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.

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. SIMILAR

CONTENT BEING VIEWED BY OTHERS DYRK1A INHIBITION RESULTS IN MYC AND ERK ACTIVATION RENDERING _KMT2A-_R ACUTE LYMPHOBLASTIC LEUKEMIA CELLS SENSITIVE TO BCL2 INHIBITION Article Open access 27

March 2025 REQUIREMENT FOR LIM KINASES IN ACUTE MYELOID LEUKEMIA Article 26 June 2020 MUTANT SETBP1 ENHANCES NRAS-DRIVEN MAPK PATHWAY ACTIVATION TO PROMOTE AGGRESSIVE LEUKEMIA Article 17 May

2021 INTRODUCTION Acute leukemias characterized 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, which 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

[3,4,5,6,7]. 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 harboring the _KMT2A_ translocations, leading to impairment of cellular differentiation [8,9,10] 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 development of new therapeutic agents, such as inhibitors of KMT2A-fusion complex [3,4,5, 13,14,15,16] and inhibitors of kinases essential for the KMT2A-signaling pathways

[17,18,19], 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 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, inhibitor 2 of Ser-Thr protein phosphatase 2A and putative histocompatibility leukocyte antigen class

II-associated protein II. Originally identified as fusion gene in acute leukemia [20], it is overexpressed in several forms of solid tumors and hematological malignancies

[21,22,23,24,25,26]. In _BCR::ABL_+ chronic myeloid leukemia (CML), SET-mediated PP2A inactivation is essential for the self-renewal of CML leukemic stem cells [27]. Silencing SET

re-activates PP2A and switches the oncogenic driver kinase BCR::ABL off, highlighting its potency as a negative regulator of PP2A and a positive regulator of oncogenic cascades [21, 26, 28].

SET has also been recognized as a prognostic marker for poor overall survival in AML [22, 24], although the exact molecular mechanisms linking a SET oncoprotein pathway with aggressive AML

outcomes remain still obscure. Interestingly, SET has been reported to interact with the N-terminal region of KMT2A, that is retained in KMT2A-fusion proteins [29]. However, the role of SET

in the pathogenesis of _KMT2A_-R leukemia is unknown. Based on this and the fundamental role played by PP2A-modulated kinases on the survival and resistance of _KMT2A_-R-leukemic cells [17,

19, 30], we herein investigated the role of the PP2A endogenous inhibitor SET in _KMT2A_-R leukemia. Our overall results provide novel insights on the role of SET in _KMT2A_-R leukemia, via

modulation of _HOXA_ gene expression and MYC stability, and a proof of concept that inhibition of SET is a promising novel strategy to treat this aggressive form of acute leukemia. RESULTS

SET IS OVER-EXPRESSED IN _KMT2A_-R CELL LINES AND PRIMARY SAMPLES We used public data repositories to characterize the role of SET across various hematopoietic contexts, to analyze its mRNA

expression in human HSCs and progenitors (_n_ = 34) [31], and in multiple independent human AML primary samples (_n_ = 384) covering the main cytogenetic subsets [10]. SET was expressed at

high levels in both HSC and myeloid progenitors (MP) compared with mature monocytes and myelocytes (Fig. 1A and Supplementary Fig. 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) [22, 24]. A PrognoScan database-based Kaplan–Meier analysis of the overall survival of 163 AML patients by high (_n_ = 57) and low (_n_ = 

106) SET levels [32], revealed that high SET expression positively correlated with poor overall survival in human AML (Fig. 1C and Supplementary Table 1), consistent with previous reports

[24]. 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 leukemic cell 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

characterized 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 localization [26, 33, 34], we analyzed its subcellular localization 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 we 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 [26, 33, 34], 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 analyzed by western blot with anti-phospho-Ser 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 and Supplementary 1D–F). Overall, these data indicate that SET is over-expressed, phosphorylated on Ser residues

and abundantly localized in the cytosol in _KMT2A_-R and -wt leukemic cells. _SET_ CORRELATES WITH THE EXPRESSION OF KMT2A TARGETS _MEIS_ AND _HOXA_ GENES To understand the role of SET in

