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Neal S Young - One of the best experts on this subject based on the ideXlab platform.

  • single cell rna seq reveals the differentiation hierarchy of normal human bone marrow and a distinct transcriptome signature of Monosomy 7 cells
    Blood, 2016
    Co-Authors: Xin Zhao, Sachiko Kajigaya, Xingmin Feng, Danielle M Townsley, Zhijie Wu, Keyvan Keyvanfar, Marie J Desierto, Xujing Wang, Neal S Young
    Abstract:

    Monosomy 7 is a frequent cytogenetic abnormality in hematopoietic malignancies and a general indicator of poor prognosis. Due to lack of distinct cell surface markers between Monosomy 7 cells and normal cells, it is not feasible to physically separate aneuploid from diploid cells. We performed single-cell RNA-seq (scRNA-seq), which allows the entire transcriptome of large numbers of single cells to be assayed in an unbiased way, to investigate hematopoietic differentiation of normal and aneuploid human hematopoietic cells. Bone marrow samples were collected from four patients (P1-P4) with myelodysplastic syndrome, and four healthy volunteers. Conventional cytogenetics showed -7/7q- in bone marrow cells from P1, P3 and P4, and dup(1)(q21q32) in cells from P2; retrospectively, P2 was found positive for Monosomy 7 as well as trisomy 8 by fluorescence in situ hybridization. Fresh CD34+CD38- and CD34+CD38+cells were sorted by flow cytometry and then subjected to Fluidigm C1 Single-Cell Auto Prep System for scRNA-seq. After excluding cells with low transcriptome coverage, 326 cells from P1 and P2 (analysis is in progress for P3 and P4), and 391 cells from healthy subjects were analyzed by comparison of transcriptomes from 17,071 genes. Nonlinear dimension reduction and visualization were achieved using t-distributed Stochastic Neighbor Embedding (tSNE). Cells from healthy controls clustered into seven subgroups based on their gene expression pattern, and each group could be associated with a previously reported hematopoietic cell type by known marker genes (Laurenti E, Nat Immunol, 2013). These cell types included hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP) (Fig 1a). Individual cells from healthy controls were ordered by Monocle software based on their expression profile similarity to uncover a differentiation hierarchy. A two-branch trajectory of development from HSC was revealed, with one branch progressing towards erythroid cell and the other to lymphoid/myeloid cells (Fig 1b). This pattern differs from the classic hematopoietic model, but is consistent with reports claiming existence of early-lymphoid-biased progenitors that retain myeloid but not erythroid potential (Doulatov S, Nat Immunol, 2010), and of dominance of multipotent and unipotent progenitors over scarce oligopotent progenitors in the adult marrow hematopoietic hierarchy (Notta F, Science, 2016). We compared single cells from patients and healthy controls for regional and chromosomal copy number differences in gene expression. We identified subclonal populations of cells from patients that showed decreased expression of chromosome 7 genes (60% in P1, and 55% in P2; Fig 1c and 1d), and increased expression of chromosome 8 (77% in P2) and chromosome 1 long arm genes (P2), at FDR=0.05 estimated with cells from health donors. Gene Ontology enrichment analysis using topGO indicated that cells with low global expression of chromosome 7 genes had dysregulated expression of immune related genes, including B cell receptor signaling pathway, T cell activation and differentiation, antigen receptor-mediated signaling pathway, as well as signal transduction and Fc-γ Receptor signaling pathway. ScRNA-seq analysis reveals a simple pattern of normal human hematopoietic development and the molecular signature of aneuploid cells from patients with developing "clonal evolution". This powerful method should improve characterization of functional changes in human cells with chromosome abnormalities. Disclosures Desierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

  • clonal evolution in aplastic anemia is driven by chromosomal instability rather than mutations in myeloid malignancy candidate gene
    Blood, 2013
    Co-Authors: Bogdan Dumitriu, Sachiko Kajigaya, Xingmin Feng, Yasutaka Ueda, Danielle M Townsley, Jun Zsu, Yoshi Wakabayashi, Yanqin Yang, Delong Liu, Neal S Young
    Abstract:

