Acute lymphoblastic leukemia (ALL) is the most frequent cancer in children. ALL is treated by multi-drug chemotherapy, which cause serious side effects. Chemotherapy is ineffective in ~20% of children, which is related to drug resistance and results in relapse (disease recurrence). Relapsed ALL is very difficult to treat and often fatal. The majority of relapses occur in patients with T-ALL subtype (T-cell acute lymphoblastic leukemia), which is more aggressive, more heterogeneous in its (epi-)genetic background and much less understood than ALL arising from B-cells. Unlike in B-cell ALL, in T-ALL, the genetic prognostic markers of the risk of relapse are still missing. Since ALL is the most frequent childhood cancer, accurate identification of patients at high-risk of relapse, relapse prevention and treatment are one of the most important challenges in pediatric oncology. Yet, this requires an in depth understanding of the mechanisms of relapse. Based on our preliminary results and literature data from other cancers, we hypothesize that the mechanisms contributing to T-ALL relapse include: processes related to drug-resistance, cell stemness (related to biological plasticity, which enables leukemic cells to dedifferentiate and alter their dependencies), senescence-like phenotype induced by chemotherapy (related to increased survival potential after recovery from the senescence-like state), and clonal heterogeneity (one of the main drivers of clonal evolution). We aim to provide novel knowledge on the genes and biological processes, which drive the survival advantage of T-ALL cells and their evolution from diagnosis to relapse. We will use: 1/ single-cell sequencing of the transcriptome of T-ALL cells (scRNA-seq) obtained from patients’ samples at diagnosis and at relapse, and 2/ genome-wide CRISPR/Cas9 dropout screen in relapsed T-ALL cell lines. The scRNA-seq will enable us to investigate clonal heterogeneity and clonal evolution based on gene expression analysis at a single-cell resolution and to identify relapse-related transcriptomic signature. The CRISPR/Cas9 dropout screen will enable to identify genes essential for cells’ survival (the disruption of these genes by CRISPR/Cas9 cause these cells ‘dropout’ from the cell culture). Thus we will provide an overview of the genes and processes indispensable for T-ALL cells’ survival in vitro and characterize T-ALL gene dependencies. Next, we will functionally investigate several genes of potential importance for T-ALL survival and relapse (selected from our preliminary results and from the results of scRNA-seq and CRISPR/Cas9-screens). Using lentiviral transduction and CRISPR/dCas9-VPR system (for gene activation) or CRISPR/dCas9-KRAB system (for gene inhibition) in T-ALL cell lines, we will investigate, how these changes of gene expression impact the survival of T-ALL cells in vitro. We will verify gene expression changes by qRT-PCR and Western blot, and we will investigate stably modified T-ALL cell lines using several functional assays (growth competition assay, apoptosis, proliferation, and viability assays). Finally, we will use a bioinformatics approach to analyse the data on gene expression (RNA-seq) and clinical outcome (including information on relapse) available in public databases. We will aim to verify, if the altered expression of the genes (a relapse-related transcriptomic signature, we aim to identify in our study) correlates with the occurrence of T-ALL relapse in other cohorts of T-ALL patients. Such a comprehensive approach to investigate the mechanisms of relapse have not been used in T-ALL research thus far. The expected project outputs: 1/overview of genes & processes essential for T-ALL cells’ survival (gene dependencies); 2/description of genes & processes contributing to T-ALL relapse, with a functional characteristics of selected genes; 3/characteristics of clonal heterogeneity & clonal evolution in T-ALL relapse; 4/description of the contribution of cell stemness, chemotherapy-induced senescence, and drug resistance to T-ALL relapse; 5/identification of relapse-associated transcriptomic signature, which might facilitate the development of T-ALL risk classifier. The ultimate goal of the project is to unravel the mechanisms of T-ALL relapse and to pave the way towards precise identification of high risk patients, which will help to determine the best treatment options to prevent leukemia relapse and improve the survival of T-ALL patients.

Project manager

Małgorzata Dawidowska

Contractors

Roman Jaksik