Mesenchymal Stromal Precursor Cells from the Bone Marrow of Acute Myeloid and Lymphoid Leukemia Patients: Characteristics in Newly Diagnosed, before and after Allogeneic Hematopoietic Stem Cell Transplantation

Abstract Introduction Hematopoiesis is maintained in close contact with bone marrow (BM) stroma. Malignant hematopoietic cells probably affect the hematopoietic microenvironment, as well as high dose chemotherapy. The goal of this study was to analyze the alterations in the characteristics of human multipotent mesenchymal stromal cells (MSC) and their more differentiated progeny – fibroblastic colony forming units (CFU-F), derived from the BM of acute myeloid and lymphoid leukemia (AML and ALL) patients. Methods 26 newly diagnosed cases (18 AML, 8 ALL) and 35 patients (20 AML, 15 ALL) before and during 1 year after allogeneic hematopoietic stem cell transplantation (alloHSCT) were involved in the study after informed consent. BM was aspirated prior to any treatment in the newly diagnosed group and before the conditioning and at 6 time points during 1st year after the alloHSCT. MSC were cultured in aMEM with 10% FCS. Cumulative MSC production was counted after 3 passages. CFU-F concentration was analyzed in BM samples by standard protocol. The relative expression level (REL) of genes was measured by RT2 Profiler PCR Array (Qiagen) and TaqMan RQ-PCR. MSC and CFU-F from 50 healthy donors of BM for alloHSCT were used as a control after informed consent. Results The CFU-F concentration in the BM of patients at the moment of diagnostics was 1/3 of the donor’s for AML and ≈1/10 of ALL (9±5.2 and 3.9±2.3 versus 31±3.5 per 106 nucleated cells in donor BM, p<0.05). Most of the patients assigned to alloHSCT were in the remission and at that moment CFU-F concentration in their BM differed insignificantly from the donors’ one. After alloHSCT CFU-F concentration in AML and ALL patients’ BM decreased 3-9 folds for the whole next year. The decrease at each time point was highly significant comparing to donors. MSC properties were also altered in patients with acute leukemia. Cumulative cell production in MSC of newly diagnosed AML and ALL patients did not differ from donors’ one. MSC production from the BM of patients after standard courses of chemotherapy (for AML patients -mainly various numbers of “7+3” blocks and for ALL patients - ALL-2009) before alloHSCT did not differ from donors, while after alloHSCT cumulative AML and ALL MSC production decreased 1.3-6.3 folds for the next year. The decrease at almost each time point was significant comparing to donors. Gene expression analysis revealed that in MSC at the moment of AML diagnosis the REL of FGF2, IL1B, IL6, JAG1, PDGFB, VCAM1, VEGFA decreased more than 2 fold. Prior to alloHSCT the REL of IL6 and IL1B were still very low, after the alloHSCT the REL of IL6, JAG1, PDGFB increased, while IL1B stayed at the low level at least for 6 months. In MSC at the moment of ALL diagnosis the REL of IFNG, IGF1, IL6, KITLG, NES, PTPRC, TBX5 decreased more than 5 fold, while these of CSF3, ICAM1, IL1B, ITGAX – increased more than 5 fold. REL of IL6 and IDO1 were elevated prior to alloHSTC and stayed at such level for a year. Prior to alloHSCT the REL of FGFR2, PDGFRA, SDF1 and FGF2 were very low, after the alloHSCT the REL of JAG1, FGF2, TGFB1 slightly increased, while FGFR2, PDGFRB, SDF1 stayed at the low level at least for 1 year. Conclusions During the leukemia development malignant cells alter the stromal precursor cells leading to the decrease in the REL of many regulatory genes and in the number of more differentiated stromal precursor cells in the BM. Chemotherapy used for induction of the remission in these patients restore functional ability of MSC and CFU-F, while gene expression levels remained altered. Conditioning regiments used for the alloHSCT significantly damage both types of studied stromal precursors, and the effect lasted at least for 1 year. So, both leukemic cells and chemotherapy affect BM hematopoietic microenvironment. Disclosures No relevant conflicts of interest to declare.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v1 ◽

2019 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow, which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at the mRNA level in the cells of the hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.Results: The results found that the bone marrow cells of patients with acute myeloid leukemia (AML) show over expression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed the positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v4 ◽

2020 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.Methods: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v2 ◽

2020 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. Methods The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML. Conclusions Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1

Blood ◽

10.1182/blood-2007-02-073486 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3706-3714 ◽

Cited By ~ 127

Author(s):

Bas J. Wouters ◽

Meritxell Alberich Jordà ◽

Karen Keeshan ◽

Irene Louwers ◽

Claudia A. J. Erpelinck-Verschueren ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Large Set ◽

Lymphoid Leukemia ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Cell Commitment ◽

Acute Myeloid

Abstract Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

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Effect of Immunization with Polyvinylpyrrolidone on the Counts of Stromal Precursor Cells in the Bone Marrow and Spleen of CBA and CBA/N Mice and Cytokine Gene Expression in Primary Cultures of These Cells

