T cell receptor (TCR) stimulation by antigen bound to major histocompatibility complex (MHC) molecules on the surface of an antigen-presenting cell (APC) induces the formation of the immune synapse (IS). The IS is a specialized cell-cell interface contact area that provides a signaling platform for integration of signals leading to intercellular information exchange, in order to ensure efficient TCR signal transduction, T cell activation, and the proper execution of diverse T lymphocyte effector functions (Dustin and Choudhuri, 2016). Comprised among these functions, IS formation triggers the convergence of T lymphocyte secretory vesicles, including multivesicular bodies (MVB) (Alonso et al., 2011; Calvo and Izquierdo, 2021), toward the microtubule-organizing center (MTOC) and the polarization of MTOC together with secretory vesicles to the IS (de la Roche et al., 2016; Huse, 2012). The canonical bullseye structure of a mature IS comprises three concentric areas referred to as supramolecular activation clusters (SMACs) that can be discerned based on the specific segregation of TCR, integrins, and co-stimulatory molecules (Monks et al., 1998). The central supramolecular activation cluster (cSMAC) is enriched in ligand-bound TCR and associated signaling molecules, the peripheral SMAC (pSMAC) in adhesion molecules such as LFA-1, and the outer distal SMAC (dSMAC) in filamentous actin (F-actin) and CD45 (Hammer et al., 2019; Blumenthal and Burkhardt, 2020). IS assembly and subsequent secretory responses are coordinated by both actin and microtubule cytoskeletons, whose interplay regulates the polarized secretory response toward the IS (Ritter et al., 2013; Hammer et al., 2019). IS formation is associated with an initial increase in cortical actin at the IS (Billadeau et al., 2007), followed by a decrease in cortical actin density at the central region of the immune synapse (cIS) including the cSMAC, that contains the secretory domain (Griffiths et al., 2010; Ritter et al., 2015). The partial depletion of F-actin at the cIS has been proposed, apart from allowing the focusing and docking of secretory vesicles for secretion (Billadeau et al., 2007), to initiate key subsequent events leading to MTOC polarization and delivery of secretory vesicles, such as lytic granules in cytotoxic T lymphocytes (CTL) (Ritter et al., 2015) and cytokine-containing secretory vesicles in T-helper (Th) lymphocytes (Chemin et al., 2012), to the secretory domain of the IS. The CTL degranulation of diverse secretory lytic granules with MVB structure results in the secretion to the synaptic cleft of Fas ligand (FasL)-containing exosomes (Peters et al., 1989; Peters et al., 1991; Martínez-Lorenzo et al., 1999; Alonso et al., 2005; Alonso et al., 2011; Mazzeo et al., 2016). These exosomes, along with perforin and granzymes, which are secreted in both soluble and nanoparticulate form in the so-called supramolecular attack particles (Bálint et al., 2020; Cassioli and Baldari, 2022), lead to the induction of target cell apoptosis. In addition, FasL-containing exosomes have been involved in autocrine FasL/Fas-dependent, activation-induced cell death (AICD) produced upon TCR triggering (Martínez-Lorenzo et al., 1999; Monleón et al., 2001; Alonso et al., 2005; Calvo and Izquierdo, 2020), an important immunoregulatory event involved in the downregulation of T cell immune responses (Krammer et al., 2007; Nagata and Suda, 1995).

