ABSTRACT
Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Cytidine Deaminase , DNA Replication , Deoxycytidine , Gemcitabine , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Cytidine Deaminase/metabolism , Cytidine Deaminase/genetics , Cell Line, Tumor , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/drug therapy , Mice , Drug Resistance, Neoplasm/genetics , Antimetabolites, Antineoplastic/pharmacology , Xenograft Model Antitumor Assays , CRISPR-Cas SystemsABSTRACT
FBXW7 is a commonly mutated tumor suppressor gene that functions to regulate numerous oncogenes involved in cell-cycle regulation. Genome-wide CRISPR fitness screens identified a signature of DNA repair and DNA damage response genes as required for the growth of FBXW7-knockout cells. Guided by these findings, we show that FBXW7-mutant cells have high levels of replication stress, which results in a genotype-specific vulnerability to inhibition of the ATR signaling pathway, as these mutant cells become heavily reliant on a robust S-G2 checkpoint. ATR inhibition induces an accelerated S-phase, leading to mitotic catastrophe and cell death caused by the high replication stress present in FBXW7-/- cells. In addition, we provide evidence in cell and organoid studies, and mining of publicly available high-throughput drug screening efforts, that this genotype-specific vulnerability extends to multiple types of cancer, providing a rational means of identifying responsive patients for targeted therapy. SIGNIFICANCE: We have elucidated the synthetic lethal interactions between FBXW7 mutation and DNA damage response genes, and highlighted the potential of ATR inhibitors as targeted therapies for cancers harboring FBXW7 alterations.
Subject(s)
DNA Repair , Neoplasms , Humans , F-Box-WD Repeat-Containing Protein 7/genetics , Ataxia Telangiectasia Mutated Proteins/genetics , Mutation , Neoplasms/genetics , Cell DeathABSTRACT
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
Subject(s)
DNA Damage , Gene Regulatory Networks/physiology , Aminoquinolines/pharmacology , Animals , CRISPR-Cas Systems/genetics , Cell Line , Cytochrome-B(5) Reductase/genetics , Cytochrome-B(5) Reductase/metabolism , DNA Damage/drug effects , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Humans , Mice , Picolinic Acids/pharmacology , RNA, Guide, Kinetoplastida/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/geneticsABSTRACT
Complete and accurate DNA replication is fundamental to cellular proliferation and genome stability. Obstacles that delay, prevent, or terminate DNA replication cause the phenomena termed DNA replication stress. Cancer cells exhibit chronic replication stress due to the loss of proteins that protect or repair stressed replication forks and due to the continuous proliferative signaling, providing an exploitable therapeutic vulnerability in tumors. Here, we outline current and pending therapeutic approaches leveraging tumor-specific replication stress as a target, in addition to the challenges associated with such therapies. We discuss how replication stress modulates the cell-intrinsic innate immune response and highlight the integration of replication stress with immunotherapies. Together, exploiting replication stress for cancer treatment seems to be a promising strategy as it provides a selective means of eliminating tumors, and with continuous advances in our knowledge of the replication stress response and lessons learned from current therapies in use, we are moving toward honing the potential of targeting replication stress in the clinic.