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

Abstract

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. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.

Fig. 1. SET is an oncogene over-expressed in acute myeloid leukemia.

A Hierarchical tree plot that shows the expression of SET mRNA in human HSCs, progenitors and differentiated blood cells. The level of expression is visualized by size and color of the nodes (data obtained from Bloodspot Gene expression profiles (GSE42519); n = 34; hematopoietic stem cells (HSC) n = 4, multipotential progenitors (MPP) n = 2, common myeloid progenitors (CMP) n = 3, granulocyte myeloid progenitors (GMP) n = 5, megakaryocyte-erythroid progenitor cells (MEP) n = 2, early promyelocyte (ea_PM) n = 3, late promyelocyte (late_PM) n = 3, myelocyte (MY) n = 2, metamyelocyte (MM) n = 3, band cell (BC) n = 4, polymorphonuclear cells n = 3. B Heatmap of SET mRNA along with other oncogenes, housekeeping genes and genes annotated as either down-regulated or up-regulated in KMT2A-R leukemia in a large AML- RNAseq dataset (n = 384) comprising 31 samples from patients carrying rearrangements of KMT2A. Meta-analyses of RNAseq data from Leucegene (GSE62190, GSE66917, GSE67039). 1: complex karyotype, 2: EVI-R; 3: intermediate; 4: inversion 16; 5: KMT2A-R; 6: monosomy 5; 7 normal karyotype; 8: t(15:17); 9: t(8;21); 10 trisomy-tetrasomy 8. C Kaplan–Meier analysis showing the survival in AML patients with low (n = 106) or high expression levels of SET (n = 57). Meta-analyses of micro-array data from the PrognoScan database (GSE12417). Log-Rank test; *p < 0.05. D, E Immunoblot of SET in KMT2A-R- AML cell lines (MV411, THP1, ML2, MLOM13, NOMO1), KMT2A-R-ALL cell lines (SEM, HB11;19, KOPN8, RS4;11), KMT2A-R-primary samples (PS) and six independent healthy bone marrow (BM) controls. The data also present the expression of SET in three KMT2A-wt cell lines K562 (BCR::ABL (t19;22) erythroleukemia cell line, Kasumi1 (AML1:ETO, t8;21 AML cell line) and U937 (CALM::AF10, AML cell line). Densitometry analysis was conducted by Li-COR Image Studio software. GAPDH was used as a loading control. Values are expressed as ratio between SET and GAPDH, relative to the expression of SET in mononuclear cells isolated from bone marrow of healthy volunteers (BM). F Immunoblot of SET in cytoplasmic and nuclear factions of KMT2A-wt (K562) and KMT2A-R-AML cell lines (THP1 and MV411). Laminin B1 and GAPDH were used as nuclear and cytoplasmic markers, respectively. G Detection of ^SerSET in KMT2A-R cell lines. Immunoblotting against phosphorylated Ser was performed after SET immunoprecipitation.

Fig. 2. SET Knock down impairs the colony forming unity (CFU) ability of KMT2A-R-leukemic cells.

