Silencing of GATA3 defines a novel stem cell-like subgroup of ETP-ALL

Background GATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL). Methods We analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3low and GATA3high expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine. Results In our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3low. DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3low compared with GATA3high ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3low ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3high ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation. Conclusions We propose GATA3low ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0324-8) contains supplementary material, which is available to authorized users.


Background
GATA3 is a transcription factor with a pivotal role in multiple developmental steps of T lymphopoiesis [1,2], including the development of early T cell precursors (ETPs), a rare subpopulation of cells sharing characteristics with multipotent hematopoietic progenitors in the bone marrow [3]. ETPs are considered the most immature thymic cells with potential for complete T cell differentiation and retain plasticity for differentiation towards dendritic, NK, B, or myeloid cells [4]. In a murine model, GATA3 was required for the development of ETPs, whereas totipotent hematopoietic stem cells (HSCs) remained unaffected by in vivo manipulation of GATA3 expression levels. Indeed, in murine HSCs, GATA3 was silenced by DNMT3A-dependent DNA hypermethylation [5]. By losing repressive epigenetic marks during T lymphopoiesis, GATA3 functions as a key regulator of T cell differentiation through the interaction with a multitude of target genes that differ in a subpopulation specific manner [6]. For example, GATA3 was reported to restrain Notch activity, repress NK-cell fate and upregulate T cell lineage genes to facilitate T cell differentiation [7].
Lack of GATA3 has been linked to cancerogenesis, as absence of GATA3 expression was associated with poor prognosis and undifferentiated tumors in breast cancer [8]. Moreover, several other cancers exhibited aberrant GATA3 expression, including urothelial carcinoma [9], renal cell carcinoma [10], pancreatic cancer [11], cervical cancer [12], or Hodgkinʼs lymphoma [13]. In childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL), specific germline variants of GATA3 were associated with a higher incidence of BCP-ALL and a higher risk of relapse [14,15].
Given GATA3's prominent role in both cancerogenesis and T cell development, we investigated GATA3 in ETP-ALL. ETP-ALL is a subtype of T-ALL characterized by a distinct gene expression profile (GEP) and a distinct immunophenotype with lack of CD1a and CD8, weak expression of CD5 and additional expression of more than 1 myeloid and/or stem cell marker [16]. ETP-ALL accounts for 11-15 % of cases with T-ALL [16][17][18] with similar distributions among pediatric and adult cohorts. We and others have characterized the mutational landscape of ETP-ALL with alterations in genes involved in cytokine and RAS signaling (e.g., NRAS, KRAS, FLT3, and JAK1), epigenetic regulation (e.g., EZH2, DNMT3A, and SUZ12), and hematopoietic development (e.g., ETV6, RUNX1, and IKZF1) [19,20]. Notably, the incidence of activating NOTCH1 mutations is considerably lower in ETP-ALL (15 %) when compared to T-ALL (higher than 50 %) [17,21]. GATA3 inactivating mutations were reported in 9 % of pediatric ETP-ALL patients predominantly affecting the DNA binding domain [19].
The prognostic relevance of ETP-ALL is controversially discussed. Comparing ETP-ALL with non-ETP-ALL, some reports indicate adverse prognosis in pediatric and adult patients with ETP-ALL with CR rates of 58-73 %, median event-free survival of 1.2 years, and 3-year overall survival of 30-60 % [16,17,22]. Other groups found similar outcome of ETP-ALL and non-ETP-ALL patients with 5-year overall survival rates of 67-93 and 77-92 %, respectively [23,24].
Given the critical role that GATA3 plays in early lymphoid development, we investigated GATA3 in ETP-ALL, a stem cell-like leukemia blocked at the crossroads of lymphoid and myeloid differentiation. We hypothesized that aberrant GATA3 expression would divert ETP-ALL from the lymphoid fate and determine a novel biological subgroup of ETP-ALL.