We have previously described that cortical actin reorganization at the IS plays an important role in MVB polarized traffic leading to exosome secretion in Th lymphocytes (Herranz et al., 2019; Bello-Gamboa et al., 2020; Calvo and Izquierdo, 2021). With respect to the molecular cues controlling this process, we have shown that TCR-stimulated protein kinase C δ (PKCδ) regulates cortical actin reorganization at the IS, thereby controlling MTOC/MVB polarization and ultimately leading to exosome secretion at the IS and AICD in Th lymphocytes (Herranz et al., 2019). Moreover, PKCδ is necessary for the polarization of lytic granules and the induction of cytotoxicity by mouse CTL (Ma et al., 2008; Ma et al., 2007), which supports a general role of PKCδ in secretory traffic leading to apoptosis in T lymphocytes. Besides PKCδ, several actin cytoskeleton regulators, including the formin FMNL1 and Diaphanous-1 (Dia1), also regulate MTOC polarization (Gomez et al., 2007; Kühn and Geyer, 2014; Kumari et al., 2014). In this context, we have shown both PKCδ-dependent phosphorylation of FMNL1 at the IS and PKCδ-dependent F-actin clearing at the cIS, which participate in MTOC/MVB polarization leading to exosome secretion in CD4⁺ Jurkat T lymphocytes forming IS (Herranz et al., 2019; Bello-Gamboa et al., 2020; Calvo and Izquierdo, 2021). However, a formal connection between FMNL1 phosphorylation, F-actin regulation, and MVB polarization/exosome secretion at the IS has not been established yet.

Regarding potential formin regulatory pathways, we have shown that FMNL1, but not Dia1, is strongly phosphorylated in T lymphocytes by PKCδ activators such as phorbol myristate acetate (PMA), but also upon TCR stimulation via an anti-TCR agonist as well as IS formation (Bello-Gamboa et al., 2020). FMNL1 phosphorylation was inhibited in PKCδ-interfered T lymphocytes (Bello-Gamboa et al., 2020), which is associated with a deficient MTOC polarization toward the IS (Herranz et al., 2019), supporting a PKCδ role in both FMNL1 phosphorylation and MTOC polarization (Herranz et al., 2019; Bello-Gamboa et al., 2020). Interestingly, FMNL2, a related formin which exhibits a high homology with FMNL1, is phosphorylated by PKCα and, to a lower extent, by PKCδ, at S1072 (Wang et al., 2015). This phosphorylation reverses FMNL2 autoinhibition mediated by interaction of N-terminal Diaphanous inhibitory domain (DID) with the C-terminal Diaphanous autoinhibitory domain (DAD), resulting in increased F-actin assembly, β1-integrin endocytosis, invasive motility (Wang et al., 2015), and filopodia elongation (Lorenzen et al., 2023). In this regard, recent evidence suggests that the mutation of a particular residue in FMNL2 impacting the DID-DAD interaction may result in functional implications for the characteristic actin-regulating activity of FMNL2, specifically in the context of podosome formation in macrophages (Trefzer et al., 2021). Out of the three FMNL1 isoforms (α, β, and γ) present in T lymphocytes and Jurkat cells (Colón-Franco et al., 2011), S1086 in FMNL1β is surrounded by a sequence displaying high homology to the one surrounding S1072 in FMNL2 (Wang et al., 2015; Bello-Gamboa et al., 2020; Figure 1). Moreover, we have shown that FMNL1β is the only FMNL1 isoform phosphorylated upon IS formation and capable of recovering MTOC polarization when it was re-expressed in cells simultaneously interfered for the three FMNL1 isoforms (Bello-Gamboa et al., 2020). Thus, we hypothesize that IS-induced, PKCδ-dependent phosphorylation at S1086, a residue located within FMNL1β DAD, may release the autoinhibition mediated by DID/DAD interaction, thereby leading to the activation of FMNL1β. Consequently, this activation would mediate the F-actin reorganization at the IS and the MTOC/MVB polarization, thereby enabling the subsequent exosome secretion. Here, we report that FMNL1β phosphorylation at S1086 regulates F-actin reorganization leading to MTOC/MVB polarization and exosome secretion.

