SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML.

doi:10.21203/rs.3.rs-2583091/v1

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SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML.

https://doi.org/10.21203/rs.3.rs-2583091/v1

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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.

SET

KMT2A

MLL

leukemia

I2PP2A

phosphatase

PP2A

MYC

FTY720

Acute leukemias characterised by chromosomal translocations involving the KMT2A gene (previously known as MLL, HRX, HTRX1 or ALL1) are a highly aggressive group of leukemias with a poor prognosis (1, 2). They account for over 80% of infant acute lymphoblastic leukemia (ALL), 5–10% of acute myeloid leukemia (AML) and over 70% of therapy related AML (2). KMT2A encodes for the Histone-Lysine-N-methyltransferase 2A that regulates gene transcription via methylation of histone H3 in the gene bodies (1, 3). The N-terminal portion of KMT2A binds to DNA, whereas the C-terminal contains the catalytic domain for histone methylation, which is essential to relax the chromatin and to drive the expression of genes during embryogenesis and hematopoiesis (1). KMT2A chromosomal translocations give rise to chimeric oncofusion proteins that retain only the N- terminal DNA binding domain of KMT2A fused with over hundred partner proteins (2). These fusions impair the normal functioning of KMT2A and cause aberrant transcriptional activation by recruitment of an epigenetic multiprotein complex that includes the histone lysine 79 (H3K79) methyltransferase Dot1L, the bromodomain proteins, the acetyltransferase HBO1 and several other proteins that acquire opportunistic oncogenic functions (3–7). The HOX genes, in particular HOXA9 and HOXA10, are primary targets of KMT2A-fusion products and, together with the cofactor MEIS1, play an important role in the transcriptional reprogramming of hematopoietic stem cells (HSCs) and progenitor cells harbouring the KMT2A translocations, leading to impairment of cellular differentiation (8–10) and resistance to DNA damage inhibitors (11). In addition, expression of the oncogene MYC contributes to KMT2A-mediated leukemogenesis by driving cell proliferation and survival (12). Despite the developments of new therapeutic agents, such as inhibitors of KMT2A-fusion complex (3–5, 13, 14) and inhibitors of kinases essential for the KMT2A-signaling pathways (15, 16), none of these are FDA approved. Consequently, patients affected by KMT2A-R-leukemia are still treated with chemotherapy followed by bone marrow transplantation, with suboptimal outcomes. Therefore, the identification of novel targets and/or targetable pathways for KMT2A-R-leukemia is of primary importance for the development of new and effective therapeutic approaches.

SET is an oncoprotein also known as template activating factor-I-β (TAF I-β), inhibitor 2 of protein phosphatase 2A (I2PP2A) and putative histocompatibility leukocyte antigen class II-Associated Protein II (PHAPII). Originally identified as fusion gene in leukemia (17), it is over-expressed in several forms of solid tumors and hematological malignancies (18–22). In BCR::ABL + chronic myeloid leukemia (CML), the inhibitory effect of SET on SER-THR phosphatase PP2A switches the oncogenic driver kinase BCR::ABL off, highlighting its potency as a negative regulator of oncogenic cascades (18, 22, 23). SET has also been recognised as a prognostic marker for poor overall survival in AML (19, 20), although the exact molecular mechanisms linking a SET oncoprotein pathway with aggressive AML outcomes remain still obscure. On these bases and also given the fundamental role played by PP2A-modulated kinases on the survival and resistance of KMT2A-R-leukemic cells (15, 16, 24), we herein investigate the role of the PP2A endogenous inhibitor SET in KMT2A-R-leukemia. Our overall results provide a proof of concept that inhibition of SET is a promising novel strategy to treat this aggressive form of acute leukemia.

Cell lines

The cells lines used for this study (supplementary table 4) were either purchased from DSMZ or a gift of Prof. Eric So (King’s College University, London), Prof. Owen Williams (University College London) or Dr David Giuliano (University of East London).

Primary Cells

Primary samples (supplementary table 5) were obtained from the Cancer Tissue Bank at the Barts Cancer Institute (London) under ethical approval (REC reference: 17/WM/0428).

PDX samples

The KMT2A-PDX were a generous gift of Prof. Owen Williams. These cells were grown in methocult H4435 (Stem Cell Technologies) with addition of 10 ng/mL human TPO1 and human FLt3 ligand (Stem Cell technologies).

