Targeting SET-PP2A complex as a new avenue for therapeutic targeting of KMT2A-driven acute leukemia

Maria Teresa Esposito
University of Roehampton

Introduction/Background & aims Acute Leukemias associated to chromosomal translocations rearranging the Histone-Lysine-N-methyltransferase 2A (KMT2A) gene are a highly aggressive group of leukemias. Despite extensive research, KMT2A-R patients have a dismal prognosis and no targeted therapy is yet available. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of KMT2A-R-leukemic cells’ survival and resistance [1]. However, kinase- targeted cancer therapies have failed to provide results of clinical translational value because of compensatory mechanisms taking place when targeting a single kinase or pathway. The phosphorylation status, and thus the oncogenic potential of any given protein, is not regulated only by kinases, but rather by an intimate balance between kinases and their neglected antagonist phosphatases. The versatility   of   phosphatase   functions   and   their   involvement in multiple feedback mechanisms makes them attractive targets for future drug development. In this project we explored the role of Ser/Thr phosphatase tumor suppressor PP2A and its endogenous inhibitor SET (also known as I2PP2A) as a potential therapeutic target for KMT2A-R leukemia.

Method/Summary of work The expression of SET was analysed in a large acute myeloid leukemia (AML)- RNAseq dataset comprising KMT2A-R (n=31) and non KMT2A-R patients (n=384) [2].  The in vitro experiments were performed on a set of 9 KMT2A-R leukemic cell lines and 8 primary samples. 3 non KMT2A-R leukemic cell lines and 6 independent bone marrow from age matched healthy volunteers were included as controls. SET gene and protein expression were analysed by real time PCR and Western blot. Genetic modulation of SET was achieved by specific shRNA expressed by lentiviral vectors. The effect of FTY720 was evaluated by analysis of proliferation, cell cycle and cell death.  The analysis of PP2A activity upon FTY720 treatment was evaluated by analysis of phosphorylation of PP2A targets by Western blot. The effect of FTY720 on gene expression was evaluated by RNAseq (vehicle n=3, FTY720 treated n=3). 

Results/Discussion Our results indicate that SET is expressed at comparable levels in all subtypes of AML analysed. Western blot analyses confirmed that the SET is over-expressed in KMT2A-R cell lines and primary samples compared to bone marrows isolated from healthy volunteers. Molecular targeting by shRNA showed that SET knock-down abolished the clonogenic ability of KMT2A-R leukemic cell lines, whereas it had no effect on  the colony formation of two independent non KMT2A-R leukemic cell lines (eK562 shScramble 585± 164 vs shSET 603± 133 p>0.999; eKasumi  shScramble 227± 54 vs shSET 247± 25 p=0.9948),  indicating that SET knock-down specifically impairs the self-renewal of KMT2A-R cells. We then tested pharmacological modulation of SET by  FTY720 (Fingolimod), a FDA approved immunosuppressive drug that has gained attention as anti-cancer and PP2A activating drug [3]. FTY720 induced cell cycle arrest and modest cell death in almost all the cell lines analysed. This effect was mirrored by a decrease in the  activation by phosphorylation of some key targets of PP2A including ERK and Akt. Pre-treatment with Okadaic Acid , an inhibitor of PP2A, followed by FTY720, restored the levels of phospho-Akt and phosphor-ERK in both non KMT2A and KMT2A-R leukemic cells whereas it specifically  decreased the percentage of dead cells  in KMT2A-R cells (THP1: fold ratio FTY720+ OA vs FTY720 0.46 p<0.0001; MV411: fold ratio FTY720+ OA vs FTY720 0.59 p<0.0001). This suggests that Okadaic acid was able to partially rescue the effect of FTY720 on cell death and that the FTY720 effects observed in KMT2A-R cells were indeed dependent on PP2A activation. In addition, FTY720 increased the response of KMT2A-R cells to standard chemotherapeutic drug Daunorubicin (p<0.0001), whereas it had a modest effect in non KMT2A-R leukemic cells (p<0.0001). Although in other models the anti-cancer properties of FTY720 have been ascribed to its ability to disrupt the binding between SET and PP2A, our RNAseq data indicate that, upon FTY720 treatment, SET is specifically down-regulated at transcriptional level in KMT2A-R cell lines (Log2Fold change -0.312 p=0.004832), a result that we confirmed by Western blot analysis.

Conclusion(s) Our data indicate that  SET is a promising therapeutic target for KMT2A-R leukemia. FTY720, an immunosuppressor approved by FDA for multiple sclerosis, induces a specific decrease in SET expression in KMT2A-R cells, leading to cell cycle arrest and cell death and it increases the reponse to standard chemotherapy.  Repositioning of FTY720 may therefore represent a novel avenue for tailored therapeutic targeting of the aggressive KMT2A-R leukaemia.

Reference(s)

1. Cruickshank, M.N., et al., Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin. Leukemia, 2017. 31(1): p. 40-50.

2. Lavallee, V.P., et al., The transcriptomic landscape and directed chemical interrogation of MLL-rearranged acute myeloid leukemias. Nat Genet, 2015. 47(9): p. 1030-7.

3. White, C., et al., The emerging role of FTY720 (Fingolimod) in cancer treatment. Oncotarget, 2016. 7(17): p. 23106-27.