Figure 1

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Our previously published results suggest that PKCδ-dependent phosphorylation of FMNL1β may regulate its function in F-actin reorganization at the IS and hence affect MTOC polarization (Herranz et al., 2019; Bello-Gamboa et al., 2020). We have previously shown that phosphorylation occurs in FMNL1 but not in a homologous formin, Dia1 (Bello-Gamboa et al., 2020), that also regulate MTOC polarization and it is a major actin regulator in T lymphocytes (Gomez et al., 2007), supporting an specific role of FMNL1 phosphorylation, but not Dia1 phosphorylation on MTOC polarization. Here, we have identified for the first time a positive regulatory role of FMNL1β phosphorylation at S1086 in cortical F-actin regulation that governs MTOC/MVB polarization and exosome secretion at the IS in Th lymphocytes. Moreover, the fact that the phosphomimetic YFP-FMNL1βS1086D expression in PKCδ and FMNL1-interfered cells did not recover the MTOC PI observed in C3 control cells (Figure 5) supports the idea that FMNL1β phosphorylation at S1086 is necessary but not sufficient for MTOC polarization, at least in cells lacking PKCδ. Thus, these results support that another signal, apart from PKCδ activation, or another PKCδ-controlled pathway not involving FMNL1β phosphorylation, evoked upon TCR triggering at the IS, is also implicated in MTOC/MVB polarization to the IS.

Our results on FMNL1β phosphorylation are certainly related to what was found in formin homology domain protein 1 (FHOD1) regulation. FHOD1 is grouped into Dia-related formins (Schönichen and Geyer, 2010). FHOD1 requires active Rac to be recruited to the plasma membrane, but its recruitment appears insufficient for FHOD1 activation, which is achieved only after being phosphorylated by Rho-dependent protein kinase (ROCK) at three serine and threonine sites included in a polybasic arginine-rich region inside the C-terminal DAD (Takeya et al., 2008), leading to disruption of FHOD1 autoinhibitory state and F-actin stress fiber formation (Takeya et al., 2008). This activation process is similar to what we have described in this article for FMNL1β. Moreover, the PKC-dependent mode of FMNL2 activation during integrin activation (Wang et al., 2015) and filopodia formation (Lorenzen et al., 2023) is comparable to what we have found in FMNL1β during polarized traffic to the IS. Furthermore, FHOD3 formin is also autoinhibited by an intramolecular interaction between its C- and N-terminal domains. Phosphorylation of the three highly conserved residues (S1406, S1412, and T1416) within the polybasic, C-terminus of FHOD3 by ROCK1/2 is sufficient for its activation and is crucial in regulating myofibrillogenesis in cardiomyocytes (Zhou et al., 2017). Thus, with the results provided here for FMNL1β, it has been reported that at least four different formins are activated through phosphorylation by three different kinases at the C-terminal polybasic regions, which are included in a common DAD. This underscores the evolutionary importance of this regulatory mechanism in formin activation, ultimately leading to very diverse cellular responses. However, although we do not have conclusive evidence supporting that PKC directly phosphorylates FMNL1β, the presence of a conserved S1086 within an RRSV/AR motif that contains a potential target for classical PKCs (Wang et al., 2015; Lorenzen et al., 2023; Nishikawa et al., 1997), along with the recognition of the phosphorylated residue by a validated anti-Phospho-Ser PKC substrate antibody (Wang et al., 2015), certainly support this possibility.

