Early T-cell Precursor ALL (ETP-ALL) is a subgroup of acute T leukemias with a peculiar immunophenotypic profile. Patients display a poor early response to conventional induction treatment, but an outcome after intensified therapy not worse than the other T-ALL. ETP-ALL was extensively characterized from a genetic point of view in the paper published by Zhang et al in 2012, but information about proteomic aberrancies is still lacking. In this study we profiled, by Reverse Phase Protein Arrays, the activation and expression of a considerable number of proteins in a significative cohort of 16 pediatric patients affected by ETP-ALL at diagnosis. We compared protein activation in these patients versus a group of 62 other T-ALL pediatric patients. Phosphoprotemic data were also related to mutational status of ETP-ALL patients. Our most interesting findings concern the hyperactivation of the LCK/Calcineurin and mTOR/STAT3 signaling pathways in ETP-ALL patients. LCK, despite of a lower expression, resulted strikingly boosted in these patients with a concomitant overexpression of the downstream target Calcineurin. The LCK/Calcineurin axis, followed by the activation of the NFAT family of transcription factors, was already demonstrated to be critical for T cell leukemogenesis. We also observed a possible feedback loop which could sustain mTOR activation through p70 S6Kinase hyperphosphorylation. mTOR, in turn, phosphorylates and activates STAT3, counteracting cell differentiation. The mTOR/STAT3 axis activity thus could sustain cell proliferation and survival while maintaining the markedly undifferentiated phenotype typical of ETP-ALL. Interestingly, the activation of the mTOR/STAT3 pathways is not influenced by the presence of FLT3 or PTEN mutations. Our paper also reports in ETP-ALL patients the hyperactivation of the RAS/MAPK and the JAK/STAT signaling pathways, that is not dependant from BRAF, NRAS, IL7R, JAK1 or JAK3 somatic mutations.\ud In conclusion, our study is the first one investigating the activation of a such number of proteins in a wide cohort of pediatric ETP-ALL patients. We identified the specific activation of signaling pathways, such as the LCK/Calcineurin and the mTOR/STAT3 ones, important for T cell leukemogenesis and for the support of cancer cell viability and block of differentiation. Our results will help to explain the mechanism responsible of the poor response to conventional treatment of these patients, and might also suggest new druggable targets for personalized and less toxic therapies

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for “Differentially Expressed Genes” (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2, TMA7, SPCS2, CDCP1, CAPZA1, FGFR1OP2, MAN1A2, NT5C3A and SRP54. In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.

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Early T-cell precursor (ETP) is an aggressive form of acute lymphoblastic leukemia (ALL), associated with high risk of relapse. This leukemia subtype shows a higher prevalence of mutations, typically associated with acute myeloid leukemia (AML), including RAS and FLT3 mutations. FLT3-ITD was identified in 35% cases of adult ETP-ALL, but data in the pediatric counterpart are lacking. ETPs frequently lack immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, used for minimal residual disease (MRD) monitoring. Among 718 T-ALL enrolled in Italy into AIEOP-BFM-ALL2000, AIEOP-ALLR2006, and AIEOP-BFM-ALL2009 consecutive protocols, 86 patients (12%) were identified as ETP and 77 out of 86 children were studied for the presence of FLT3-ITD. A total of 10 out of 77 (13%) ETP cases were FLT3-ITD positive. IG/TR MRD monitoring was feasible only in four cases. FLT3-ITD MRD monitoring was performed using real-time PCR in all FLT3-ITD positive ETP cases. A comparison between IG/TR and FLT3-ITD resulted in comparable findings. Our study demonstrated that the FLT3-ITD prevalence in children was lower (13%) than that reported in adult ETP-ALL. FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL when IG/TR markers are not available, potentially selecting those patients who should spare allogeneic hematopoietic stem cell transplantation (HSCT). Finally, the FLT3 pathway is a robust druggable target in this aggressive form of leukemia.