Phosphoproteomic analysis reveals hyperactivation of mTOR/STAT3 and LCK/Calcineurin axes in pediatric early T-cell precursor ALL

STAT3 and

Arrays (RPPA).The whole blood blast percentage for all samples was between 78% and 97%.
Protein lysates were freshly made from bone marrow mononuclear cells separated by Ficoll-Hypaque technique.RPPA analysis was performed as previously described, 9 and the activation and/or expression of 50 different proteins was studied (Supplementary Table S2) with a particular focus on AKT/mTOR, JAK/STAT, RAS/MAPK and TCR signaling pathways.Statistical analyses were performed using Prism (GraphPad Software, Inc) and identification of differentially activated or expressed proteins in ETP-ALL patients was obtained through Mann-Whitney test.The activation status of selected pathways was evaluated through Global test analysis 10 performed with R (www.r-project.org).This latter analysis revealed that the AKT/mTOR and the JAK/STAT pathways are hyperactivated in ETP-ALL compared to both the whole cohort of analyzed T-ALL (Supplementary Figure S1a) and the subgroup of Early-T ALL only, not ETP (Supplementary Figure S1b).Besides, ETP-ALL patients showed also the RAS/MAPK pathway more activated when compared to the Early-T ALL subgroup (Supplementary Figure S1b).This comprehensive analysis well supports the RAS/MAPK and JAK/STAT signaling deregulation reported by other groups. 2,7,8Moreover, in ETP-ALL patients we also found the hyperactivation of the RAS/MAPK pathway members B-RAF, the main ERK activator, phosphorylated in S445 (Supplementary Figure S1c), and ERK1/2 phosphorylated in T202/Y204 (Supplementary Figure S1d), while activated MEK1/2 in S217/221 is upregulated in ETP only when compared to the Early-T subgroup (Supplementary Figure S1e).S1f-h).Somatic mutations in the NRAS and FLT3 genes were detected respectively in 3 and 5 of the 16 ETP patients (Supplementary Table S3), a result which correlates very well with the observed hyperactivation of downstream signaling in the same patients.However, somewhat unexpectedly, we also found a similar hyperactivation in non mutated patients.Importantly, no differences were observed between FLT3 mutated and non mutated patients at the level of AKT/mTOR signaling.