With regard to potential regulatory pathways involved in formin activation and subcellular localization, it is remarkable that the most abundant formins in T lymphocytes, Dia1 and FMNL1 (Gomez et al., 2007), are constitutively inactive in the cytoplasm due to intramolecular DAD-DID binding, which blocks formin ability to nucleate and elongate actin filaments (Hammer et al., 2019). Regarding a possible control of formin subcellular location by PKC, it has been shown that both relocation of FMNL2 from the plasma membrane to the endosomal compartment and activation are dependent on C-terminal S1072 phosphorylation by PKCα (Wang et al., 2015). However, FMNL2 location in filopodia seems to be independent of the phosphorylation state (Lorenzen et al., 2023). We have previously described PKCδ-dependent phosphorylation of FMNL1β (Bello-Gamboa et al., 2020), and here we have demonstrated that this phosphorylation occurs at S1086, leading to F-actin reorganization at the IS, and resulting in MTOC polarization. However, in contrast to FMNL2 relocation, which is dependent on PKCα, we have shown that transient YFP-FMNL1βWT relocation from cytosol to the IS occurred in the absence of PKCδ (Figure 6), in conditions where neither F-actin reorganization (Herranz et al., 2019) nor MTOC polarization (Figure 5) occurred. Moreover, both YFP-FMNL1βS1086A and YFP-FMNL1βS1086D translocated to the IS (Video 3 and Figure 6—figure supplement 2), demonstrating that S1086 phosphorylation is not required for IS location. However, as it happens during S1072 phosphorylation in FMNL2 (Wang et al., 2015), it is possible that blocking phosphorylation at S1086 through the introduction of single point mutations (YFP-FMNL1βS1086A or YFP-FMNL1βS1086D) may not block FMNL1β translocation to the IS per se, but may alter the kinetics of the process. For this reason, in future approaches, a thorough study on the kinetic considerations on S1086 PKCδ-mediated phosphorylation contribution to FMNL1β translocation to the IS will need to be addressed.

Jurkat T cells may not fully recapitulate the regulation of polarization in primary T cells, and the evidences from activating surfaces acting as artificial synapses show distinct actin cytoskeleton behavior (presence or not of F-actin foci) in primary versus immortalized T cells, at least in CD4⁺ cells, that may reflect mechanistic distinctions in the T cell models systems utilized to study cytoskeletal actin dynamics (Kumari et al., 2019; Colin-York et al., 2019). However, to date, no clear evidence has been obtained regarding the existence of such an F-actin foci network in cell-cell IS models based on primary T lymphocytes or Jurkat cells. F-actin foci have been exclusively observed in primary T cells engaged with artificial activating surfaces (planar lipid bilayers or coated glass), which does not apply to our data obtained from cell-cell synapses. However, to solve any potential differences in FMNL1 localization between primary versus immortalized T cells, we have performed experiments analyzing the subcellular localization of FMNL1 in CD19/CD22 dual-targeted CAR T cells forming synapses with CD19⁺/CD22⁺ Raji cells used as CTL targets and T lymphoblasts forming synapses with SEE and SEB-pulsed Raji cells. We observed, in fixed-cell images, the presence of endogenous FMNL1 colocalizing with F-actin-enriched areas in the IS region of both CAR T cells and primary T lymphoblasts, which also exhibited MTOC polarization toward the IS (Figure 6—figure supplement 1). The presence of FMNL1 at the IS developed by primary T lymphoblasts was transient, as occurred in Jurkat cells (not shown). These observations extend our results on FMNL1 localization at the IS in the Jurkat-Raji plus SEE synapse model to the IS developed by primary human T lymphocytes. Regarding exosome secretion in primary human T lymphocytes, additional experiments involving the expression of the diverse FMNL1β variants will be necessary to assess whether FMNL1β and its phosphorylation at S1086 controls exosome secretion in primary T lymphocytes, as occurred in Jurkat cells. These experiments will require the development of new transduction protocols to improve the otherwise inefficient expression in primary T lymphocytes, that it is even worse using the bi-cistronic large plasmids (>15 kb) that we have employed in our studies. In addition, it is well known that the mere expression of large proteins such as the 180 kDa (30+150 kDa) YFP-FMNL1 chimeric variants constitutes a significant challenge. Thus, we have been unable to achieve enough transfection efficiency to perform these experiments, which would have allowed us to fully extend the effect of FMNL1β and its phosphorylation at S1086 on exosome secretion to primary T cells. However, FMNL1 is necessary for MTOC/MVB polarization in Jurkat cells (Gomez et al., 2007; Bello-Gamboa et al., 2020), as confirmed in this manuscript, and this finding has already been extended to primary CD8⁺ T cell clones (Gomez et al., 2007). All of this, together with the fact that exosome secretion requires MTOC/MVB polarization both in Jurkat cells and primary T lymphoblasts (Alonso et al., 2011; Bello-Gamboa et al., 2020), suggests FMNL1 may also control exosome secretion in primary T cells, although the formal demonstration will require further research. Regarding the effect of the diverse FMNL1β variants expression on MTOC/MVB polarization and exosome secretion in primary T lymphocytes, the data from activating surfaces clearly shows that the synaptic actin architecture of the IS from primary CD8⁺ T cells is essentially indistinguishable and thus unbiased from that of Jurkat T cells, but different to that of primary CD4⁺ cells (Murugesan et al., 2016). Thus, it is expected that all our data in Jurkat T cells could probably be extrapolated to the synaptic architecture of primary CD8⁺ cells, although more experiments involving primary CD4⁺ are necessary to extend all our results to the last cell type.

