Gain-of-function mutation of GATA-2 in acute myeloid transformation of chronic myeloid leukemia

Abstract

Acquisition of additional genetic and/or epigenetic abnormalities other than the BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast crisis (BC). To gain insights into the underlying mechanisms of progression to BC, we screened DNA samples from CML patients during blast transformation for mutations in a number of transcription factor genes that are critical for myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. A L359V substitution within zinc finger domain (ZF) 2 of GATA-2 was found in eight cases with myelomonoblastic features, whereas an in-frame deletion of 6 aa (delta341-346) spanning the C-terminal border of ZF1 was detected in one patient at myeloid BC with eosinophilia. Further studies indicated that L359V not only increased transactivation activity of GATA-2 but also enhanced its inhibitory effects on the activity of PU.1, a major regulator of myelopoiesis. Consistent with the myelomonoblastic features of CML transformation with the GATA-2 L359V mutant, transduction of the GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring murine cells disturbed myelomonocytic differentiation/proliferation in vitro and in vivo, respectively. These data strongly suggest that GATA-2 mutations may play a role in acute myeloid transformation in a subset of CML patients.

Fig. 1.

GATA-2 mutations in myeloid blast transformation of CML. (A) DNA sequencing analyses detect a recurrent T/G heterozygosity at 1,075 nt (coding region sequence) of GATA-2 exon 5 in both cDNA and genomic DNA samples isolated from BM cells of CML patients during AP/BC. (B) An 18-bp deletion of the GATA-2 gene from nucleotide 1021 to nucleotide 1038 specifically detected at BC phase in a BM sample of patient UPN6. (C) MALDI-TOF MS confirms a GATA-2 T-to-G mutation within BM cells at BC phase. (D) The detection of the nucleotide 1075 T-to-G mutation in BM samples of three patients by allele-specific PCR at AP/BC but not at CP. (E) The GATA-2 gene deletion results in a deletion of amino acids 341–346 (Δ341–346) around the ZF1 domain border. L359V substitution is within ZF2 domain. (F) Morphological and histochemical examinations of BM samples from patient UPN3 with L359V or from patient UPN6 with Δ341–346. (a) Wright's staining of a BM cellular smear from patient UPN3 at CP. (b–g) Examination on the BM samples from UPN3 at BC. (b) Wright's staining. (c) Myeloperoxidase staining, a specific marker of myeloid cells. Note the presence of strong positive, weak positive, or even negative contingents, suggesting the identities of myeloblasts and monoblasts. (d) Periodic acid Schiff staining. (e) Naphthol AS-chloracetate esterase staining, a marker of granulocytes. (f) Naphthol AS-D acetate esterase (NAS-DAE) staining. (g) Inhibition of NAS-DAE staining by sodium fluoride, a test to distinguish granulocytes from monocytes. (h) Wright's staining of BM samples from UPN 6 at BC shows the presence of eosinophilia. (G) Disease progression of CML patients with GATA-2 mutation compared with those without GATA-2 mutation.

Fig. 2.

GATA-2 L359V has enhanced transcriptional activity on target genes. (A) Transfection assays were conducted in 293T cells using a GATA-2 response element-coupled luciferase reporter, with wtGATA-2-, GATA-2 L359V-, or Δ341–346 mutant-expressing plasmid. (B–D) Examination of mRNA levels of EpoR, EDN-1, and P-selectin using real-time RT-PCR in BM samples isolated at either the CP or BC stage of indicated patients (n = 3). (E) Comparison of the binding affinities of three different GATA-2 proteins with CBP or HDAC1 in a GST pull-down assay. (F) Gel-shift assay on the specific binding of the three GATA-2 proteins with a DNA probe containing a classic GATA-2 response element.

Fig. 3.

GATA-2 mutants exhibit enhanced inhibition on the transcriptional activity of PU.1. (A) GATA-2 mutants physically associate with PU.1 in vivo. Flag-tagged PU.1 cDNA plus one of three c-Myc-tagged GATA-2 cDNAs were cotransfected into Cos-7 cells. Left shows that the immunoprecipitation of whole-cell lysates by anti-Myc antibodies coprecipitates the PU.1 protein. Right shows that, in a reverse way, PU.1 antibody-mediated precipitates contain three GATA-2 proteins, as probed by an anti-c-Myc antibody. (B Left) Different doses of GST-wtGATA-2, GST-GATA-2 L359V, or GST-Δ341–346 mutant and GST were used to pull down the lysates of the PU.1-transfected Cos-7 cells. (B Right) Coomassie blue staining confirms the quantification of GST fusion proteins by Bradford assay. (C) The M-CSFR promoter-coupled luciferase reporter plasmid was cotransfected into Cos-7 cells, with different doses of wtGATA-2 or GATA-2 mutant expression plasmids as indicated. The relative luciferase units are expressed as mean ± SD. *, P < 0.05. (D) G-CSFR promoter-coupled luciferase reporter was cotransfected with different doses of plasmids as shown in C. (E) GATA-2 L359V competes with c-JUN on binding to PU.1. Combinations of different amounts of ³⁵S-labeled GATA-2 and c-JUN as indicated were incubated with the GST-PU.1 protein. Pull-down efficiencies of GATA-2 and c-JUN by PU.1 were examined by autoradiography.

Fig. 4.

Enhanced activities of GATA-2 interfere with myelomonocytic differentiation/proliferation. wtGATA-2- and GATA-2 mutant-expressing plasmids were stably transfected into HL-60 myeloid leukemia cells. One set of representative results from four independent transfection experiments is shown. (A) CD11b (Left) or CD14 (Right) levels were monitored along with 1 × 10⁻⁶ M ATRA-induced granulocytic differentiation of indicated HL-60 cells by flow cytometry (means ± SD). P values indicate the comparisons between GATA-2 mutants and wtGATA-2. (B) CD11b and CD14 levels were analyzed at 24 or 48 h after treatment with 1 × 10⁻⁷ M VitD₃ as in A. (C) Wright's staining of indicated HL-60 subclones before and after 48 h of treatment with ATRA or VitD₃. (D) GATA-2 L359V+BCR/ABL/GFP induced a myelomonocytic leukemia-like disease in transduced cell-transplanted mice. Morphological pictures of peripheral blood were taken from deceased recipients under a ×100/1.0 oil-immersion objective lens.