Whole-genome sequencing study for the origins of mutational signatures associated with aging, repair deficiency, and cancer therapy

Abstract

The comprehensive analysis of mutational signatures in DNA repair-deficient cell lines provides fundamental insights into genomic stability, cancer progression, cancer therapy, and resistance to chemotherapeutic agents such as temozolomide (TMZ). By utilizing whole-genome sequencing (WGS) of various human cancer cell lines, this analysis demonstrates the essential role of DNA repair pathways, including mismatch repair (MMR), base excision repair (BER), and DNA polymerase theta- mediated end joining (TMEJ), in shaping the mutational landscape. This thesis consists of three chapters unraveling the relationship between mutational signatures and these fundamental insights. The first chapter investigates spontaneous mutagenesis in TK6 lymphoblast cell lines and identifies the aging- related mutational signatures SBS5 and SBS40, which are prominently detected in older patients’ cancers. Additionally, the mismatch repair deficiency (MMRd)-associated signatures SBS44 and SBS26 were observed in different MMRd cell lines, emphasizing the influence of specific MMRd conditions on mutational patterns. The second chapter focuses on the relationship between mutational patterns and resistance acquisition in TMZ. An accumulation of SBS11 mutations was observed in the MMRd cell lines. Under MMRd conditions, BER-deficient cells exhibited increased drug sensitivity. The third chapter describes the role of POLQ, a major enzyme in TMEJ, in inducing mutational signatures such as SBS3 and ID6 in BRCA-mutated cancers. Furthermore, the mutational signature of POLQ upon exposure to ionizing radiation therapy is identified in this chapter. This proposes that POLQ is a potential therapeutic target for BRCA-mutated cancers. Chapter 1 explores spontaneous mutational patterns by employing 38 isogenic DNA repair- deficient human TK6 lymphoblast cell lines. This chapter illustrates that the background mutational pattern of wild-type TK6 cells is similar to the flat mutational signatures SBS5 and SBS40. The mutational signatures SBS5 and SBS40 are associated with aging, particularly in tumor samples from older patients, suggesting that these clock-like signatures accumulate over time. REV7-defective cell lines had lower mutation numbers and a lower contribution of the SBS40 signature. This result indicates that aging-associated mutagenesis is mainly caused by polymerase zeta-mediated translesion synthesis (TLS). Combinations of MSH2-/- MMRd with additional repair knockouts yielded distinct and meaningful mutational patterns. While SBS26 and ID12 were induced in the MSH2-/- FANCD2-/- cell line, SBS44 and ID1 were observed in the MSH2-/- ATAD5-/- cell line. Notably, microsatellite instability increased most in the MSH2-/- FANCD2-/- cell line. Overall, this study provides a comprehensive view of mammalian cell background mutagenesis. Chapter 2 investigates mutational patterns and responses of TMZ, which is a DNA- methylating chemotherapeutic agent commonly used to treat glioblastomas. Through the analysis of 427 genomes, mutational patterns in a collection of ~40 isogenic DNA repair-deficient human TK6 lymphoblast cell lines were determined. The mutational patterns were correlated with the TMZ sensitivity of the respective lines. This study highlights how TMZ leads to unique mutational signatures following sequential inactivation of DNA repair pathways, reflecting the development of chemotherapy resistance in glioblastomas. MMR induces cell death and restricts mutagenesis in MGMT-/- cells. Therefore, MMRd enables cell lines to develop resistance to TMZ, leading to the accumulation of SBS11 mutations. In MMRd conditions, only the knockout of BER after abasic site formation contributed to re-sensitizing cells to TMZ. Blocking BER before abasic site formation resulted in an accumulation of T > A substitutions by TLS without impacting cell survival. These findings reveal potential vulnerabilities of TMZ-resistant tumors. Chapter 3 defines the mutational signatures of POLQ, a versatile enzyme playing a role in DNA polymerase theta-mediated end joining (TMEJ), especially within BRCA-mutated cancers. TMEJ functions as an alternative pathway to repair double-strand breaks (DSBs) when the repair mechanisms HR and NHEJ are unavailable. TMEJ is essential for the survival of cancer cells with BRCA mutations. Measuring POLQ activity in BRCA mutated cancers is vital because these tumors are deficient in other DSB repair pathways and are likely sensitive to POLQ inhibition. Therefore, this study identifies mutational signatures associated with POLQ in human cancers, characterized by short insertions and deletions occurring in specific microhomology regions. By examining 82 COSMIC signatures, BRCA- mutated cancers with higher POLQ expression exhibited a notably higher contribution of ID6, ID8, and SBS3. Moreover, the role of POLQ in X-ray (low Linear Energy Transfer (LET)) and carbon-ion (high LET) irradiation was investigated in this chapter. ID12, related to MSI and detected in the MSH2-/- FANCD2-/- cell line, was the most common indel mutational signature in irradiations. Cells exposed to carbon-ion irradiation had comparatively low ID12, but cells exposed to X-ray irradiation displayed high ID12. This finding suggests that whereas both carbon-ion and X-ray irradiation increase MSI in the genome, the effects of X-ray exposure are more significant. Compared to WT and POLQ-/- cells, LIG4-/- knockout cells showed decreased ID8 in both X-ray and carbon ion irradiation. Consequently, it was discovered that both irradiations contribute to ID8 through NHEJ. In contrast to X-rays, only carbon-ion irradiated LIG4-/- cells resulted in deletions with a length greater than 200 bp and less than 10,000 bp and deletions with a microhomology 3-6 bp length due to POLQ. This chapter points out the importance of identifying POLQ-enriched tumors, since these tumors may be more responsive to therapies focused on interfering with TMEJ. In conclusion, this thesis provides a broad understanding of the role that DNA repair deficiencies contribute to determining the mutational landscape of cancer cells. New opportunities for developing targeted cancer treatments are enabled by defining distinct mutational signatures related to DNA repair deficiencies in MMR, BER, and POLQ. Specifically, a BER-targeting drug in MMRd- resistant tumors could re-sensitize them to TMZ. POLQ inhibition could be a novel therapeutic option for BRCA-mutated or carbon-ion irradiated cancers. These observations highlight the important role of DNA repair pathways in cancer progression, aging, and treatment efficiency, offering promising strategies to improve therapeutic outcomes.