Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant

Abstract

Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures:No relevant conflicts of interest to declare.