Patient samples
Additional file 1: Figure S1 provides an overview over the sample cohorts and subsequent experiments.
Based on immunophenotyping of diagnostic samples at the central diagnostic reference laboratory of the German Multicenter Study Group for Acute Lymphoblastic Leukemia (GMALL) in Berlin, Germany, we identified additional 70 ETP-ALL samples [17]. Sufficient RNA for GATA3 mRNA expression analysis was available for all 70 samples, and sufficient genomic DNA (gDNA) for methylation assays was available for 69 samples of these adult ETP-ALL cases. As reference cohort, we used 112 non-ETP-ALL patients, of which 21 (19 %) had an immunophenotype of early T-ALL, 20 (18 %) of mature T-ALL, and 71 (63 %) of thymic T-ALL.
All patients, including the two independent cohorts of T-ALL, and normal controls gave written informed consent to participate in the study according to the Declaration of Helsinki. The studies were approved by the ethics board of the Johann Wolfgang von Goethe University, Frankfurt/Main, Germany.

Nucleic acid preparation and molecular characterization
Pretreatment bone marrow and peripheral blood samples from patients were used for gDNA and total RNA extraction using TRIzol (Life Technologies, Grand Island, NY, USA) according to the manufacturer's protocol with minor modifications. Complementary DNA (cDNA) was synthesized using 500 ng of total RNA and avian myeloblastosis virus reverse transcriptase (RT-AMV; Roche, Mannheim, Germany) in the presence of RNase inhibitor (RNasin; Roche, Mannheim, Germany).
Samples of patients with ETP-ALL (n = 70) and non-ETP-ALL (n = 112) were investigated by comparative multiplex real-time PCR (RT-PCR) for expression of GATA3 (FWD: 5′-ACTACGGAAACTCGGTCAG-3′, REV: 5′-GTAGGGATCCATGAAGCAG-3′, Probe: 5′-CG GTGCAGAGGTACCCTCCG-3′) and glucose-6-phosphate isomerase (GPI) as a housekeeping gene. Relative GATA3 expression values of ETP-ALL (n = 70) and non-ETP-ALL (n = 112) were normalized to GATA3 expression in the human T-ALL cell line Jurkat. We identified a bimodal distribution of GATA3 mRNA expression levels by K-means clustering and defined a cutoff at an expression level of 0.2 relative to Jurkat and defined all samples with GATA3 expression below that cutoff as GATA3 low and samples with higher expression as GATA3 high (Fig. 1).

Western blot
GATA3 protein levels were measured using standard western blotting techniques using the GATA3 antibody HG3-35 (Santa Cruz Biotechnology Inc., Dallas, TX, USA).

Gene expression profiling
A GATA3-associated GEP was generated from data of 83 adult T-ALL patients (including 30 ETP-ALL and 53 non-ETP-ALL samples defined by hierarchical clustering using a list of genes reported as differentially expressed in pediatric ETP-ALL [16], GEO accession number GSE78132). For analysis, common probe sets between HG-U133 Plus 2.0 and HG-U133 A + B (Affymetrix, Santa Clara, CA, USA) were identified and quantile normalized. For ANOVA analysis, the type of chip was integrated as a random effect to take the batch effect into account. GATA3 expression was calculated from signals obtained from probe sets 209602_s_at, 209603_at, and 209604_s_at, respectively. As for the RT-PCR expression levels, we identified a bimodal distribution of GATA3normalized expression values and defined a cutoff at 8.2 on a logarithmic scale to categorize patients below that cutoff as GATA3 low and with higher expression as GATA3 high . A GATA3-dependent GEP was generated by the comparison of the expression profiles from GATA3 low (n = 11) and GATA3 high (n = 72) samples. Lists of genes with at least 1.5-fold under-or overexpression comparing GATA3 low and GATA3 high were generated (Additional file 2: Table S1), and statistical significance was calculated by ANOVA with a FDR ≤0.05. Data analysis was carried out with Partek Genomic Suite v6.6 Software (Partek Inc., St. Louis, MO, USA).
For gene set enrichment analysis (GSEA), GATA3-supervised GEPs were examined for enrichment of curated gene sets representing ETP-ALL [19], HSC [30], T cell differentiation [30], granulocyte/macrophage progenitors (GMP) [30], and myeloid/lymphoid progenitors (MLP) [31] Fig. 1 GATA3 mRNA expression in patient samples. a Affymetrix-based mean differential expression of GATA3 based on normalized expression values in normal controls (NC) and selected hematological disorders. Horizontal lines indicate mean GATA3 expression AE s.e. Note the segmented y-axis. b RT-PCR-based analyses of GATA3 mRNA expression relative to Jurkat on a logarithmic scale revealed lower GATA3 expression in ETP-ALL (n = 70) than in non-ETP-ALL (n = 112) (4.82 vs. 6.29, p = 0.0005 indicated by asterisk). We identified a bimodal distribution of GATA3 expression by K-means clustering with a cutoff at a relative expression of 0.2 (indicated by the dotted line). The GATA3 low cohort contained only cases with ETP-ALL (i.e., GATA3 low ETP-ALL) comparing GATA3 low ETP-ALL (n = 11) and GATA3 high ETP-ALL (n = 19) cases. Additionally, we used decitabineinduced changes of GEP as a discriminator to analyze enrichment of these curated gene lists in PER-117 cells. Data analyses were carried out with the GSEA desktop application version 2.0.12 [32,33] from the Broad Institute (http://www.broadinstitute.org/gsea).

