Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2038-2038
Author(s):  
Hein Than ◽  
Naoto Nakamichi ◽  
Anthony D. Pomicter ◽  
John O'Shea ◽  
Orlando Antelope ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Model Systems ◽  
Juvenile Myelomonocytic Leukemia ◽  
Functional Screening ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Myelomonocytic Leukemia

Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML. Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts. Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented. These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

scholarly journals Immunoexpression of CD34, CD117, and p53 in Hypocellular Bone Marrow Disorders

2021 ◽  
Author(s):  
Pooja Sharma ◽  
Anshu Palta ◽  
Anita Tahlan ◽  
Manveen Kaur ◽  
Ram Singh
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
The Other ◽  
Immunohistochemical Expression ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Blast Count ◽  
Bone Marrow Disorders ◽  
Acute Myeloid

Abstract Objectives Hypocellular bone marrow (BM) disorders comprise heterogeneous entities associated with peripheral cytopenias and decreased production of hematopoietic cells in BM. This study was undertaken to analyze immunohistochemical expression of CD34, CD117, and p53 in morphologically diagnosed patients of hypocellular BM (aplastic anemia [AA], hypocellular myelodysplastic syndrome [h-MDS], and hypocellular acute myeloid leukemia [h-AML]). Materials and Methods BM specimens were obtained from patients presenting with pancytopenia/bicytopenia. On 30 patients diagnosed as hypocellular BM, immunohistochemistry (IHC) for CD34, CD117, and p53 was performed. Results BM cellularity was < 30% in all (100%) patients. Blast count was increased in h-MDS and h-AML. Features of dysplasia were noted in six (20%) patients. Out of these, three patients were diagnosed as h-MDS having bilineage/trilineage dysplasia, and the other three patients were of AA (11.5% patients) displaying only dyserythropoiesis. On IHC, percentage of BM CD34+ cells was increased in h-MDS+ h-AML (3.87 ± 0.86) as compared with AA (0.19 ± 0.15) and controls (0.81 ± 0.21), p = 0.01. Percentage of BM p53+ cells was also increased in h-MDS+ h-AML (2.9 ± 2.07) as compared with AA and controls, which did not show any p53+ cells, p = 0.0. No statistically significant difference was observed in the expression of CD117 in h-MDS+ h-AML (4.95 ± 3.40) compared with AA (4.49 ± 1.07), p = 0.99. Conclusion The study demonstrates the usefulness of CD34 and p53 immunoexpression as an important ancillary method in distinguishing various hypocellular BM disorders, especially h-MDS and AA. However, the role of CD117 remains unclear and needs to be evaluated further by larger studies.