Bulletin of Experimental Biology and Medicine ◽

10.1007/s10517-012-1644-5 ◽

2012 ◽

Vol 153 (1) ◽

pp. 64-67 ◽

Cited By ~ 8

Author(s):

U. F. Gorskaya ◽

T. A. Danilova ◽

M. V. Mezentzeva ◽

M. M. Shapoval ◽

V. G. Nesterenko

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Primary Cultures ◽

Precursor Cells ◽

Cytokine Gene Expression ◽

Cytokine Gene ◽

Stromal Precursor Cells

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CEBPα Is a Transcriptional Repressor of T-Cell Related Genes Explaining the Myeloid/T-Lymphoid Features of CEBPα-Silenced AML

Blood ◽

10.1182/blood.v118.21.554.554 ◽

2011 ◽

Vol 118 (21) ◽

pp. 554-554

Author(s):

Erdogan Taskesen ◽

Roberto Avellino ◽

Meritxell AlberichJorda ◽

Daniel G. Tenen ◽

Jeroen Ridder de ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

T Cell ◽

Differentially Expressed Genes ◽

Target Genes ◽

Differentially Expressed ◽

Lymphoid Leukemia ◽

Hematopoietic Stem ◽

Knock Out ◽

Molecular Abnormalities

Abstract Abstract 554 Acute myeloid leukemia (AML) is a heterogeneous disease of different subtypes characterized by distinct cytogenetic or molecular abnormalities. We recently identified a previously unrecognized subtype with a unique epigenetic feature, i.e. silencing of the gene that encodes CCAAT-enhancer binding protein alpha (CEBPα) by DNA hypermethylation (denoted as the CEBPαsilenced group). The leukemic blast cells of these patients express myeloid as well as T-lymphoid markers. Moreover, gene expression and DNA methylation profiling put these leukemias in between AML and T-lymphoid leukemia (T-ALL). CEBPα is a transcription regulator that is essential for normal neutrophil development. We hypothesize that methylation and consequently silencing of the gene encoding CEBPα is abnormal and an important hit in the transformation of this unique leukemia subtype. The mechanism by which CEBPα silencing plays a role in transformation of cells with myeloid/T-lymphoid features is the aim of our study. We carried out gene expression profiling of the CEBPαsilenced group (n=10) and compared gene expression levels to normal CD34+ bone marrow cells (n=11) and the remaining AML group (n=506) using a three-way ANOVA and a post-hoc test (tukey-kramer method). We detected 686 differentially expressed genes with P < 0.05 after multiple testing. Of these 686 genes, 288 were up- and 401 down-regulated in the CEBPαsilenced group. We next asked the question which of those genes might be bona fide CEBPα targets and whether expression has been altered as the result of CEBPα silencing. We transduced estrogen-inducible C/EBPα in 32D cells and carried out ChIP-on-chip analysis using ER specific antibodies. The analysis yielded a collection of 529 CEBPα target genes that are significant enriched for C/EBPα binding (P < 0.05) using Hypergeometric Analysis of Tiling-arrays (HAT). We hypothesized that the direct target genes of CEBPα, derived from the 32D model system, were also present among deregulated genes in CEBPαsilenced human AMLs. We therefore overlaid the detected direct binding targets of CEBPα in 32D cells, with the differentially expressed genes in the CEBPαsilenced group and identified 49 overlapping genes (P=1×10−7) as putative direct targets. Among these 49 genes, 25 were down-regulated and 24 were upregulated in the primary CEBPαsilenced AML group. Both groups of genes were highly enriched for the CEBPα consensus binding sequence, i.e. 16/25 and 20/24 promoter regions respectively. The 25 downregulated genes, among which ACSL1, MYCT1 or Slc7a11, represent targets that are most likely normally activated by CEBPα, but are not transcribed in CEBPαsilenced human AMLs due to the absence of CEBPα. Among the genes that were upregulated in CEBPαsilenced leukemias are BCL2, CCR9, CEBPG or CD47. These putative target genes are repressed in CEBPα expressing cells and activated when CEBPα is silenced. This observation suggests that the transcription factor CEBPα may also acts as a repressor of gene transcription. We therefore studied the expression of two of those genes, i.e. CEBPG and CCR9 in Lin-Sca+Kit+ (LSK) bone marrow hematopoietic stem cells from wild type versus conditional Mx-Cre/CEBPα knock-out mice. Similar to what we observed in human CEBPαsilenced leukemias, we found that these two genes were switched off in LSK cells from CEBPα knock-out animals. Ingenuity pathway analysis of the 24 upregulated genes detected high enrichment (P < 0.001) of pathways involved with T-cell development. Our data clearly suggest that CEBPα may act as repressor of T-cell related genes through direct promoter interaction. We therefore propose that silencing of CEBPα by promoter hypermethylation is one of the transforming events that driving towards mixed myeloid/T-lymphoid leukemia. Disclosures: No relevant conflicts of interest to declare.

