Despite the progress made in targeted anticancer therapies in recent years, challenges remain. The identification of new potential targets will ensure that the arsenal of cancer therapies continues to expand. FAM83B was recently discovered in a forward genetic screen for novel oncogenes that drive human mammary epithelial cell (HMEC) transformation. We report here that elevated FAM83B expression increases Phospholipase D (PLD) activity, and that suppression of PLD1 activity prevents FAM83B-mediated transformation. The increased PLD activity is engaged by hyperactivation of epidermal growth factor receptor (EGFR), which is regulated by an interaction involving FAM83B and EGFR. Preventing the FAM83B/EGFR interaction by site-directed mutation of lysine 230 of FAM83B suppressed PLD activity and MAPK signaling. Furthermore, ablation of FAM83B expression from breast cancer cells inhibited EGFR phosphorylation and suppressed cell proliferation. We propose that understanding the mechanism of FAM83B-mediated transformation will provide a foundation for future therapies aimed at targeting its function as an intermediary in EGFR, MAPK, and mTOR activation.

SUMMARY Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2′-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.

Telomeres are nucleoprotein complexes that reside at the ends of linear chromosomes and help maintain genomic integrity. POT1 and TPP1 are telomere-specific proteins that bind as a heterodimer to single-stranded telomere DNA to prevent illicit DNA damage responses and to enhance telomerase-mediated telomere extension. Telomere DNA is guanosine-rich and, as such, can form highly stable secondary structures including G-quadruplexes. G-quadruplex DNA folds into different topologies that are determined by several factors including monovalent ion composition and the precise sequence and length of the DNA. Here, we explore the influence of DNA secondary structure on POT1-TPP1 binding. Equilibrium binding assays reveal that the POT1-TPP1 complex binds G-quadruplex structures formed in buffers containing Na+ with an affinity that is 5-fold higher than for G-quadruplex structures formed in the presence of K+. However, binding of a second heterodimer is insensitive to DNA secondary structure, presumably due to unfolding resulting from binding of the first POT1-TPP1. We further show that the rate constant for POT1-TPP1-induced unfolding of DNA secondary structure is substantially faster for G-quadruplex topologies formed in the presence of Na+ ions. When bound to DNA, POT1-TPP1 forms complexes with similar CD spectra and enhances telomerase activity for all DNA substrates tested, regardless of substrate secondary structure or solution monovalent ion composition. Together, these data indicate that binding of POT1-TPP1 unfolds telomere secondary structure to assist loading of additional heterodimers and to ensure efficient promotion of telomerase-mediated extension.

Pancreatic ductal adenocarcinomas (PDAC) are deadly on account of the delay in diagnosis and dearth of effective treatment options for advanced disease. The insurmountable hurdle of targeting oncogene KRAS, the most prevalent genetic mutation in PDAC, has delayed the availability of targeted therapy for PDAC patients. An alternate approach is to target other tumour-exclusive effector proteins important in RAS signalling. The Family with Sequence Similarity 83 (FAM83) proteins are oncogenic, tumour-exclusive and function similarly to RAS, by driving the activation of PI3K and MAPK signalling. In this study we show that FAM83A expression is significantly elevated in human and murine pancreatic cancers and is essential for the growth and tumorigenesis of pancreatic cancer cells. Elevated FAM83A expression maintains essential MEK/ERK survival signalling, preventing cell death in pancreatic cancer cells. Moreover, we identified a positive feed-forward loop mediated by the MEK/ERK-activated AP-1 transcription factors, JUNB and FOSB, which is responsible for the elevated expression of oncogenic FAM83A. Our data indicates that targeting the MEK/ERK-FAM83A feed-forward loop opens up additional avenues for clinical therapy that bypass targeting of oncogenic KRAS in aggressive pancreatic cancers.

SignificanceTelomere length homeostasis is an important mechanism for maintaining genomic stability. Telomere length is regulated by numerous events that include protein–DNA interactions, the length and structure of telomere DNA, and recruitment of telomerase. Here we used hydroxyl radical footprinting to identify environmental changes in the telomere end-binding heterodimer, POT1-TPP1, as a function of telomere length. Our data identified a specific residue (histidine 266) of the POT1 protein that reports differences in solvent accessibility as a function of telomere DNA length. We further show that the chronic lymphocytic leukemia-related H266L POT1 mutation disrupts the ability of POT1-TPP1 to negatively regulate telomerase activity in vitro and in cancer cells.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Telomeres are specialized nucleoprotein complexes that comprise the ends of linear chromosomes. Human telomeres end in a short, single-stranded DNA (ssDNA) overhang that is recognized and bound by two telomere proteins, POT1 and TPP1. Whereas POT1 binds directly to telomere ssDNA, its interaction with TPP1 is essential for localization of POT1 to the telomere. TPP1 also provides enhanced binding and sequence discrimination that regulates POT1-TPP1 interactions exclusively with telomere ssDNA. Finally, TPP1 recruits telomerase, the enzyme responsible for synthesis of telomere DNA, to the telomere. While the oligosaccharide–oligonucleotide-binding (OB)-fold domain of TPP1 has been solved by X-ray crystallography, the molecular interactions within the POT1-TPP1-ssDNA ternary complex and the conformational changes that contribute to its diverse functions remain ambiguous. We employed hydrogen/deuterium exchange combined with mass spectrometry to identify three peptides, all residing within the OB-fold of TPP1, that exhibit altered exchange rates upon complex formation or ssDNA binding. Mutation of these regions combined with functional assays revealed the diverse contributions of each moiety in protein–protein interactions, regulating telomerase activity or DNA-binding. Together, these functional data combined with biophysical analyses and homology modeling provide a molecular understanding of the diverse contributions of TPP1 in telomere maintenance.

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