Background KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. We explored the role of SET, the endogenous inhibitor of SER/THR phosphatase PP2A in KMT2A-R leukemia. Material and Methods The expression of SET was analysed in a large acute myeloid leukemia (AML)- RNA-seq dataset and in primary KMT2A-R samples and aged matched-controls. Stable SET knockdown (KD) was established by RNA interference in three KMT2A wild-type (wt) and four KMT2A-R leukemic cell lines. Gene and protein expression were analysed by RT-qPCR, ChiP, IP and western blot. RNA-seq and phospho-proteomics were employed to evaluate the effect of the SET-PP2A inhibitor FTY720 on global protein phosphorylation and gene expression. The cellular impact of FTY720 was evaluated by analysing proliferation, cell cycle and apoptosis in leukemic cell lines and by colony formation assay in two patient-derived xenograft (PDX). Results SET mRNA was found expressed in blasts from KMT2A-R-patients and in leukemic stem cells. SET protein interacted with both KMT2A wt and fusion proteins. Knockdown of SET inhibited the transcription of KMT2A target genes HOXA9 and HOXA10and abolished the self-renewal of KMT2A-R leukemic cells. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest, apoptosis and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic and western blot analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3b, ARKB, and led to degradation of MYC, supporting the hypothesis of a feedback loop among SET, PP2A and MYC. The RNA-seq indicated that FTY720 reduced the activity of signalling pathways implicated in gene transcription and it compromised the expression of several genes belonging to the KMT2A-R leukemia signature. Conclusions Taken together our results identify SET as a novel player in KMT2A-R leukemia and provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.

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.