer overall daily functioning: odds ratio=44, 95% CI=4-500, p=0.002), suggesting that larger proportions of mutated cells may have greater effects on gene expression profiles. Accordingly, there were linear correlations between the VAFs of the mutated cell populations and the levels of differentially expressed genes (Figure 1C). CONCLUSIONS: The connection between mutant clones of CHIP and disease remains poorly elucidated. For the first time, to our knowledge, we studied gene expression in CHIP leukocytes. We report that the most prominent gene expression differences between people with CHIP and those without CHIP involve neutrophil degranulation and the innate immune system. Additionally, higher VAFs may have a greater influence on gene expression levels and health than lower VAFs. We plan to validate these candidate genes in a larger cohort. These novel data warrant further investigation of the cellular pathways perturbed by somatic mutations of CHIP. Disclosures Buckstein: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals EXPRESSION OF micro RNAs (miRNAs) IN MYELODYSPLASTIC SYNDROME (MDS)

2019 ◽  
Vol 141 (7-8) ◽  
pp. 226-232
Keyword(s):  
Gene Expression ◽  
Myelodysplastic Syndrome ◽  
Healthy Volunteers ◽  
Myelogenous Leukemia ◽  
University Hospital ◽  
Gene Expressions ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Micro Rnas ◽  
Significant Difference

Myelodisplasia or myelodysplastic syndrome (MDS) is the name for a group of heterogeneous clonal hematological disorders of hematopoietic stem cells followed by ineffective hematopoesis of one or more cell lines and the emergence of consequent cytopenias with increased risk of progression to acute myelogenous leukemia (AML). Micro Messenger Ribonucleic Acids (miRNAs) are short, non-coding RNA molecules that, apart from contributing to MDS pathogenesis, act as regulators of epigenetic mechanisms and also are recognized as potential prognostic markers for early diagnosis and classification of MDS. The aim of the study was to examine the levels of gene expression of specific miRNAs (hsa-miR-125a, hsa-miR-99b, hsa-miR-126 and hsa-miR-125b) in healthy volunteers plasma and MDS diagnosed patients. Gene expressions of miRNAs were determined at the Clinical Institute of Medical Biochemistry and Laboratory Medicine, Merkur University Hospital, accredited according to EN ISO 15189:2012, in plasma samples of four healthy volunteers and 33 MDS patients diagnosed at the Institute of Hematology of the Clinic for Internal Diseases of Merkur University Hospital, Reference Center of the Ministry of Health of the Republic of Croatia for Diagnosis and Treatment of MDS. Statistically significant difference in gene expression of miRNA in healthy volunteers compared to the MDS patients was not found (P [hsa-miR-125a] = 0.398; P [hsa-miR-99b] = 0.134; P [hsa- miR-126] = 0.305; P [hsa-miR-125b] = 0.079). MiRNA ratios of hsa-miR-125a and hsa-miR-99b in MDS patients were almost twice as high compared to normalized levels of gene expression in healthy volunteers (2.30 versus 1.90), and the level of change of miRNAs hsa-miR-125 and hsa-miR-99b was more than two times higher than the level of change of miRNA hsa-miR-125b. Finally, the results of the research indicate that the gene expression of miRNAs hsa-miR-125a and hsa-miR-99b could be regulated by the same mechanism and could be clinically relevant in MDS patients.

scholarly journals Changes in the Multipotent Stromal Mesenchymal Cells from the Bone Marrow of the Patients with Hematological Diseases in Debut and after the Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5014-5014
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Nina J. Drize ◽  
Aminat A. Magomedova ◽  
Ekaterina A. Fastova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Marker Gene ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Stromal Microenvironment ◽  
Expression Of Genes

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.

scholarly journals Clonal hematopoiesis in donors and long-term survivors of related allogeneic hematopoietic stem cell transplantation

Blood ◽  
2020 ◽  
Vol 135 (18) ◽  
pp. 1548-1559 ◽  
Author(s):  
Steffen Boettcher ◽  
C. Matthias Wilk ◽  
Jochen Singer ◽  
Fabian Beier ◽  
Elodie Burcklen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Increased Risk ◽  
Long Term Survivors

