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The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.

Keywords
5-Azacytidine, Celastrol, microRNA miR-155, transcription factor PU.1,
MeSH
Leukemia, Myeloid, Acute * drug therapy genetics MeSH
Cell Differentiation genetics MeSH
Humans MeSH
MicroRNAs * genetics metabolism MeSH
Proto-Oncogene Proteins genetics metabolism MeSH
Gene Expression Regulation, Leukemic MeSH
Trans-Activators metabolism MeSH
Check Tag
Humans MeSH
Publication type
Journal Article MeSH
Names of Substances
MicroRNAs * MeSH
MIRN155 microRNA, human MeSH Browser
proto-oncogene protein Spi-1 MeSH Browser
Proto-Oncogene Proteins MeSH
Trans-Activators MeSH

Article

Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

Cells. 2022 Jan 11 ; 11 (2) : . [epub] 20220111

ISSN 2073-4409
Source

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.

Keywords
Azacytidine, CDX mice, PI3K/AKT signaling, myelodysplastic syndrome, resistance,
MeSH
Molecular Sequence Annotation MeSH
Azacitidine pharmacology MeSH
Models, Biological * MeSH
Drug Resistance, Neoplasm * drug effects genetics MeSH
DNA, Neoplasm genetics MeSH
Phosphatidylinositol 3-Kinases metabolism MeSH
Mice, SCID MeSH
Mice MeSH
Proto-Oncogene Proteins c-akt metabolism MeSH
Reproducibility of Results MeSH
Signal Transduction drug effects MeSH
Transcriptome genetics MeSH
Animals MeSH
Check Tag
Mice MeSH
Animals MeSH
Publication type
Journal Article MeSH
Research Support, Non-U.S. Gov't MeSH
Names of Substances
Azacitidine MeSH
DNA, Neoplasm MeSH
Proto-Oncogene Proteins c-akt MeSH

Article

Dynamic alterations of bone marrow cytokine landscape of myelodysplastic syndromes patients treated with 5-azacytidine

Oncoimmunology. 2016 ; 5 (10) : e1183860. [epub] 20160513

ISSN 2162-4011
Source

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis frequently progressing into acute myeloid leukemia (AML), with emerging evidence implicating aberrant bone marrow (BM) microenvironment and inflammation-related changes. 5-azacytidine (5-AC) represents standard MDS treatment. Besides inhibiting DNA/RNA methylation, 5-AC has been shown to induce DNA damage and apoptosis in vitro. To provide insights into in vivo effects, we assessed the proinflammatory cytokines alterations during MDS progression, cytokine changes after 5-AC, and contribution of inflammatory comorbidities to the cytokine changes in MDS patients. We found that IL8, IP10/CXCL10, MCP1/CCL2 and IL27 were significantly elevated and IL12p70 decreased in BM of MDS low-risk, high-risk and AML patients compared to healthy donors. Repeated sampling of the high-risk MDS patients undergoing 5-AC therapy revealed that the levels of IL8, IL27 and MCP1 in BM plasma were progressively increasing in agreement with in vitro experiments using several cancer cell lines. Moreover, the presence of inflammatory diseases correlated with higher levels of IL8 and MCP1 in low-risk but not in high-risk MDS. Overall, all forms of MDS feature a deregulated proinflammatory cytokine landscape in the BM and such alterations are further augmented by therapy of MDS patients with 5-AC.

Keywords
5-azacytidine, DNA damage, bone marrow plasma, cytokines, inflammation, myelodysplastic syndromes,
Publication type
Journal Article MeSH
Research Support, Non-U.S. Gov't MeSH

Article

GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia

PloS one. 2016 ; 11 (3) : e0152234. [epub] 20160324

PLoS One
ISSN 1932-6203
Source

GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.

Article

Epigenetic control of SPI1 gene by CTCF and ISWI ATPase SMARCA5

PloS one. 2014 ; 9 (2) : e87448. [epub] 20140203

PLoS One
ISSN 1932-6203
Source

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement in leukemia remains elusive. Here, we show that Ctcf binds to the imprinting control region of H19/Igf2 in AML blasts. We also demonstrate that Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding to its target sites on DNA. Furthermore, Smarca5 supports Ctcf functionally and is needed for enhancer-blocking effect at ICR. We next asked whether CTCF and SMARCA5 control the expression of key hematopoiesis regulators. In normally differentiating myeloid cells both CTCF and SMARCA5 together with members of the Cohesin complex are recruited to the SPI1 gene, a key hematopoiesis regulator and leukemia suppressor. Due to DNA methylation, CTCF binding to the SPI1 gene is blocked in AML blasts. Upon AZA-mediated DNA demethylation of human AML blasts, CTCF and SMARCA5 are recruited to the -14.4 Enhancer of SPI1 gene and block its expression. Our data provide new insight into complex SPI1 gene regulation now involving additional key epigenetic factors, CTCF and SMARCA5 that control PU.1 expression at the -14.4 Enhancer.

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5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity

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