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

Abstract

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.

Figure 1.

SET is deregulated in samples from patients with AML, and confers a poor outcome in AML. (A) Kaplan-Meier analyses of overall survival for SET overexpression in a series of 146 patients with AML and clinical follow-up data available who received induction therapy. (B) Kaplan-Meier analyses of overall survival for SET overexpression in the subgroup of patients with normal karyotype. (C) Western blot analysis showing SET expression levels in 16 AML patients at diagnosis.

Figure 2.

SET induces cell growth and restores proliferation in ectopically PP2A-expressing AML cells. (A) MTS assay showing proliferation in HEL cells transfected with SET compared to cells transfected with an empty vector; (B) MTS assay showing proliferation in HEL cells transfected with PP2A alone or in combination with SET or with an empty vector; (C) MTS assay showing the effect of SET in cells treated with the PP2A activator forskolin (40 μM). *P<0.05; ** P<0.01.

Figure 3.

(A) Western blot showing increased levels of SET after transfection with EVI1. (B) Specific DNA binding of EVI1 to the SET promoter was detected by ChIP. Real time PCR was performed on fragmented chromatin precipitated by anti-EVI1 antibody (gray bars) or normal rabbit IgG (black bars) from an EVI1-positive (EVI1+) TF1 cell line and the EVI1-negative (EVI1−) MOLM13 cell line. Results are expressed as percentage of input as calculated by qRT-PCR. The EVI1-positive target gene PBX1 was used as a positive control. (C) Luciferase reporter assay in HEK293 cells co-transfected with pGL3-SETpromoter and pCMV6-EVI1 or an empty vector. Firefly luciferase activity was normalized to Renilla luciferase activity.

Figure 4.

Western blot analysis showing EVI1 and SET levels in 12 AML patients at diagnosis. A protein extract of the K562 cell line was used as the positive control for SET and EVI1.