“Our lab is generally interested in discovering and characterizing previously unknown or poorly understood factors that are essential for genome maintenance. This is particularly important in the context of cancers with hallmark dysregulation of genome maintenance mechanisms,” explains Dr. Richard Adeyemi, Assistant Professor in Fred Hutch’s Basic Sciences Division. In a recently published Nature Communications study from the Adeyemi Lab, the researchers investigated the role of FLIP, a protein they previously identified as a factor that promotes resistance to the widely used chemotherapeutic agent cisplatin. Dr. Adeyemi adds, “in this work we sought to determine how loss of this gene increases the sensitivity of cells to various agents that damage DNA particularly during replication.”
“Inter-strand crosslinks are relatively rare but deadly lesions that typically form when nucleotides on opposing strands of DNA are covalently linked,” says Dr. Adeyemi. Part of the problem is that when these opposing strands of “DNA form a bond, it distorts the DNA and causes a lot of problems,” states Jessica Tischler, a research technician in the Adeyemi lab and co-author of the study. This type of DNA damage can occur through endogenous metabolic events or can be caused by crosslinking agents, like cisplatin, that exploit the toxicity of this form of DNA damage to kill rapidly dividing cancer cells. “However, resistance [to such chemotherapies] can develop and there is also the problem of toxicity to dividing non-cancerous cells. Furthermore, exactly how such lesions are repaired is not well understood,” notes Dr. Adeyemi. “Understanding the details of the inter-strand crosslink repair system could result in safer treatments of cancer, as pharmaceutical drugs inducing inter-strand crosslinks in cancer cells could affect non-cancer cells,” adds Dr. Hiroshi Tsuchida, a senior scientist in the Adeyemi lab and co-author on this study.
Following up their previous study identifying novel genes required for inter-strand crosslink repair and replication stress response using a genome-wide CRISPR/Cas9 knockout screen, the Adeyemi team focused on Clorf112 (FLIP), an uncharacterized gene that was one of the top genes identified in their screen. To validate the role of FLIP in DNA damage response, the researchers created mutant cell lines lacking FLIP expression. Through treatment with inter-strand crosslink and replication stress inducing agents, the authors found that FLIP was important for promoting cell viability and genomic instability after DNA damage was induced. To understand how FLIP protects cells from DNA damage, the Adeyemi group considered if it could interact with other known DNA repair proteins. RAD51 is an enzyme that performs the critical steps in homologous recombination, a process that repairs DNA in a relatively error-free manner and has been shown to interact with another less well studied protein, FIGNL1. Interestingly, published proteomic datasets and work done in plants has shown that FLIP and FIGNL1 can interact. Validating this in mammalian cells, the researchers demonstrated that FLIP and FIGNL1 do physically interact and that loss of FIGNL1 led to increased sensitivity to cisplatin that was almost as high as in FLIP knockout cells. As the group was performing these knockout and validation experiments, they got some unanticipated results. “When we silenced the gene FLIP in the cell, another protein FIGNL1 also disappeared, and we eventually found the two form a complex and both proteins need to be present in the cell to stabilize each other. It was a really cool piece of data that we stumbled upon,” exclaimed Tischler.
Finding that FLIP and FIGNL1 interact, and knowing FIGNL1 interacts with RAD51, the logical next step was to ask if FLIP interacts with RAD51. Indeed, the authors found that FLIP interacts with RAD51 and can do so independently of FIGNL1. The researchers then sought to understand the purpose of this interaction during DNA damage or replication stress. “A critical step during recombination-mediated repair is the formation of DNA bound by RAD51 (this bound form is called a nucleofilament). We knew a lot about the factors that were important for forming the RAD51 filament but thought that factors that oppose filament formation would not be important for recombination. Our work unexpectedly shows that even though FLIP/FIGNL1 opposed filament formation, they were still essential for recombination, and we think this is because recombination cannot be completed without these factors,” Dr. Adeyemi explains. These unexpected results were uncovered by identifying that FLIP loss leads to increased RAD51 on chromatin both with and without exogenous DNA damage. The authors speculate that FLIP is essential for limiting RAD51 levels on chromatin in the absence of damage and that FLIP is also required for RAD51 dissociation from nucleofilaments, thus allowing proper completion of homologous repair. Future work will be aimed at biochemically characterizing the exact step by which this regulation occurs. Tischler remarks that “this study is one piece of the larger puzzle in elucidating the DNA repair pathways in cells.” As there is still much to uncover here, Dr. Tsuchida adds that the group is currently working to “understand the details of how FLIP functions as an important factor of inter-strand crosslink repair,” which will help deepen our understanding of the entire DNA repair system.
This work identifies a protein complex that is critical for repairing various kinds of DNA damage and “ensures homologous recombination can be completed in a timely fashion. In the absence of this complex, recombined chromosomes do not detach properly leading to various pathologies that are commonly found in cancer,” says Dr. Adeyemi. Tischler adds, “understanding how a process works is the first step in developing any new therapies. By characterizing what FLIP does in the cell, we uncover more of the picture of the different mechanisms of DNA repair and uncover new potential targets for chemotherapy and other cancer treatments.”
This work was supported by the National Institutes of Health and the Ovarian Cancer Research Alliance.
Fred Hutch/UW/Seattle Children’s Cancer Consortium member Dr. Richard Adeyemi contributed to this work.
Tischler JD, Tsuchida H, Bosire R, Oda TT, Park A, Adeyemi RO. FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress. Nature Communications, 2024.