Although the mechanism(s) involved in FMNL1β recruitment to the IS remains unclear, it is probably that it involves Rac1 and Cdc42 small G proteins (Kühn and Geyer, 2014; Gomez et al., 2007; Favaro et al., 2013), which are activated at the IS upon TCR triggering (Deckert et al., 2005). However, we cannot exclude the participation of another PKC isoform or a different protein kinase that may induce FMNL1β phosphorylation in different residues upon previous FMNL1β relocation to the IS primed by small G protein, as was shown for FMNL2 relocation (Wang et al., 2015). Further experiments are needed to establish this important point.

Taken together, our results support that IS-induced, PKCδ-dependent phosphorylation of S1086 in FMNL1β DAD, most probably executed by PKCδ itself, activates FMNL1β, which in turn regulates cortical actin at the IS and hence controls MTOC/MVB polarization and exosome secretion. The behavior of non-phosphorylatable YFP-FMNL1βS1086A (equivalent to FMNL2-S1072A described in Wang et al., 2015) confirms this hypothesis, since its expression inhibits F-actin reorganization at the IS, MTOC polarization, and exosome secretion. In this respect, it has been reported that the phosphorylation of FMNL2 by PKCα in S1072 is necessary for the secretion of the pro-metastatic factor Angiopoietin-like 4 (ANGPTL4) in breast cancer cells (Frank et al., 2023), which constitutes the first description for an activated formin involvement in secretion during cell invasion. In addition, ANGPTL4 can be secreted into exosomes by lung cancer cells (Mo et al., 2020; Zhang et al., 2022) and FMNL2 controls exosome secretion (He et al., 2023). Thus, although it is not clear whether ANGPTL4 is released in exosomes or in soluble form (Frank et al., 2023), both FMNL2 and FMNL1β appear to control PKC-regulated exosome secretion. However, the extracellular signals evoking exosome secretion are strikingly different in the various cell types, since in tumor cells hypoxia and radiation can amplify the otherwise constitutive and multidirectional exosome secretion, whereas T lymphocytes constitutively secrete very low levels of exosomes and none in a polarized manner unless activated through TCR or synaptic contact (Calvo and Izquierdo, 2020). Taken together, these considerations support a more general role of the formin-actin cytoskeleton axis in directional exosome secretion. Given all these considerations, it will be interesting to analyze the role of FMNL2 in inducible exosome secretion at the IS in T lymphocytes, but also the role of FMNL1β in exosome secretion by cancer cells during cell invasion. This is particularly relevant as exosome secretion is required for directionally persistent and efficient in vivo movement of cancer cells (Sung et al., 2015), and it is known that FMNL1 stimulates both leukemia cell proliferation and migration (Favaro et al., 2013; Thompson et al., 2020).