    ![Graphic][1] The pathophysiology of human aplastic anemia (AA) is immune-mediated destruction of bone marrow stem and progenitor cells. Most patients respond to immunosuppressive therapies (IST), which markedly improved survival in this disease. However, a minority of patients undergoes transformation to malignant hematologic disease, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), usually accompanied by the cytogenetic abnormality Monosomy 7. Clonal evolution in AA confers a poor prognosis in the clinic but is an opportunity to assess early events in oncogenesis in the setting of inflammation and tissue regeneration. We previously reported that low mean telomere length of leukocytes at the time of diagnosis of AA ( Scheinberg et al., JAMA 2010 ) was associated with increased risk of progression to MDS. In the current study, we directly compared acquired mutations in candidate genes and chromosomal instability, as measured by telomere length, in a cohort of AA patients that had progressed to MDS. Thirteen AA patients who developed Monosomy 7 were compared with 30 AA patients who had received similar treatments but did not progress to MDS. Leukocytes telomere content was measured by qPCR in samples obtained at different time points from the diagnosis of AA and chromosome-specific telomere length was assessed by single telomere length assay (STELA) for Xp, Yp, 12q, and 17p . In the AA patients who had evolved to MDS and AML, analysis of acquired mutations in myeloid-specific genes was performed by comparison with control “germline” DNA from purified CD3 lymphocytes by exome sequencing. Cells from AA patients with clonal evolution showed marked progressive telomere attrition, 419 bp/year during the period preceding development of Monosomy 7. Telomere attrition was progressive from the time of diagnosis of AA. By STELA, accumulation of very short telomere fragments was apparent at 6 months after IST. In contrast, for the AA control group, patients whose disease was stable, telomere attrition was not accelerated in serial qPCR determinations, nor was there increased accumulation of short telomere fragments by STELA. A similar pattern of increased telomere attrition was reproduced in vitro by cultivation of bone marrow cells obtained six months after IST in all AA patients who developed MDS, while none of the AA control bone marrow cultured cells developed shorter telomeres. We examined bone marrow myeloid cells at the time of Monosomy 7 for acquired mutations in 125 candidate genes reported to be recurrently mutated in AML and MDS. Exome sequencing was performed using Agilent SureSelect Target Enrichment System. The raw reads were mapped to UCSC Human Genome hg19 by BWA software with default setting. With an average of 111-fold coverage on selected exon regions, somatic mutations were identified between paired samples using SAMtools and Shimmer software for SNP detection. Acquired mutations in myeloid cells were found in two cases. One patient had a heterozygous mutation in DNMT3A (K829T) present since diagnosis of AA. The other patient also had a heterozygous mutation in DNMT3A (P904S) as well as mutations in DOTL1, ASXL1, SETBP1, and STAT3 . All these mutations were identifiable after IST as neutrophils recovered. Despite the presence of multiple mutations, this patient had shown a good hematologic response to IST; evolution was manifest as recurrent pancytopenia and stable marrow myeloblasts at about 5% for over 2 years after first detection of Monosomy 7. The remaining 11 patients, all of whom lacked candidate gene mutations, had progressive increase in bone marrow myeloblast numbers; the only other three survivors in this cohort had received hematopoietic stem cell transplant. In conclusion, telomere shortening rather than accumulation of point mutations in hematopoietic cells preceded aneuploidy and malignant transformation at an early stage of oncogenesis in this group of patients. These results from AA may be generalizable to other cancers arising in the setting of inflammation and tissue regeneration in other organs. Identification of critically short telomeres before the development of cytogenetic abnormalities may allow for improved management of patients at risk of clonal evolution, and pharmacologic strategies to increase telomerase activity might mitigate the risk of cancer in these settings Disclosures: No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif

  • very short telomeres of peripheral blood leukocytes precede clinical progression to myelodysplasia with Monosomy 7 in aplastic anemia patients
    Blood, 2012
    Co-Authors: Bogdan Dumitriu, Sachiko Kajigaya, Yasutaka Ueda, Danielle M Townsley, Neal S Young
    Abstract:

