Het karakteriseren van de complexiteit van acute lymfatische leukemie: inzichten uit single-cell genomics en transcriptomics

The most predominant type of cancer affecting young children is acute lymphoblastic leukemia (ALL), which arises from uncontrolled proliferation of developing B or T cells. Despite the overall survival rate of children now generally surpassing 90%, adults and patients with relapsed or refractory disease still face an unfavourable prognosis. In addition, current treatment protocols rely on intensive chemotherapy which is accompanied by significant side effects. Extensive whole-genome sequencing efforts have uncovered a wide variety of mutations in patients with ALL, underscoring the inherent heterogeneity of the disease. Still, there remains a pressing need for an improved understanding of the underlying disease mechanisms. Advancements in single-cell sequencing technology are now enabling researchers to obtain incredible high-resolution insights into cancer biology. In this work, we aimed to characterize the clonal dynamics of a cohort of 12 B-ALL patients at single-cell resolution. A custom amplicon library was used to evaluate somatic mutations in mutational hotspot regions and demonstrated that patients with PAX5 alterations or hyperdiploid karyotype displayed a larger number of subclones with different mutations, often in small abundance. Studying additional samples during the first weeks of chemotherapy treatment revealed that these subclones dissappeared with varying kinetics and small numbers of leukemic cells were detectable at the end of induction treatment. In addition, single-cell CRISPR screening in primary pro-T cells enabled us to interrogate the effects of gene pertubation on transcriptional regulation and cell proliferation in T-ALL. Various recurrent perturbations were clustered based on their effect on STAT, NOTCH, E2F and MYC signaling pathways. In particular, CRISPR screening identified Bcl11b as a potent tumor suppressor gene in immature T cells, associated with deregulated inflammatory signaling and JAK/STAT hyperactivation. In summary, our findings demonstrate a significant degree of clonal heterogeneity in B-ALL, with potential implications regarding treatment resistance and relapse risk. Moreover, we showed single-cell CRISPR screening to be a valuable tool for characterizing the mechanisms of transcriptional deregulation driven by recurrent genetic alterations in T-ALL. Together, this work sought to offer fundamental insights in the biology of ALL, ultimately aiming to contribute to improved risk stratification and better patient outcomes.