Abstract Clonal hematopoiesis (CH) is associated with age and an increased risk of myeloid malignancies, cardiovascular risk, and all-cause mortality. We tested for CH in a setting where hematopoietic stem cells (HSCs) of the same individual are exposed to different degrees of proliferative stress and environments, ie, in long-term survivors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their respective related donors (n = 42 donor-recipient pairs). With a median follow-up time since allo-HSCT of 16 years (range, 10-32 years), we found a total of 35 mutations in 23 out of 84 (27.4%) study participants. Ten out of 42 donors (23.8%) and 13 out of 42 recipients (31%) had CH. CH was associated with older donor and recipient age. We identified 5 cases of donor-engrafted CH, with 1 case progressing into myelodysplastic syndrome in both donor and recipient. Four out of 5 cases showed increased clone size in recipients compared with donors. We further characterized the hematopoietic system in individuals with CH as follows: (1) CH was consistently present in myeloid cells but varied in penetrance in B and T cells; (2) colony-forming units (CFUs) revealed clonal evolution or multiple independent clones in individuals with multiple CH mutations; and (3) telomere shortening determined in granulocytes suggested ∼20 years of added proliferative history of HSCs in recipients compared with their donors, with telomere length in CH vs non-CH CFUs showing varying patterns. This study provides insight into the long-term behavior of the same human HSCs and respective CH development under different proliferative conditions.

scholarly journals Aging, hematopoiesis, and the myelodysplastic syndromes

2017 ◽  
Vol 1 (26) ◽  
pp. 2572-2578 ◽  
Author(s):  
Stephen S. Chung ◽  
Christopher Y. Park
Keyword(s):  
Myelodysplastic Syndromes ◽  
Bone Marrow Failure ◽  
Hematologic Malignancies ◽  
Relative Increase ◽  
Extrinsic Factors ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Increased Risk ◽  
The Many ◽  
The Relationship

Abstract The aging hematopoietic system undergoes numerous changes, including reduced production of red blood cells and lymphocytes as well as a relative increase in the production of myeloid cells. Emerging evidence indicates that many of these changes are due to selection pressures from cell-intrinsic and cell-extrinsic factors that result in clonal shifts in the hematopoietic stem cell (HSC) pool, resulting in predominant HSC clones that exhibit the functional characteristics associated with HSC aging. Given the recent descriptions of clonal hematopoiesis in aged populations, the increased risk of developing hematologic malignancies in individuals with clonal hematopoiesis, and the many similarities in hematopoietic aging and acquired bone marrow failure (BMF) syndromes, such as myelodysplastic syndromes (MDS), this raises significant questions regarding the relationship between aging hematopoiesis and MDS, including the factors that regulate HSC aging, whether clonal hematopoiesis is required for the development of MDS, and even whether BMF is an inevitable consequence of aging. In this article, we will review our current understanding of these processes and the potential intersections among them.

Trisomy 21 Driven Pro-Inflammatory Signalling in Fetal Bone Marrow May Play a Role in Perturbed B-Lymphopoiesis and Acute Lymphoblastic Leukemia of Down Syndrome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1206-1206 ◽  
Author(s):  
Sorcha Isabella O'Byrne ◽  
Natalina Elliott ◽  
Gemma Buck ◽  
Siobhan Rice ◽  
David O'Connor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Down Syndrome ◽  
Trisomy 21 ◽  
Lymphoblastic Leukemia ◽  
B Lymphopoiesis ◽  
Increased Risk ◽  
Cd34 Cells ◽  
Inflammatory Signalling