    Abstract 1265 BACKGROUND: Acquired severe aplastic anemia (SAA) is a human disease characterized by severe pancytopenia due to marked reduction in the numbers of hematopoietic progenitors and stem cells. Immunosuppressive treatment with antithymoglobulin and cyclosporine results in improvement in blood counts in above 2/3 of patients. The most serious long-term complication in SAA is progression to myelodysplasia (MDS) and acute myeloid leukemia, usually associated with the cytogenetic abnormality Monosomy 7. Clonal evolution usually requires a hematopoietic stem cell transplant and it is the major cause of morbidity and mortality. OBJECTIVE: Decreased average telomere length in leukocytes on presentation has been associated with increase risk of progression to MDS (Scheinberg et. al., 2010 JAMA, vol. 304(12):1358–64). Single telomere length assay (STELA) uses single molecule polymerase chain reaction (PCR) to amplify chromosome-specific telomeres based on the specificity of their subtelomeric region (Baird et. al., 2003 Nature Genetics, vol. 33(2):203–7). We have utilized STELA for identify SAA patients at risk of progression to MDS prior to development of the cytogenetic abnormality. METHODS: Peripheral blood samples were obtained at diagnosis and sequentially after immunosuppressive treatment from SAA patients who fulfilled entry criteria for protocols at the National Institutes of Health. Chromosome-specific PCR amplification of telomere using primers specific for Xp/Yp subtelomeric regions was performed. A non-radioactive, digoxin-labeled telomeric probe was used for Southern blotting to identify discrete bands, allowing quantification of very short telomeres. Results were confirmed using chromosome-specific primers for 2p, 11q, 12q, and 17p. RESULTS: Initially, multiple serial samples from five patients who had progressed to Monosomy 7 MDS were analyzed by STELA of DNA extracted from peripheral blood leukocytes at the diagnosis of SAA and again at progression to MDS. All five patients had increase in very short telomeres ( CONCLUSIONS: This novel, non-radioactive method of quantifying chromosome-specific telomere length is capable of identifying very short telomeres in SAA patients progressing to MDS with Monosomy 7. Previous studies have associated average short telomere length with chromosomal instability and progression to malignant phenotype. Identifying very short telomere in a subpopulation of circulating cells by STELA regardless of the average telomere length by qPCR is a more sensitive method. Chromosome-specific primers and analysis of end-to-end fusion of different chromosomes will allow for better characterization of the chromosomal instability leading to malignant transformation in these patients. Allogeneic stem cell transplant is the definitive treatment for MDS with Monosomy 7 but requires time for identification of a suitable donor. Identification of critically short telomere before development of the cytogenetic abnormality will allow for timely management of patients at risk of clonal evolution. Therapeutic telomerase upregulation by androgens might reduce very short telomeres and potentially decrease the transformation risk. Disclosures: No relevant conflicts of interest to declare.

  • effects of granulocyte colony stimulating factor on Monosomy 7 aneuploidy in healthy hematopoietic stem cell and granulocyte donors
    Transfusion, 2012
    Co-Authors: Matthew J Olnes, Andrea Poon, Zachary Tucker, Loretta Pfannes, Susan J Miranda, Kelsey Loeliger, Hesed Padillanash, Thomas Ried, Susan F Leitman, Neal S Young
    Abstract:

    Background—Reports of Monosomy 7 in patients receiving granulocyte colony stimulating factor (G-CSF) have raised concerns that this cytokine may promote genomic instability. However, there are no studies addressing whether repeated administration of G-CSF produces Monosomy 7 aneuploidy in healthy donors. Study Design and Methods—We examined chromosomes 7 and 8 by fluorescent in situ hybridization (FISH) in CD34+ cells from 35 healthy hematopoietic stem cell transplant (HSCT) donors after G-CSF administration for 5 days, and by spectral karyotyping analysis (SKY) in four individuals to assess chromosomal integrity. We also studied 38 granulocyte donors who received up to 42 doses of G-CSF and dexamethasone (Dex) using FISH for chromosomes 7 and 8. Results—We found no abnormalities in chromosomes 7 and 8 in G-CSF mobilized CD34+ cells when assessed by FISH or SKY, nor did we detect aneuploidy in G-CSF/Dex treated donors. Conclusion—G-CSF does not promote clinically detectable Monosomy 7 or trisomy 8 aneuploidy in HSCT or granulocyte donors. These findings should be reassuring to healthy HSCT and granulocyte donors.

  • jak2 inhibition with tg101348 inhibits Monosomy 7 myelodysplastic syndromes mds bone marrow cells in vitro a potential targeted therapy for Monosomy 7 mds
    Blood, 2010
    Co-Authors: Matthew J Olnes, Andrea Poon, Zachary Tucker, Neal S Young, Elaine M. Sloand
    Abstract:

    Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that Monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on Monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of Monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in Monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases Monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with Monosomy 7 MDS and AML. ![Figure][1] Disclosures: No relevant conflicts of interest to declare. [1]: pending:yes

Mark R. Litzow - One of the best experts on this subject based on the ideXlab platform.

  • independent prognostic significance of Monosomy 17 and impact of karyotype complexity in monosomal karyotype complex karyotype acute myeloid leukemia results from four ecog acrin prospective therapeutic trials
    Leukemia Research, 2017
    Co-Authors: Stephen A Strickland, Larry D Cripe, Hugo Fernandez, Gary A Hicks, Rodney R Higgins, Athena M Cherry, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Mark R. Litzow
    Abstract:

    Abstract The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (Monosomy 5, Monosomy 7, and Monosomy 17) within MK+/CK+ AML is not well defined. We analyzed data from 1,592 AML patients age 17–93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥ 5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p

  • independent prognostic significance of Monosomy 17 and impact of karyotype complexity in monosomal karyotype complex karyotype acute myeloid leukemia results from four ecog acrin prospective therapeutic trials
    Leukemia Research, 2017
    Co-Authors: Stephen A Strickland, Larry D Cripe, Hugo Fernandez, Gary A Hicks, Rodney R Higgins, Athena M Cherry, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Mark R. Litzow
    Abstract:

    Abstract The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (Monosomy 5, Monosomy 7, and Monosomy 17) within MK+/CK+ AML is not well defined. We analyzed data from 1,592 AML patients age 17–93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥ 5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p

  • monosomal karyotype mk in older patients with acute myeloid leukemia aml on eastern cooperative oncology group ecog therapeutic trials poor prognostic impact of mk but not of Monosomy 7
    Blood, 2010
    Co-Authors: Stephen A Strickland, Larry D Cripe, Rodney R Higgins, Athena M Cherry, Mark R. Litzow, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Selina M Luger, Elisabeth Paietta
    Abstract:

    Abstract 579 Background: In AML, age and cytogenetics remain among the major prognostic factors. Age above 55 years and a complex karyotype (CK; ≥ 3 abnormal clones) or the presence of Monosomy 7 (M7) are universally associated with unfavorable prognosis. Recent data from HOVON-SAKK ( J Clin Onc 26: 4791) suggest that in patients under the age of 60 years, subgroups with poor risk versus very poor risk can be delineated based on the presence of a MK. MK was defined as either the presence of at least two autosomal monosomies or one Monosomy plus at least one structural abnormality. Limited data exists regarding the impact of MK in AML patients over the age of 55 years. Methods: Data from 935 AML patients above age 55 enrolled on three prospective ECOG therapeutic clinical trials (E1490, E3993, E3999) between 1990 and 2005 were retrospectively reviewed to identify those with MK with single and/or multiple monosomies, MK in the presence or absence of a CK, CK with/without MK, MK with/without M7, and normal karyotype (CN). CK was defined as ≥ 3 clonal abnormalities for the purposes of this study. Complete remission (CR), disease-free survival (DFS), and overall survival (OS) were assessed. Results: The median age of evaluable patients was 68 years (range 56 – 93) with a male/female ratio of 391/299. Cytogenetic data were evaluable in 690 (74%) subjects. Clonal cytogenetic abnormalities occurred in 399 (58%). Core-binding factor (CBF) abnormalities accounted for 16/399 (4%) whereas CK was detected in 181/399 (45%). MK was present in 172 (25%) evaluable cases, predominantly in the setting of CK (140/172, 81%). Overall CR was 44%. CR was achieved in 49% MK−, 29% MK+, 31% CK+, and 49% of CK−. Data for the combinations of MK and CK are presented in Table 1. Median follow-up for survivors was 5.0 years. OS at 4-years was 9% for all evaluable patients and 4y-DFS was 12% in those achieving CR. Median OS of MK+/CK+ (0.3y) was significantly worse (p Conclusions: AML patients above the age of 55 years have an unfavorable prognosis with the majority of patients harboring adverse clonal cytogenetic abnormalities and failing to achieve a CR. Fewer than 10% survive 4-years from diagnosis. Results from the ECOG experience demonstrate the prognostic predictive value of MK in older AML patients. In contrast to the data presented by the HOVAN-SAKK group, the prognostic value of MK in patients over 55 years is limited to those with MK in combination with CK. These results also demonstrate the failure of Monosomy 7 to provide prognostic value in the setting of MK. Disclosures: No relevant conflicts of interest to declare.

Ayalew Tefferi - One of the best experts on this subject based on the ideXlab platform.

  • sole abnormalities of chromosome 7 in myeloid malignancies spectrum histopathologic correlates and prognostic implications
    American Journal of Hematology, 2012
    Co-Authors: Fareeda Taher Nazer Hussain, Ryan A. Knudson, Edward P Nguyen, Sania S Raza, Animesh Pardanani, Curtis A Hanson, Daniel L Van Dyke, Ayalew Tefferi
    Abstract:

    Among 6,565 consecutive abnormal cytogenetic reports at our institution, 3,192 (49%) constituted sole abnormalities, of which 230 (7%) involved chromosome 7: Monosomy 7 (n = 98), 7q- (n = 51), der(1;7)(q10;p10) (n = 44), balanced translocations (n = 15), ring 7 (n = 13), and 7p- (n = 9). The most frequent histopathologic correlates were myelodysplastic syndromes (MDS; 28%), acute myeloid leukemia (AML; 17%), secondary or therapy-related MDS/AML (13%), primary myelofibrosis (PMF; 7%), and chronic myelomonocytic leukemia (6%). Monosomy 7 was the most frequent in each one of these disease categories except PMF where 7q- was more frequent. In primary MDS, patients with der(1;7)(q10;p10) (n = 13), compared to those with Monosomy 7 (n = 30) or 7q- (n = 15), were less likely (P = 0.04) to display excess blasts or multilineage dysplasia but overall and leukemia-free survival adjusted for these variables revealed no significant difference between the three groups (P = 0.57 and 0.81, respectively). The current study does not prognostically distinguish Monosomy 7 from 7q- or der(1;7), in MDS.

  • monosomal karyotype in myelodysplastic syndromes with or without Monosomy 7 or 5 is prognostically worse than an otherwise complex karyotype
    Leukemia, 2011
    Co-Authors: Mrinal M Patnaik, Janice M Hodnefield, C A Hanson, Daniel L Van Dyke, Ryan A. Knudson, Ayalew Tefferi
    Abstract:

