Nuclear Factor of Activated T cells signaling as a key regulator of glucocorticoid resistance in pediatric T leukemias

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display glucocorticoid (GC) treatment resistance. These patients, classified as Prednisone Poor Responders (PPR), have poorer outcome compared to the other T-ALL patients receiving a high-risk adapted therapy. Since GC are administered to ALL patients during all the different therapy phases, GC resistance represents an important challenge to be addressed in order to improve PPR patients outcome. Mechanisms underlying GC resistance are not yet fully unraveled, however form our previous research we demonstrated that in PPR patients the kinase LCK is hyperactivated and its targeting with Dasatinib, in combination with Dexamethasone, is able to revert GC resistance both in vitro and in vivo. Our findings also revealed that LCK in PPR T-ALL cells controls the Calcineurin/NFAT signaling activation. Since we already demonstrated that the LCK/Calcineurin/NFAT pathway contributes to GC resistance, we believe that focusing on differentially regulated NFAT targets between GC resistant and sensitive T-ALL cells will unravel molecular mechanisms responsible of GC resistance. This will be also key to identify early GC resistance biomarkers that could be easily translated into clinics to promptly identify and efficiently treat GC resistant patients. The first aim is to deeply characterize molecular mechanisms accounting for NFAT-induced GC resistance in T-ALL by integrating ChIP-Seq, Gene Expression Profiling (GEP) and Reverse Phase Protein Arrays (RPPA) data. The second aim is to identify an early biomarker of GC resistance, already detectable at diagnosis, to propose an early phase clinical trial considering the Dexamethasone+Dasatinib treatment from the first day of therapy for resistant patients identified by biomarker expression. By ChIP-Seq/GEP data integration we will identify the differentially regulated NFAT DNA-binding sites and genes in GC resistant T-ALL cell lines and patients compared to the sensitive ones. Next, by RPPA and validation experiments we will further profile the activation state of signaling pathways correlated with identified genes to assess their functional role in GC resistance. By (phospho-)flow cytometry we will test the putative GC resistance biomarker in T-ALL samples and, in order to confirm its prognostic value, we will treat Patient-Derived Xenograft (PDX) mice with Dexamethasone+Dasatinib selecting PDX according to the new biomarker expression. This approach will also provide a therapy stratification criteria already at diagnosis to be included in a future clinical trial. From the accomplishment of this project we expect to identify which NFAT family members are primarily involved in GC resistance and which/how NFAT target genes/pathways are responsible of this unresponsiveness. We also expect to identify the best diagnostic GC resistance biomarker and to validate the Dexamethasone+Dasatinib treatment in a suitable number of PDX mouse models selected according to the biomarker expression. Results from this project will not only unravel important mechanisms responsible of therapy resistance, but will also implement patients stratification strategy by providing new drug resistance molecular markers that can be easily introduced in current diagnostic protocols. Moreover, this will point out a more specific therapeutic approach based on the introduction of new drugs that will be particularly relevant to improve GC resistant T-ALL patients outcome.