MN1 Expression Predicts Prognosis of Acute Myeloid Leukemia with Normal Cytogenetics.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2351-2351
Author(s):  
Michael Heuser ◽  
Gernot Beutel ◽  
Jürgen Krauter ◽  
Nils von Neuhoff ◽  
Brigitte Schlegelberger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Performance Status ◽  
Ecog Performance Status ◽  
Free Survival ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Abstract Cytogenetic aberrations are important prognostic factors in acute myeloid leukemia (AML). However, approximately half of adult AML patients lack cytogenetic abnormalities and identification of predictive molecular markers might improve therapy. Fusion of meningioma-1 (MN1) to TEL (ETV6) has been found in AML and MDS with t(12;22)(p13;q11). However, expression levels of MN1 have not been reported previously in AML. We evaluated MN1 expression as a prognostic marker in 142 AML patients aged 18–60 years with normal cytogenetics, who were uniformely treated according to the AML-SHG 1/99 trial. Patients received intensive, cytarabine-based induction and consolidation treatment including allogeneic progenitor cell transplantation if an HLA-compatible sibling was available, or in case of relapse. Specimens were obtained at diagnosis, and routine cytogenetic, FLT3-mutation, and MLL-PTD analyses were performed. MN1 expression was quantified by real-time RT-PCR on a LightCycler using QuantiTect SYBR Green. AML samples were dichotomized at the median value resulting in two groups: a low MN1 group and a high MN1 group. Baseline characteristics and outcome parameters were compared between these two groups. In addition, CD34+ cells were immunomagnetically enriched from mobilized blood of a healthy donor using MACS CD34 isolation kit. Cells were cultured in IMDM medium with various cytokines including either G-CSF, M-CSF or EPO. At various time points, cells were harvested and analyzed for MN1 expression. There were no significant differences between low MN1 and high MN1 expressing patients with respect to age, gender, ECOG performance status, diagnosis of de novo or secondary AML, FAB morphology, white blood cell count, percentage of blasts in blood or bone marrow, FLT3 mutations, or MLL-PTD. Low MN1 expressing patients significantly more often achieved a good response to the first course of induction treatment defined as blasts in bone marrow below 5%, no blasts in peripheral blood, and no extramedullary manifestation at day 15 compared to high MN1 expressing patients (87.3% vs. 71.8%, p=.02). There was no significant difference for remission status between the two groups. High MN1 expression predicted significantly shorter event-free survival (19% vs. 45.8% at 3 years, log-rank p=.0009), shorter relapse-free survival (23% vs. 52.8% at 3 years, log-rank p=.001), and shorter overall survival (38.2% vs. 58.8% at 3-years, log-rank p=.03). The high MN1 group relapsed significantly more often compared to the low MN1 group (56.7% vs. 35%, p=.02), and thus received an allogeneic transplant significantly more often (50.7% vs. 33.8%, p=.04). In multivariate analysis including known risk factors only MN1 expression, age (above the median compared to below the median age), and ECOG performance status (0 or 1 compared to 2) remained significant (hazard ratio: 2 (p=.01), 2.1 (p=.005) and 2.8 (p=.005), respectively). MN1 expression in CD34+ cells was 37-fold higher compared to the CD34− cell fraction. However, by in vitro differentiation of CD34+ cells using various cytokines including either G-CSF, M-CSF or EPO, MN1 expression dropped to levels found in the CD34− fraction within 7 days of culture. In conclusion, high MN1 expression predicts adverse prognosis and may define an important risk factor in AML with normal cytogenetics. Its upregulation in hematopoietic progenitor cells hints at a functional role of MN1 in blocking differentiation.

scholarly journals 5-Azacytidine Is Effective for Targeting Leukemia-Initiating Cells in Juvenile Myelomonocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4342-4342
Author(s):  
Christopher Felix Krombholz ◽  
Lorena Gallego Villar ◽  
Pritam Kumar Panda ◽  
Sushree Sangita Sahoo ◽  
Marcin W. Wlodarski ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Epigenetic Therapy ◽  
Juvenile Myelomonocytic Leukemia ◽  
Antileukemic Activity ◽  
Cpg Sites ◽  
Cd34 Cells ◽  
Xenograft Mice ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of young children that originates from early hematopoietic stem/progenitor cells. We have previously developed an in vivo disease model using xenotransplantation of primary JMML cells into Rag2-/-γc-/- mice (Haematologica 2016;101:597). The model reproduces a characteristic JMML phenotype including myelomonocytic proliferation, hepatosplenomegaly, and lung infiltration. Case-specific driver mutations and DNA methylation patterns are unchanged after xenologous engraftment, indicating their origin in leukemia-initiating cells. We and others recently discovered a tight link between prognosis and differential DNA methylation in JMML (Nat Commun 2017;8:2126; Nat Commun 2017;8:2127; Blood 2018;131:1576). We also reported that the DNA methyltransferase inhibitor 5-azacytidine (azacitidine, 5AC) has unprecedented clinical activity in JMML and induces complete or partial remissions before allogeneic HSCT (Blood 2015;125:2311). Cytosine arabinoside (araC) is a structurally related nucleoside which is commonly used for cytoreduction in JMML but lacks the ability to induce remissions. Here we employed the xenotransplantation model to investigate the antileukemic activity and epigenetic effects of 5AC on JMML in comparison to araC. After eight weeks of leukemic expansion, 15 xenograft mice were treated with two cycles of 5AC (3 mg/kg/d x 5 days every two weeks). Control groups included mice treated with araC 20 mg/kg/d (N=15) or carrier solution (0.9% NaCl, N=20). The experimental animals maintained stable body weight, and no major toxicity on murine hematopoiesis was observed. 5AC and araC exhibited antileukemic activity and substantially reduced the human JMML cell content in bone marrow, spleen, liver, and lung. However, we noted that CD34+ stem/progenitor cells within the human leukemia population were depleted after treatment with 5AC but not after araC (5AC, 20.2% +/- 7.3%; araC, 35.6% +/- 6.1 %; carrier, 39.4% +/- 3.5%; p<0.01). To demonstrate that the selective reduction of CD34+ cells impaired the leukemia-initiating capacity of the xenograft, we treated a subsequent series of mice as above and retransplanted the bone marrow into secondary recipient mice. JMML cells obtained from 5AC-treated primary recipients sustained engraftment in only one of 9 secondary recipients at 30 weeks after retransplantation whereas JMML xenografts treated with araC or carrier engrafted in 8/13 or 4/8 secondary mice, respectively (p=0.03). We then examined the genome-wide DNA methylation in 5AC-treated xenografts (N=5) using Infinium 450K arrays. The JMML genomes exhibited global and profound DNA demethylation with near-complete loss of fully methylated CpG sites. A focused analysis of approximately 5,000 CpG sites with JMML-specific methylation illustrated that the profiles of 5AC-treated JMML cells were more similar to healthy human CD34+ cells than untreated JMML cells. As expected, no change in DNA methylation was observed in xenografts treated with araC. Next we studied the early effects of 5AC on the transcriptome and epigenome of JMML. Xenograft mice were treated as above, and JMML cells were harvested from bone marrow on days 0, 2, 4, and 6. RNA sequencing readily identified non-random changes in gene transcription that progressed over time from days 2 to 6 and were reproducible across replicate mice. Between days 0 and 6 we observed >2fold upregulation of 856 transcripts and downregulation of 958 transcripts (<0.01 false discovery rate, multitest-corrected). CpG-rich 5' regions (putative promoters) of corresponding genes were invariably demethylated. Gene Ontology enrichment analysis linked upregulated transcripts to myeloid differentiation whereas downregulated transcripts were involved in nucleosome assembly/organization and chromatin silencing. In summary, the xenograft experiments highlight the therapeutic potential of 5AC in JMML and thus encourage the further clinical development of epigenetic therapy with hypomethylating agents for this disease. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Effect of Dioxin (TCDD) on Gene Transcription of Human CD34+ Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4033-4033
Author(s):  
Nicola S. Fracchiolla ◽  
Federica Servida ◽  
Pier A. Bertazzi ◽  
Paolo Corradini ◽  
Antonio Colombi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiovascular Disease ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Juvenile Myelomonocytic Leukemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hodgkin's Lymphomas ◽  
Vitro Effect

