Mapping the pathways to PARP inhibitor resistance by single cell sequencing

Targeted chemotherapeutics show great promise for treating cancer with minimal side effects, however these agents have substantial problems with the development of drug resistance. The tendency for rapid relapse with a new, drug resistant cancer limits the application of these agents, and the mechanisms of drug resistance are therefore of substantial interest. In many cases it is not clear how drug resistant cells arise; these may be pre-existing random mutants that emerge under drug selection, but the extreme stress placed on cancer cells during extended drug treatment can also give rise to novel mutations. If treatment-induced mutations are important in creating drug resistance, this would offer attractive pathways for combination therapies that both target the cancer cells and the mechanisms underlying drug-induced mutation. Unfortunately, the pathways by which novel mutations arise under drug stress remain largely uncharacterized.

The genetic and epigenetic changes that occur during extended drug stress are hard to study as the cell populations are highly heterogeneous, however recent developments in single cell technologies allow the determination of gene expression, genetic and epigenetic states simultaneous in many individual cells. Here, in collaboration with Artios Pharma, we will apply single cell technologies to study heterogeneous cell populations under long-term treatment with PARP inhibitors to provide new insights into the mutational pathways acting during chemotherapy.

Aim
To determine the extent to which PARP inhibition causes novel mutations which confer PARP inhibitor resistance.

Objectives
- Determine the spectrum of genome rearrangements caused by PARP inhibition in BRCA deficient cancer cells
- Understand the role of Pol theta as an instigator of genome heterogeneity
- Elucidate the gene expression determinants required for survival of PARP inhibitors.

Hypothesis
Cells that survive PARP inhibition will have been subject to mutagenesis resulting in substantial genomic rearrangements and copy number variation. This may be the Achilles Heel of therapies based on PARP inhibition due to the massively increased chance of novel resistance mutations arising. We hypothesise that the mutational spectrum will be highly non-random because sites of replication stress are also non-random, leading to hotspots of copy number variation at a subset of loci. Importantly, mutations arising through defined pathways such as microhomology-mediated repair by pol theta should be preventable. Furthermore, initial survival of PARP inhibitor treatment is required for cells to regain proliferation through resistance mutations, providing another route to preventing the emergence of PARP inhibitor-resistant clones.

Experimental plan
Initial characterization of the PARP treated cell population will involve mammalian cell culture and drug treatment assays. Flow cytometry will be used to characterize sub-populations in terms of cell cycle dynamics and long term survival.
Single cell genome re-sequencing will be applied to these heterogenous populations at various times during drug treatment to determine the extent to which PARP inhibition drives genome rearrangements. Combination studies will be performed with inhibitors of pol theta, an enzyme important for double strand break repair.
Population and single-cell RNAseq will then be applied to understand the transcriptional changes required for initial survival in the presence of PARP inhibitors and the transition from drug tolerance to drug resistance.

This project will involve lab work with an emphasis on high through-put techniques and single cell methods, combined with a substantial bioinformatic component required for the analysis of single-cell data.