Their total forms do not differ between patients groups (Supplementary Figure
Although this may be due to the limited number of patients analyzed, these data allow to hypothesize that the high signaling observed in ETP-ALL has grounds other than the solely somatic mutations, in keeping with the observation that the AKT/mTOR hyperactivation is neither © 2017 Macmillan Publishers Limited.All rights reserved. ascribable to the presence of PTEN mutations, and that the JAK/STAT pathway hyperactivation cannot be associated to somatic mutations in IL7R, JAK1, JAK2 or JAK3 genes (Supplementary Table S3).
A striking finding, then, concerned the SRC family non-receptor tyrosine kinase LCK, which resulted dramatically underexpressed in ETP-ALL compared to non-ETP T-ALL or Early-T ALL only (Figure 1a).This is in accordance with LCK mRNA downregulation observed in ETP-ALL patients by Zhang et al. 2 However, activated LCK (measured through SRC Y416) normalized on total LCK clearly indicated that LCK activity is significantly higher in ETP-ALL patients (Figure 1b).This observation is supported by results concerning the inhibited form of LCK (Supplementary Figure S2a) which does not differ between patients, suggesting a functionally leading role for the activating phosphorylation site.A well known function of LCK, upon TCR engagement, is to trigger through the PLCɣ1 phosphodiesterase activity the release of Ca 2+ from the endoplasmic reticulum, which in turn leads to the activation of the calcium-and calmodulin-activated serine/threonine phosphatase Calcineurin.We therefore analyzed the activation of PLCɣ1 in Y783 and confirmed its upregulation in ETP-ALL patients (Figure 1c), along with the overexpression of Calcineurin A (Figure 1d).Among Calcineurin substrates are the NFAT transcription factors, which upon dephosphorylation redistribute to the nucleus and modulate the expression of target genes involved in the pathogenesis of cancer and in many aspects of the immune response.The activation of the Calcineurin-NFAT pathway was already demonstrated to be crucial for the maintenance of the T-cell leukemic phenotype in vivo, 11 and all these findings suggest that in ETP-ALL the upregulation of the LCK/Calcineurin pathway could contribute to leukemogenesis.To support our hypothesis we treated primary cultures from 3 ETP-ALL and 4 non-ETP T-ALL pediatric patients with the LCK inhibitor Dasatinib.MTT test revealed a significative reduction of cell viability in ETP-ALL cells compared to non-ETP already at nanomolar concentrations of the drug (Figure 1e).Only one ETP-ALL specimen had enough cells available to perform additional experiments, thus we confirmed, after Dasatinib treatment, the dephosphorylation of SRC Y416 (Figure 1f) and the downmodulation of the whole LCK/Calcineurin axis through the decrease of mRNA levels of the NFAT target IL-2 (Figure 1g).
© 2017 Macmillan Publishers Limited.All rights reserved.
mTOR signaling was also investigated.Remarkably, while expression of total mTOR protein did not differ between patients groups (Supplementary Figure S2b), we noticed a dramatic increase of mTOR phosphorylation at S2448 in ETP-ALL patients (Figure 2a).Cellular mTOR can form two distinct complexes, called mTORC1 and mTORC2, which display substrate specificity.
Phosphorylation at S2448 is known to be a marker of mTORC1, rather than mTORC2, integrity/activity. 12mTORC1 keeps tight control of initiation of translation, protein synthesis and cell cycle progression through its specific target, the Ribosomal Protein S6 kinase (p70 S6K).
Consistently with the above result, p70 S6K was also found hyperphosphorylated, and therefore activated, at the mTORC1 site T389 in ETP-ALL patients (Figure 2b).In turn, phosphorylation by p70 S6K of the 40S ribosomal protein S6 at S235/236 was boosted in ETP-ALL patients (Figure 2c).Since p70 S6K has been identified as the mTOR S2448 kinase, it is tempting to speculate that in ETP-ALL patients a permanent circuit is at work, where p70 S6K may mediate phosphorylation of mTOR at S2448 once it has been activated by mTOR phosphorylation at T389 in a possible feedback loop.To test our hypothesis, PI3K/mTOR signaling was modulated with the mTORC1 inhibitor Rapamycin.Its effect on cell viability was assessed by MTT (Figure 2d) in 3 ETP-ALL and 4 non-ETP T-ALL primary samples.Results show that direct mTOR inihibition by Rapamycin drops cell viability significantly at all concentrations tested only in ETP-ALL cells, while exerting only a very weak effect in T-ALL samples.To dissect what step of the cascade is boosted in ETP-ALL samples, vertical targeting was performed by means of the PI3K delta inhibitor CAL-101, which has already been shown to be cytotoxic to ETP-ALL-like cell models, 13 as well as of the dual pan-PI3K and mTOR inhibitor PF-04691502.Compared to mTOR single targeting by Rapamycin, neither CAL-101 nor PF-04691502 displayed as much efficacy (Figure 2e).However, it is worth noting that while CAL-101 was completely ineffective, dual inhibition by PF-04691502 showed some efficacy likely due to mTOR inhibition.This strongly support our hypothesis that mTOR S2448 and p70 S6K T389 phosphorylation may constitute a self-sustaining loop that contributes to the higher signaling phenotype of ETP-ALL.
In keeping with the observed constitutive activation of mTOR signaling is also the striking  ) and, to a lesser extent, S705 (Supplementary Figure S2c), whereas total STAT3 protein level was unaffected (Supplementary Figure S2d).Interestingly, mTOR-driven phosphorylation of STAT3 at both sites has been reported in human cancer cells and monocytes, 14 and has been claimed to promote viability and maintenance of cancer stem cells while counteracting differentiation. 15,16,17It is therefore conceivable that mTOR and STAT3 could work in tandem to maintain a markedly undifferentiated phenotype of ETP-ALL patient cells, while sustaining cell proliferation and survival.
In conclusion, our study demonstrates that ETP-ALL patients display a peculiar proteomic activation pattern which differs from that of the other pediatric T-ALL patients.Indeed, not only we confirmed that the JAK/STAT and the RAS/MAPK signaling pathways are upregulated, but we also demonstrated for the first time that the LCK/Calcineurin and the mTOR/STAT3 axes are boosted (Figure 2g).In particular, the hyperactivation of LCK triggers the Calcineurin/NFAT-mediated transcription which is critical for T cell leukemogenesis.Moreover, the hyperactivation of mTOR, probably not a consequence of FLT3 mutations but of an aberrant feedback loop through p70 S6K, increases STAT3 and 40S ribosomal protein S6 phosphorylation promoting cancer cells viability.
The abnormal signaling activation observed in ETP-ALL will be extremely helpful to elucidate the mechanism behind the poor response to induction treatment of these patients, and might also suggest new druggable targets for personalized and less toxic therapies (i.e.LCK 18,19 or mTOR [20][21][22] inhibitors).

Figure 2 .
Figure 2. mTOR/STAT3 axis in pediatric ETP-ALL.(a) mTOR is hyperphosphorylated at S2448 in assay, of ETP-ALL patients' cells (n=3).(f) STAT3 is hyperactivated at S727 in ETP-ALL patients (mean 17790 ± 1570) compared to the other T-ALL (mean 10620 ± 761.1) (p<0.0001) and Early-T ALL only (mean 9555 ± 776.8) (p<0.0001).(g) Schematic figure of the mostly activated pathways in ETP-ALL patients.On the left, the LCK/Calcineurin pathway in which SRC activation at Y416 is followed by PLCγ1 activation at Y783, promoting Ca 2+ release from the endoplasmic reticulum which in turn leads to the activation of the calcium-and calmodulin-activated serine/threonine phosphatase Calcineurin.Calcineurin promotes IL-2 gene transcription through the activation of the NFAT transcription factors family.On the right, the activated mTOR pathway leads to phosphorylation of STAT3 at S727 and Y705 and to the activation of p70 S6 kinase at T389 and downstream S6 Ribosomal Protein at S235/236.Activation of mTORC1 is boosted by a positive feedback loop with p70 S6 kinase.* p<0.05, ** p<0.01, *** p<0.001.