leukemic stem cells (LSC), we analyzed the expression of _SET mRNA_ in 12 high-LSC frequency mouse _KMT2A-_R AMLs and 22 low-LSC frequency _KMT2A-_R-AMLs, using a public dataset [35], as

described in material and methods. 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. We then used cBioPortal database to explore the potential correlation between the expression of _SET_ mRNA

and genes identified as LSC markers in human AML by Gentles et al. [36]. Out of the 52 genes identified by Gentles et al., 39 were found in the cBioPortal database for all the cohorts

included in the study. Our in-silico analysis showed a significant positive correlation between _SET_ expression and some LSC genes, such as _FAIM_ and _STAR_ (Fig. 2B). In addition, we

checked whether there was a correlation between SET expression and the self-renewal associated genetic signature identified in _KMT2A_-R-AML by Krivtsov et al. [37]. In this case, out of the

34 described by Krivtsov et al., we found 29 genes in the cBioPortal database. _SET_ expression significantly correlated with some primary targets of _KMT2A_-R-AML such as _MEIS_, _HOXA5_,

_HOXA9_ and _HOXA10_ (Fig. 2C). These data indicate that SET expression is significantly higher in high-LSC frequency mouse KMT2A-R AML and that _SET_ expression significantly correlated

with the expression of some LSC marker genes and with KMT2A targets _MEIS_ and _HOXA_ genes. SET IS ESSENTIAL FOR _KMT2A_-R LEUKEMIC CELLS’ CLONOGENIC ABILITY 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. Analysis of protein by western blot (Fig. 2D) and of gene expression by

RT-qPCR (Fig. 2E) confirmed the knock down (KD) in all the cell lines investigated. We then assessed the effect of SET KD on the clonogenic ability of the cells. _SET_ KD completely

abolished the clonogenic ability of _KMT2A_-R leukemic cell lines (THP1, MV411, and SEM, Fig. 2H, I and K and Supplementary Fig. 2), whereas it had little or no effect on the colony

formation of three independent _KMT2A_-wt leukemic cell lines (K562, Kasumi1, and REH Fig. 2F, G, and J). Then, we determined the impact of SET KD on cell proliferation of KMT2A-wt leukemic

cells by monitoring GFP fluorescence and by MTS assay, as described in [38] and in Supplementary material and methods. The correlation between the GFP fluorescence and the OD values

determined for the MTS assay are reported in supplementary Fig. 3. The correlation between two assays were above 0.8, indicating that the GFP monitoring of the newly generated GFP-expressing

lines can be used as a readout of cell proliferation in these experimental conditions. SET KD attenuated the proliferation of K562 (Fig. 2L, Supplementary Fig. 3 and Supplementary Table 2),

in line with a previous report [21]. SET KD had a stronger impact on _KMT2A_-wt AML cell line Kasumi1 (Fig. 2M, Supplementary Fig. 3 and Supplementary Table 2) and on _KMT2A_-wt ALL cell

line REH (Fig. 2N, Supplementary Fig. 3 and Supplementary Table 2). Interestingly, whereas the GFP monitoring indicates that eGFP-Kasumi1-shSET proliferate significantly more slowly than

eGFP-Kasumi1-shScramble, the MTS assay indicates that eGFP-Kasumi-shSET are still metabolically active. The discrepancy between these results might be due to the emergence of growth arrested

cells that are still highly metabolically active [39]. Overall, these data indicate that SET-KD specifically abolishes the clonogenic ability in _KMT2A_-R-leukemic cells and it attenuates

the proliferation of _KMT2A-_wt leukemic cells; whereas in K562 the effect on proliferation is mild, in Kasumi1 and REH SET KD induces a significant arrest in proliferation. 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) [40], 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 [28, 41,42,43]. 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 μM (Supplementary Fig. 4A), which were

reported as non-toxic to healthy bone marrow mononuclear cells [27, 28]. We then assessed the effect of FTY720 on proliferation, cell cycle and cell death of _KMT2A_-wt (K562, Kasumi1 and

REH) and _KMT2A_-R-leukemic cells (THP1, MV411 Hb119, and SEM) (Fig. 3A–G and Supplementary Fig. 3 and Table 2). We observed that 5 µM FTY720 had a variable but significant effect on the

proliferation of all the analyzed cell lines, ranging from modest for the _KMT2A_-wt-cell lines K562 and Kasumi1 to severe for eGFP-REH (Fig. 3G and Supplementary Fig. 3 and Table 2), as

reported [28, 44, 45]. In addition, FTY720 severely halted the proliferation of all the _KMT2A_-R-cell lines. Cell cycle analyses revealed that treatment with FTY720 for 48 h induced a

significant increase of cells in G1 and a reduction in S and G2-M phase, in _KMT2A_-R-cells (Fig. 3H and Supplementary Fig. 4B), indicating cell cycle failure. Moreover, we investigated the

fraction of leukemic cells undergoing cell death upon FTY720 treatment by FACS, by gating the GFP negative (GFP-) cells, as described previously [38] and in the Supplementary material and

methods. FTY720 induced a statistically significant increase in cell death in two _KMT2A_-wt-cell lines K562 and REH whereas this was not significant for the _KMT2A-_wt cell line

eGFP-Kasumi1. In contrast, FTY720 induced a consistent and statistically significant increase in cell death in _KMT2A_-R-cells (Fig. 3I and Supplementary Fig. 4C). These results indicate

that the SET inhibitor FTY720 induces heterogenous effects in leukemic cells; in K562 FTY720 has a modest effect on proliferation and cell death; in Kasumi1 FTY720 has a modest effect on

proliferation and it does not induce cell death; in REH, FTY720 significantly impairs the proliferation and it induces cell death; in _KMT2A_-R-leukemic cells, FTY720 significantly impairs

the proliferation, causes cell cycle arrest in G1 and increases the rate of cell death. 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 [40, 46]. 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) [28, 41,42,43]. 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 [47] by western blot. In K562 cells, we observed reduction in phospho-ERK1/2 (Thr202/Tyr204), which was

instead unchanged in Kasumi1; in these two cell lines, phospho- GSK3β (Ser9) expression decreased, upon FTY720 treatment. Notably, in _KMT2A_-R-leukemic cells, we observed a dramatic and

stable decrement in the expression of the PP2A target phospho- ERK1/2 (Fig. 4B). In addition, we observed a sustained reduction in the abundance of phospho-AKT1(Ser473), a phosphosite

identified as marker of active AKT1 (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). We first tested OA in K562 cells and confirmed that the range of concentrations used did not impact the cell proliferation (Supplementary

Fig. 5A). Treatment with 5 nM OA for 2 hours induced a significant increase in pAKT Ser473, a phosphosite that is not reported as regulated by PP2A (PhosphositePlus), indicating that this

concentration might alter the activity of other phosphatases. At lower concentrations, equal to 2.5 nM, OA caused a significant increase in three PP2A targets, phospho-GSK3β Ser9,

phospho-ERK1 (Thr202/Tyr204) and phospho-AKT Thr308 with maximal expression after 2–4 hours of treatment (Supplementary Fig. 5B, C), indicating that, at this concentration, OA preferentially

inactivates PP2A. We therefore used this concentration of OA for our combination experiments with FTY720. Western blot analysis of PP2A targets in the cells pre-treated with OA for 4 h and

then treated with FTY720 for 20 hours, revealed that pre-treatment with OA restored phospho-AKT and phospho-ERK1/2 levels in KMT2A-R- leukemic cells (Fig. 4C). Whereas pre-treatment with OA

did not have any effect on the percentage of dead cells in eGFP-K562 and eGFP-Kasumi1, it significantly decreased the percentage of cell death in _KMT2A_-R-cells (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 α 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) [48]. 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 3. 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 signaling in eGFP-THP1 (Fig. 5C and Supplementary Fig. 7E). In

contrast, the data indicate a strong decrease in cell division-related kinase signaling, but a subtler increase in apoptosis eGFP-MV411 (Fig. 5C and Supplementary Fig. 7F). In addition,

FTY720-modulated phosphosites implicated in transcription regulation, chromatin organization, DNA damage repair, mRNA processing, microtubule and actin cytoskeletal organization (Fig. 5C).

We then used Kinase-Substrate Enrichment Analysis (KSEA) for the characterization of kinase activity from the phospho-proteomic dataset. The mitosis-regulating kinase Aurora kinase B (AURB),

a critical regulator of MYC protein stability and transcriptional activity [49], was the kinase most significantly impacted by FTY720 in both cell lines (Fig. 5D, E). Indeed, FTY720

decreased the phosphorylation of several AURB targets, including PLK1 (Thr210) (Fig. 5F, G, H). As expected, FTY720 inhibited MAPK3 (ERK1) and phosphorylation of ERK1 on Thr202 and Tyr204

was significantly reduced in both cell lines (Supplementary Fig. 7G); furthermore, FTY720 significantly inhibited Abl1 and CDK2 in eGFP-MV411 (Fig. 5I and Supplementary Fig. 7I). Although

the overall effect of FTY720 resulted in a significant increase in GSK3β 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 GSK3β-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 (HR), such as BRCA1 (Fig. 5K, L and Supplementary Fig. 7K). Taken together, these

data indicate that FTY720 reduced the activity of phospho-signaling associated to cell division and MYC stability and increased apoptosis-related and DNA damage kinase signaling 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 of _KMT2A_-R cell line THP1 treated with FTY720 for 24 h, to capture early changes in genetic expression, preceding cell cycle arrest and apoptosis driven by FTY720 over

48–72 h. Volcano plot filtering was used to identify differentially expressed genes between vehicle control group and FTY720 -treated group (Fig. 6A). 980 genes were significantly

upregulated and 898 genes were significantly downregulated by FTY720 (fold change >1.3, _p_adj < 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 signaling pathway, extrinsic apoptotic signaling 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 [44, 45, 50, 51]. To investigate whether there was any overlap between the phospho-proteomic data and the RNA-seq data, we intersected

the two datasets. The results indicate that 59 out of 980 genes upregulated by FTY720, code for proteins that undergo changes in phosphorylation and 98 out of 898 genes downregulated by

FTY720, code for protein that undergo changes in phosphorylation (Supplementary Fig. 8C, D and Supplementary Tables 4 and 5). Among the genes down-regulated by FTY720 and that encode for

proteins that are also hypo-phosporylated by FTY720, we validated PLK1, a downstream target of Aurora kinase B and mediator of MYC stability [49, 52, 53] (Fig. 6B). The western blot analysis

indicates that PLK1 is downregulated in Kasumi1, THP1, and MV411 upon FTY720 treatment (Fig. 6B). Phospho-PLK1 (T210), a phosphosite regulated by AURB, which was identified in our

phospho-proteomics, was undetectable in these cell lines. In K562, PLK1 expression and phosphorylation on T210 did not change upon treatment with FTY720 or when SET was KD (Supplementary

Fig. 9A). More importantly the RNA-seq data indicated that, upon FTY720 treatment, several genes overexpressed in cancer, including _MYC_ and _SET_ were downregulated (Supplementary Table

6), 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, 8,9,10]

(Supplementary Table 7). We performed RT-qPCR and western blot to validate the decreased expression of _MYC_ and _SET_ upon FTY720 treatment (Fig. 6C, D and Supplementary Fig. 9B-C). Upon

FTY720 treatment, _MYC_ mRNA and protein expression was down-regulated in Kasumi1 and in the _KMT2A_-R cell lines, but not in K562 (Fig. 6C, D and Supplementary Fig. 9B-C). _SET_ mRNA was

down-regulated in all leukemic cells (Fig. 6C) but, SET protein was significantly reduced only in _KMT2A_-R-cells (Fig. 6D and Supplementary Fig. 9B). RT-qPCR also confirmed a specific

decrease in the expression of the _KMT2A_ target genes _HOXA9_ and _HOXA10_, in _KMT2A_-R-cells, upon FTY720 treatment (Fig. 6C); these genes were also specifically down-regulated in

_KMT2A_-R-cells, but not in Kasumi1, when SET was knocked down (Fig. 6E, F), suggesting that SET might regulate the expression of _HOXA_ 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 [54,55,56]. 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. 6G and Supplementary Figs. 9E and 10). Immunoprecipitation experiments revealed that SET interacted with both KMT2A and KMT2A-fusion protein

(Fig. 6H–K and Supplementary Fig. 9F–G). 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 5 µM FTY720 with 10 nM 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 dead cells was

significantly high for all the AML cell lines (Fig. 7A). The cell death increase was modest only for eGFP-Kasumi1 cell line, but the effect was very strong in _KMT2A_-R-cell lines, namely

eGFP-THP1 and eGFP-MV411 (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.