A Expression of mouse SET mRNA (probe1421819_a_at) in 12 high LSC frequency KMT2A-R AMLs (KMT2A::MLLT3 and KMT2A:MLLT1) and 22 low LSC frequency KMT2A-R-AMLs (KMT2A::AFF1p, KMT2A::AF10 and KMT2A::GAS7). Meta-analyses of micro-array data from GSE13690. Unpaired t test; **p < 0.01. B Spearman correlation matrix for SET mRNA and human LSC genetic signature obtained from ref. [36]. C Spearman correlation matrix for SET mRNA and the self-renewal associated genetic signature identified in human KMT2A-R AML cells by [37]. The genes in red have a significant positive correlation with SET (Rs >0.2 and p < 0.05), while the genes in blue have a significant negative correlation with SET (Rs < −0.2 and p < 0.05). The genes marked with a cross do not correlate with SET expression. D Immunoblot for SET in K562, Kasumi1, and REH stably expressing shSCRAMBLE, RFP or shSET. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. Values are expressed as ratio between SET and GAPDH relative to untransduced cells. E qRT-PCR showing the expression of SET in eGFP-THP1, eGFP-MV411, and eGFP-SEM expressing either shScramble or shSET. Gene expression was normalized to GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to shScramble controls. Data represent mean ± SD of three independent experiments. Unpaired t test **** p < 0.0001. F–K Effect of shSET on colony-forming unit ability. Data show mean ± SD of three independent experiments. 2-way Anova Tukey’s multiple comparative tests **p < 0.01; ****p < 0.0001. K–N Proliferation curve of K562 and Kasumi1 and REH, stably expressing eGFP and shScramble or shSET. GFP expression was used as quantitative reporter of cell proliferation. For each cell line, the same number of cells was plated at t0 and the GFP signal was measured by a fluorescent microplate reader at each time point. Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Tukey’s multiple comparison test *p < 0.05; **** p < 0.0001.

Fig. 3. FTY720 induces cell cycle arrest and drives apoptosis in KMT2A-R leukemic cell lines.

A–G Proliferation curve of K562, Kasumi, THP1, MV411, SEM, Hb1119, and REH stably expressing eGFP, upon treatment with FTY720 for 6 days. GFP expression was used as quantitative reporter of cell proliferation. For each cell line, the same number of cells was plated at t0 and the GFP signal was measured with a fluorescent microplate reader every 2 days. Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Sydak’s multiple comparison test *p < 0.05; ** p < 0.01; **** p < 0.0001. H Cell cycle analysis performed on K562, Kasumi1, MV4,11 and THP1, upon 5 µM FTY720 treatment for 48 h. Data show mean ± SD of triplicate wells and are representative of two independent experiments. Two-tailed unpaired t test *p < 0.05. I Fraction of cells undergoing cell death (GFP-) upon 5 µM FTY720 treatment for 48 h. Data show mean ± SD of triplicate wells and are representative of three independent experiments. Two-tailed unpaired t test *p < 0.05; ****p < 0.0001.

Fig. 4. The Effect of FTY720 on KMT2A-R cells is dependent on PP2A activation.

A Protein complex -immunoprecipitation showing SET—PP2A interaction in KMT2A-R- AML cell lines upon FTY720 treatment for 24 h. Immunoblotting against SET (39 kDa) was performed after Immunoprecipitation of PP2A (FT: Flow through control; IgG HC: IgG high chains; IgG LC: IgG low chains). B Immunoblot for phospho-AKT1/2 (Ser473) (60 kDa), AKT (pan)(60 kDa), phosphoGSK3β (Ser9) (42 kDa), GSK3β (42 kDa), phosphoERK1/2 (Thr402/Tyr404) (42–44 kDa), ERK1/2 (42–44 kDa) and GAPDH (37 kDa) in K562, Kasumi1, THP1 and MV411 upon 5 µM FTY720 treatment for 24 and 48 h. Densitometry analysis was conducted by Li-cor Image Studio software. GAPDH was used as a loading control. Values are expressed relative to the vehicle at 24 h. C Immunoblot for phospho-AKT1/2 (Ser473) (60 kDa), AKT (pan) (60 kDa), phosphoGSK3β (Ser9) (42 kDa), GSK3β (42 kDa), phosphoERK1/2 (Thr402/Tyr404) (42–44 kDa), ERK1/2 (42–44 kDa) and GAPDH (37 kDa) in K562, Kasumi1,THP1 and MV411 upon 5 µM FTY720 treatment for 24 h. Cells were pre-treated with 2.5 nM Okadaic Acid for 4 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. Values are expressed relative to the vehicle. D Analysis of cell death upon FTY720 treatment. Cells were pre-treated with 2.5 nM Okadaic Acid for 4 h and then treated with 5 µM FTY720 for 72 h. GFP signal was used as quantitative reporter of alive, non-apoptotic cells and measured by fluorescent- activated cell sorting (FACS). Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Dunet’s multiple comparison test **p < 0.01; ***p < 0.001; ****p < 0.0001. E Analysis of cell death of eGFP-MV411 transfected with either shSCRAMBLE or shPP2A upon FTY720 treatment for 72 h. GFP signal was used as quantitative reporter of alive cells and measured by flow cytometry. Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Tukey’s multiple comparison test **p < 0.01; ***p < 0.001. F qRT-PCR showing the expression of PPP2CA and PPP2CB in eGFP-MV411 shPP2A. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to shSCRAMBLE controls. Data represent mean ± SD of three replicas. Two tailed paired t test *** p < 0.001.