    Monosomal karyotype (MK) refers to the presence of two or more distinct autosomal monosomies or a single Monosomy associated with a structural abnormality. In acute myeloid leukemia, MK has been shown to be prognostically worse than an otherwise complex karyotype. The current study examines whether the same holds true for myelodysplastic syndromes (MDS). A total of 127 MDS patients (median age 70 years) with a complex karyotype were considered; 106 (83%) met the above-stipulated criteria for MK and 21 (17%) had a complex karyotype without monosomies. Survival was significantly inferior in patients with MK compared with those with a complex karyotype without monosomies (P=0.01; HR 1.9, 95% confidence interval (95% CI), 1.1–3.3). Multivariable analysis identified MK (P=0.002), advanced age (P=0.0004) and bone marrow blast percentage (0.04) as independent risk factors for survival. There was no difference in survival among MK patients further substratified by the presence or absence of Monosomy 7 and/or Monosomy 5. Although not statistically significant, leukemia-free survival was also worse with MK compared with complex karyotype without monosomies (P=0.09; HR 2.7, 95% CI 0.8–9.0). MK in MDS identifies a prognostically worse subgroup of patients with a complex karyotype, regardless of whether Monosomy 7 or 5 is part of the MK component.

Elaine M. Sloand - One of the best experts on this subject based on the ideXlab platform.

  • jak2 inhibition with tg101348 inhibits Monosomy 7 myelodysplastic syndromes mds bone marrow cells in vitro a potential targeted therapy for Monosomy 7 mds
    Blood, 2010
    Co-Authors: Matthew J Olnes, Andrea Poon, Zachary Tucker, Neal S Young, Elaine M. Sloand
    Abstract:

    Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that Monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on Monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of Monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in Monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases Monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with Monosomy 7 MDS and AML. ![Figure][1] Disclosures: No relevant conflicts of interest to declare. [1]: pending:yes

  • a non atp competitive dual inhibitor of jak2v617f and bcr ablt315i kinases elucidation of a novel therapeutic spectrum based on substrate competitive inhibition
    Genes & Cancer, 2010
    Co-Authors: Shashidhar S Jatiani, Matthew J Olnes, Elaine M. Sloand, Loretta Pfannes, Stephen C Cosenza, M Ramana V Reddy, Stacey J Baker, Ajoy K Samanta, Ralph B Arlinghaus, Premkumar E Reddy
    Abstract:

    Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non–ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2V617F-positive leukemic cells and blocks the IL-3–mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABLT315I and JAK2V617F. Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from Monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including Monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

  • suppression of cyclin d1 by on 01910 na is associated with decreased survival of trisomy 8 myelodyplastic bone marrow progenitors a potential targetted therapy
    Blood, 2007
    Co-Authors: Elaine M. Sloand, Loretta Pfannes, Ramana M V Reddy, Premkumar E Reddy, Jerome S Groopman, Neal S Young
    Abstract:

    Abstract Trisomy 8 CD34 cells persist and even expand in patients with bone marrow failure despite a potent specific immune response against them(Sloand EM et al; Blood 2005; 106(3):841). We previously demonstrated dramatic increases in c-myc, survivin, and CD1 in trisomy 8 CD34 cells by microarray analysis, realtime PCR, and immunoblot (Sloand et al; Blood 2007; 109:2399) and postulated that upregulation of c-myc located on chromosome 8 was responsible for increases in CD1 and survivin, an anti-apoptotic protein. Knock-down of either survivin or c-myc resulted in selective trisomy 8 apoptosis and death. The styryl sulfones (Reddy, M et al Acta Chim Hungarica 1984; 115:269), are novel small molecule anticancer agents that inhibit cell cycle progression in cancer cells, and phase I studies for solid tumors have demonstrated little toxicity in humans (Donehower, R et al, J. Clin. Oncol. 24. 2006 abstract #13026). Initial studies in our laboratory demonstrated significant decreases in CD1 measured after trisomy 8 cells were co-cultured with increasing concentrations (10–30 nM). In this study, we examined the effect 01910.Na, a styryl sulfone, on trisomy 8 cell growth and survival. Short-term culture of mononuclear cells from four normal bone marrows with increasing concentrations of the drug (10–200nM) showed no adverse effect on hematopoietic colony formation (N=4). MDS bone marrow cells from ten trisomy 8 patients; 4 with RAEB and 6 with RA and two Monosomy 7 patients with RAEB were studied. All patients with cytogenetics demonstrating trisomy 8 either had it as the sole karyotypic abnormality or with Monosomy 7 in the same clone. Bone marrow was cultured with drug for 2 weeks at four different drug concentrations and fluorescent in situ hybridization performed by three blinded investigators counting 300 cells each. There was a substantial decline in the number and percent of aneuploid cells containing trisomy 8 (p=0.03; N=6) Fig 1. Other cells, including those with Monosomy 7 as the sole abnormality and diploid cells were relatively unaffected. Continued treatment of the cells with a 100–250nM concentration for a 14 day period resulted in optimal killing of aneuploid cells when compared to one treatment at the beginning of the two week period. The number of blasts also decreased over the two week period when slides were prepared, stained with HE this effect was dose-dependent (N=3; p=0.02; Fig 2). Flow cytometric examination of a fourth patient confirmed these findings, demonstrating increased proportions of mature CD15 positive myeloid cells and decreased number of immature CD33 cells or blasts staining with CD34 concomitant with decreased numbers of trisomy 8 cells (Fig3). ON 01910.Na may prove a targeted therapy for patients with MDS and a trisomy 8 clone. A phase I/II trial is set to commence shortly. Download : Download high-res image (103KB) Download : Download full-size image Figure

