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420 The Transcription Factors NFATc1 and NFATc2 Control Glucocorticoid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Targeted Therapy in Lymphoid Leukemias
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Translational Research, Combination therapy, Diseases, Therapies, Lymphoid Malignancies
Sunday, December 10, 2023: 10:45 AM

Giulia Veltri, PhD1,2*, Alberto Peloso1,2*, Alice Cani, PhD3*, Elena Mariotto1,2*, Giulia Borile, PhD3*, Loris Russo4*, Chiara Frasson1,2*, Matilde Cescon4*, Egidio Iorio5*, Sonia Minuzzo6*, Panagiotis Ntziachristos, PhD7, Alessandra Biffi, MD, PhD1,8*, Barbara Buldini, MD, PhD1,9, Martina Pigazzi, PhD3,8*, Stefano Indraccolo, MD6,10*, Silvia Bresolin, PhD1,2* and Valentina Serafin, PhD1

1Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
2Division of Pediatric Hematology, Oncology and Stem Cell Transplant, Maternal and Child Health Department, University of Padova – Italy, University of Padua, Padova, Italy
3Foundation Istituto Ricerca Pediatrica (IRP), Padova, Italy
4Department of Molecular Medicine, University of Padova, Padova – Italy, University of Padua, Padova, Italy
5High Resolution NMR Unit Core Facilities, Istituto Superiore Di Sanità, Roma, Italy, Roma, Italy
6Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padova, Italy
7Ghent University, Corneel Heymanslaan 10, Ghent, Belgium
8Department of Women's and Children's Health, Onco-hematology lab and clinic, University of Padova, Padova, Italy
9Department of Pediatric Hematology and Oncology, University of Padova, Padua, Italy
10Istituto Oncologico Veneto - IRCCS, Padua, PD, ITA

Background: The overall survival of pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is significantly improved (˜75-80%) with the current therapeutic approaches. Nevertheless, for those patients that do not respond to conventional treatment and experience relapse, the prognosis is extremely poor. In this context, it is well defined that resistance to glucocorticoids (GCs), pillar drugs in the treatment protocol, can predispose pediatric T-ALL patients to a poor outcome. A pivotal role of LCK kinase has been elucidated in supporting GC resistance in T-ALL cells in our previous study and recently confirmed by other colleagues. However, to date, the biological processes modulated by LCK in this context are not yet defined.

Aim: the identification of new mechanisms underlying GC resistance can lead to alternative therapeutic approaches to prevent or overcome GC resistance and ameliorate the outcome of this subgroup of patients.

Methods and Results: we uncovered the involvement of NFATc1 and NFATc2 transcription factors, that act downstream LCK kinase, in guiding GC resistance in T-ALL cells. Of note, a high NFATc1 and NFATc2 transcriptional activity characterizes pediatric GC resistant T-ALL patients at diagnosis, that in turns show a low Glucocorticoid Receptor (GR) activity. In agreement, NFATc1 or NFATc2 specific gene silencing in 3 T-ALL GC resistant cell line models and primary cells increases dexamethasone response, by restoring GR canonical transcriptional activity through the increase expression of BIM GR target gene (p value <0.05). Conversely, NFATc1 or NFATc2 overexpression in a murine T-ALL GC sensitive cell line confers resistance to dexamethasone treatment. Interestingly, by Gene Expression Profile (GEP) and Nuclear Magnetic Resonance (NMR) analysis, we observed that NFATc1 gene silencing in GC resistant cells significantly downregulates intracellular cholesterol abundance. Additionally, by Chromatin Immunoprecipitation (ChIP) we revealed that NFATc1 can directly control the transcription of HMGCS1, EBP and DHCR7, key enzymes of cholesterol biosynthesis process (n≥3, p value <0.05 for all the three genes). In agreement, exogenous cholesterol addition to NFATc1 knock-down cell lines rebuild dexamethasone resistance. Conversely, by GEP and flow cytometry analysis we observed that NFATc2 gene silencing in T-ALL GC resistant cells leads to a downregulation of the Wnt/β-catenin signaling pathway and to an increased T-cell differentiation. Furthermore, by ChIP analysis we revealed that in T-ALL GC resistant cells NFATc2 can directly affect the transcription of LRP6, a key Wnt/β-catenin signaling component (n=3, p value <0.05). In agreement, the Wnt/β-catenin signaling activation, by Wnt3a stimulation, restores dexamethasone resistance in NFATc2 knock-down T-ALL cells. Finally, we observed that the inhibition of cholesterol biosynthesis by simvastatin or of Wnt/β-catenin by PRI-724 increases GC sensitivity in T-ALL GC resistant cells by the High-Throughput drug synergism Screening (HTS) and the Highest Single Agent (HSA) approach.

Conclusions: Overall, we revealed for the first time the involvement of NFATc1 and NFATc2 transcription factors in supporting GC resistance in T-ALL cells by the modulation of cholesterol biosynthesis and Wnt/β-catenin signaling, both processes well-described in sustaining chemotherapy resistance, paving the rationale to alternative therapeutic options for T-ALL GC resistant pediatric patients.

Disclosures: No relevant conflicts of interest to declare.

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