Methylation analysis
We assessed global DNA methylation analyses in 12 ETP-ALL and 14 BCP-ALL samples by the Illumina Infinium® HumanMethylation450 BeadChip platform. Hybridization was performed according to the manufacturer's protocol. The signals generated for unmethylated and methylated cytosine nucleotides by single-nucleotide extension of locus-specific methylation probes were transformed into β values ranging from 0 to 1 (representing 0 to 100 %) for each of the 450,000 interrogated CpG residues. We assumed differential methylation, if more than three differentially methylated sites (DMS) with a p value <0.05 were present for each gene and the absolute difference of the corresponding β values ð △ βÞ was greater than 0.17. Data analysis was carried out with Partek Genomic Suite v6.6 Software (Partek Inc., St. Louis, MO, USA).
Sufficient amounts of gDNA for bisulfite conversion was available for 69 ETP-ALL and 48 AML samples, which was carried out using the EpiTect Bisulfite Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. For validation of the differentially methylated region of GATA3 detected by global methylation analysis, primers were designed for amplification and pyrosequencing based on the bisulfite converted sequence of GATA3 (genomic location: GRCh37: chr10:8097750-8098004) and used in the Pyrosequencing Assay Design Software v1.0 (Biotage, Uppsala, Sweden) for assay design. Amplification of a 255-bp sequence was carried out in all 69 bisulfite converted ETP-ALL samples using a 5′-GGAGGAGGTGGATGTGTTTTTTAAT-3′ forward and a 3′-AACCCCAATTTTTTTATAAATAAAC CA-5′reverse biotinylated primer. Additionally, 13 representative samples of the non-ETP-ALL cohort were selected for analysis by pyrosequencing; 100 ng of bisulfite-converted gDNA was used per reaction with Taq-DNA-polymerase (Hot Start Mix S, peqlab, Erlangen, Germany). Samples were analyzed for specificity and correct size by 2 % agarose gel electrophoresis.
For pyrosequencing, a 5′-GTTACGGTGTAGAGGTA TTTT-3′ sequencing primer was used. The percentage of CpG site methylation was calculated through the ratio of the relative content of thymine (i.e., unmethylated cytosine) and the relative content of cytosine (i.e., methylated cytosine) using the Pyro Q-CpG Software version 1.0.9 (Biotage, Uppsala, Sweden). Four of 12 CpG sites covered by the sequencing primer failed quality control due to the reference sequence pattern at the end of the amplicon. The remaining eight CpG sites were included to calculate the mean percentage of methylation for each sample.
Additionally, the ETP-ALL cell line Loucy (with high GATA3 expression, GATA3 high ETP-ALL) and the non-ETP-ALL cell lines, Jurkat, Molt4, BE13, and RPMI8402 were obtained from the German Resource Center for Biological Material, DSMZ (Braunschweig, Germany) and previously characterized on a molecular level [35]. 5-Azacytidine and 5-aza-deoxycytidine were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell proliferation assay
Cell proliferation was measured with the WST-1 reagent according to the manufacturer's instructions (Roche Diagnostics GmbH, Germany). Cell lines were treated with various concentrations of 5-azacytidine (Sigma-Aldrich, St. Louis, USA) and 5-aza-deoxycytidine (Sigma-Aldrich, St. Louis, USA), and absorbance was measured after 48, 72, and 96 h by optical density absorption analyses at a wavelength of 450 nm using an ELISA multiplate reader.