Virus Production And Cell Transduction

Knock-down of SET was conducted in vitro using the lentiviral viruses purchased from Sigma (supplementary table 7). The viruses are based on the plasmid vector pLKO.p1 co-expressing a puromycin resistance cassette to enable transduction selection of mammalian cells and ensure the establishment of stable clones. As a transduction negative control, we used non targeting shRNA control (shScramble), designed to target no known gene sequence in the cells being used. The pLKO, 1-puro CMV-tag RFP, with no shRNA insert and expressing the Red Fluorescent Protein, was used as a transduction positive control.

Cell proliferation and apoptosis analysis

The cell lines were stably transduced with a lentivirus vector expressing the enhanced Green Fluorescent Protein (eGFP) (Arroyo and Calle, unpublished data). Cell proliferation was measured by analysis of GFP fluorescence at fluorescent microplate reader at 495nm. Apoptosis was measured by flow cytometry using a BD Accuri™ C6 Flow cytometer using the GFP signal, measured in FL1-A channel (533/30), to discriminate GFP negative (-) (apoptotic cells) from GFP positive (+) (alive cells).

Reagents

FTY720 was purchased from Selleckchem (S5002). The antibodies used in this study are listed in supplementary table 8 and 9. Quantitative real-time PCR (RT-qPCR) was performed using specific primers from SIGMA (supplementary table 10). The primers used for ChiP are listed in supplementary table 11.

Phospho-proteomic Experiments

Phospho-proteomic experiments were performed using mass spectrometry with some technical modifications as reported (25).

Microarray, Rna-seq And Bioinformatic Analysis

The expression profile of SET in human HSC and blood cells was obtained from Bloodspot Gene expression profiles (GSE42519) (26). The expression profile of SET mRNA in AML patients was obtained from Leucegene gene expression profiles (GSE62190, GSE66917, GSE67039) (10). The expression of mouse SET mRNA (probe1421819_a_at) was analysed by using the micro-array data from GSE13690 (27). Meta-analyses of micro-array data from the PrognoScan database (GSE12417) were used to determine the prognosis of patients expressing high or low levels of SET (28). The RNA-seq in FTY720- vs vehicle-treated THP1 cells was performed by Novogene. Downstream analysis was performed using a combination of programs including STAR, HTseq, Cufflink and Novogene wrapped scripts. Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using the DESeq2 R package (2_1.6.3).

Statistical analysis

Data are expressed ad mean +/- standard deviation (SD) from at least 3 independent experiments, unless stated otherwise. Statistical significance was determined using GraphPad Prism 7 with the tests reported in the figure legends.

Additional methods are in the supplementary methods.

SET is over-expressed in KMT2A-R cell lines and primary samples

To characterise the role of SET across various hematopoietic contexts, we used public data repositories to analyse its mRNA expression in human HSCs and progenitors (n=34) as well as in multiple independent human AML primary samples (n=384) covering the main cytogenetic subsets (10, 26). SET was expressed at high levels in both HSC and myeloid progenitors (MP) compared with mature monocytes and myelocytes (Fig. 1A and supplementary Figure 1A), thereby indicating that it is a gene expressed during early hematopoietic development. In silico evaluation of SET mRNA expression across all AML was uniformly high and it did not show segregation with any distinct molecular group as previously suggested (Fig. 1B and supplementary Fig 1A-C) (19, 20). A PrognoScan database-based Kaplan–Meier analysis of the overall survival of 117 AML patients by high (n=11) and low (n=106) SET levels (28), revealed that high SET expression positively correlated with poor overall survival in human AML (Fig. 1C), consistent with previous reports (20). To further substantiate the role of SET in AML we evaluated its protein levels by western blot in a panel of KMT2A-R-AML (THP1, MV411, ML2, MOLM13, NOMO-1) and KMT2A-R-ALL (SEM, Hb1119, KOPN8, RS411) cell lines and primary samples (PS), KMT2A wild-type (wt) cell lines (K562, BCR::ABL+ erythroleukemia, Kasumi1 AML1::ETO+ AML, REH TEL::AML+ ALL, U937 CALM::AF10+ AML), mononuclear cells isolated from the bone marrow (BM) and peripheral blood (PB) of healthy adult volunteers. SET was significantly up-regulated in all the leukemic cells lines and in all KMT2A-R primary samples, irrespective of tumor lineage, compared to BM controls (Fig. 1D-E). Given the poor outcomes of the KMT2A-R-leukemias and the relatively well characterised cellular context driving leukemogenesis, we further investigated the mechanistic role of SET in this group. As SET oncoprotein has several distinct roles depending on its subcellular localisation (22), we analysed its subcellular localisation in two KMT2A-R-cell lines (THP1 and MV411) and one KMT2A-wt cell line (K562) by nuclear/cytoplasm fractionation followed by western blot and showed that SET was relatively more abundant in the cytoplasm than in the nucleus of these cells (Fig. 1F). As phosphorylation of SER9 and SER25 of SET inhibits its nuclear import (29, 30), we also investigated the phosphorylation status of SET. As no specific antibodies against phospho-SET are commercially available, SET was immunoprecipitated and protein samples were analysed by western blot with anti-pSER antibodies. Consistent with the prevalent cytoplasmatic localization, our results showed that, in KMT2A-R-AML cell lines, SET is phosphorylated on Serine residues (Fig. 1G). Overall, these data indicate that SET is over-expressed, phosphorylated and abundantly localised in the cytosol in KMT2A-R and -wt cells.