DISCUSSION SET is an endogenous PP2A inhibitor overexpressed in several types of solid tumors and hematological malignancies [21,22,23,24,25,26]. Mechanistically, SET over-expression and the

resulting PP2A inhibition is critical for the maintenance of the leukemogenic program by BCR::ABL in CML [21, 27]. 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 and it positively correlates with the expression of

_MEIS_ and _HOXA_ genes. 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 [26, 33, 34]. We demonstrate that SET silencing has a detrimental effect on the

proliferation of _KMT2A_-wt- leukemic cells, as also observed in CML and solid tumors [21, 57,58,59]. More importantly, we show for the first time that SET silencing completely abolishes the

ability of _KMT2A_-R-leukemic cells to form colonies in semi-solid medium, indicating that SET plays an important role in the clonogenic ability of _KMT2A_-R-cells. 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 [28, 40,41,42,43], that had been shown to induce cell death in several solid tumors and leukemia models by various mechanisms [44, 45, 50, 51]. In agreement with

previous reports on CML [28], we show that FTY720 has a modest negative impact on the proliferation of _BCR:ABL_ + _KMT2A_-wt- leukemic cell line K562 and promotes cell death. In _KMT2A_-wt-

leukemic cell line REH, FTY720 has a greater cytostatic and cytotoxic impact, in agreement with data previously published [44]. In contrast to [45], in Kasumi1, FTY720 has a modest effect

on proliferation and it does not induce cell death. Whereas both FTY720 and SET KD have a similar impact on _KMT2A_-wt cell lines K562 and REH, FTY720 does not fully mimic the effect of SET

KD in Kasumi1, as these cells are more sensitive to SET KD than to FTY720 treatment. This might be explained by the residual level of SET. Indeed, whereas the KD induces a 90% reduction in

SET protein levels, FTY720 does not reduce the expression of SET protein in Kasumi1. In _KMT2A_-R cells, FTY720 causes cell cycle arrest and promotes cell death. Accordingly, our

phospho-proteomic and RNA-seq analyses in KMT2A-R-models 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 signaling mediated by AURB, PLK1, ERK1 and MYC, suggesting reactivation of their upstream

negative regulator PP2A [47]. 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 some of

the effects of FTY720 on _KMT2A_-R-cells are dependent on PP2A activation. We are aware that some of the proteomic changes observed might not be due to PP2A activation or they might be

compensatory effects. Further studies in PPP2CA and PPP2R1A KD cells are needed to validate PP2A as mediator of each of these aforementioned signaling pathways. Significantly,

phospho-proteomic analyses indicate that FTY720 affects pathways implicated in gene transcription. 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. Interestingly, 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 [41, 42, 46]. The effect of FTY720 on SET transcription could be explained

by the modulation of MYC, which is a transcriptional activator of SET [60]. Previous studies recognized MYC as a critical substrate of PP2A complex in cancer [58, 59, 61]. 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 [58, 59]. Our data support the hypothesis of a feedforward loop between PP2A, AURB, PLK1, MYC, and SET whereby FTY720 reduces MYC transcription, by reducing the activity of AURB,

an upstream regulator of MYC [49], and it compromises MYC stability, by reducing the activity of AURB, PLK1, ERK1 and GSK3β, which modulate MYC by phosphorylation on S62 and T58 [49, 52,

53, 61]. Proteasomal degradation of MYC suppresses MYC-dependent gene transcription and therefore expression of downstream targets PLK1 [53] and SET [60] which, in turn, might feed into the

activation of PP2A, with profound effects on survival and proliferation of leukemic cells (Fig. 8). What our model does not fully explain is how SET KD or pharmacological inhibition by

FTY720 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 to the promoter of

_HOXA10_, suggesting that SET might recruit KMT2A to the promoter of this gene or vice versa. This is supported by a yeast two hybrid screening that reported KMT2A as a SET interactor [29]

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 [54]. 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_ [55, 56]. 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 SET, PP2A, and gene transcription [62]. In addition, a study identified CDK9, an

important regulator of RNAPII and critical player in KMT2A-fusion-mediated transcription [18], as a PP2A target, corroborating the crosstalk between phosphorylation and transcription [63].