Apoptosis assay
Apoptosis was measured using Annexin V Apoptosis Detection Kit (BD Pharmingen, Heidelberg, Germany). Cells were labeled with Annexin V and 7-amino-actinomycin D (7-AAD) after treatment with 5-azacytidine and 5-azadeoxycytidine. Analyses were performed by FACS Calibur (Becton-Dickinson) to determine the percentage of apoptotic cells from combined 7-AAD incorporation and Annexin V binding.

Statistical analysis
The statistical difference of gene expression between two independent groups was tested by the non-parametric Mann-Whitney U test. For non-parametric correlation of mRNA expression and DNA methylation, Spearman's rank correlation coefficient was calculated. Fisher's exact test was used to test for the association between two kinds of classifications (e.g., 2 × 2 contingency table).
To further explore GATA3 expression in T-ALL, we analyzed GATA3 mRNA expression by quantitative RT-PCR in larger cohorts of ETP-ALL (n = 70) and non-ETP-ALL (n = 112). The mean relative expression of GATA3 was lower in ETP-ALL than in non-ETP-ALL (4.82 ± 0.78 vs. 6.29 ± 0.60, mean ± s.e., p = 0.0005). Interestingly, we found a bimodal distribution of GATA3 expression with one third of ETP-ALL patients lacking GATA3 expression (23/70, 33 %, GATA3 low ETP-ALL). In contrast, none of 112 non-ETP-ALL samples lacked GATA3 expression, which consisted of 71 thymic, 21 early, and 20 mature T-ALL patient samples (Fig. 1b). In agreement with this, the non-ETP-ALL cell lines Molt4, Jurkat, RPMI8402, and BE13 all expressed GATA3, while PER-117 [34], a cell line with an ETP-ALL immunophenotype and GEP (Additional file 3: Figure S2) lacked GATA3 expression. Western blotting revealed that differential GATA3 mRNA expression translated into differential protein expression levels (Additional file 4: Figure S3).

GATA3 silencing is mediated by aberrant DNA methylation
To explore the regulation of GATA3 expression, we investigated global DNA methylation on the Illumina HumanMethylation 450 k platform in 12 ETP-ALL samples (Fig. 2), which were selected according to GATA3 mRNA expression (GATA3 low vs. GATA3 high ) and mutational status of DNMT3A. The genomic locus of GATA3 (NC_000010.10) was represented by 72 CpG sites.

GATA3 low ETP-ALL is associated with FLT3 mutations
Our group previously assessed the mutational landscape of ETP-ALL by whole exome sequencing [20] and targeted NGS re-sequencing [37]. Within this cohort of ETP-ALL, we have investigated the mutational pattern with respect to GATA3 expression (Additional file 6: Table S2). In contrast to pediatric cohorts, we found no GATA3 mutations in this cohort, including a screen for hotspot mutations of exon 4 in an additional expansion cohort of 70 samples of adult ETP-ALL.

Distinct transcriptional program of GATA3 low ETP-ALL
To explore differences of the transcriptional program, microarray expression data of 83 T-ALL patients were available; 11 of 83 patients were defined as GATA3 low , while the remaining 72 patients were classified as GATA3 high . Including probe sets with at least 1.5-fold overexpression, we detected 1435 differentially expressed probes sets in GATA3 low compared to GATA3 high T-ALL cases (Additional file 2: Table S1). Hierarchical clustering with this gene list revealed a GATA3 low -derived gene expression signature (Fig. 3a). Importantly, this GATA3 low GEP identified all but one case of ETP-ALL in an independent cohort of pediatric T-ALL (Additional file 7: Figure S5) [16]. Annotation of the top 267 DEG (i.e., genes with fold change ≥3×) using the KEGG pathway and mean DNA methylation (lower panel) of GATA3 low ETP-ALL (n = 4, blue) and GATA3 high ETP-ALL (n = 8, red). Comparing GATA3 low ETP-ALL (n = 4, blue) with GATA3 high ETP-ALL (n = 8, red), 35 differentially methylated sites were located within a 6-kb segment of GATA3 (indicated by the gray box), including the CpGs that were analyzed by pyrosequencing in a larger cohort of patients. b GATA3 DNA methylation as assessed by pyrosequencing (of CpGs within the gray box in a) was negatively correlated to GATA3 mRNA expression in ETP-ALL (n = 64, r = −0.73, p < 0.0001). The dotted line indicates the cutoff to distinguish GATA3 low (empty dots) and GATA3 high (solid dots) samples. c Pyrosequencing revealed higher GATA3 DNA methylation in ETP-ALL (n = 69) than in non-ETP-ALL (n = 13) (28 vs. 5 %, p < 0.0001 indicated by asterisk). Empty and solid dots indicate GATA3 low and GATA3 high ETP-ALL, while triangles indicate non-ETP-ALL database demonstrated significant enrichment of upregulated genes associated to cancer and, notably, AML, while genes associated to T cell signaling were downregulated in GATA3 low samples (Additional file 8: Table S3).
We analyzed global gene expression of PER-117 cells by Affymetrix microarrays before and after treatment with decitabine at a final concentration of 5 μM at three time points (0, 24, and 48 h). At both 24 and 48 h after decitabine treatment, we detected significant changes in global gene expression compared to untreated cells (Fig. 4c) with 2019 differentially expressed probe sets (fold change of ≥1.5 and FDR <0.05) after 48 h of exposure to decitabine (Fig. 4d). Principal component analysis revealed differential changes of global gene expression after 24 and 48 h: GEP changes represented by the first principal component expanded up until 48 h, while GEP changes subsumed by the second and third principal components were nearly completely reversible after 48 h (Fig. 4c).

