Systems Approaches to Understanding the Impact of Cell-Cell Fusion on Therapeutic Resistance

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PROJECT SUMMARY Ploidy altering mechanisms such as cell-cell fusion and whole genome doubling (WGD) can drive intratumoral heterogeneity and alter chemotherapeutic response. Understanding the causes and consequences of these mechanisms is especially important in triple negative breast cancer (TNBC) which accounts for 15-20% of diagnosed breast cancers and does not have targeted treatment options. Broad spectrum chemotherapies remain the standard of care for TNBC, but their efficacy is dampened by high cellular heterogeneity and 40% of treated TNBC patients will relapse. We hypothesize that ploidy altering mechanisms are a driving force behind intratumoral heterogeneity and can function as active escape or damage recovery mechanisms to allow cells to resist chemotherapy. An updated version of the ClonMapper barcoding system which includes nucleotide- indexed expressed DNA barcodes on GFP and mCherry will be used to enable parallel tracking of cell-cell fusion events in fluorescent live-cell imaging and determination of detailed transcriptomic and karyotypic clonal trajectories in ploidy-separated longitudinal single-cell sequencing. The ClonMapper expressed DNA barcodes will be added to TNBC cell lines and patient-derived cell cultures. In a series of controlled experiments we will (1) determine the rates and distinguish the effects of pre-existing aneuploidy and therapy-induced aneuploidy in clinically relevant TNBC models across different chemotherapeutic treatments, (2) follow subclonal transcriptomic and karyotypic trajectories in scRNA-seq and scDNA-seq under normal and chemotherapeutic conditions to determine if different chemotherapies select for or generate specific transcriptomic or karyotypic patterns, and (3) elucidate the molecular factors which activate cell-cell fusion, WGD, or other ploidy altering mechanisms. The results of these experiments will be rich in longitudinal single cell data on transcriptomic and chromosomal transitions utilized by TNBC cells across chemotherapeutic perturbation and recovery. As the degree of tumor aneuploidy can be easily determined from patient samples, we will experimentally parameterize and validate an agent-based model which includes aneuploidy fraction, spontaneous and stress-induced mechanisms of ploidy alteration, and genomic stability to predict tumor evolution under different therapeutic schedules. This study will be the first to systematically investigate spontaneous and chemotherapy-induced mechanisms of ploidy alteration in a longitudinal single-cell framework. In elucidating the cellular states which predispose cancer cells for ploidy alteration, identifying the phenotypic and chromosomal transitions induced by these events, and quantifying the effects of pre-existing and de novo generated aneuploidy on chemotherapeutic resistance, the causes and consequences of cell-cell fusion and WGD may be revealed as primary contributors to chemoresistance and inspire novel treatment strategies to improve TNBC patient outcomes.