Moreover, in a recent study, AAkula et al. combined the data from two phospho-proteome studies, including the one performed on HeLa where SET was downregulated by siRNA [62], to identify

phospho-proteins co-regulated by RAS and PP2A [64]. The study indicates that PP2A- and RAS-mediated phosphorylation converge on epigenetic complexes, including the DOT1L complex. The DOT1L

phosphosites Ser900 and Ser1104, identified as dephosphorylated by siSET in the study by Aaakula et al., were also found dephosphorylated by FTY720 in THP1 (Supplementary Table 2),

supporting the concept that SET mediates its anti-tumor effects though modulation of relevant epigenetic factors. Another SET-dependent DOT1L phosphosite identified by Aakula et al.,

Ser1001, was found to correlate positively with _HOXA_ gene expression in _KMT2A_-R-AML patients [65]. Therefore, the molecular mechanisms implicated in the SET-mediated regulation of KMT2A

transcriptional signature warrant further investigation. Integrating these published phospho-proteomes and validating the functional role of some of these phosphorylation sites might might

reveal further mechanistic insights into the role of SET and PP2A inhibition in leukemic transcription and chromatin accessibility and should be explored in future studies. Consistent with

its anti-proliferative effect, FTY720 treatment was shown to be synergistic with cytotoxic chemotherapeutic agent, doxorubicin, in solid tumors [66, 67]. 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. This effect might be

partially dependent on the impact of FTY720 on the DNA damage response barrier; by hyperactivating error-prone DDR pathways, such as the NHEJ via PRKDC (DNA-PK), and by simultaneously

inhibiting HR-mediated DNA repair via modulation of BRCA1 phosphorylation, FTY720 might offset the DDR HR- mediated, essential to repair faithfully the DNA damage lesions induced by

daunorubicin, and drive the leukemic cells to death. 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. In THP1-based mouse models, we observed immunosuppression, lymphopenia and severe weight

loss (data not shown). As FTY720 exerts its immunosuppressive properties after being phosphorylated in vivo in FTY720-P, a mimetic of sphingosine-1-phosphate (S1P) critical for lymphocyte

trafficking [40], and several studies indicate that the phosphorylation of FTY720 is not essential to exert anti-leukemic effects [27, 28, 50], studies have been focused on the development

of FTY720 analogs with anti-cancer but no immunosuppressive properties. 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 [46, 68, 69]. Furthermore, SH-RF-177, a FTY720 analog that is efficiently phosphorylated but that does not activate S1P receptor 1,

shows anti-leukemic activity on ALL cells [70]. Therefore, further studies are needed to evaluate the potential of these analogs in _KMT2A_-R-leukemia. In conclusion, our study demonstrates

for the first time that the expression of PP2A inhibitor SET positively correlates with _MEIS_ and _HOXA_ genes. SET is critical for the clonogenic ability of _KMT2A-_R-leukemic cells and

can be pharmacologically targeted by using FTY720, an immunosuppressive sphingosine mimetic that reactivates PP2A by suppressing SET. We provide the evidence that silencing and

pharmacological inhibition of SET in _KMT2A_-R-leukemic cells promote cell cycle arrest and apoptosis, rewire the transcriptional program, down-regulate the expression of _HOXA_ genes and

direct MYC toward proteasomal degradation. These data shed light on SET as a new therapeutic target of _KMT2A_-R leukemia. MATERIAL AND METHODS CELL LINES The cells lines used for this study

and the experimental conditions are indicated in supplementary material and methods. Some of the cell lines used in this study were stably transduced with a lentivirus vector expressing the

enhanced green fluorescent protein (eGFP) and positive clones were sorted by FACS, as described [38]. The cell lines eGFP-K562, eGFP-Kasumi1, eGFP-THP1, eGFP-MV411, eGFP-REH, eGFP-SEM,

eGFP-Hb1119, and eGFP-U937 were further tested for authenticity by STR profiling (Eurofin Genomics). PRIMARY CELLS Primary samples, described in supplementary material and methods, 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, described in supplementary material

and methods and in [71, 72], were a generous gift of Prof. Owen Williams. VIRUS PRODUCTION AND CELL TRANSDUCTION Knock-down of _SET_ was conducted in vitro using the lentiviral viruses

purchased from Sigma-Merck, as described in supplementary material and methods. 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. The pLKO, 1-puro CMV-tag red fluorescent protein (RFP), with no shRNA insert and expressing the RFP, was used as a transduction positive control. CELL