SET is essential for KMT2A-R leukemia self-renewal

To understand the role of SET in leukemic stem cells (LSC), we analysed the expression of SET mRNA in 12 high LSC frequency mouse KMT2A-R AMLs (KMT2A::MLLT3 and KMT2A:MLLT1) and 22 low LSC frequency KMT2A-R-AMLs (KMT2A::AFF1p, KMT2A::AF10 and KMT2A::GAS7), using public data repositories (32). Interestingly, we found that SET mRNA expression was significantly higher in high LSC frequency KMT2A-R AML than in low LSC frequency KMT2A-R AML, (Fig. 2A), suggesting a potential role of SET in KMT2A-R leukemia self-renewal. Therefore, to determine whether SET has a functional role in KMT2A-R- leukemia, we knocked down SET gene in human cell lines by using RNA interference. SET knockdown (KD) completely abolished the clonogenic ability of KMT2A-R leukemic cell lines (for THP1 and MV411 almost 100% reduction in colony number, for SEM 90.5% reduction) (Fig 2B, E, F and H and supplementary 2), whereas it had little or no effect on the colony formation of three independent KMT2A-wt leukemic cell lines (K562 shScramble 554± 126 vs shSET 604± 83 p>0.05; Kasumi1 shScramble 227± 54 vs shSET 167±140 p>0.05; REH shScramble 479±54 vs shSET 271±115 p>0.05) (Fig. 2B, C, D, G). SET KD attenuated the proliferation of K562 (eGFP-K562 fold ratio shSET vs shScramble 0.89 p<0.05) (Fig 2I, K and supplementary Figure 3), in line with a previous report (18) and similarly to KMT2A-wt AML cell line Kasumi1 (eGFP-Kasumi fold ratio shSET vs shScramble 0.55 p<0.0001) (Fig. 2J-K and supplementary Figure 3). Overall, these data indicate that SET KD specifically abolishes the self-renewal of KMT2A-R-leukemic cells and, consistently, it attenuates the proliferation of KMT2A-wt leukemic cells.

The SET inhibitor FTY720 induces cell cycle arrest and drives cell death in KMT2A-R leukemic cells

