Browsing by Subject "Genome instability"

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases is one of the most common endogenous sources of single base substitution mutations in human cancer. Accordingly, APOBEC enzymes represent a major source of intratumor genetic heterogeneity and have been associated with immunotherapy response in diverse cancer types. However, the consequences of APOBEC mutagenesis on tumor progression in vivo are not well understood. To address this, I developed several murine tumor models with inducible APOBEC3B expression and studied the contribution of APOBEC activity to tumor evolution and immunogenicity. First, I explored the effects of APOBEC activity on tumor relapse using a murine model of mammary tumor recurrence. APOBEC activity led to a significant acceleration in tumor recurrence following the strong selective pressure of oncogenic driver signaling loss. Recurrent APOBEC tumors had undifferentiated histological features and large, irregularly shaped nuclei containing defects like micronuclei, multinucleation, and chromatin bridges. I found that recurrent APOBEC tumors amplified the therapy resistance-associated oncogene, c-Met, on circular extrachromosomal DNA, likely driving the proliferation of the recurrent cancer cells. Second, because APOBEC mutational signatures are enriched in the majority of HER2-positive breast cancer patients, I used a syngeneic HER2-driven mammary tumor model to study the effects of APOBEC activity on the tumor immune microenvironment. I found that APOBEC activity induced an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. Consistent with the observed immune infiltration in murine APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive breast cancers. In conclusion, I’ve identified a novel role for APOBEC activity in generating chromosomal instability, consisting of mitotic errors, oncogene amplification, and extrachromosomal DNA formation to promote tumor recurrence. Moreover, APOBEC activity also stimulated an antitumor adaptive immune response and sensitized tumors to immunotherapy.

A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.

I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.

To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.