PROLIFERATION AND CELL DEATH ANALYSIS Cell proliferation and cell death were monitored by GFP fluorescence and by measuring the percentage of GFP negative cells as described in ref. [38] and

in supplementary material and methods. REAGENTS FTY720 was purchased from Selleckchem (S5002). The antibodies used in this study are listed in supplementary material and methods. RT-QPCR

AND CHIP Quantitative real-time PCR (RT-qPCR) was performed using specific primers from Sigma-Merck. ChIP assay was performed as previously reported [6, 73]. The primers are listed in

supplementary material and methods. PHOSPHO-PROTEOMIC EXPERIMENTS Phospho-proteomic experiments were performed using mass spectrometry as reported in refs. [48, 74] and in supplementary

material and methods. 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) [31]. 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 analyzed by using the micro-array data from GSE13690 [35]. This dataset was generated by Somervaille et al., by analyzing the transcriptional profiles of 34

mouse _KMT2A_-R-AML. In this study, Somervaille et al. assessed the relative LSC frequency of these AML by determining the colony-forming cell (CFC) frequencies of spleen and bone marrow

cells collected from leukemic mice. The transcriptional profiles were split in two groups: a group labeled as high-LSC frequency (comprising AML initiated by _KMT2A::MLLT3_ and

_KMT2A:MLLT1_) and one labeled as low-LSC frequency (comprising AML initiated by _KMT2A::AFF1p_, _KMT2A::AF10_ and _KMT2A::GAS7_). Meta-analyses of micro-array data from the PrognoScan

database (GSE12417) were used to determine the prognosis of AML patients expressing high or low levels of SET [32]. The data represent the analysis of 163 patients treated in the German

AMLCG 1999 trial. The survival analysis in PrognoScan is based the minimum p-value approach to find the cutpoint in continuous gene expression measurement for grouping patients. The publicly

accessible online database cBioPortal (https://www.cbioportal.org/, accessed on 29 May 2023) was used to explore the correlation of SET with the self-renewal leukemic stem cell (LSC) marker

genes published by Gentles et al. [36] and by Krivtsov et al. [37]. We checked four AML cohorts: OHSU (Cancer cell 2022, _n_ = 942), OHSU (Nature 2018, _n_ = 672), TCGA (Firehose Legacy,

_n_ = 200) and TCGA (NEJM 2013, _n_ = 200). The co-expression of SET with the genes of interest was tested by the Spearman’s correlation coefficient (Rs), that summarize the strength and

direction (positive or negative) of a relationship between two variables. A significant correlation of co-expression with SET was considered according to the absolute value of the Spearman’s

correlation coefficient as weak (0.20–0.39), moderate (0.40–0.59), strong (0.60–0.79) or very strong (0.80–1) when the _p_ value was lower than 0.05 (_p_ < 0.05). The graphs were made

using GraphPad Prism 9 software. 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), as described in supplementary material and methods. STATISTICAL ANALYSIS Data are expressed as mean ± standard deviation (SD) from at least three independent

experiments, unless stated otherwise. Statistical significance was determined using GraphPad Prism 7 with the tests reported in the figure legends. Additional methods are available in the

supplementary materials and methods. DATA AVAILABILITY The FTY720-RNASeq datasets generated during the current study are available in the GEO repository (GSE218708). The three plot shown in

Fig. 1 has been drawn based on data obtained from Bloodspot Gene expression profiles (GSE42519). The heatmap shown in Fig. 1 has been generated by analyzing 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

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heterogeneity of signaling pathway activation in leukemia cells. Sci Signal. 2013;6:rs6. Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to thank

