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.

Background:Mixed lineage leukemia (MLL) is the most common leukemia in infant patients. This leukemia is due to chromosomal translocations at locus 11q23 and is associated with poor clinical outcome. Novel, targeted therapeutic approaches are needed. Protein phosphatase 2A (PP2A) is a serine threonine phosphatase which regulates the phosphorylation of several kinases, including Erk, Akt, GSK3 which are fundamental for MLL cells’ survival. PP2A is a trimeric protein complex in which a core dimer formed between the scaffold subunit and the catalytic subunit is associated with one of the many regulatory subunits that facilitate and direct the interaction of the trimer with substrate proteins. PP2A activity is regulated by interaction with PP2A inhibitor proteins (PIPs), as well as by post‐translational modifications of PP2A complex components.Aims:In this context, our aim is to understand how PP2A post‐translational modifications affect the activity of PP2A on its downstream targets and to investigate whether PP2A re‐activation, by preventing phosphorylation and activation of collateral pathways, might represent a valid therapeutic strategy for MLL.Methods:12 different leukemic cell lines and 8 primary samples from MLL patients were included in this study. We measured the PP2A phosphatase activity by a colorimetric assay using a synthetic phospho‐peptide and malachite green reagent. The protein levels of PP2A, phospho‐PP2A, demethyl‐PP2A, LCMT‐1, PME‐1, Akt, phospho‐Akt, Erk, and phospho‐Erk were determined by western blot using specific antibodies.Results:Compared to healthy bone marrow, we found an increase in the phosphorylation at Tyrosine 307 (Tyr307) and a decrease in the methylation at Leucine 309 (Leu309) of the catalytic subunit of PP2A in MLL samples. These changes in the post‐translational modifications of the catalytic subunit correlated with a decrease in the phosphatase activity of PP2A. Methylation at Leu309 has been described as an absolute requirement for the binding of the regulatory subunit B55α to PP2A core. B55α was found up‐regulated in MLL cell lines and primary samples compared to healthy bone marrow. The methylation of the catalytic subunit of PP2A is catalyzed by leucine carboxyl methyltransferase (LCMT‐1), which we found down‐regulated in MLL samples, whereas demethylation is catalyzed by the phosphatase methylesterase (PME‐1) that was found up‐regulated in MLL. In line with the inactivation of PP2A in the MLL samples, PP2A downstream targets such as Akt and Erk were found hyper‐phosphorylated in MLL samples.Summary/Conclusion:Our results suggest that PP2A phosphatase activity inhibition might sustain in MLL samples a persistent serine/threonine phosphorylation of PP2A substrates, such as Akt and Erk that mediate pro‐survival and anti‐apoptotic signals. This inhibition might be explained, at least in part, by phosphorylation and demethylation of PP2A catalytic subunit. A better understanding of the mechanisms that regulate PP2A activity could provide new strategies to rescue PP2A phosphatase activity and target fundamental mechanisms of leukemic survival and chemotherapy resistance.