Discussion
Here, we discovered a novel, molecularly distinct subgroup of T-ALL patients lacking GATA3 expression (GATA3 low ). All GATA3 low T-ALL patients exhibited an immunophenotype of ETP-ALL, while GATA3 high T-ALL patients were of thymic, early, or mature subtypes. The subgroup of GATA3 low ETP-ALL is molecularly and clinically relevant as it lacks T lineage commitment in favor of a sustained myeloid gene expression signaling and a high rate of FLT3 mutations.
Clustering analysis revealed a third of our cohort's ETP-ALL samples to be GATA3 low . To study mechanisms of silenced GATA3 mRNA expression, we investigated DNA methylation. We identified a CpG island of GATA3 with consistently higher GATA3 DNA methylation in GATA3 low ETP-ALL compared to GATA3 high ETP-ALL including more than 30 DMS. This GATA3 CpG island was differentially methylated in renal cell carcinoma [10] and thyroid adenocarcinoma. In fact, cg01255894, a hypermethylated CpG site in ETP-ALL, was among the top 25 methylation probes that were most negatively correlated with gene expression [36]. Notably, GATA3 DNA hypermethylation was absent in non-ETP-ALL indicating that GATA3 silencing was a distinct mechanism in ETP-ALL. It is tempting to relate this finding to reports of murine DNMT3A-deficient mice, where GATA3 silencing was associated with DNMT3A-dependent DNA hypermethylation in HSC [5]. Indeed, when we compared DNMT3A mutated and DNMT3A wild-type ETP-ALL, we found lower GATA3 DNA methylation in samples with mutated DNMT3A, but GATA3 mRNA expression was not different between DNMT3A wild-type and mutated ETP-ALL. Thus, DNMT3A contributes to GATA3 DNA methylation; however, redundant mechanisms are likely required for GATA3 silencing in GATA3 low ETP-ALL. Importantly, hypermethylation of GATA3 was found only in the subset of GATA3 low ETP-ALL, but not in other leukemic subtypes such as typical T-ALL or BCP-ALL. Notably, in 49 samples from patients with AML, GATA3 expression was similarly low as in GATA3 low ETP-ALL (mean 0.2 vs. 0.03), but DNA hypermethylation was absent in AML (17 vs. 46 %). Thus, GATA3 low ETP-ALL may reflect the transformed precursor stage of yet non-committed ETP that physiologically harbor GATA3 DNA hypermethylation.
In order to explore the cell of origin of GATA3 low ETP-ALL, we identified a GATA3 low -specific GEP in a cohort of T-ALL, including ETP-ALL and non-ETP-ALL patient samples. GATA3 low and GATA3 high samples generated distinct gene expression clusters in a supervised analysis. The biological significance of this observation was underscored when we validated our GATA3 low signature by identifying cases with ETP-ALL in an independent cohort of pediatric patients with T-ALL [16] by unsupervised hierarchical clustering. Moreover, pathway annotation of DEG indicated upregulation of myeloid genes and downregulation of T cell differentiation. Perhaps unsurprisingly, we found depletion of T cell signaling and enrichment of myeloid signaling when we performed GSEA comparing GATA3 low ETP-ALL with T-ALL. By restricting the analysis to ETP-ALL only, we confirmed enrichment of GMP and MLP signatures and depletion of T cell differentiation in GATA3 low samples compared to GATA3 high ETP-ALL, which pointed at a specific molecular bracket within ETP-ALL. The specificity of GATA3 in this regard was further underscored when we analyzed other relevant transcription factors involved in T cell differentiation. Other transcription factors, such as MEF2C, PU.1, BCL11B, LMO1-3, HOXA1, TCF-1, or LYL1 failed to identify subsets with meaningful gene set enrichment in neither "typical" T-ALL nor ETP-ALL. Only the transcription factor LEF1 segregated cases into subgroups with similar gene set enrichment patterns as GATA3 subgroups, albeit with significant overlap of GATA3 low /LEF1 low cases. LEF1 is an important effector of WNT signaling and, like GATA3, known to be essential for early stages of T cell development. In T cell malignancy, LEF1 was implicated in transforming T cells in the absence of TCF1 [38].
