ABSTRACT
Although poly(ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after restoration of HR. Here, we report that dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 5, and 9, additionally has potent activity against CDK12, a transcriptional regulator of HR. In BRCA-mutated triple-negative breast cancer (TNBC) cells and patient-derived xenografts (PDXs), dinaciclib ablates restored HR and reverses PARP inhibitor resistance. Additionally, we show that de novo resistance to PARP inhibition in BRCA1-mutated cell lines and a PDX derived from a PARP-inhibitor-naive BRCA1 carrier is mediated by residual HR and is reversed by CDK12 inhibition. Finally, dinaciclib augments the degree of response in a PARP-inhibitor-sensitive model, converting tumor growth inhibition to durable regression. These results highlight the significance of HR disruption as a therapeutic strategy and support the broad use of combined CDK12 and PARP inhibition in TNBC.
Subject(s)
BRCA1 Protein/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Resistance, Neoplasm/drug effects , Mutation/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Triple Negative Breast Neoplasms/enzymology , Triple Negative Breast Neoplasms/pathology , Amino Acid Sequence , Animals , BRCA1 Protein/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cyclic N-Oxides , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , DNA Damage/genetics , DNA Repair/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockout Techniques , Homologous Recombination/drug effects , Humans , Indolizines , Mice , Protein Kinase Inhibitors/pharmacology , Pyridinium Compounds/pharmacology , RNA, Small Interfering/metabolism , Transcription, Genetic/drug effects , Triple Negative Breast Neoplasms/genetics , Xenograft Model Antitumor AssaysABSTRACT
Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Directed DNA Polymerase/metabolism , Homologous Recombination , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Amino Acid Motifs , Animals , Carcinoma, Ovarian Epithelial , Cell Cycle , Cell Death , Cell Line, Tumor , DNA End-Joining Repair/genetics , DNA Replication , DNA-Directed DNA Polymerase/deficiency , Embryo Loss , Fanconi Anemia Complementation Group D2 Protein/deficiency , Fanconi Anemia Complementation Group D2 Protein/genetics , Female , Genomic Instability , Homologous Recombination/genetics , Humans , Mice , Molecular Targeted Therapy , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/pathology , Protein Binding , Rad51 Recombinase/antagonists & inhibitors , Rad51 Recombinase/metabolism , Recombinational DNA Repair/genetics , DNA Polymerase thetaABSTRACT
The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1(+/-) mice were small at birth and exhibited reduced fertility, thus resembling Usp1(-/-) mice. Unexpectedly, homozygous Uaf1(-/-) embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1(+/-) murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1(-/-) mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.
Subject(s)
Embryo Loss/genetics , Gene Deletion , Homologous Recombination , Mice/embryology , Mice/genetics , Nuclear Proteins/genetics , Animals , Carcinogenesis/genetics , Carcinogenesis/pathology , Cells, Cultured , Chromosomal Instability , DNA Repair , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Homozygote , Humans , Infertility/genetics , Male , Mice, Inbred C57BL , Mutagens/pharmacology , Nuclear Proteins/metabolism , Proliferating Cell Nuclear Antigen/metabolism , UbiquitinationABSTRACT
Fanconi anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras-oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.