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to aryl hydrocarbon receptor (AhR), a member of the erb-A family, allowing, after DNA binding, gene expression regulation. TCDD has a large number of biological effects, as skin and cardiovascular disease, diabetes and cancer. An increase in myeloid leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, was observed after 15 years in the population exposed to TCDD after the 1976 accident in Seveso, Italy. In the present study, we analyzed the in vitro effect of TCDD exposure on human CD34+ progenitor cells from G-CSF stimulated leukapheresis of 4 normal donors. Gene expression modulation induced by TCDD was analysed on highly purified (>96%) CD34+ cells, after exposure to 10 nM of TCDD for 12 hrs. Gene expression profiles have been generated by high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed with a supervised approach (DNA-Chip Analyzer, dChip 2006). The differential expression of 257 transcripts (150 up regulated and 106 down-regulated) distinguished the 4 TCDD treated from the 4 untreated control samples. Interestingly, a number of the differentially expressed genes were involved in skin and cardiovascular diseases, diabetes, and different cancers, all of which have been associated with TCDD exposure. Among skin diseases, defects in laminin beta 3, ALOX12B and keratin 2a, all downregulated in TCDD exposed CD34+ cells, are associated with development of epidermolysis bullosa, erythroderma ichthyosiform, and Siemens ichthyosis bullosa, respectively. As far as diabetes and cardiovascular disease pathogenesis are concerned, defects of SLC2A4 gene, dowregulated by TCDD, is associated with the development of non insulin dependent diabetes, while epoxide hydrolase 2 and EGR2 are associated with cardiovascular disease. Among cancer related genes, ARHGAP26 and ABL2 are associated with juvenile myelomonocytic leukemia and acute myeloid leukemia with eosinophilia, respectively. Nevertheless, the expression of numerous other genes potentially involved in hemopoiesis/leukemogenesis, was modulated by TCDD exposure. Among these examples are c-kit ligand, LIF receptor, pre B lymphocyte gene 1, piwi-like 2, FLT3 ligand, chemokine ligands 14 and 15, and cdk2, that were all up regulated, while MLL4, wnt inhibitory factor 1, chemokine ligand 7, and lymphoid blast crisis oncogene, were down regulated. In conclusion, the gene expression pattern induced by TCDD exposure on the CD34+ normal progenitor cells is consistent with the spectrum of TCDD induced toxicities/diseases. In particular, it provides the basis for a possible role of TCDD in the neoplastic transformation of hemopoietic stem cells and support the epidemiologic data of increased hematologic cancer risk in the population exposed accidentally to the substance.

