ABSTRACT
Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.
Subject(s)
DNA Helicases/metabolism , DNA Replication , Gene Expression Regulation, Neoplastic , R-Loop Structures , Telomere Homeostasis , Telomere/physiology , BRCA2 Protein/metabolism , Biomarkers, Tumor/metabolism , Cell Line, Tumor , DNA Damage , DNA Polymerase III/metabolism , DNA Repair , DNA, Single-Stranded , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group N Protein/metabolism , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Phenotype , Rad51 Recombinase/metabolismABSTRACT
In the era of precision medicine, cancer researchers and oncologists are eagerly searching for more realistic, cost effective, and timely tumor models to aid drug development and precision oncology. Tumor models that can faithfully recapitulate the histological and molecular characteristics of various human tumors will be extremely valuable in increasing the successful rate of oncology drug development and discovering the most efficacious treatment regimen for cancer patients. Two-dimensional (2D) cultured cancer cell lines, genetically engineered mouse tumor (GEMT) models, and patient-derived tumor xenograft (PDTX) models have been widely used to investigate the biology of various types of cancers and test the efficacy of oncology drug candidates. However, due to either the failure to faithfully recapitulate the complexity of patient tumors in the case of 2D cultured cancer cells, or high cost and untimely for drug screening and testing in the case of GEMT and PDTX, new tumor models are urgently needed. The recently developed patient-derived tumor organoids (PDTO) offer great potentials in uncovering novel biology of cancer development, accelerating the discovery of oncology drugs, and individualizing the treatment of cancers. In this review, we will summarize the recent progress in utilizing PDTO for oncology drug discovery. In addition, we will discuss the potentials and limitations of the current PDTO tumor models.