We next tested pharmacological modulation of SET by FTY720 (Fingolimod) (31), a FDA-approved immunosuppressive drug that has gained further attention as anti-cancer and PP2A activating drug, due to its ability to disrupt the binding between SET and PP2A (32-34). We first carried out a dose-response titration assay and determined that the half maximal inhibitory concentration of FTY720 in vitro ranged between 1 and 5 mM (Supplementary Fig. 4A), which were reported as non-toxic to healthy bone marrow mononuclear cells (23, 35). We then assessed the effect of FTY720 on proliferation, cell cycle and cell death of KMT2A-wt and KMT2A-R-leukemic cells. As in our model the GFP fluorescence is proportional to the number of alive cells, we used GFP as a reporter of cell viability (Fig. 3A). We observed that 5 µM FTY720 had a variable but significant effect on the proliferation of all the analysed cell lines, ranging from modest for the KMT2A-wt-cell lines (vehicle vs FTY720 fold ratio eGFP-K562= 1.19; eGFP-Kasumi1= 1.85) to severe for the KMT2A-R-cell lines (vehicle vs FTY720 fold ratio eGFP-THP1= 3.17; eGFP-MV411= 4.18; eGFP-SEM= 2.32). In addition, FTY720 severely halted the proliferation of KMT2A-wt eGFP-REH (vehicle vs FTY720 fold ratio= 6.17), as previously reported (36). Cell cycle analyses revealed that treatment with FTY720 for 48 hours induced a significant increase of cells in G1 and a reduction in S and G2-M phase, in KMT2A-R-cells (Fig. 3B and supplementary Fig. 4B), indicating cell cycle failure. Moreover, we investigated the faction of leukemic cells undergoing apoptosis upon FTY720 treatment by FACS, by gating the GFP negative (GFP-) cells. FTY720 induced a statistically significant increase in apoptosis in two KMT2A-wt-cell lines (fraction of GFP- cells in FTYT720 treated vs vehicle for eGFP-REH 0.9± 0.028 vs 0.016± 0.002; for eGFP-K562: 0.393±0.059 vs 0.147±0.027), whereas this was not significant for the KMT2A-wt cell line eGFP-Kasumi1 (0.124± 0.036 vs 0.147±0.062). In contrast, FTY720 induced a consistent and statistically significant increase in apoptosis in KMT2A-R-cells (eGFP-THP1:0.43±0.08 vs 0.10±0.061; eGFP-MV411: 0.285±0.136 vs 0.12±0.072; eGFP-SEM: 0.12±0.01 vs 0.04±0.004; eGFP-Hb1119: 0.391± 0.028 vs 0.038± 0.004) (Fig. 3C and supplementary Fig. 4C). These results indicate that the SET inhibitor FTY720 induces heterogenous effects in leukemic cells; specifically, in KMT2A-R-leukemic cells, FTY720 causes cell cycle arrest in G1 and significantly increases the rate of apoptosis.

The effect of FTY720 is dependent on PP2A activation

FTY720 is a SET inhibitor able to rescue the activity of PP2A towards its target pathways (31, 37). By immunoprecipitation, we showed that FTY720 treatment for 24 hours disrupted the binding between SET and PP2A in KMT2A-R cells, confirming the molecular mechanism reported in other leukemic models (Fig.4A) (23, 32-34). To investigate whether the observed effects of FTY720 on KMT2A-R cells were due to the activation of PP2A, we analyzed the phosphorylation of some of PP2A targeted pathways (38) by western blot. 24 hours after treatment we observed a reduction in the abundance of phospho-AKT1(SER473) in KMT2A-wt cells (Fig. 4B); in Kasumi1 cells, the reduction in phospho-AKT1 was sustained over 48 hours, whereas in K562, phospho-AKT1 expression returned to the levels of vehicle-treated cells, indicating that the effect of FTY720 was temporary. In K562 cells, we also observed reduction in phospho-ERK1 (THR202/TYR04), which was instead unchanged in Kasumi1; in these cell lines, also phospho- GSK3β (SER9) expression decreased. Notably, in KMT2A-R-leukemic cells, we observed a dramatic and stable decrement in the expression of the PP2A targets phospho- AKT1 and phospho- ERK1 (Fig. 4B). To confirm that the effect of FTY720 was specifically dependent on PP2A activation, we performed the same experiments by pre-treating cells with the phosphatase inhibitor okadaic acid (OA) used at a concentration that inhibits PP2A but no other phosphatases (19). We first tested OA in K562 cells and confirmed that the range of concentrations used did not impact the cell proliferation (Supplementary Fig. 5A). As expected, OA treatment caused a significant increase in the PP2A targets, phospho-GSK3β, phospho-ERK1 and phospho-AKT1, with maximal expression after treatment with 2.5nM OA for 4 hours and 5nM for 2 hours (Supplementary Fig. 5B). We therefore used the lower concentration of OA for our combination experiments with FTY720. Western blot analysis of PP2A targets in the cells pre-treated with OA for 4 hours and then treated with FTY720 for 20 hours, revealed that pre-treatment with OA restored phospho-AKT1 and phospho-ERK1 levels in all the leukemic cells analysed (Fig. 4C). Whereas pre-treatment with OA did not have any effect on the percentage of apoptotic cells in eGFP-K562 and eGFP-Kasumi1, it significantly decreased the percentage of apoptosis in KMT2A-R-cells (fraction GFP- cells FTY720+ OA vs FTY720 eGFP-THP1: 0.11 ± 0.047 vs 0.32 ± 0.090; eGFP-MV411 0.10 ± 0.039 vs 0.26 ± 0.14) (Fig. 4D and supplementary Fig. 6), suggesting that OA rescues the effect of FTY720 in KMT2A-R-leukemic cells. A similar effect was obtained by knocking down PPP2CA, the gene encoding for the catalytic a subunit of PP2A (Fig 4E-F). These data indicate that the effects of FTY720 on KMT2A-R cells are dependent on PP2A activation.