The fact that FMNL1- and PKCδ-interfered cells have a similar phenotype to that of FMNL1- or PKCδ-interfered cells (Bello-Gamboa et al., 2020; Herranz et al., 2019) (this paper) supports the notion that PKCδ and FMNL1β participate in the same F-actin regulatory pathway controlling MTOC/MVB polarization. Additional experiments involving in vitro measurements of F-actin reorganizing/severing activities of the diverse FMNL1β variants, phosphorylated or not in vitro by PKCδ, will be necessary to address whether PKCδ directly phosphorylates FMNL1β at S1086.

Our findings show that FMNL1 colocalizes with F-actin at the IS edges which are part of the dSMAC (Figures 6 and 8). These data are aligned with the previous observation that Dia1 and FMNL1 are intrinsically inactive because they undergo intramolecular folding and remain in the cytoplasm (Hammer et al., 2019) and that after IS formation, they are enriched at the tips of F-actomyosin spikes at the outer edge of the dSMAC, giving rise to the actomyosin arcs at the pSMAC in the IS formed by Jurkat cells (Murugesan et al., 2016; Hammer et al., 2019). Dia1 is not a target of PKC, whereas FMNL1 is phosphorylated by PKC activation (Bello-Gamboa et al., 2020). Thus, in this paper we have focused on FMNL1. High-resolution, spatiotemporal analysis of FMNL1β localization at the different subsynapic locations, together with superresolution techniques (Murugesan et al., 2016; Calvo and Izquierdo, 2018), may provide new clues regarding the subsynaptic F-actin network targeted by phosphorylated FMNL1β. Even though the nature of the downstream effectors and consequences of FMNL1β phosphorylation on synaptic F-actin architecture can only be speculated at this point, concentric actomyosin arcs generated by FMNL1 propel TCR microclusters toward the IS and reinforce cytotoxicity (Murugesan et al., 2016; Hammer et al., 2019). Thus, high-resolution spatiotemporal analysis of actomyosin arcs structure and function during TCR microcluster movement in FMNL1-interfered cells, and/or cells expressing the FMNL1β mutants described here, will provide important clues on this important matter. It is noteworthy that a majority of the image analysis of IS organization and F-actin dynamics is based on planar synapses obtained using artificial APC substitutes, by either coating stimulatory and co-stimulatory molecules on glass or plastic surfaces or by embedding these molecules into lipid bilayers (Dustin, 2009; Hammer et al., 2019; Dupré et al., 2021). These artificial synapses can indeed be imaged and analyzed with the highest possible resolution (Carisey et al., 2018; Murugesan et al., 2016; Calvo and Izquierdo, 2018). However, the cell-cell synapse model analyzed here mimics the complex interactions and irregular, 3D stimulatory surface of a physiological synapse better than artificial synapses do (Dustin, 2009; Hammer et al., 2019). In this important sense, the data presented here are indeed closer to a physiological situation. In addition, since actomyosin arcs arise from active, formin-dependent nucleation sites at the outer edge of the dSMAC (Murugesan et al., 2016), it will be interesting to analyze, using superresolution techniques, whether phosphorylated FMNL1β is located at these sites. Indeed, actomyosin arc network spanning from dSMAC along the pSMAC is the most likely source of T cell-based force that augments cytotoxicity by straining the target cell plasma membrane (Hammer et al., 2019). Thus, it will be interesting to study the effect of FMNL1β mutants on actomyosin arcs formation and analyze functional deficiencies (i.e. decreased T cell-APC adhesion frequency, IS size, and IS formation efficiency) in IS formed by cells interfered in FMNL1β and/or expressing the FMNL1β variants described here. Visualization of actin and actomyosin networks, TCR microclusters, and secretion granules dynamics in ‘real’ live T cell-APC conjugates at the superresolution level (Hammer et al., 2019) is clearly needed to advance in this area.

