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Investigation of TET2 and DNMT3A expression in myelodysplastic syndrome and acute myeloid leukemia

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Author(s):
Renata Scopim Ribeiro
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
Defense date:
Examining board members:
Fabíola Traina; Elvira Deolinda Rodrigues Pereira Velloso; Erich Vinicius De Paula
Advisor: Fabíola Traina
Abstract

Myeloid neoplasms comprise a heterogeneous group of hematologic malignancies that originate from a common myeloid precursor at different stages of differentiation. Cellular changes that lead to development of malignancies may occur through epigenetic mechanisms or genetic alterations. DNMT3A encodes methyltransferases that add methyl groups to cytosine residues in DNA, TET2 promotes hydroxylation of methylated cytosine, and both proteins are important elements in epigenetic control. TET2 and DNMT3A are recurrently mutated in myeloid malignancies, but the prognostic consequence of TET2 and DNMT3A mutation is still controversial. The functional consequences of DNMT3A mutation has not been defined, but the protein silencing in murine progenitor cells promotes self-renewal and reduces cell differentiation. TET2 mutation results in loss of function and participates in the neoplastic transformation of myeloid cells, favoring the proliferation of granulomonocytic cells. However, the expression of TET2 and DNMT3A in these diseases has been rarely addressed. Then, the aims of this study were (1) to evaluate TET2 and DNMT3A gene expression in hematopoietic cells from healthy individuals and from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); (2) to correlate TET2 and DNMT3A expression with clinical phenotype and outcomes of MDS patients; (3) to investigate TET2 and DNMT3A expression during hematopoietic cell differentiation; and (4) to evaluate the effect of DNMT3A silencing in the phenotype of leukemia cell lines. In this study, the expression of TET2 was decreased in cells from patients with MDS and AML compared to healthy donors (p<.001) and reduced high-risk MDS compared to low risk MDS (p=.02). The results in sequential samples from five patients with MDS indicate reduced expression of TET2 at the time of disease progression. By univariate analysis, clinical factors that significantly affected both event free survival (EFS) and overall survival (OS) included risk stratification by WHO 2008 (high vs. low, p<.0001), IPSS (int-2/high vs. low/int-1, p <.0001), hemoglobin (<10 vs. ? 10, p<.05), white blood cell counts (< 3 vs. ? 3 x109/L, p<.05), absolute neutrophil counts (< 1.5 vs. ? 1.5, p<.05) and bone marrow blast percentage (? 5 vs. <5 or ? 10 vs. <10, p<.0004). Furthermore, low TET2 expression negatively impacted both EFS (HR: 6.51 [2.42-17.49], p=.0002) and OS (HR: 7.25 [2.77-18.99], p<.0001). Multivariate analyses indicated that low TET2 expression (p <.0001), along with IPSS high/intermediate-2 risk group (p <.0001), and hemoglobin <10 g/dL (p<.03) were independently prognostic for worse EFS and OS. During erythroid differentiation of CD34+ cells from normal individuals and patients with low-risk MDS, we observed an increased expression of TET2 (p=.03). During cell differentiation of leukemic cell lines, we observed a significantly increase in the expression of TET2 during granulocytic and megakaryocytic differentiation (p=.04 and p=.03, respectively); there was also an increased expression during erythrocytic differentiation, but this was not statistically significant. The expression of DNMT3A was similar between patients with AML, MDS and healthy donors, and it did not impact survival outcomes in MDS patients. DNMT3A expression was not modulated during erythroid differentiation of CD34+ cells from normal individuals and patients with MDS. In leukemic cell lines models of differentiation, we observed a significantly increase in the DNMT3A expression during granulocytic differentiation, but not in erythrocytic and megakaryocytic differentiation. The DNMT3A silencing did not result in significant changes in apoptosis, proliferation and cell cycle in leukemic cell lines HL60 and U937. PTEN gene and protein expression was not modulated in leukemic cell lines submitted to inhibition of DNMT3A. The findings reported here suggest that, similarly to the presence of TET2 mutations, the low expression of TET2 can participate in the process of cell transformation in high risk MDS and AML. Clinical studies should consider the investigation of TET2 expression together with the studies of TET2 mutation to defining prognosis. Our results of expression and function suggest that DNMT3A mutation, instead of the expression, should be the main mechanism by which DNMT3A participates in neoplastic transformation and that DNMT3A function may vary according to the cell line studied (AU)

FAPESP's process: 11/15905-3 - DNMT3A mutations, expression and function in myelodysplasic syndromes and acute myeloid leukemia.
Grantee:Renata Scopim Ribeiro
Support Opportunities: Scholarships in Brazil - Master