The phospho-proteome reveals FTY720-mediated effects on cell division, apoptosis and gene transcription

To reveal the global impact of FTY720 on leukemic cells, we performed a phospho-proteomic analysis on two KMT2A-R cell lines (eGFP-THP1 and eGFP-MV411) treated with FTY720, using in gel digestion and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (25). LC–MS/MS analysis showed a differential pattern of phospho-protein abundance in eGFP-THP1 and eGFP-MV411 cells, with 2276 phosphosites up-regulated and 1862 phosphosites down-regulated (>2 fold and p<0.01) in eGFP-THP1, and with 1428 phosphosites up-regulated and 743 phosphosites down-regulated ( >2 fold and <0.01) in eGFP-MV411 (Fig 5A and B and Supplementary Fig. 7), in comparison to vehicle-treated cells. The complete list of identified proteins and phospho-peptides are provided in Supplementary Table 1. To gain deeper biological insights, we categorized the phospho-proteins using Gene Ontology (GO) (Fig 5C), revealing that treatment with FTY720 led to a robust decrease in cell division and an increase in apoptosis-related kinase signalling in eGFP-THP1 cells (Fig 5C and Supplementary Fig. 7E). In contrast, the data indicate a strong decrease in cell division-related kinase signalling, but a subtler increase in apoptosis eGFP-MV411 (Fig 5C and Supplementary Fig 7F). In addition, FTY720-modulated phosphosites implicated in transcription regulation, chromatin organisation, DNA damage repair, mRNA processing, microtubule and actin cytoskeletal organization (Fig 5C). We then used Kinase-Substrate Enrichment Analysis (KSEA) for the characterisation of kinase activity from the phospho-proteomic dataset. The mitosis-regulating kinase Aurora Kinase B was the kinase most significantly impacted by FTY720 in both cell lines (Fig. 5D- E). Indeed, FTY720 decreased the phosphorylation of several Aurora Kinase B targets, including PLK1 (THR210) (Fig 5 F-G-H). As expected, FTY720 inhibited MAPK3 (ERK1) and phosphorylation of ERK1 on THR202 and TYR204 was significantly reduced in both cell lines (Fig Supplementary Fig 7G); furthermore, FTY720 significantly inhibited Abl1 and CDK2 in eGFP-MV411 (Fig 5I and supplementary Figure 7I). Although the overall effect of FTY720 resulted in a significant increase in GSK3b activity, the analysis of the phosphosites regulated by this kinase indicated that this effect was mostly due to the increased phosphorylation of a single target RCAN1 (SER163), whereas GSK3b-dependent phosphorylation of MYC on THR58 was significantly inhibited by FTY720 (Fig 5J and supplementary Fig 7J). FTY720 significantly increased the activity of DNA damage kinase ATM, with an overall activation of targets implicated in DNA repair by Non homologous end joining (NHEJ), such as PRKDC (DNA-PK), and inhibition of sensors involved in transcription and DNA repair by homologous recombination, such as BRCA1 (Fig 5K-L and supplementary Fig7K). Taken together, these data indicate that FTY720 reduced the activity of phospho-signalling associated to cell division and increased apoptosis-related and DNA damage kinase signalling in KMT2A-R cells.