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Expression of BECN1 gene in Acute Myeloid Leukemia: Association with Hematological and Clinical Parameters

QJM ◽

10.1093/qjmed/hcab091.007 ◽

2021 ◽

Vol 114 (Supplement_1) ◽

Author(s):

Mohamed Moustafa Ahmed ◽

Manal Fawzy Ghozlan ◽

Walaa Ali Mohamed ◽

Nesma Ahmed Safwat ◽

Noha Bassiouny Hassan

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Control Group ◽

Fish Analysis ◽

Newly Diagnosed ◽

Significant Difference ◽

Acute Myeloid

Abstract Background In acute myeloid leukemia (AML), there is copy number loss in autophagic genes such as BECN1. Accordingly, decreased autophagy and the development of AML are related. BECN1 is a critical mediator that influences the onset and progress of autophagy. Objective To investigate the expression status of BECN1 gene in newly diagnosed adult AML patients and its association with various hematological parameters and clinical outcomes. Methods Case control study to study BECN1 gene expression variability between 50 newly diagnosed adult AML patients and 20 healthy age and sex matched controls, with follow up of the patients to detect its effect on induction therapy. All AML patients underwent full history taking, through clinical examination, laboratory investigations such as complete blood count (CBC) with examination of peripheral blood and bone marrow Leishman stained films, immunophenotyping, cytogenetic analysis (karyotyping/FISH analysis) and BECN1 gene expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR). Results In our study, a highly significant difference was found as regards reduced expression of BECN1 gene in patients group compared to control group. We also found reduced BECN1 gene expression in both intermediate and adverse risk groups compared to favorable risk group. Reduced expression of BECN1 gene was associated with increasing age and total leukocytic count (TLC), peripheral blood (PB) and bone marrow (BM) blasts, the presence of FLT3-ITD mutation, CD34 and CD117 and in non-responders group. No statistically significant difference was found as regards haemoglobin (Hb) level, platelet (PLT) count and FAB subtypes. Conclusion Autophagy plays an important role in the pathogenesis of AML. Furthermore; the reductive regulation of the BECN1 gene may carry a poor prognosis and is associated with many well established bad prognostic factors especially FLT3-ITD mutation. Targeting autophagy pathways especially its major regulator (BECN1 gene) may become an effective and promising new line of therapy for AML patients.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v3 ◽

2020 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

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Gene Expression Profiling Predictive of Tipifarnib (ZARNESTRA®, R115777) Response in Patients with Newly Diagnosed Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.2785.2785 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2785-2785 ◽

Cited By ~ 1

Author(s):

Mitch Raponi ◽

Yi Zhang ◽

Tim Jatkoe ◽

Jack Yu ◽

Grace Lee ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Genetic Markers ◽

Myeloid Leukemia ◽

Phase 2 ◽

Newly Diagnosed ◽

Gene Expression Signatures ◽

Acute Myeloid

Abstract The farnesyltransferase inhibitor (FTI), tipifarnib (ZARNESTRA®, R115777), has demonstrated clinical response in patients with hematological disease. While the inhibition of protein farnesylation is the primary mechanism of action (MOA), the level of farnesyl inhibition is not a reliable pharmacodynamic marker of response, nor is it clear what genetic markers can be employed to predict response. This prospectively designed study was conducted to identify potential genetic markers and expression signatures that may be surrogate predictors of response for tipifarnib in patients with acute myeloid leukemia (AML). Bone marrow samples were collected and gene expression profiles analyzed from a single arm phase 2 clinical study of tipifarnib in poor-risk patients with newly diagnosed AML (Lancet, et al. Blood. 2004;104:249a). In total, 79 samples were obtained before (n = 25), during (n = 30), and after (n = 24) tipifarnib treatment. Bone marrow samples were analyzed using the Affymetrix U133A GeneChip® array. Global gene expression signatures revealed that tipifarnib treatment resulted in gene expression changes that were maintained for up to 120 days following treatment termination. Pretreatment vs posttreatment samples identified approximately 500 genes that had significant changes (False Discovery Rate [FDR] <0.005) in gene expression following farnesyltransferase inhibition, including several genes associated with farnesylation (eg, K-ras, FNTA). Many of the modulated genes were identified as those significantly involved in protein biosynthesis, DNA replication, intracellular signaling, and cell cycle pathways, thus, reflective of inhibition of cellular proliferation. A subset of 27 genes (including genes associated with signal transduction and cell cycle) was also identified as being differentially modulated between responders and nonresponders (P <0.01). Gene expression signatures previously identified from a phase 2 clinical trial in relapsed and refractory AML were also tested in pretreatment samples to examine their ability to predict response (Raponi, et al. Blood. 2004;104:246a). A combination of 6 genes was found to have significant predictive accuracy in this independent set of samples (P = 0.05). The genes identified from these studies might be used as surrogate biomarkers of tipifarnib activity.

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