Acute lymphoblastic leukemia is the most common childhood tumor and about 85% of cases are due to the expansion of a clone of B-cell precursors (BCP-ALL). Steady progress in development of effective treatments has led to an elevated rate of success in treating this disease. To date, even thought about 75% of patients are cured, 25% of cases having a relapse has a survival probability of only 30%. Of note, more than 50% of relapses concern patients not classified in high-risk groups based on assessment of prognostic factors at diagnosis or on measurement of Minimal Residual Disease (MRD), a surrogate parameter of individual response to therapy. The understanding of the molecular pathogenesis of the disease and the biological basis for explaining different clinical response represent the most relevant challenges in the field that could open new perspectives in the identification of either new prognostic factors or new molecules for targeted therapeutic approaches. In this setting we focused our studies on a poor prognosis subtype of BCP-ALL, bearing rearrangements in the Cytokine Receptor Like Factor 2 (CRLF2) gene. Alterations in the CRLF2 gene (CRLF2r) are present in about 10% of childhood BCP-ALL, 50% of Down Syndrome ALL and 50% of Ph-like ALL and are responsible of the overexpression of Thymic Stromal Lymphopoietin Receptor (TSLPR).We first studied the phenotypic expression of TSLPR and its associated molecular and phosphosignaling profile in a large and prospective cohort of patients, demonstrating that the TSLPR screening at diagnosis can be successfully performed by standardized flow cytometry protocols. We further found an activation of both JAK/STAT and PI3K/mTOR pathways in the CRLF2 rearranged patients giving the rationale to test targeted tyrosine kinase inhibitors (TKIs) to treat this subgroup of patients. Starting from these evidences we investigate the TSLPR-driven pathway by using a new high dimensional single cell approach, called mass cytometry (CyTOF), that allow the simultaneous measurement of about 50 parameters per cell. By applying this technology we studied 15 BCP-ALL (9 CRLF2r and 6 CRLF2wt) primary samples treating the cells with 3 different TKIs (Dasatinib, Ruxolitinib and BEZ235) and 2 anti-TSLPR monoclonal antibodies. We demonstrated a strong signaling inhibition with Dasatinib, Ruxolitinib and one anti-TSLPR mAb in CRLF2r BCP-ALL samples associated with a synergic in vitro efficacy of JAK/STAT inhibitors (Ruxolitinib and anti-TSLPR mAb) with Dasatinib and BEZ235 in BaF3 CRLF2/IL7Rα expressing cells. We also studied the MRD cells at Day 8 and Day 15 showing that the MRD cells of CRLF2r patients maintain their TSLPR positivity and responsiveness to both stimulation and drug treatment. Meanwhile, we also tested a histone deacetylase inhibitor, Givinostat, as a new therapeutic tool to use with conventional chemotherapy for childhood CRLF2r BCP-ALL. We demonstrated an in vitro inhibition and cell death induction of BCP-ALL CRLF2r cell lines at very low doses confirmed by a cytotoxic effect also in ex-vivo experiments where Givinostat was able to kill blast cells preserving the normal hematopoietic counterpart as demonstrated by a CyTOF analysis. In vivo Givinostat was able to markedly reduce the engraftment of leukemic blasts in the bone marrow of treated mice causing a transcriptional modulation of genes involved in JAK/STAT pathway leading to the inactivation of the signaling network. In the last part of our project we studied, in collaboration with Dr.Kara Davis at Stanford University, 60 primary diagnostic samples with known outcome in order to investigate possible features correlated with relapse. By using CyTOF we identified 8 predictors that separate patients who will relapse from whose who will not and these features suggested a high basal activation of IL-7 signaling node in late pro-B cells and poor response following pre-BCR engagement in pre-BI cells in patients going to relapse.

(2016). Cell signaling in high risk childhood B cell precursor acute lymphoblastic leukemia: high-throughput dissection and targeting strategies.. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Cell signaling in high risk childhood B cell precursor acute lymphoblastic leukemia: high-throughput dissection and targeting strategies.

SARNO, JOLANDA
2016

Abstract

Acute lymphoblastic leukemia is the most common childhood tumor and about 85% of cases are due to the expansion of a clone of B-cell precursors (BCP-ALL). Steady progress in development of effective treatments has led to an elevated rate of success in treating this disease. To date, even thought about 75% of patients are cured, 25% of cases having a relapse has a survival probability of only 30%. Of note, more than 50% of relapses concern patients not classified in high-risk groups based on assessment of prognostic factors at diagnosis or on measurement of Minimal Residual Disease (MRD), a surrogate parameter of individual response to therapy. The understanding of the molecular pathogenesis of the disease and the biological basis for explaining different clinical response represent the most relevant challenges in the field that could open new perspectives in the identification of either new prognostic factors or new molecules for targeted therapeutic approaches. In this setting we focused our studies on a poor prognosis subtype of BCP-ALL, bearing rearrangements in the Cytokine Receptor Like Factor 2 (CRLF2) gene. Alterations in the CRLF2 gene (CRLF2r) are present in about 10% of childhood BCP-ALL, 50% of Down Syndrome ALL and 50% of Ph-like ALL and are responsible of the overexpression of Thymic Stromal Lymphopoietin Receptor (TSLPR).We first studied the phenotypic expression of TSLPR and its associated molecular and phosphosignaling profile in a large and prospective cohort of patients, demonstrating that the TSLPR screening at diagnosis can be successfully performed by standardized flow cytometry protocols. We further found an activation of both JAK/STAT and PI3K/mTOR pathways in the CRLF2 rearranged patients giving the rationale to test targeted tyrosine kinase inhibitors (TKIs) to treat this subgroup of patients. Starting from these evidences we investigate the TSLPR-driven pathway by using a new high dimensional single cell approach, called mass cytometry (CyTOF), that allow the simultaneous measurement of about 50 parameters per cell. By applying this technology we studied 15 BCP-ALL (9 CRLF2r and 6 CRLF2wt) primary samples treating the cells with 3 different TKIs (Dasatinib, Ruxolitinib and BEZ235) and 2 anti-TSLPR monoclonal antibodies. We demonstrated a strong signaling inhibition with Dasatinib, Ruxolitinib and one anti-TSLPR mAb in CRLF2r BCP-ALL samples associated with a synergic in vitro efficacy of JAK/STAT inhibitors (Ruxolitinib and anti-TSLPR mAb) with Dasatinib and BEZ235 in BaF3 CRLF2/IL7Rα expressing cells. We also studied the MRD cells at Day 8 and Day 15 showing that the MRD cells of CRLF2r patients maintain their TSLPR positivity and responsiveness to both stimulation and drug treatment. Meanwhile, we also tested a histone deacetylase inhibitor, Givinostat, as a new therapeutic tool to use with conventional chemotherapy for childhood CRLF2r BCP-ALL. We demonstrated an in vitro inhibition and cell death induction of BCP-ALL CRLF2r cell lines at very low doses confirmed by a cytotoxic effect also in ex-vivo experiments where Givinostat was able to kill blast cells preserving the normal hematopoietic counterpart as demonstrated by a CyTOF analysis. In vivo Givinostat was able to markedly reduce the engraftment of leukemic blasts in the bone marrow of treated mice causing a transcriptional modulation of genes involved in JAK/STAT pathway leading to the inactivation of the signaling network. In the last part of our project we studied, in collaboration with Dr.Kara Davis at Stanford University, 60 primary diagnostic samples with known outcome in order to investigate possible features correlated with relapse. By using CyTOF we identified 8 predictors that separate patients who will relapse from whose who will not and these features suggested a high basal activation of IL-7 signaling node in late pro-B cells and poor response following pre-BCR engagement in pre-BI cells in patients going to relapse.
BIONDI, ANDREA
GAIPA, GIUSEPPE
leukemia, cell signaling, therapeutic strategies
MED/06 - ONCOLOGIA MEDICA
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
(2016). Cell signaling in high risk childhood B cell precursor acute lymphoblastic leukemia: high-throughput dissection and targeting strategies.. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).
File in questo prodotto:
File Dimensione Formato  
phd_unimib_744675.pdf

embargo fino al 30/06/2019

Descrizione: tesi dottorato
Tipologia di allegato: Doctoral thesis
Dimensione 29.27 MB
Formato Adobe PDF
29.27 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/116109
Citazioni
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
Social impact