Fig. 5. Phospho-proteomic profile analysis of FTY720 treated KMT2A-R leukemic cells.

Characterization of the impact of FTY270 on kinase signaling networks in eGFP-THP1 and eGFP-MV411 cells. A, B Volcano plot showing the most highly enriched phosphosites from the Phospho-proteomic analysis of cells treated with 5 μM FTY720 for 48 h relative to vehicle. Color coded quadrants represent phopshopeptides that possess a fold change >2 and p value < 0.01. C Over-representation analyses of Gene Ontology Biological Processes (GO_BP) enriched in cells treated with 5 μM FTY720 for 48 h relative to vehicle. Representative labeled terms were determined using affinity propagation, nodes represent individual phophoproteins allocated to the GO_BP and color coded to represent Log2(FTY720/Vehicle). Attribute circle layout was used based on Log2(FTY720/Vehicle) and total numbers of enriched phosphoproteins are shown within the center circle for each term. D, E Kinase-Substrate Enrichment Analysis (KSEA) of cells treated with 5 μM FTY720 for 48 h relative to vehicle. KSEA was performed using KSEA App. For a given upstream kinase, the m threshold was set to 5, the NetworKIN PhosphoSitePlus threshold was set to 5 and the p value cut off was <0.1. F Hierarchical clustering of phosphosites allocated to specific kinases by KSEA analysis. To create groups of phosphorylation sites that share similar patterns of abundance changes targets of specific kinases were grouped together by KSEA analysis. Log2 fold-change of >1 and p value < 0.1 filters were applied to exclude phosphosites that were unchanged in abundance and phosphosite abundances were z-transformed by row. Euclidean distance average linkage clustering was then applied to these datapoints and clustered heatmaps shown. G–L Interaction network analysis. Phosphosites allocated to kinases with enriched or inhibited activity due to FTY270 were mapped via network interaction analysis. Nodes represent proteins with their phosphosites mapped on. Color code represents Log2(FTY720/Vehicle) and edges represent known protein-protein interactions. G, K show data for eGFP-THP1 cells and H, I, J and L show data for eGFP-MV411 cells.

Fig. 6. Gene expression profile analysis of FTY720 treated KMT2A-R leukemic cells.

