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The impact of cytotoxic chemotherapy on somatic mutation in normal human cells


Type

Thesis

Change log

Authors

Dunstone, Eleanor 

Abstract

Since their advent in the mid-twentieth century, cytotoxic chemotherapy drugs have been used to treat hundreds of millions of people with cancer. These drugs remain the most effective form of treatment in many cases, with over half of newly-diagnosed patients requiring chemotherapeutic intervention. Unfortunately, a small proportion of cancer survivors will go on to develop second tumours as a result of the treatment they received for their first diagnosis. These tumours are usually genetically unrelated to the patient’s first cancer, suggesting that cytotoxic chemotherapy may increase the chance of normal cells becoming malignant.

Many chemotherapy drugs are thought to exert their effects on tumours by inducing DNA damage, which can generate somatic mutations in any surviving tumour cells. However, the systemic nature of many treatments means that the patient’s normal cells are also at risk of chemotherapy-induced mutagenesis. Patterns of mutations or ‘mutational signatures’ associated with chemotherapy have been seen in patient tumours, in cells exposed to drugs in vitro, and in some normal tissue samples from cancer patients. However, a comprehensive study of the impact of chemotherapy on the somatic mutational landscape of a wide range of normal human tissues has not yet been performed. In this project, I investigate the mutational impact of cytotoxic chemotherapy using two complementary approaches: the identification of chemotherapy-associated mutational signatures in vitro using organoid models; and the sequencing of samples of a wide range of tissue types from chemotherapy-treated patients. This work was enabled by recent developments in highly error-corrected duplex sequencing approaches, facilitating accurate detection of mutations at single molecule level.

The results of this study show that many widely-used chemotherapy drugs are mutagenic in normal cells, generating distinctive patterns of single-base substitutions (SBS), doublet-base substitutions (DBS), and small insertions and deletions (indels). Treatment of normal human cells with chemotherapy drugs and environmental agents in vitro demonstrated mutational signatures associated with a wide range of agents. Alkylating and platinum-based agents were the most mutagenic classes of chemotherapeutics, with all 13 alkylating agent drugs and all four platinum-based drugs tested generating SBS signatures. Many alkylating agents and all platinum-based agents also generated an increase in DBS and indels. Previously undescribed signatures discovered include SBS signatures associated with mitomycin C, lomustine/carmustine, busulfan and thiotepa, alongside a DBS signature of mitomycin C and an indel signature associated with a broad range of platinum-based and alkylating agents. Additionally, indel signatures of topoisomerase II inhibitors and bleomycin were described for the first time. The antimetabolite drugs tested generally did not show significant mutagenesis when applied as single treatments; however, a previously-observed signature associated with 5-fluorouracil was recovered when samples were exposed to repeated treatments.

The in vitro studies also highlighted the complex relationships between compound concentration, cytotoxicity and mutagenicity, with many compounds showing non-linear dose responses or differing relationships between dose and mutagenicity between different organoid tissues-of-origin. Treatment- associated mutagenesis was also shaped by DNA repair, demonstrated by investigating the relationship between in vitro temozolomide-induced mutagenesis and the activity of the DNA repair enzyme O-6- methylguanine-DNA methyltransferase (MGMT). Temozolomide exposure was shown to generate ten-fold higher SBS burdens in MGMT-knockout human induced pluripotent stem cells (hiPSCs) than in MGMT-wild-type hiPSCs, showing a different mutational signature depending on MGMT status.

The sequencing of normal tissue samples from chemotherapy-treated patients showed that many of these drugs are also mutagenic in vivo, with eight of the signatures identified in vitro being observed in patient samples. Many alkylating and platinum-based agents were shown to have a major impact on SBS burden in normal human tissue samples, with some patients carrying several times as many mutations as would be expected for a person of their age. High excess SBS burdens are seen across many tissue types, including those composed predominantly of post-mitotic cells such as cardiac muscle, skeletal muscle and the neuron-rich cerebellar granular layer, which have historically been considered not to acquire substantial numbers of somatic mutations during adult life. These burdens are associated with signatures of platinum-based drugs, thiotepa, temozolomide and other alkylating agents. Additionally, DBS signatures associated with platinum-based agents and mitomycin C were observed across many normal human tissue samples, as were indel signatures associated with thiotepa and other cross-linking/alkylating agents. Conversely, some agents that generated signatures in vitro appeared not to be mutagenic in vivo, including 5-fluorouracil/capecitabine and topoisomerase II inhibitors. Alongside the requirement for repeated treatment in vitro, this suggests that antimetabolite- associated mutagenesis may be restricted to dividing cells, suggesting that mechanism of action influences the capacity of different drugs to generate mutations in normal cells.

The prevalence of high chemotherapy-associated mutation burdens in normal tissues from cancer patients presents one possible factor contributing to the increased risk of second tumour development in chemotherapy-treated survivors of cancer, and the widespread DNA damage observed in post-mitotic tissues may inform research into other long-term side effects of chemotherapy treatment such as cardiac and neurological dysfunction. The work also generates insights into the fundamental mechanisms of mutation in human cells and provides a compendium of signatures of exposures to chemotherapy drugs and other environmental mutagens, facilitating future efforts to further characterise the distribution of these mutational patterns in different patients and tissue types.

Description

Date

2023-07-01

Advisors

Stratton, Michael
Campbell, Peter

Keywords

Carcinogen, Chemotherapy, Genomics, Mutagen, Somatic mutation

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge