Gene re-activation mechanisms in the context of acquired drug resistance

Abstract

In this thesis we aim to better understand the molecular mechanisms that cells employ to upregulate genes undergoing chemotherapeutic drug treatments. We use the ABCB1 gene as our model gene to study this process as its role in Taxol resistance in cell lines in culture and mice is well characterized. In Chapter 2 we demonstrate that ABCB1 upregulation is the major mechanism of acquired taxane-related drug resistance in the untransformed human retinal pigment epithelial (RPE-1) cells. In Chapter 3, we characterize the molecular mechanisms underlying ABCB1 upregulation in this cell line. We show that Nuclear Lamina (NL) interactions are required to maintain ABCB1 repressed. Perturbations in NL lead to a heterogeneous population with increased number of cells acquiring Taxol resistance. In Chapter 4, we aim to understand whether chromatin and three-dimensional genome changes, previously reported in literature upon DSB induction, could lead to upregulation of ABCB1. We also investigate whether the observed epigenetic alterations could be stably inherited and consequently contribute to Taxol resistance. Chapter 5 describes an unexpected event leading to ABCB1 gene activation following CRISPR-Cas9 genome editing. When inducing a DSB with a lentiviral-based sgRNA vector, this can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. Finally, in Chapter 6, we aim to further understand the DNA damage response in the context of chromatin. We demonstrate that CRISPR-Cas9 screens can be successfully implemented in the recently reported multiplexed reporter assay from Schep et. al to uncover the role of DDR factors on regulating repair pathway choice in the context of chromatin. Lastly, in Chapter 7 we summarize and review the results described in this thesis. While the role of ABCB1 in acquired multi drug resistance in patients is still debatable, we propose a set of novel molecular mechanisms that lead to upregulation of this gene.