The CXCR4 Inhibitor Plerixafor Effectively Mobilizes Primary AML in NODscid IL2Rγc−/− Xenografts and Markedly Reduces but Does Not Eradicate Leukemia in Combination with Cytarabine +/− Clofarabine Chemotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1432-1432
Author(s):  
Sylvia Chien ◽  
Xin Zhao ◽  
Thalia Papayannopoulou ◽  
Frederick R. Appelbaum ◽  
Pamela S. Becker
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Fold Increase ◽  
White Blood Count ◽  
Saline Control ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Total Body

Abstract Abstract 1432 Background: The origin of relapse in AML is believed to be related to persistence of resistant “leukemia stem cells.” in the bone marrow microenvironment where adhesion confers drug resistance. Engraftment of human AML in immunodeficient mice is dependent on CXCR4 (Tavor et al 2004). CXCR4 inhibitors, such as AMD3100 (plexiglass,hereafter P), overcome adhesion mediated chemotherapy resistance (Zeng et al 2006) and mobilize human leukemia engrafted in immunodeficient mice (Zeng et al 2009). P also mobilized leukemia in an APL murine model and in combination with chemotherapy reduced tumor burden (Nervi et al 2009). Methods: We studied the combination of P 5mg/kg daily sc X 3, cytarabine (araC=A) 300mg/kg IP X 3 and clofarabine (C), 20mg/kg IP X 3 in the NODscid IL2R γc−/− mouse engrafted with primary patient AML CD34+ cells after 350 cGy total body irradiation. We could first detect circulating human CD45+ or human CD34+ cells, denoting engraftment, by flow cytometry as early as 5–13 weeks. We then injected plerixafor to assess mobilization capability at 8–16 weeks, followed by the combination of plerixafor and chemotherapy. Animals were sacrificed by 14–38 days after chemotherapy, and assessed for AML in blood, marrow, and spleen. Results: A single 5mg/kg dose of P, produced a 2.26 ± 0.94 (SD) fold increase in peak mobilization (at 2 hours) compared to saline control, p=0.026. P-induced mobilization was directly related to expression of CXCR4, with a patient exhibiting 10.3% CXCR4 showing 0.86× baseline, as compared to a patient with 24.7% CXCR4 exhibiting a 2.2-fold increase, and 84.9% CXCR4, a 3-fold increase. Chemotherapy,described above, was given 2 hours after plerixafor. For animals that received P/A vs. P/A/C, there was no statistically significant difference in leukemic burden (in millions of human CD34+ AML cells ± SD) of the animals sacrificed 14 days after initiation of treatment: bone marrow six bones 116.3 ± 33.7 vs. 111.7 ± 29.2 (p=0.86), spleen 50.8 ± 10 vs. 43.7 ± 19.1 (p=0.59), blood 10.9 ± 9.6 vs. 3.1 ± 1.4 (p=0.16), or estimated total body burden 178.0 ± 45.3 vs. 158.5 ± 30 (p=0.52). A comparison of 4 groups of animals, P/A/C vs. P/A vs. A/C vs. A demonstrated a statistically significant difference between certain groups, at certain time points. For example, on day 10, P/A/C treated animals had a lower human PB CD34+ count than P/A, 0.07 vs. 0.24 × 109/L (p=0.034). On day 14, P/A/C had a lower CD34+ count than A/C, 0.08 vs. 0.16 (p=0.047). But at day 38, the leukemia had already recurred, and there was no statistically significant difference in the organ or total body involvement by the leukemia. There was a very low white blood count days 5–24 post chemotherapy, with only minimal residual disease detectable by flow cytometry (analogous to a period of remission in humans), but by day 38, the leukemia had recurred (Figure 1), and there was no statistically significant difference in the organ or total body involvement by the leukemia amongst the groups (Figure 2). Conclusion: This model demonstrates the efficacy of chemotherapy in reducing circulating leukemia, but resistant cells appear to remain sheltered and give rise to relapse. Despite the beneficial effects of CXCR4 inhibitors in vitro and in vivo in a murine APL model, the combination of plerixafor with chemotherapy did not prevent or postpone leukemic relapse. These results could be attributed to either inefficient scheduling of plerixafor (for example, continuous infusion may have worked better), or non-cycling cells may be preferentially mobilized (Bonig H et al., 2009), and thus less susceptible to cytarabine treatment. These concepts are currently being explored. Alternatively, concomitant inhibition of other adhesion receptors may be necessary to prevent leukemia from homing back to the marrow. Precise timing and degree of mobilization in combination with chemotherapy may be required to optimize this approach in the clinic. Disclosures: Becker: Sanofi-Oncology (Genzyme): Research Funding.

