Abstract 5688: RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers

Abstract Genome instability has long been considered the primary driver of most cancer types. A double strand break (DSB) in DNA can have deleterious consequences for a cell, which if not repaired faithfully, can lead to mutations and chromosomal rearrangements, or even cell death. DSBs can be processed by several DNA repair pathways, of which homologous recombination (HR) is the preferred method due to its error-free nature. HR uses an intact homologous DNA sequence as a template for recovering the information lost at the break site. A significant proportion of all cancers, especially triple-negative breast, ovarian pancreatic and prostate cancers, have loss of function alterations affecting genes involved in HR-mediated DNA repair. Alternate repair pathways operate when HR is defective in tumors, but the pathways operative in this context remain a matter of contention. Previous work in vivo in yeast and in vitro systems has established a new role of RNA in DNA repair. Owing to its abundance in the cell and its sequence similarity to parental DNA, we sought to define whether RNA can act as a template for the repair of DSBs in human cells. We developed a novel high throughput assay to test if DNA breaks can be repaired using RNA as an alternative template in mammalian cells. Human cells were transfected with a guide RNA cloned in a Cas9 expression vector to generate a site-specific DSB at the AAVS1 locus, a safe harbour, in the human genome. Furthermore, a donor template in the form of DNA or RNA (homologous to the DSB locus) containing a unique mutational signature was provided at the time of transfection. The unique mutational signature enables us to determine if the donor has been utilized as a template for DNA repair. Using this assay, we demonstrate that cells can use a spliced RNA transcript as a functional template to repair a DSB. We have identified that Rev3L, a key component of the translesion synthesis polymerase Pol Zeta (ζ), has a novel reverse-transcriptase activity in human cells and can help repair the DSB using RNA as a template. Further characterization of this repair pathway and its associated mutational scar will provide new insights into the mutational signatures seen in HR-defective cancers, enabling a better understanding of the DNA repair pathways upregulated in these tumours. The proposed studies could help prioritize novel therapeutic approaches by exploiting synthetic lethality in HR-deficient cancers as well as HR-proficient cancers when used in combinatorial cancer therapy. Citation Format: Manisha Jalan, Juber Patel, Kyrie S Olsen, Sana Ahmed-Seghir, Daniel S Higginson, Jorge S Reis-Filho, Nadeem Riaz, Simon N Powell. RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5688.