Mortality from pancreatic ductal adenocarcinoma cancer (PDAC) is among the highest of any cancer and frontline therapy has changed little in years. Activation of endothelial nitric oxide synthase (eNOS or NOS III) has been implicated recently in the pathogenesis of PDAC. In this study, we used genetically engineered mouse and human xenograft models to evaluate the consequences of targeting eNOS in PDAC. Genetic deficiency in eNOS limited the development of pre-invasive pancreatic lesions and trended towards an extended lifespan in mice with advanced pancreatic cancer. These effects were also observed upon oral administration of the clinically evaluated NOS small molecule inhibitor L-NAME. Similarly, other transgenic models of oncogenic KRas-driven tumors responded to L-NAME treatment. Finally, these results were recapitulated in xenograft models of human pancreatic cancer, in which L-NAME was found to broadly inhibit tumorigenic growth. Taken together, our findings offer preclinical proof-of-principle to repurpose L-NAME for clinical investigations in treatment of PDAC and possibly other KRas-driven human cancers.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.Telomeres are DNA-protein complexes that protect the ends of linear eukaryotic chromosomes from illegitimate recombination, degradation, and recognition as DNA doublestrand breaks. The DNA portion of human telomeres is composed of tandem arrays of the short G-rich repetitive sequence TTAGGG in which the G-rich strand extends beyond the Crich strand, forming a 3Ј single-stranded DNA (ssDNA) overhang. Electron microscopic analysis suggests that the 3Ј overhang can loop back and invade telomeric double-stranded DNA (dsDNA) to form a large lariat structure, effectively hiding the end of the telomere (16). The protein portion of telomeres is composed of two telomeric dsDNA-binding proteins, TRF1 and TRF2 (6,25,42,46,47), and one telomeric ssDNA-binding protein, POT1 (4, 5), which form a large complex, mediated by proteins such as TIN2 and TPP1 (24,26,28,30,31,39,56,57), that maintains chromosome end stability (21,22,43,48).POT1 is a 634-amino-acid protein comprised of an N-terminal evolutionarily conserved pair of oligonucleotide/oligosaccharide (OB) folds responsible for telomeric ssDNA binding (4,5,33), with the remaining C terminus serving to bind TPP1 (31, 57) and TRF2 (55). With regard to TPP1, both recombinant (49) and purified (52) human TPP1 proteins form a heterodimer with POT1 in vitro, which enhances the association of POT1 with a G-strand telomere oligonucleotide. Moreover, in the lower eukaryote Oxytricha nova, the POT1 and TPP1 homologues TEBP␣ (4, 5) and TEBP␤ (49, 52) form a ternary complex with ssDNA (12, 23) that protects chromosome ends (15). Heterodimerization of POT1 with TPP1 thus appears to enhance POT1 function at telomeric ssDNA. With regard to TRF2, POT1 coimmunoprecipitates with TRF2 (55) and has been isolated by gel filtration in several protein subcomplexes containing TRF2 (27,30,39). POT1 thus interacts directly with telomeric ssDNA, in association with TPP1, and presumably indirectly with telomeric dsDNA, thr...

