Defining the role of ribosomal alterations in T-cell acute lymphoblastic leukemia

Background T-cell Acute Lymphoblastic Leukemia (T-ALL) development can be driven by different genetic lesions; among these, somatic mutations in genes encoding for ribosomal proteins (RPs), mainly RPL5 and RPL10, are found with a frequency close to 10%. Several studies suggest that in T-ALL the process of ribosome biogenesis is potentially altered at many different levels, and that translation deregulation may play an essential role in its development. Importantly, it has been demonstrated both in physiology and in pathology, that sub-populations of ribosomes, characterized by diverse compositions, may co-exist within the same cell, and that they can modulate the translation of specific transcripts, thereby adding a new level of gene expression control. Hypothesis Altogether these observations suggest the hypothesis that alterations of the translational apparatus and, in particular, of the ribosome itself, may play an important functional role in supporting the neoplastic phenotype of T-ALL. Aims To test our hypothesis we have organized our project in different aims, that will be completed in a 5-year time period. First, we will characterize the ribosomes in cell-line models of T-ALL, both structurally (i.e. presence of the mutant protein) and functionally. Second, we will characterize the mutations affecting ribosome biogenesis in T-ALL patients. Third, we will analyze the alterations of translational targets in T-ALL cell lines, to understand how mutations in RPL5 and RPL10 can condition the cellular translatome. Then, we will test the efficacy of different compounds targeting the translational machinery, both in cell line models of T-ALL and in patients' derived cells. Finally, we will generate iPS from RPL5 and RPL10 mutants TALL cells to have a model that can be used to study the impact of these mutations on tumorigenesis. The last two aims will be pursued during the last two years, upon funding confirmation. Experimental Design In task 1 we will look for the presence of mutant RPL5 and RPL10 in ribosomes highly purified from T-ALL cell lines, by Mass Spectrometry. Furthermore, we will functionally test the same ribosomes, by a novel cell-free translation assay recently set-up in our lab. In task 2, we will perform whole-exome sequencing of a group (40-60) of pediatric T-ALL patients, and study the molecular and functional features of the ribosomes highly purified from those cases with RPL5/10 mutations. In task 3, we will study, by ribosome profiling, how the translatome may change in a T-ALL model of RPL5 and RPL10 mutations. In task 4, we will screen several compounds targeting the translational apparatus for their efficacy in T-ALL, both in cell lines and ex-vivo. In task 5, we will generate iPS starting from T-ALL cells carrying the RPL5/10 mutations, to generate a model useful to study the impact of these mutations on tumorigenesis. Expected Results We expect to understand how the somatic RP mutations frequently found in RPL5 and RPL10 in pediatric T-ALL can contribute to drive leukemogenesis.