FTY720 affects the core transcriptome of KMT2A-R leukemia

As the phospho-proteomics indicated phosphorylation changes in targets involved in transcription (Fig 5C), we performed RNA-seq analysis to determine the gene expression profile of KMT2A-R cell line THP1 treated with FTY720 for 24 hours. Volcano plot filtering was used to identify differentially expressed genes between vehicle control group and FTY720 treated group (Fig. 6A). According to the results, 980 genes were significantly up-regulated and 898 genes were significantly downregulated by FTY720 (fold change >1.3, padj<0.05). Functional annotation clustering of these differentially expressed genes using the Gene ontology (GO), Kyoto Encyclopedia of gene and genomes (KEGG) and Reactome annotation databases indicated that sets of down-regulated genes were highly enriched in functional groups that related to ribosome biogenesis and rRNA processing (Supplementary Fig. 8A), whereas up-regulated genes were highly enriched in functional groups that related to myeloid cell activation (Supplementary Fig. 8B). In addition, upon treatment with FTY720, genes involved in autophagy, intrinsic apoptotic signalling pathway, extrinsic apoptotic signalling pathway and cell cycle arrest resulted upregulated (Supplementary Fig. 8B). Thus, these data corroborate our previous findings on cell cycle arrest and apoptosis and also indicate that FTY720 can induce cell death by multiple mechanisms, as reported in other models (36, 39-41). More importantly, several genes over-expressed in cancer, including SET and MYC were down-regulated (Supplementary table 2), as well as genes associated with histone methyltransferase activity, among which several members of the KMT2A- fusion epigenetic complex, and HOXA9/MEIS1 target genes (3, 8-10) (Supplementary table 3). We performed RT-qPCR and western blot to validate the decreased expression of SET and MYC upon FTY720 treatment (Fig. 6B and supplementary Fig. 9A-B). Interestingly, western blot analyses indicated that SET protein was significantly reduced only in KMT2A-R-cells (Fig. 6C). RT-qPCR also confirmed a specific decrease in the expression of the KMT2A target genes HOXA9 and HOXA10, in KMT2A-R-cells (Fig. 6B); these genes were also down-regulated in KMT2A-R-cells, when SET was knocked down (Fig. 6D), suggesting that SET might regulate the expression of these genes in KMT2A-R-cells. Previous reports indicated that SET and the oncofusion protein SET::NUP214 modulate the expression of HOXA gene cluster in HeLa cell line and in T-ALL primary samples (42-44). To identify whether SET was enriched on HOXA9 and HOXA10 promoters, we performed chromatin immunoprecipitation (ChIP) experiments. As expected, KMT2A localized on the promoters of both HOXA9 and HOXA10; in contrast, SET was enriched only on HOXA10 promoter (Fig. 6E and supplementary Fig 9C and 10). Immunoprecipitation experiments revealed that SET interacted with both KMT2A and KMT2A-fusion protein (Fig. 6F and supplementary Fig. 9D). Collectively, our results indicate that genetic and pharmacological modulation of SET rewires the KMT2A-R- core gene expression signature and reduces the expression of key genes critical for sustaining this disease.

FTY720 leads to increased sensitivity of KMT2A-R leukemia to chemotherapy

Daunorubicin is an anthracycline included in the intensive multiagent chemotherapy to induce remission in KMT2A-R-AML patients (1). We investigated whether SET targeting via FTY720 could enhance daunorubicin-induced cytotoxicity in KMT2A-R-cells. To this aim, we tested 5µM FTY720 with 10nM daunorubicin, a concentration that had shown a very modest effect on the proliferation and survival of AML cells, including those carrying KMT2A-translocations. Whereas the combination treatment FTY720 + Daunorubicin did not have any effect on eGFP-K562, the percentage of apoptotic cells was significantly high for all the AML cell lines (Figure 7A). The apoptosis increase was modest only for eGFP-Kasumi1 cell line (fraction GFP- cells in FTY720 vs FTY720 +DNR 0.12±0.06 vs 0.22± 0.082), but the effect was very strong in KMT2A-R-cell lines, namely eGFP-THP1 (0.25±0.13 vs 0.55± 0.23, p<0.0001) and eGFP-MV411 (0.24 ±0.18 vs 0.57±0.19, p<0.001) (Fig. 7A and supplementary Fig. 11). To assess the biological importance and therapeutic relevance of SET targeting via FTY720 in KMT2A-R-leukemia, we tested FTY720 in combination with daunorubicin in two KMT2A-R-patient-derived-xenograft (PDX) models in vitro. Combination treatment resulted in a significant reduction of colony number compared with vehicle and with single drug treatment of all samples (74% reduction in comparison to vehicle) (Fig. 7B-C), suggesting that FTY720 treatment enhances the response to daunorubicin.