Introduction: Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL), suggesting that trisomy 21 (T21) has specific effects on hematopoietic stem and progenitor cell (HSPC) biology in early life. Data from human fetal liver (FL) indicates that T21 alters fetal hematopoiesis, causing multiple defects in lympho-myelopoiesis. The impact of T21 on fetal B lymphopoiesis and how this may underpin the increase in ALL is not well known. We have recently found that fetal bone marrow (FBM) rather than FL is the main site of B lymphopoiesis; with a marked enrichment of fetal-specific progenitors (early lymphoid progenitors, ELP and PreProB progenitors) that lie upstream of adult type ProB progenitors (O'Byrne et al, Blood, in press). Previous preliminary data suggested that B progenitors were also reduced in T21 FBM (Roy et al, Blood. 124, 4331). Aim: To dissect putative molecular mechanisms responsible for the defects in T21 FBM B-lymphopoiesis and its association with childhood DS ALL. Methods: Second trimester human FBM and paediatric ALL samples were obtained from the Human Developmental Biology Resource and UK Childhood Leukaemia Cell Bank respectively. Multiparameter flow cytometry/sorting, transcriptome analysis by RNA-sequencing and microarray, and stromal co-culture assays were used to characterize HSPC and mesenchymal stromal cells (MSC) from normal (NM) disomic (n=21-35) and T21 (n=7-12) human FBM; RNASeq was performed on cytogenetically matched non-DS (n=13) and DS ALL (n=7). Results: In contrast to NM FBM, fetal specific progenitors were virtually absent (CD34+CD10-CD19-CD127+ ELP 2.8±0.4% vs. 0.8±0.4% of CD34+ cells) or very severely reduced (CD34+CD10-CD19+ PreProB 12.8±1 vs 2.6±0.7%) in T21 FBM. This was despite a >4-fold increase in the frequency of immunophenotypic HSC (4.2±1.2% vs 0.9±0.2% of CD34+ cells) and similar frequencies of MPP and LMPP in T21 FBM. As in adult BM, the vast majority of B progenitors in T21 FBM were CD34+CD10+CD19+ ProB progenitors with a frequency (28.8±8.3%) similar to NM FBM (30.3±2.3% of CD34+ cells). Thus, T21 causes a severe block in B-progenitor commitment at the LMPP stage, in tandem with a compensatory expansion of ProB progenitors. Consistent with this, T21 FBM HSC, MPP and LMPP had reduced B cell potential in vitro compared to NM FBM in MS5 co-cultures. RNAseq of NM (n=3) and T21 (n=3) FBM HSPC demonstrated global transcriptomic disruption by T21, with increased gene expression in HSC, MPP, LMPP and ProB progenitors. Cell cycle genes were enriched in T21 ProB progenitors. Despite these functional and global gene expression differences, expression of key B-lineage commitment genes was maintained suggesting the defect in B-lymphopoiesis may be secondary to lineage skewing of multipotent progenitors towards a non-B lymphoid fate and/or mediated by extrinsic factors. GSEA pointed to a role for multiple inflammatory pathways in T21 hematopoiesis with dysregulation of IFNα, IL6 and TGFβ signalling pathways in T21 HSC/LMPP. To investigate the role of the T21 microenvironment, we co-cultured NM HSC, MPP and LMPP with T21 or NM primary FBM MSC. T21 FBM MSC (n=3) had reduced capacity to support B cell differentiation in vitro consistent with perturbation of MSC function by T21. Similar to T21 FBM HSPC, transcriptomic analysis of T21 FBM MSC by microarray showed enrichment for IFNα signalling compared to NM; and T21 HSPC and MSC both showed increased gene expression for IFNα receptors IFNAR1 and IFNAR2, which are encoded on chromosome 21. Since IFNα was undetectable by ELISA of conditioned media from NM and T21 MSC, differences in secreted IFNα from MSC are unlikely to fully explain the increased IFN signalling in T21 HSPC and MSC. This suggests that T21 may drive autocrine rather than paracrine IFN signalling in FBM cells. Finally, RNASeq showed perturbed inflammatory signalling in DS ALL compared to non-DS ALL, suggesting a role for T21-driven inflammatory pathways in the biology of DS ALL. Conclusions: These data show that T21 severely impairs B lymphopoiesis in FBM and is associated with expression of proinflammatory gene expression programs in T21 FBM HSPC and MSC and DS ALL. The compensatory expansion of T21 FBM ProB progenitors, through self-renewal or via an alternative differentiation pathway; with concomitant T21-driven proinflammatory signalling may underpin the increased risk of B progenitor ALL in childhood. Disclosures No relevant conflicts of interest to declare.

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