A Volcano plot of gene expression differences for THP1 cells treated with FTY720 for 24 h. The dots to the left of 0 represent gene probes with adjusted p value for multiple testings (padj) < 0.05 and fold change >1.3 (log2fold change < 0). The dots to the right of 0 represent gene probes with padj < 0.05 and fold change >1.3 (log2fold change > 0). B Immunoblot for phospho-pLK1 (Thr210) (58 kDa), PLK1 (58 kDa) and GAPDH (37 kDa) in K562, Kasumi1, THP1 and MV411 upon 5 µM FTY720 treatment for 24 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. C qRT-PCR showing the expression of SET, MYC, HOXA9, and HOXA10 in leukemic cells upon 5 µM FTY720 treatment for 24 h. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to vehicle controls. Data represent mean ± SD of three independent experiments. Two tailed paired t test *p < 0.05; **p < 0.01; ***p < 0.001. D Immunoblot for SET and MYC in leukemic cells upon FTY720 5 µM treatment for 48 h. Densitometry analysis was conducted by LI-COR Image Studio software. GAPDH was used as a loading control. Values are expressed relative to vehicle control. E, F qRT-PCR showing the expression of SET, MYC, HOXA9, and HOXA10 in leukemic cells transduced with lentiviral vectors expressing shSET and selected with puromycin for 72 h. Gene expression was normalized by GAPDH control and analyzed by Pfaffl equation. Values are expressed relative to shSCRAMBLE controls. Data represent mean ± SD of two independent experiments. Two tailed paired t-test ***p < 0.001; ****p < 0.0001. G Chromatin immunoprecipitation (ChIP) experiments were performed by using anti-MLL (KMT2A/MLL) and anti-SET (SET) antibodies (Ab); ChIP with anti-IgG (IgG) represents the negative control. ChIP data are expressed as percentage of specific target gene promoter elements (pr) (i.e., HOXA9-pr, HOXA10-prE1, HOXA10-prE2, HOXA10-prE3, ACTB-pr) in precipitated chromatin compared with the INPUT (% INPUT), where ACTB-pr (the promoter of ACTIN) was the negative control. Results represent the mean ± SD average of three independent experiments. H–K Co-immunoprecipitation experiments were performed using whole cell extracts (WCE), anti-MLL or anti-SET antibodies (Ab). MLL, MLL-AF9 and MLL-AF4 proteins were immunoprecipitated as baits with the anti-MLL Ab (IP MLL) in the respective cell lines and the presence of SET was revealed by western blot (WB) (left panels); SET was immunoprecipitated as bait (IP SET) with the anti-SET Ab and the presence of wild type MLL or MLL-AF9 or MLL-AF4 was analyzed by WB with the anti-MLL Ab (right panels). Immunoprecipitation with anti-IgG (IP IgG) was used as negative control.

Fig. 7. FTY720 increases the response to Daunorubicin in KMT2A-R leukemic cells and PDX.

A Analysis of cell death. Cells were treated with 5 µM FTY720, 10 nM Daunorubicin or combination for 72 h. GFP signal was used as quantitative reporter of alive, non-dead cells and measured by FACS. Data show mean ± SD of triplicate wells and are representative of three independent experiments. 2-Way Anova Tukey’s multiple comparison test *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. B Effect of 5 µM FTY720, 10 nM Daunorubicin or combination on colony-Forming Unit ability of KMT2A-PDX samples. Data show the percentage of colonies in comparison to vehicle treated cells and the mean ± SD of duplicate wells and are representative of two independent samples ** p < 0.01. C Colony morphology of KMT2A-R-PDX. Cells were treated with the drugs in methocult for 14 days. Digital microscope images were captured using Evos FL Digital Inverted Fluorescence Microscope (magnification 40×).

Fig. 8. Molecular mechanisms underlying FTY720 effects in KMT2A-R-leukemic cells.

Schematic cartoon representing the molecular mechanism underlining the effect of FTY720 in KMT2A-R- leukemic cells. The survival and proliferation of KMT2A-R leukemic cells is maintained by the activation of signaling pathways regulated by multiple kinases including ERK1, GSK3β and AURB (left). FTY720 disrupts the binding between SET and PP2A, re-activating PP2A, that leads to inactivation of multiple kinases (right). The restored activity of PP2A affects MYC transcription via AURB, and MYC protein stability, via ERK1, GSK3β and PLK1. The latter is also a downstream effector of AURB. The overall effect is suppression of MYC transcription and MYC stability. This, in turn, affects the transcription of genes regulated by MYC, including PLK1 and SET. The inhibition of SET feeds into the activation of PP2A, with profound effects on survival and proliferation of KMT2A-R-leukemic cells.