SET is an endogenous PP2A inhibitor overexpressed in several types of solid tumours and hematological malignancies (18–20, 22, 35). Mechanistically, SET over-expression, and the resulting PP2A inhibition, is critical for the maintenance of the leukemogenic programme by BCR::ABL in chronic myeloid leukemia (CML) (18, 35), in contrast, the exact molecular mechanisms linking a SET oncoprotein pathway with aggressive AML outcomes are not known. Here we report that SET is over-expressed in the KMT2A-R subtypes of AML and ALL. We also show that, in KMT2A-R-cell lines, SET is relatively more abundant in the cytoplasm than in the nucleus and is phosphorylated on Serine residues, which is in line with the notion that the nuclear import of SET is inhibited by phosphorylation on SER9 and SER24 (29, 30). We demonstrate that SET KD has a detrimental effect on the proliferation of KMT2A-wt- leukemic cells, as also observed in CML and solid tumours (18, 45–47). More importantly, we show for the first time that SET KD completely abolishes the ability of KMT2A-R-leukemic cells to form colonies in semi-solid medium, suggesting that SET plays an important role in the self-renewal of KMT2A-R-cells. In order to provide a proof of concept that pharmacological modulation of SET is a promising strategy for the treatment of KMT2A-R-leukemia, we tested in vitro FTY720 (Fingolimod), a proven inhibitor of the interaction between SET and PP2A (23, 31–34), that had been shown to induce cell death in several cancer and leukemia models by various mechanisms, including autophagy (36, 41). In agreement with previous report on CML and AML (18, 23, 39, 40), we show that FTY720 has a negative impact on the proliferation of KMT2A-wt- leukemic cells, causes cell cycle arrest and promotes apoptosis. Accordingly, our phospho-proteomic and RNA-seq analyses reveal decreased activity of kinases implicated in cell division and enhanced expression of genes promoting cell cycle arrest and apoptosis. In particular, phospho-proteomic data show that FTY720 has an inhibitory effect on the signalling mediated by ERK1 and MYC, suggesting reactivation of their upstream negative regulator PP2A (23, 35, 39). Rescue experiments with the PP2A inhibitor okadaic acid, as well as by silencing of PPP2CA, the gene encoding for PP2A catalytic subunit α, prove that the effects of FTY720 on KMT2A-R-cells are dependent on PP2A activation. Significantly, phospho-proteomic analyses indicate that FTY720 affects pathways implicated in gene transcription. On the other hand, RNA-seq analysis shows that FTY720 reduces the expression of SET mRNA, a result confirmed by RT-qPCR in KMT2A-R as well as KMT2A-wt-leukemic cells. However, western blot experiments show a sustained and specific reduction of SET protein expression following FTY720 treatment only in KMT2A-R-leukemic cells. This latter result uncovers a novel molecular mechanism underlying FTY720 action, distinct from the current understanding of FTY720 as a sphingosine mimetic capable of disrupting the interaction between SET and PP2A without affecting the levels of SET (33, 34, 38). The effect of FTY720 on SET transcription could be explained by the modulation of MYC, which is a transcriptional activator of SET (48). Previous studies recognized MYC as a critical substrate of PP2A complex in cancer (46, 47, 49). In pancreatic and breast cancer cells, PP2A activation by KD of SET or by the SET antagonist OP449 decreases SER62 and THR58 phosphorylation of MYC and directs MYC towards ubiquitin-mediated proteasomal degradation (46, 47). Our phospho-proteomic data support the hypothesis of a feedback loop between SET, PP2A and MYC, whereby FTY720 reduces the stability of MYC by reducing the activity of ERK1 and GSK3β, the MYC-upstream regulators. Proteasomal degradation of MYC suppresses gene transcription and therefore protein expression of SET, which in turn, might lead to further activation of PP2A, with profound effects on survival and proliferation of leukemic cells (Fig. 7D). What our model does not fully explain is how SET KD or pharmacological inhibition regulate the expression of KMT2A signature genes. Our data indicate that SET interacts with both KMT2A wt and KMT2A fusion proteins and that it is recruited on the promoter of HOXA10, suggesting that SET might recruit KMT2A on the promoter of this gene or vice versa. This is supported by a yeast two hybrid screening that reported KMT2A as a SET interactor (50) and by experiments performed in HeLa cells showing that the interaction between SET and KMT2A has a synergistic effect on the activation of HOXA gene expression (42). More recent studies report that the oncofusion protein SET::NUP214, which has been found in a subset of T-ALL patients with abnormal expression of HOXA gene cluster, recruits both KMT2A and DOT1L to the promotor regions of HOXA9 and HOXA10 (43, 44). Notably, a phospho-proteomic study in HeLa cells, where SET was knocked down by RNAi, identified several targets directly involved in RNA Polymerase II (RNAPII)-mediated transcription and RNA processing, providing a possible link between PP2A and gene transcription (51). In addition, a recent study identified CDK9, an important regulator of RNAPII and critical player in KMT2A-fusion-mediated transcription, as a PP2A target, corroborating the crosstalk between phosphorylation and transcription (52). Therefore, the molecular mechanisms implicated in the SET-mediated regulation of KMT2A transcriptional signature warrant further investigation.