  • granulocyte colony stimulating factor preferentially stimulates proliferation of Monosomy 7 cells bearing the isoform iv receptor
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Elaine M. Sloand, Agnes S M Yong, Tullia C Bruno, Monika Fuhrer, Elena E. Solomou, Sachiko Kajigaya, Shakti Ramkissoon, John A Barrett, Neal S Young
    Abstract:

    Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of Monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on Monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with Monosomy 7 were cultured with 400 ng/ml GCSF, all samples showed significant increases in the proportion of Monosomy 7 cells. In contrast, bone marrow from karyotypically normal aplastic anemia, myelodysplastic syndrome, or healthy individuals did not show an increase in Monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and Monosomy 7, GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA, CD34 cells showed increased expression of the GCSFR class IV mRNA isoform, which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in Monosomy 7 CD34 cells, with increased phosphorylated signal transducer and activation of transcription protein, STAT1-P, and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting Monosomy 7 cells. The abnormal response of Monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated Monosomy 7 clones expressing the class IV GCSFR, which is defective in signaling cell maturation.

  • distinctive gene expression profiles of cd34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities
    Blood, 2004
    Co-Authors: Guibin Chen, Sachiko Kajigaya, Elaine M. Sloand, Jaroslaw P Maciejewski, Weihua Zeng, Akira Miyazato, Eric M Billings, Neal S Young
    Abstract:

    Aneuploidy, especially Monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with Monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with Monosomy 7 and trisomy 8 using Affymetrix GeneChips. Two methods were employed: standard hybridization and a small-sample RNA amplification protocol for the limited amounts of RNA available from individual cases; results were comparable between these 2 techniques. Microarray data were confirmed by gene amplification and flow cytometry using individual patient samples. Genes related to hematopoietic progenitor cell proliferation and blood cell function were dysregulated in CD34 cells of both Monosomy 7 and trisomy 8 MDS. In trisomy 8, up-regulated genes were primarily involved in immune and inflammatory responses, and down-regulated genes have been implicated in apoptosis inhibition. CD34 cells in Monosomy 7 showed up-regulation of genes inducing leukemia transformation and tumorigenesis and apoptosis and down-regulation of genes controlling cell growth and differentiation. These results imply distinct molecular mechanisms for Monosomy 7 and trisomy 8 MDS and implicate specific pathogenic pathways.

Stephen A Strickland - One of the best experts on this subject based on the ideXlab platform.

