Differential effects on gene transcription and hematopoietic differentiation correlate with GATA2 mutant disease phenotypes

Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1 more strongly than WT potentially perturbing differentiation via sequestration of PU.1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema.C-E Chong, P Venugopal, PH Stokes, YK Lee, PJ Brautigan, DTO Yeung, M Babic, GA Engler, SW Lane, M Klingler-Hoffmann, JM Matthews, RJ D'Andrea, AL Brown, CN Hahn, and HS Scot

Contribution of the GATA2 transcription factor to the development and progression of myeloid disorders

The GATA2 transcription factor has an essential role in the proliferation and differentiation of hematopoietic cells. GATA2 contributes to myeloid malignancies through various mechanisms, including GATA2 overexpression in acute myeloid leukemia (AML), somatic GATA2 mutations in chronic myeloid leukemia in blast crisis and AML with biallelic CEBPA mutations, and germline mutations of GATA2, associated with four related familial syndromes that predispose to AML, among other myeloid malignancies. We hypothesized that deregulation of either GATA2 expression or GATA2 aberrant function caused by mutations could alter key downstream target genes, contributing to the pathogenesis of these diseases. Our general aim was to identify and functionally characterize novel target genes of the GATA2 transcription factor in AML, and to assess the effect of the GATA2 mutated proteins in the transcription of known target genes of GATA2. The results indicate that MYB is a key transcriptional target of GATA2 in AML, and that leukemic cells depend on MYB to maintain their proliferative phenotype. Moreover, MYB and GATA2 expression is positively correlated in three independent cohorts of patients with AML. On the other hand, human GATA2 contributes to its own transcription, and GATA2 mutations p.Thr354Met, p.Thr355del and p.Arg396Gln are unable to activate the GATA2 promoter, causing a haploinsufficiency that may be responsible for GATA2 deficit in affected individuals. Moreover, the expression of genes with important functions in myeloid development is deregulated in a patient with p.Arg396Gln mutation, including GATA2. Finally, in silico and in vitro studies indicate that p.Arg396Gln is unable to retain the immature phenotype of hematopoietic stem and progenitor cells, and that p.Arg396Gln might contribute to alter the leukocyte profile in affected individuals

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

BACKGROUND: Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. DESIGN AND METHODS: We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. RESULTS: SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. CONCLUSIONS: These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators

Activation of innate-adaptive immune machinery by Poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer

Objective Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. Design To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. Results By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). Conclusion This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC