Mechanisms of CNS infiltration in T-cell leukemia: a molecular study of zebrafish and mouse models

In 1970s, the introduction of presymptomatic central nervous system (CNS) therapy changed the prognosis of pediatic Acute Lymphoblastic Leukemia (ALL). Before that, more than half of complete remissions obtained with systemic chemotherapy experienced CNS relapse. The contemporary use of risk-directed treatment has improved 5-year event-free survival to rates about 80% for childhood ALL. However, still CNS relapse remains an important cause of mortality occurring in about 3-8% of ALL patients. Recent clinical trials focus in reducing CNS relapse with the use of individualized therapy that can avoid both over- and under-treatment. Moreover, several studies aim to minimize secondary negative effects related to use of cranial irradiation, a treatment still recommended for ALL children at high risk of CNS relapse (i.e: patients with CNS leukemia at diagnosis or T-immuniphenotype with high White Blood Cell (WBC) count). This project aims to deep insight into the mechanisms used by blast cells to infiltrate the CNS. We focused on T-ALL as in this leukemia the phenomenon of CNS disease is more recurrent than in B-ALL. To identify conserved molecular mechanisms that could be at the basis of CNS infiltration we started the analysis on animal models to move subsequently to human patients. To goal in this aim we used a gene expression profiling (GEP) approach. Chapter 1 reports a study performed on two zebrafish models both developing T-ALL but that are genetically different; hMYC-ER line overexpresses human C-MYC, while hlk model carries an unknown mutation. We demonstrate for the first time that both these zebrafish model can mimic the phenomenon of CNS disease in T-ALL. Moreover, we found a different predisposition for hMYC-ER and hlk cancers to infiltrate the CNS. A whole transcriptome analysis of hMYC-ER and hlk T-ALL helped us to identify different molecular mechanisms that could control motility of blast cells. Particularly, we identified on cxcr4/cxcl12 axis, an important mechanisms that could predispose T-lymphoblasts to infiltrate the CNS environment. In fact, we found a positive correlation between cxcr4 expression levels and seriousness of CNS infiltration. The importance of CXCR4/CXCL12 axis in determining extramedullary infiltration is still debated, as studies on pediatric ALL patients reveal conflicting results. In Chapter 2, we investigated the association of CXCR4/CXCR4 expression and CNS infiltration, using murine models xenoengrafted with human patients T-ALL cells. Also in this animal model we found diverse degree of CNS infiltration on xenografted mice derived by different human patients. Preliminary results seem to confirm that higher levels of CXCR4 expression favour the migration of T-lmphoblasts towards the CNS environment. Further analyses of validation are ongoing on a larger cohort. In Chapter 3 we switch to study the phenomenon of CNS infiltration on ALL patients. GEP analysis was used to compare patients with (CNS+) or without (CNS-) disease. However, this approach on human patients failed to find a strong signature that could identify CNS+ and CNS- patients. This result underlines the difficulty to study this phenomenon directly on human patients, as a high heterogeneity is present inside both T- and B-ALL group. Moreover, we could not find an association between CXCR4 expression and CNS infiltration in a T-ALL cohort and that stress the idea that blasts cells can use different mechanisms to infiltrate the CNS and the high expression of CXCR4 might be a predisposing factor that requires interaction with others mechanisms. T-test analysis performed on zebrafish gene expression results revealed a significant different gene expression profile existing inside the zebrafish group overexpressing hCMYC (hMYC-ER). This observation challenges us to clarify the importance and the role of C-MYC oncogene in T-ALL leukemogenesis. In Chapter 4 we were able to find a strong signature insight the group of patients with high expression of C-MYC that can discriminate patients with- and without- C-MYC rearrangements. In both Chapter 4 and 5 we tested the ability of gene expression profiling approach in identifying signature within specific subgroup of T-ALL patients. In fact also in Chapter 5 we showed the presence of a gene expression profile that characterize patients carrying MLLT10 rearrangements inside the HOXA category of pediatric T-ALL