GATA3 Silencing Defines a Distinct Subset of ETP-ALL.

The transcription factor GATA3 plays an important role in normal T cell development. Its role in mature T cells is well understood, but its function in earlier stages of T cell development remains unclear. Whereas GATA3 levels are precisely regulated for the T cell differentiation program, aberrant expression of GATA3 has been linked to tumorigenesis. Based on these observations, we investigated the role of GATA3 in Early Thymic Progenitor Acute Lymphoblastic Leukemia (ETP-ALL), a newly defined high-risk subgroup of T-ALL, characterized by a specific gene expression profile and distinct immunophenotype.

Eighty-six bone marrow samples from adult patients with newly diagnosed T-ALL, including ETP-ALL (n=17) enrolled into the German Multicenter Acute Lymphoblastic Leukemia (GMALL) trials, were studied for GATA3 expression by oligonucleotide expression arrays (HG-U133 plus 2.0) within the Microarray Innovations in LEukemia study. We identified additional 71 ETP-ALL adult patients and 94 T-ALL patients enrolled on the GMALL protocol, in which GATA3 mRNA expression was measured by quantitative polymerase chain reaction (RT-PCR). Combining ETP-ALL and T-ALL cases (n=165), we defined two GATA3 expression groups GATA3^null and GATA3^high based on a biological gap (GATA3 expression of 0.2). DNA methylation was analyzed in both T-ALL (n=11) and ETP-ALL (n=69) samples by pyrosequencing with primers designed to include seven CpG sites of Exon 2/Intron 3 of GATA3. Samples were grouped into GATA3 high vs. low methylation according to their mean methylation being below or above 40%.

Based on gene expression arrays we observed a high proportion of ETP-ALL (11/17) that lacked GATA3 expression, whereas only a small fraction of the remaining T-ALL cases (3/69) had no GATA3 expression. These results were validated by RT-PCR in a larger cohort: 26% of ETP-ALL (19/71) were GATA3^null, but only 2% of T-ALL (2/94) were in the GATA3^null expression group. To explore the regulation of this specific expression pattern, epigenetic regulation of GATA3 was analyzed by pyrosequencing. While unselected T-ALL samples were hypomethylated (< 6% methylated CpG), ETP-ALL samples had a higher GATA3 methylation status (28% methylated CpG, p<0.001). ETP-ALL cases were further categorized into high methylated (18/69) and low methylated samples (51/69) and correlated to mRNA expression. GATA3^null samples showed a higher degree of GATA3 methylation (41% methylated CpGs) compared to GATA3^high samples (8% methylated CpGs, p < 0.001). In an in-vitro assay of T-cell leukemia cell lines demethylating agents increased GATA3 mRNA expression by up to 5-fold. In murine hematopoetic stem cells it was shown that loss of DNMT3A induced GATA3 expression via hypomethylation. In ETP-ALL, we identified 11 DNMT3A mutations in 69 samples (16%) and correlated the DNMT3A mutation status to GATA3 methylation. Ten of 11 (91%) DNMT3A mutated samples showed low level GATA3 methylation, whereas 17 (29%) of the 58 DNMT3A wildtype cases had high methylation.

ETP-ALL is a subgroup of adult T-ALL with a distinct molecular profile. Here we show that within ETP-ALL a separate molecular entity can be defined by GATA3 silencing due to DNA methylation. In-vitro studies showed that GATA3 expression can be restored by the use of demethylating agents. As loss of function mutations in DNMT3A correlate with low GATA3 methylation in ETP-ALL, a potential role of DNMT3A in the epigenetic silencing of GATA3 is suspected. So far, the number of targeted drugs available for T-ALL is limited. Therefore, incorporating demethylating agents may resolve the T-cell differentiation block in T-ALL by increasing GATA3 expression. Future work will explore downstream effects of GATA3 in acute leukemia.

No relevant conflicts of interest to declare.