Consistent with its anti-proliferative effect, FTY720 treatment has been shown to be synergistic with cytotoxic chemotherapeutic agents in solid tumors (31). We demonstrated that FTY720 increases the response of KMT2A-R-cells to daunorubicin, a standard chemotherapeutic agent used in induction and consolidation treatment for KMT2A-R patients. Since the anti-cancer effect of FTY720 is an off-target effect and the dose used as an anticancer drug is much higher than the dose used as immune-suppressor, it has been postulated that FTY720 might be too toxic to be used in clinics. As FTY720 exerts its immunosuppressive properties after being phosphorylated in vivo in FTY720-P, a mimetic of sphingosine-1-phosphate (S1P) acting as an agonist of G protein coupled receptor family S1PR_1 − 5, that is critical for lymphocyte trafficking (31), some studies have been focused on the development of FTY720 analogues with anti-cancer but no immunosuppressive properties. As several studies indicate that the phosphorylation of FTY720 is not essential to exert anti-leukemic effects (23, 35, 39) analogues of FTY720 that cannot be phosphorylated might retain the anti-cancer effect without inducing immunosuppression. Such molecules (AAl-149 (S), (S)-FTY720-OMe, (S)-FTY720-regioisomer, and OSU- 2S, MP07-66, CM-1231) have been shown to activate PP2A and induce apoptosis in leukemia models (30, 35, 53).Furthermore, SH-RF-177, a FTY720 analogue that is efficiently phosphorylated but that does not activate S1P1R, shows anti-leukemic activity on ALL cells (54). Therefore, further studies are needed to evaluate the potential of these analogues in KMT2A-R-leukemia.

Our study demonstrates for the first time that the PP2A inhibitor SET is critical for the self-renewal of KMT2A-R-leukemic cells and can be pharmacologically targeted by using FTY720, an immunosuppressive sphingosine mimetic that reactivates PP2A by inhibiting SET. We provide the evidence that knockdown and pharmacological inhibition of SET in KMT2A-R-leukemic cells promote cell cycle arrest and apoptosis, rewire the transcriptional programme, down-regulate the expression of HOXA genes and direct MYC toward proteasomal degradation. Pharmacological targeting of SET via FTY720 can be used, in KMT2A-R-leukemia, to augment the effect of the conventional chemotherapeutic agent daunorubicin. These data shed light on SET as a new therapeutic target of KMT2A-R leukemia.

Acknowledgments

We would like to thank Leukemia UK, the University of Roehampton, the British Society of Hematology and the Institute of Biomedical Science for funding this study, the patients for donating their samples for research purposes and Prof Kamil Kranc for providing constructive feedback on the manuscript. The figure 7D was created in BioRender.com.

Ethical approval

Primary samples (supplementary table 5) were obtained from the Cancer Tissue Bank at the Barts Cancer Institute (London) under ethical approval (REC reference: 17/WM/0428).

Competing interests

The authors have no competing financial interests to declare.

Author’s contributions

MTE, BW and YC conceived the experimental design; ADM, YA, TF and MTE performed the experiments and analysed data; MA performed the in vivo experiment; MR, VR and PC performed phospho-proteomic experiments and data analysis; LC performed RNAseq data analysis and visualization with input from JP; AC and GE supervised and interpreted the ChiP and IP data; OW, BW and JG gathered patients’ material and provided clinical data; CL contributed intellectually to the interpretation of the data; BW contributed critical intellectual input in design and interpretation of data; MTE conceptualized, supervised the study, provided the funding and wrote and edited the manuscript; all the authors critically reviewed and edited the manuscript.

Funding

This project has been supported by Leukemia UK (John Goldman fellowship), the British Society of Hematology, the Institute of Biomedical Science and the University of Roehampton.

Availability of data and materials

The FTY720-RNASeq datasets generated during the current study are available in the GEO repository (GSE218708). The three plot shown in Figure 1 has been drawn based on data obtained from Bloodspot Gene expression profiles (GSE42519). The heatmap shown in Figure 1 has been generated by analysing data available from Leucegene (GSE62190, GSE66917, GSE67039). The expression of SET mRNA in murine KMT2A-LSC has been obtained by meta-analyses of micro-array data from GSE13690. Kaplan–Meier analysis of AML patients with high and low expression levels of SET is based on micro-array data from the PrognoScan database (GSE12417). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD038288 and 10.6019/PXD038288.

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No competing interests reported.

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SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML.

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