A Highly Selective SF3B1-Targeted Splicing Inhibitor Reduces Human CD34+ Cell Survival and Self-Renewal In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1653-1653
Author(s):  
Larissa Balaian ◽  
Leslie A Crews ◽  
Marianna Zipeto ◽  
Kulidjian Anna ◽  
Edward D. Ball ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cord Blood ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Self Renewal ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Before And After ◽  
Acute Myeloid

Abstract Introduction Myelodysplastic syndromes (MDS) result from ineffective hematopoietic stem cell (HSC) maintenance in an aged bone marrow microenvironment and have a proclivity for evolution to acute myeloid leukemia (AML). Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Seminal next-generation DNA sequencing Results suggest that MDS evolution is controlled by mutations in splicing related genes and epigenetic modifiers of gene expression. However little is known about the cell type and context specific functional effects of these mutations on LSC transcriptional alterations that have been shown to promote MDS/AML progression and resistance to therapies such as 5-azacytidine (Vidaza). Therefore, we investigated the effect of splicing inhibitors on LSC survival and self-renewal 1) during progression of MSD to AML and 2) before and after clinical Vidaza treatment in a bone-marrow stromal co-cultures that recapitulates key aspects of the human LSC niche. Methods Mouse bone marrow cell lines, transfected to producehuman SCF,IL3 and G-CSF, were used as a stromal monolayers. Then human CD34+ cellswere selected from MDS (n=1) and AML primary samples (n=6). As normal controls, CD34+cells from cord blood (CB, n=3) or aged bone marrow (n=3) were utilized for the co-culture experiments. Survival and self-renewal of the CD34+ cells were investigated by colony forming and replating assays. Two SF3B1-targeted splicing inhibitors: FD 895 and a FD-analog were added at the initiation of co-culture at concentrations ranging from 0.1 to10 uM. Results After 2 weeksof stromal co-culture, none of the compounds demonstrated inhibition of the cell viability. Meanwhile, the splicing inhibitors demonstratedno reduction in survival in cord blood, and minor cytotoxicity toward aged bone marrow, MDS and AML samples showed a dose- and time-dependent significant (up to 80%) inhibition of colony formation. To analyzethe effect of splicing inhibitors on LSC self-renewal, replatingassayswere performed. While compounds at high doses mediated only a slight decrease in colony formation in normal CB and a-BM samples, MDS and AML samples exhibited a dose dependent inhibition of 38.2+/-8.1% of LSC survival (p<0.001) for FD895 and considerably lower 13.8+/-3.6 % of LSC survival for FD analog (p<0.001). Analysis of pre- and post progression samples from the same patient revealed the capacity of splicing inhibitors to diminish LSC survival. In CMML, FD895 induced significantly less cytotoxicity (35% compared with 75%) after progression to AML. Notably, in aged-BM both compounds reduced only CFU-GM survival, but not HSC self-renewal. In sequential primary samples from AML patients collected before and after clinical treatment with Vidaza, naïve samples exhibited similar sensitivity to FD-895 treatment in stromal co-culture models, and LSC survival and self-renewal capacity was reduced following incubation with FD-895. In contrast, following clinical treatment in patients that were responsive to Vidaza, these samples acquired resistance to splicing inhibition. However, patients that were non-responders to Vidaza treatment retained sensitivity to FD-895 treatment. Conclusions These data indicate that RNA editing and splicing activities represent novel regulators of LSC self-renewal and survival in LSC supportive niches. These properties can be inhibited using novel splicing inhibitors with minimal toxicity toward normal progenitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A review of current knowledge of myeloproliferative disorders in the horse