The observation of a myeloid gene expression signature was further supported by the high frequency of FLT3 mutations in GATA3 low ETP-ALL. It is important to note that neither of the investigated cases fulfilled the diagnostic criteria for leukemia of ambiguous lineage or acute myeloid leukemia. Therefore, these findings point to T-lymphoblastic precursors with multilineage potential as cells of origin of GATA3 low ETP-ALL. Indeed, enrichment of ETP-ALL genes in GATA3 low compared with GATA3 high ETP-ALL reinforced this assumption as ETP-ALL by itself is characterized by upregulation of stem cell genes and myeloid-derived gene expression [19].
Ultimately, the significance of GATA3 low ETP-ALL as a subgroup of ETP-ALL will depend on the implementation of distinct therapeutic interventions. In our ETP-ALL cohort, we found no significant outcome differences comparing GATA3 low and GATA3 high ETP-ALL (1-year OS 75 vs. 79 %) in a retrospective analysis of 52 patients. In general, the clinical outcome of ETP-ALL remains controversial, as reports of adverse risk in pediatric and adult ETP-ALL [16,22,39] have been challenged by reports indicating no outcome differences between ETP-ALL and non-ETP-ALL patient cohorts [23,24]. This controversy might be in part due to the definition of ETP-ALL by GEP or flow cytometry as well as differences in treatment intensities, especially MRD-directed approaches to treatment intensification [16,19,23,24,40].
In any case, the mutational and transcriptional profile of GATA3 low ETP-ALL provides rationale for implementing targeted therapies in patients with failure of lymphoid-directed therapies. Low incidence of NOTCH1 mutations in GATA3 low ETP-ALL will likely render NOTCH-targeted therapies (e.g., γ-secretase inhibitors) ineffective. On the other hand, FLT3 mutations were detected in more than 75 % of GATA3 low ETP-ALL and our group has previously shown in vitro efficacy of FLT3 inhibitors in human T-ALL cell lines [17]. Importantly, GATA3 DNA hypermethylation implicates epigenetic therapies in GATA3 low ETP-ALL, such as decitabine, a hypomethylating agent approved for the treatment of myelodysplastic syndrome, chronic myelomonocytic leukemia, and AML. Our data demonstrate that decitabine induced apoptosis in PER-117 cells while lowering GATA3 DNA methylation. Subsequent induction of GATA3 expression may function as a surrogate of T cell differentiation which we also observed in PER-117 cells upon decitabine treatment. This is in line with murine breast cancer, where lack of GATA3 is associated with undifferentiated tumors [8]. The IC 50 of decitabine in our experiments was comparable to AML cell lines [41,42], and current dosing of decitabine in AML results in a similar range of steady state plasma levels [41]. Although further experiments are necessary to evaluate in vivo efficacy of HMA in T-ALL, it is likely that similar doses of decitabine will be required in T-ALL and AML. It is important to note that forced GATA3 overexpression alone failed to induce relevant changes in proliferation, apoptosis, or differentiation in PER-117 cells, which we attributed to GATA3 dose sensitivity. Instead, subtle changes of GATA3 expression are needed to divert aberrant DNA hypermethylation towards an equilibrium of optimum methylation in T lymphoblasts [43], for which GATA3 induction upon treatment with decitabine serves as yet another example.