  • independent prognostic significance of Monosomy 17 and impact of karyotype complexity in monosomal karyotype complex karyotype acute myeloid leukemia results from four ecog acrin prospective therapeutic trials
    Leukemia Research, 2017
    Co-Authors: Stephen A Strickland, Larry D Cripe, Hugo Fernandez, Gary A Hicks, Rodney R Higgins, Athena M Cherry, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Mark R. Litzow
    Abstract:

    Abstract The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (Monosomy 5, Monosomy 7, and Monosomy 17) within MK+/CK+ AML is not well defined. We analyzed data from 1,592 AML patients age 17–93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥ 5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p

  • independent prognostic significance of Monosomy 17 and impact of karyotype complexity in monosomal karyotype complex karyotype acute myeloid leukemia results from four ecog acrin prospective therapeutic trials
    Leukemia Research, 2017
    Co-Authors: Stephen A Strickland, Larry D Cripe, Hugo Fernandez, Gary A Hicks, Rodney R Higgins, Athena M Cherry, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Mark R. Litzow
    Abstract:

    Abstract The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (Monosomy 5, Monosomy 7, and Monosomy 17) within MK+/CK+ AML is not well defined. We analyzed data from 1,592 AML patients age 17–93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥ 5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p

  • monosomal karyotype mk in older patients with acute myeloid leukemia aml on eastern cooperative oncology group ecog therapeutic trials poor prognostic impact of mk but not of Monosomy 7
    Blood, 2010
    Co-Authors: Stephen A Strickland, Larry D Cripe, Rodney R Higgins, Athena M Cherry, Mark R. Litzow, Gordon W Dewald, Rhett P Ketterling, Hillard M Lazarus, Selina M Luger, Elisabeth Paietta
    Abstract:

    Abstract 579 Background: In AML, age and cytogenetics remain among the major prognostic factors. Age above 55 years and a complex karyotype (CK; ≥ 3 abnormal clones) or the presence of Monosomy 7 (M7) are universally associated with unfavorable prognosis. Recent data from HOVON-SAKK ( J Clin Onc 26: 4791) suggest that in patients under the age of 60 years, subgroups with poor risk versus very poor risk can be delineated based on the presence of a MK. MK was defined as either the presence of at least two autosomal monosomies or one Monosomy plus at least one structural abnormality. Limited data exists regarding the impact of MK in AML patients over the age of 55 years. Methods: Data from 935 AML patients above age 55 enrolled on three prospective ECOG therapeutic clinical trials (E1490, E3993, E3999) between 1990 and 2005 were retrospectively reviewed to identify those with MK with single and/or multiple monosomies, MK in the presence or absence of a CK, CK with/without MK, MK with/without M7, and normal karyotype (CN). CK was defined as ≥ 3 clonal abnormalities for the purposes of this study. Complete remission (CR), disease-free survival (DFS), and overall survival (OS) were assessed. Results: The median age of evaluable patients was 68 years (range 56 – 93) with a male/female ratio of 391/299. Cytogenetic data were evaluable in 690 (74%) subjects. Clonal cytogenetic abnormalities occurred in 399 (58%). Core-binding factor (CBF) abnormalities accounted for 16/399 (4%) whereas CK was detected in 181/399 (45%). MK was present in 172 (25%) evaluable cases, predominantly in the setting of CK (140/172, 81%). Overall CR was 44%. CR was achieved in 49% MK−, 29% MK+, 31% CK+, and 49% of CK−. Data for the combinations of MK and CK are presented in Table 1. Median follow-up for survivors was 5.0 years. OS at 4-years was 9% for all evaluable patients and 4y-DFS was 12% in those achieving CR. Median OS of MK+/CK+ (0.3y) was significantly worse (p Conclusions: AML patients above the age of 55 years have an unfavorable prognosis with the majority of patients harboring adverse clonal cytogenetic abnormalities and failing to achieve a CR. Fewer than 10% survive 4-years from diagnosis. Results from the ECOG experience demonstrate the prognostic predictive value of MK in older AML patients. In contrast to the data presented by the HOVAN-SAKK group, the prognostic value of MK in patients over 55 years is limited to those with MK in combination with CK. These results also demonstrate the failure of Monosomy 7 to provide prognostic value in the setting of MK. Disclosures: No relevant conflicts of interest to declare.