Abstract
Background:
Mixed Lineage Leukemia (MLL) is an aggressive form of acute leukemia caused by chromosomal translocation involving the
MLL
gene at locus 11q23. Despite novel therapeutic approaches, there are no efficient targets for MLL treatments and patients present a very poor prognosis. PP2A is a serine‐threonine phosphatase that downregulates crucial target proteins such as Akt, Erk, and c‐Myc, responsible for proliferation and survival of MLL leukemic cells. De‐regulation of PP2A is a recurring event in many cancers.
SET is an endogenous binding partner of PP2A mostly localised in the nucleus. In many diseases including cancer, PP2A inactivation is attributed to cytoplasmic accumulation of SET which inhibits PP2A through direct interaction with PP2A catalytic subunit.
FTY720, a sphingosine analogue drug, prevents the formation of SET‐PP2A complex, binding SET and rescuing PP2A activity (Pippa et al., 2014).
Aims:
To determine the role of SET‐mediated inactivation of PP2A in MLL, in order to define whether this might be an axis amenable to therapeutic target.
Methods:
The study is performed
in vitro
using a set of MLL cell lines (n = 8) and primary samples (n = 8). SET gene expression and protein level profile were investigated by real time PCR and Western blot.
We analysed SET localization by nucleus‐ cytoplasm separation assay and SET‐PP2A interaction by co‐immunoprecipitation assay. The effect of FTY720 on human cell lines was evaluated by analysis of survival by Trypan Blue exclusion and phosphorylation of PP2A targets by Western blot.
Results:
Analysis of
SET
gene expression did not show any significant difference between MLL cell lines and primary samples compared to healthy bone marrow samples. In contrast, SET protein levels were selectively increased in MLL primary samples and cell lines. Analysis of nuclear and cytosolic extracts in MLL cell lines showed that SET was mostly localised into the cytoplasm. Furthermore, immunoprecipitation experiments showed that in MLL cell lines SET is phosphorylated on a serine residue. This modification has been previously shown to be crucial for PP2A binding and inhibition. We evaluated the effect of FTY720 on MLL survival and growth of BCR‐ABL+ cell line K562 by trypan blue exclusion. The results indicate an IC50 ranging from 1.6 to 4.2 μM. The effects of FTY720 on PP2A was evaluated by analysing the phosphorylation of PP2A targets (Akt, Erk, GSK3) in MLL cell lines 24 and 48 hours after treatment. Preliminary data showed no significant changes in phosphorylation of the above targets. These results may suggest that targeting SET alone might not be sufficient to rescue the activity of PP2A in MLL and that redundant mechanisms might occur. The effect of potential combination of FTY720 and standard chemotherapy treatment will be evaluated in future studies.
Summary/Conclusion:
We showed for the first time that SET is overexpressed in MLL samples and mostly localised into the cytoplasm. Given the implication of SET as an endogenous PP2A inhibitor, we aim to understand the mechanism by which it interacts with PP2A in MLL to target this complex with the aim of designing novel therapeutic approaches for this challenging disease.