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Kabeer, Farhia

University of British Columbia

Cancer is an ecosystem of genetically diverse evolving clones, which emerge in time and space as a result of genomic and non-genomic instability. Consequently, clonal evolution provides a basis for fluctuating cell fitness, which impacts etiology and drug resistance. However, progress in defining clonal fitness by copy number alterations (CNA), has been impeded by a lack of perturbation experiments with timeseries sampling. This dissertation is focused on understanding and measuring clonal fitness determined by copy number genomic instability in breast cancer to single cell resolution. First, I established a series of transplantable human breast cancers in immunodeficient mice strains that were tolerant to DNA damaging chemotherapy. I selected four PDXs (patient derived xenografts) for detailed analysis of clonal fitness under no treatment and after treatment with DNA damaging agents, cisplatin and CX5461. Subsequently, I optimized methods of tumor dissociations, and determined the effects of digestion time and temperature on single cell gene expression. Next, I established that single cell whole genome sequencing of CNA in PDX passaged over time resulted in positive fitness over clones that gradually sweep the population. I established a mixture re-transplant paradigm to demonstrate that the fitness predictions of a Wright-Fisher population genetics model applied to the data, were reproducible experimentally. I described that fitness landscapes of all TNBC tumors were inverted under drug because low fitness clones under no treatment gave rise to drug resistant clones. Drug holiday experiments showed that cisplatin resistance had a fitness cost. I demonstrated using CX5461, that drug resistance could arise in a common background, suggesting common drug resistant states. Finally, I examined the impact of CNA on transcriptional phenotypes. By making assignments of single cells RNA-seq, to CNA defined clones, I observed that in cis, CNA mediated clonal gene expression impacts between 5-50% of the transcriptome. Time series analysis of expression revealed reversible, but time dependent expression reprogramming, thus defining the non-CNA dependence of clonal transcriptomes. Pathway analysis revealed common sets of pathways associated with late drug resistance to platinum in TNBC. These comprehensive measurements and analysis of clonal structure will advance interpretation of polyclonal resistance to therapy.

Text

2021-06-07

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/78546

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/