Characterizing genetic dependencies induced by Fanconi anemia pathway loss

About This Grant

PROJECT SUMMARY The Fanconi anemia (FA) pathway is involved in several key processes required to maintain genome stability. It promotes the repair of DNA lesions, such as inter-strand crosslinks (ICLs) and DNA double strand breaks. It is also involved in protecting nascent DNA from degradation and preventing the accumulation of single-strand DNA (ssDNA) gaps during DNA replication. FA pathway proteins also favor the resolution of RNA-DNA hybrids (R-loops), which form as a result of transcription. Mutations in FA pathway genes cause Fanconi anemia, a genetic disorder associated with bone marrow failure, developmental abnormalities, and an increased risk of hematological malignancies and solid tumors. Mutations in FA pathway genes also predispose to hereditary breast and ovarian cancer, and they have been observed in more than a third of somatic tumors. Current treatments for cancers with FA pathway defects often involve the use of DNA crosslinking agents or PARP inhibitors. However, these treatments can lose effectiveness over time as cancer cells develop resistance. To improve therapeutic outcomes, it is therefore critical to systematically define factors required for the survival of FA pathway-deficient cancer cells. In preliminary work, we conducted high-throughput genetic interaction studies to identify dependencies of mammalian cells deficient for FA pathway genes. This work identified synthetic lethal interactions between the FA pathway and the DNA repair genes GEN1, CIP2A and RHNO1. The main goals of this proposal are to characterize mechanistically the synthetic lethal relationships between these genes and the FA pathway and determine their relevance in cancer. In particular, we propose 1) to investigate the genetic dependency of cells deficient in the FA pathway on GEN1, CIP2A and RHNO1; 2) to define the endogenous sources of replication stress that sensitize cells deficient in the FA pathway to the loss of GEN1, CIP2A and RHNO1; 3) to explore the loss of GEN1, CIP2A or RHNO1 as a cancer vulnerability in the context of FA pathway deficiency and FA gene mutations. Our approach will utilize state-of-the-art CRISPR combinatorial knockout and base editing screens, coupled with functional characterization of genetic interactions and mutations using molecular and cell biology assays. We expect that these studies will advance our understanding of the interactions of the FA pathway with other DNA damage response pathways and identify vulnerabilities that could be exploited for the development of personalized therapies for FA pathway-deficient tumors.