Does inappropriate DNA replication provide a mechanism for the de novo acquisition of drug resistance?

Abstract

EGFR is the most common driver mutation in non-small cell lung cancer (NSCLC) and is a common drug target across several cancers. Targeted therapeutics such as those that target EGFR (e.g. Osimertinib) have brought significant patient benefit, however ultimately patients tumours develop resistance to targeted treatments. Initial tumour response followed by a period of dormancy before progressing in patients treated by targeted therapies has been modelled in vitro through the drug tolerant persister (DTP) phenotype system. It Is vitally important that the underlying mechanisms by which cancer DTP cells gain resistance mutations/mechanisms to targeted treatments is better understood to aid the further development of therapeutics and the directed design of highly effective combination treatments. This work initially investigates the ability of DTP cells to undergo DNA replication across multiple in vitro NSCLC DTP model systems. Interestingly, all DTP cells were able to undergo DNA replication rather than a specific subpopulation, and a large proportion of DTP cells are undergoing DNA replication at a given time. This ability of DTP cells to undergo DNA replication under targeted treatment appears to be necessary for the survival of the DTP culture. Through collaboration with AstraZeneca, I then investigated the effect of several combination therapies on the cell number, replicative fraction, and DNA damage repair (DDR) markers in multiple in vitro NSCLC DTP model systems. Osimertinib treatment led to the emergence of a subset of cells with a high DDR marker burden, which was further heightened when combined with PARP inhibitors. Notably, combining Osimertinib with either Palbociclib (a CDK4/6 inhibitor) or AZD1390 (an ATM inhibitor) enhanced cell killing beyond single-agent Osimertinib, with only the Palbociclib combination reducing the replicative fraction of DTP cells. I then explored the fate of DTP cells. Interestingly, although they complete the cell cycle prolonged exposure increased the likelihood of daughter cell death and was associated with a three fold rise in aberrant mitoses. I then investigated the replicative fitness and their transcriptomic landscape of DTP cells undergoing DNA replication while under targeted treatment. Interestingly, DNA replication initiation becomes dysregulated in DTP cells with replication fork polarity being lost around canonical DNA replication initiation zones. The transcriptome of replicative DTP cells closely resembles that of dormant G1-phase DTP cells, characterized by a reduced demand for protein synthesis. Notably, 13 DNA replication and DDR factors remain downregulated in S-phase DTP cells compared to treatment-naïve cells. Interestingly, there is an enrichment for replication forks at highly expressed genes compared to average or lowly expressed genes in replicative DTP cells suggesting there are replication-transcription conflicts in DTP cells. Finally, I sought to investigate any role of circular DNAs in the emergence and/or maintenance of the DTP state or in the emergence of acquired resistance. Interestingly, while there were no typical cancer related ecDNAs (≥1 Mb) there were several unique CNV events across multiple resistant clones. I then developed and optimised a low cell number input method to detect rare circular DNAs and their heterogeneity across a population of cells. Interestingly, this approach identified circular DNAs of ~1-10 Kb which were 6-fold more common in DTP cells. There is a bias toward larger and more abundant circle species in DTP cells. Taken together this work highlights the value of investigating the replicative capacity and health of DTP cells under targeted treatment and exploring how inappropriate replication could give rise to acquired resistance