2021 ◽  
Vol 63 (1) ◽  
Author(s):  
Katy Satué ◽  
Juan Carlos Gardon ◽  
Ana Muñoz
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Current Knowledge ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Disorders ◽  
World Health ◽  
Chronic Granulocytic Leukemia ◽  
Current State ◽  
Multiple Cell ◽  
Health Organization

AbstractMyeloid disorders are conditions being characterized by abnormal proliferation and development of myeloid lineage including granulocytes (neutrophils, eosinophils and basophils), monocytes, erythroids, and megakaryocytes precursor cells. Myeloid leukemia, based on clinical presentation and proliferative rate of neoplastic cells, is divided into acute (AML) and myeloproliferative neoplasms (MPN). The most commonly myeloid leukemia reported in horses are AML-M4 (myelomonocytic) and AML-M5 (monocytic). Isolated cases of AML-M6B (acute erythroid leukemia), and chronic granulocytic leukemia have also been reported. Additionally, bone marrow disorders with dysplastic alterations and ineffective hematopoiesis affecting single or multiple cell lineages or myelodysplastic diseases (MDS), have also been reported in horses. MDSs have increased myeloblasts numbers in blood or bone marrow, although less than 20%, which is the minimum level required for diagnosis of AML. This review performed a detailed description of the current state of knowlegde of the myeloproliferative disorders in horses following the criteria established by the World Health Organization.

scholarly journals Korszakváltás a gyermekkori szerzett csontvelő-elégtelenséggel járó kórképek kezelésében Magyarországon

2018 ◽  
Vol 159 (42) ◽  
pp. 1710-1719
Author(s):  
Krisztián Kállay ◽  
Judit Csomor ◽  
Emma Ádám ◽  
Csaba Bödör ◽  
Csaba Kassa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Survival Rate ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Severe Aplastic Anemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Reduced Intensity Conditioning ◽  
Myelomonocytic Leukemia ◽  
Reduced Intensity

Abstract: Introduction: Acquired bone marrow failures are rare but fatal diseases in childhood. Since 2013, Hungary has been participating as a full member in the work of the European Working Group on uniform diagnostics and therapy in patients with acquired bone marrow failure syndromes. Hypocellular refractory cytopenia of childhood has been emphasized as a frequent entity, transplanted by reduced intensity conditioning with excellent outcomes. Aim: To analyse and compare the results of treatment before and after our joining. Method: A total of 55 patients have been treated in the 8 centres of the Hungarian Pediatric Oncology Network during 5 years between 2013 and 2017 (severe aplastic anemia: 9, myelodysplastic syndrome: 41, juvenile myelomonocytic leukemia: 5 patients). Allogeneic hematopoietic stem cell transplantation was performed in severe aplastic anemia in 7 cases, while antithymocyte globulin was administered in one case and one patient died before diagnosis. In patients with myelodysplastic syndromes, watch and wait strategy was applied in 4, while transplantation in 37 cases. Reduced intensity conditioning was used in 54 percent of these cases. Transplantation was the treatment of choice in all 5 patients with juvenile myelomonocytic leukemia. Results: In the whole patient cohort, the time from diagnosis to treatment was median 92 (3–393) days, while in severe aplastic anemia median 28 (3–327) days only. Grade II–IV acute graft versus host disease occurred in 22.6%, grade III–IV in 6.8% and chronic in 11.2%. All the patients treated with severe aplastic anemia are alive and in complete remission (100%). The overall estimated survival rate is 85.1% in myelodysplastic syndrome, while 75% in juvenile myelomonocytic leukemia. The median follow-up was 30.4 (1.1–62.5) months. There was a remarkable increase in overall survival comparing the data before (1992–2012) and after (2013) joining the international group, 70% vs. 100% (p = 0.133) in severe aplastic anemia and 31.3% vs. 85.1% (p = 0.000026) in myelodysplastic syndrome. Conclusion: Due to a change in the paradigm of the conditioning regimen in hypocellular refractory cytopenia of childhood, the overall survival rate has significantly increased. Orv Hetil. 2018; 159(42): 1710–1719.

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