LeukaemiaUK, 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, Prof Kamil Kranc for providing constructive feedback on the manuscript, Prof. Fulvio D’Acquisto and Prof. Jolanta Opacka-Juffry for their mentoring. Figure 8 was created

in BioRender.com. 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. AUTHOR INFORMATION Author notes * Maria Teresa Esposito Present address: School of Biosciences, University of Surrey, Guildford, UK * These authors contributed equally:

Antonella Di Mambro, Yoana Arroyo-Berdugo. * These authors jointly supervised this work: Bela Patel, Maria Teresa Esposito. AUTHORS AND AFFILIATIONS * School of Life and Health Sciences,

University of Roehampton, London, UK Antonella Di Mambro, Yoana Arroyo-Berdugo, Yolanda Calle & Maria Teresa Esposito * CEINGE Biotecnologie Avanzate, Via Gaetano Salvatore, Napoli,

Italy Tiziana Fioretti, Armando Cevenini & Gabriella Esposito * Chester Centre for Leukaemia Research, Chester Medical School, University of Chester, Chester, UK Michael Randles & 

Claire M. Lucas * Centre Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Luca Cozzuto & Julia Ponomarenko * Barts Cancer Institute, Queen

Mary University of London, London, UK Vinothini Rajeeve, Michael J. Austin, Pedro Cutillas, John Gribben & Bela Patel * Department of Molecular Medicine and Medical Biotechnologies,

University of Naples Federico II, Via Pansini 5, 80131, Naples, Italy Armando Cevenini & Gabriella Esposito * University Pompeu Fabra (UPF), Barcelona, Spain Julia Ponomarenko * Great

Ormond Street Institute of Child Health London, UCL, London, UK Owen Williams Authors * Antonella Di Mambro View author publications You can also search for this author inPubMed Google

Scholar * Yoana Arroyo-Berdugo View author publications You can also search for this author inPubMed Google Scholar * Tiziana Fioretti View author publications You can also search for this

author inPubMed Google Scholar * Michael Randles View author publications You can also search for this author inPubMed Google Scholar * Luca Cozzuto View author publications You can also

search for this author inPubMed Google Scholar * Vinothini Rajeeve View author publications You can also search for this author inPubMed Google Scholar * Armando Cevenini View author

publications You can also search for this author inPubMed Google Scholar * Michael J. Austin View author publications You can also search for this author inPubMed Google Scholar * Gabriella

Esposito View author publications You can also search for this author inPubMed Google Scholar * Julia Ponomarenko View author publications You can also search for this author inPubMed Google

Scholar * Claire M. Lucas View author publications You can also search for this author inPubMed Google Scholar * Pedro Cutillas View author publications You can also search for this author

inPubMed Google Scholar * John Gribben View author publications You can also search for this author inPubMed Google Scholar * Owen Williams View author publications You can also search for

this author inPubMed Google Scholar * Yolanda Calle View author publications You can also search for this author inPubMed Google Scholar * Bela Patel View author publications You can also

search for this author inPubMed Google Scholar * Maria Teresa Esposito View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS MTE, BP, and YC

conceived the experimental design; ADM, YAB, TF, and MTE performed the experiments and analyzed 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, BP, and JG gathered

patients’ material and provided clinical data; CL contributed intellectually to the interpretation of the data; BP 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. CORRESPONDING AUTHOR

Correspondence to Maria Teresa Esposito. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY DATA AND MATERIAL AND METHODS SUPPLEMENTARY TABLE 1

SUPPLEMENTARY TABLE 3 SUPPLEMENTARY TABLE 4 SUPPLEMENTARY TABLE 5 SUPPLEMENTARY FIGURE 1 SUPPLEMENTARY FIGURE 2 SUPPLEMENTARY FIGURE 3 SUPPLEMENTARY FIGURE 4 SUPPLEMENTARY FIGURE 5

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al._ SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML. _Oncogene_ 42, 3670–3683 (2023). https://doi.org/10.1038/s41388-023-02840-1 Download citation * Received: 21

February 2023 * Revised: 07 September 2023 * Accepted: 13 September 2023 * Published: 27 October 2023 * Issue Date: 08 December 2023 * DOI: https://doi.org/10.1038/s41388-023-02840-1 SHARE

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