Remarkably, although it has already been shown that FMNL1 is important for MTOC polarization in Jurkat cells and for target cell lysis in CTL (Gomez et al., 2007), our data provide the first molecular basis underpinning this essential FMNL1 function. Here, we demonstrate that FMNL1β and its phosphorylation at S1086 participate in F-actin-controlled, polarized MVB traffic secreting effector exosomes in Jurkat T lymphocytes developing IS (Alonso et al., 2011; Alonso et al., 2005), which deliver pro-apoptotic signals to target cells (Bálint et al., 2020; Chang et al., 2022; Cassioli and Baldari, 2022). In addition, FMNL1 promotes proliferation and migration of leukemia cells (Favaro et al., 2013) and mediates posterior perinuclear actin polymerization to promote T lymphocyte effector cell migration to inflammatory sites to enable T cell-mediated autoimmunity (Thompson et al., 2020). PKCδ is crucial for directional T cell migration (Fanning et al., 2005; Volkov et al., 1998) and this process requires MVB polarized traffic and exosome secretion in the direction of migration (Sung et al., 2015). For that reason, it will be interesting to analyze the effects of the FMNL1β mutants we have described here during T lymphocyte migration and tumorigenesis, as well as to study the role of FMNL1β phosphorylation in several autoimmune disorders. In addition, improving our understanding of the molecular bases underlying the traffic events involved in polarized secretion of pro-apoptotic exosomes as we have performed here, provided that the FMNL1 effect on exosome secretion in Jurkat cells can be extended to primary T lymphocytes, will deliver clues to modify crucial immune functions involving apoptosis, such as cytotoxicity by CTLs and immunoregulatory AICD and their associated pathologies. This may allow to design therapeutic strategies to modify CAR T lifespan and/or polarized secretory function, in order to improve their efficiency for the treatment of certain cancers (Alonso et al., 2005; Alonso et al., 2007; Alonso et al., 2011; Mazzeo et al., 2016; Herranz et al., 2019; Stinchcombe et al., 2004; de Saint Basile et al., 2010; Davenport et al., 2018).

We declare we have deposited our Source data (Excel dataset) corresponding to all the dot plot data presented in this contribution with Dryad, a generic public repositorie, and thus the materials described in the manuscript, including all relevant raw data, will be freely available to any researcher wishing to use them for non-commercial purposes, without breaching participant confidentiality. Supporting files: source data files have been provided for Figures 2, 3, 6 and 9, containing images of WB blots that are provided as original Source data of uncropped, unedited WB, associated to each corresponding figure. Both PDF of WB and original TIF files are provided. In addition, Source data 1 contains p-values after applying Tukey's method (POST HOC) to the one-way ANOVA of dot plots from Figures 4, 5 and 7 are provided.

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Author details

1. Javier Ruiz-Navarro

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0005-8393-0450

2. Sara Fernández-Hermira

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Irene Sanz-Fernández

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Pablo Barbeito

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0758-0012

5. Alfonso Navarro-Zapata

   1. Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, La Paz University Hospital, Madrid, Spain
   2. Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Center (CNIO), Madrid, Spain

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Antonio Pérez-Martínez

   1. Translational Research in Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ, La Paz University Hospital, Madrid, Spain
   2. Pediatric Onco-Hematology Clinical Research Unit, Spanish National Cancer Center (CNIO), Madrid, Spain
   3. Department of Pediatric Hemato-Oncology, La Paz University Hospital, Madrid, Spain
   4. Pediatric Department, Autonomous University of Madrid, Madrid, Spain

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Francesc R Garcia-Gonzalo

   1. Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain
   2. CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
   3. Instituto de Investigación Sanitaria del Hospital Universitario La Paz (IdiPAZ), Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Resources, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9152-2191

8. Víctor Calvo

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

9. Manuel Izquierdo Pastor

   Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), CSIC-UAM, Madrid, Spain

   Present address

   Departamento de Bioquímica, Facultad de Medicina, UAM, Madrid, Spain

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   manuel.izquierdo@inv.uam.es

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7701-1002

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