Genetic Determinants and Evolutionary Trajectories of Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL), the most common adult B cell malignancy, is known to have a variable disease course and multiple modes of therapeutic resistance. Recent large-scale sequencing efforts have uncovered numerous putative CLL drivers, many of which have yet to be functionally characterized. Further, serial sequencing of tumor samples has revealed a complex interplay of clonal dynamics that dictate rate of progression and drug response. This dissertation applies two approaches towards characterizing these intricate networks: 1) a gene-directed approach, whereby a specific gene is perturbed, and its cellular impact measured; and 2) a systems-based approach, whereby cellular responses to a selective pressure are systematically characterized. Amongst the novel putative drivers identified in CLL is RPS15, a ribosomal protein that is recurrently mutated in patients and associates with poor prognosis and relapse. In generating RPS15-mutant cell lines and conditional knock-in mouse models, and through analysis of primary patient samples, we identified key alterations induced by RPS15 mutation in ribosome assembly, mRNA translation, and B cell proliferation. Our findings suggest that RPS15 mutation, alone and in the presence of other CLL drivers, can drive leukemogenesis. To systematically analyze the evolutionary trajectories of a CLL model, we developed a novel functionalized lineage tracing approach, ClonMapper, that integrates DNA barcoding with single-cell RNA-sequencing and clonal isolation to characterize thousands of clones within a mixed population. In applying this approach, we demonstrated that CLL clones survive therapy by relying on pre-existing gene expression states that confer differential drug tolerance; that Wnt, NOTCH and CXCR4 signaling is associated with greater drug tolerance; and that inter-cellular interactions can mediate long-term clonal equilibrium within a heterogeneous population. Further, we observed remarkable genetic heterogeneity and the persistence of unique transcriptomic signatures amongst clones throughout treatment exposure. By developing innovative gene-directed and systematic approaches towards characterizing disease mechanisms, we uncovered novel facets of the complex interplay between genetic and transcriptomic signatures in multi-clonal disease. We show that as our technical capabilities improve, our biological insights become more nuanced - enabling our ability to develop more effective therapies towards CLL and cancer as a whole.