Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia.

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance.

Absence of aberrant myeloid progenitors by flow cytometry is associated with favorable response to azacitidine in higher risk myelodysplastic syndromes.

BACKGROUND: In intermediate-2 (Int-2) and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological improvement and prolonged overall survival (OS) in patients who respond to therapy. However, only half of the patients who are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients. METHODS: Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia, or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS). RESULTS: The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p = 0.02). A low pretreatment FCSS was associated with significantly better OS compared with a high pretreatment FCSS (p = 0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p = 0.006). CONCLUSIONS: Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine.

Absence of aberrant myeloid progenitors by flow cytometry is associated with favorable response to azacitidine in higher risk myelodysplastic syndromes.

Background: In intermediate-2 and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological responses and prolonged overall survival in patients who respond to therapy. However, only half of the patients that are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients. Methods: Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS). Results: The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p=0.02). A low pretreatment FCSS was associated with significantly better overall survival compared with a high pretreatment FCSS (p=0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p=0.006). Conclusions: Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine. © 2014 Clinical Cytometry Society.

Redistribution of H3K27me3 and acetylated histone H4 upon exposure to azacitidine and decitabine results in de-repression of the AML1/ETO target gene IL3.

Human acute myeloid leukemia is characterized by a block in maturation caused by genetic and epigenetic alterations. We studied the effects of low concentrations of the DNA methyltransferase (DNMT) inhibitors 5-azacitidine and decitabine on apoptosis and on chromatin remodeling in an AML1/ETO inducible model of human AML. While both DNMT inhibitors induced apoptosis, only azacitidine did so via caspase activation, possibly through its exclusive non-DNA depending effects. We evaluated histone marks for permissive chromatin, H3K4me3, and acetylated histone H4, and for non-permissive chromatin, H3K9me2, and H3K27me3, at the promoter of the IL3 gene, which is under the direct control of AML1/ETO and is critical for myeloid maturation. We observed that low concentrations of DNMT inhibitors induced a loss of H3K27me3 and gain of acetylated histone H4 at the IL3 promoter exclusively in AML1/ETO-positive cells, which was associated with transcriptional reactivation of the IL3 gene.

Hypermethylation of Wnt antagonist gene promoters and activation of Wnt pathway in myelodysplastic marrow cells.

We observed aberrant gene methylation of Wnt antagonists: sFRP1, sFRP2, sFRP4, sFRP5 and DKK1 in marrow cells of 55 MDS cases. Methylation of Wnt antagonist genes was associated with activation of the Wnt signaling pathway, consistent with the up-regulation of the Wnt downstream genes TCF1 and LEF1. Azacitidine exposure induced demethylation of Wnt-antagonist gene promoters and reduction of the non-phosphorylated ß-catenin (NPBC) which is prevalent during Wnt pathway inactivation. Presence of ≥5% of bone marrow blasts was associated with methylation of sFRP1 and DKK1 and with methylation of more than two of the five Wnt antagonist genes.

Distinct signal transduction abnormalities and erythropoietin response in bone marrow hematopoietic cell subpopulations of myelodysplastic syndrome patients.

PURPOSE: Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. EXPERIMENTAL DESIGN: Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34(+), CD45(+), or CD71(+)CD45(-) bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. RESULTS: In CD71(+)CD45(-) cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71(+)CD45(-) cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34(+) and CD45(+) MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34(+) cells. CONCLUSIONS: Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71(+)CD45(-) cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations.

Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine.

Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias. To further characterize different mechanisms between these two agents, cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis. Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS. Cyclophilin A, Catalase, Nucleophosmin and PCNA were decreased exclusively by azacitidine, TCP1 and hnRNP A2/B1 by both drugs; alpha-Enolase and Peroxiredoxin-1 by decitabine. Interestingly, the expression of the proinflammatory protein Cyclophilin A, also suggested as marker of cell necrosis, was stimulated by decitabine. Finally, a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors.

Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid.

Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.

Comparative proteomic analysis of chronic myelogenous leukemia cells: inside the mechanism of imatinib resistance.

Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.