Deleting the OB folds encoding the telomeric single-stranded DNA (ssDNA)-binding activity of the human telomeric protein POT1 induces significant telomere elongation, suggesting that at least one critical aspect of the regulation of telomere length is disrupted by this POT1 ⌬OB mutant protein. POT1 is known to associate with two proteins through the protein interaction domain retained in POT1⌬OB -the telomeric doublestranded DNA-binding protein TRF2 and the telomere-associated protein TPP1. We report that introducing a mutation that reduces association of POT1 with TRF2, but not a mutation that reduces the association with TPP1, abrogates the ability of POT1 ⌬OB to promote telomere elongation. Mechanistically, expression of POT1 ⌬OB reduced the association of TRF2 with POT1, RAP1, and TIN2; however, of these proteins, only ectopic expression of POT1 suppressed the telomere elongation induced by POT1 ⌬OB . Lastly, replacing endogenous POT1 with a full-length POT1 mutant defective in the association with TRF2 induced telomere elongation. Thus, we conclude that the association of POT1 with both ssDNA and TRF2 is critical for telomere length homeostasis.Telomeres are DNA-protein complexes that protect the ends of eukaryotic chromosomes from degradation and detection as sites of DNA damage (reviewed in reference 23). Telomeric DNA is composed of tandem arrays of repetitive double-stranded DNA (dsDNA), wherein the G-rich strand extends beyond the C-rich strand. The resultant 3Ј singlestranded DNA (ssDNA) overhang can invade the dsDNA, forming a lariat structure termed the t-loop (28).The regulation of telomere length affects mammalian biology at both the cellular and organismal levels. In normal human somatic cells, telomeres progressively shorten (32, 33) to a critical length before entering a state of permanent growth arrest termed senescence (1,12,21,34,35). Abnormally short telomeres in humans and mice are associated with various anemias, cirrhosis of the liver, and other disorders due to the premature induction of senescence, particularly in highly proliferative tissues (7,11,61). Conversely, de novo elongation of the telomere by the reverse transcriptase telomerase (27, 54) ensures maintenance of telomere length in the germ line and endows cultured cells with an immortal life span (8,17,18,20,30,36,70,78). Moreover, telomerase activation and subsequent stabilization of telomere length occur in the vast majority of cancer cells (41, 62) and are required for cellular immortalization and the tumorigenic conversion of normal human cells (19,30,31,36,78).The dsDNA portion of telomeres is bound directly by two proteins, TRF1 and TRF2 (9, 23). In turn, TRF1 and TRF2 are bridged by the protein TIN2 (23, 43, 76) and bind other telomere-associated proteins such as tankyrase 1 (23, 63, 64) and RAP1 (48, 56). Telomeric ssDNA is bound directly by the protein POT1 (4, 5, 23, 52). POT1 acts in a heterodimer with the protein TPP1; this heterodimer promotes POT1 binding to telomeric ssDNA at least in vitro (72, 73), protects tel...

Mammalian telomeres are composed of G-rich repetitive double-stranded (ds) DNA with a 3 single-stranded (ss) overhang and associated proteins that together maintain chromosome end stability. Complete replication of telomeric DNA requires de novo elongation of the ssDNA by the enzyme telomerase, with telomeric proteins playing a key role in regulating telomerase-mediated telomere replication. In regards to the protein component of mammalian telomeres, TRF1 and TRF2 bind to the dsDNA of telomeres, whereas POT1 binds to the ssDNA portion. These three proteins are linked through either direct interactions or by the proteins TIN2 and TPP1. To determine the biological consequence of connecting telomeric dsDNA to ssDNA through a multiprotein assembly, we compared the effect of expressing TRF1 and POT1 in trans versus in cis in the form of a fusion of these two proteins, on telomere length in telomerase-positive cells. When expressed in trans these two proteins induced extensive telomere elongation. Fusing TRF1 to POT1 abrogated this effect, inducing mild telomere shortening, and generated looped DNA structures, as assessed by electron microscopy, consistent with the protein forming a complex with dsDNA and ssDNA. We speculate that such a protein bridge between dsDNA and ssDNA may inhibit telomerase access, promoting telomere shortening.Telomeres are DNA-protein structures that cap and protect the ends of eukaryotic chromosomes from illegitimate recombination and degradation. In humans, the DNA portion of this structure is composed of the G-rich sequence TTAGGG repeated in tandem hundreds of times (1). This G-strand extends beyond the complementary C-strand to form a ssDNA overhang. Electron microscopy revealed that this ssDNA extension appeared to invade the dsDNA, 2 forming a D-loop, with the intervening dsDNA looping out in what is termed a T-loop. This structure has been speculated to impart some of the replicative and protective functions of telomeres (2).In terms of the protein components of telomeres, telomeric DNA-binding proteins fall into two classes, ssDNA-or dsDNAbinding proteins. In humans, the primary dsDNA telomerebinding proteins are TRF1 (3) and TRF2 (4), whereas the principle ssDNA-binding protein is POT1 (5). Disruption of TRF2 by expression of a dominant-negative version of the protein (6, 7) and disruption of POT1 expression by RNAi or genetic knock out (8 -11) can lead to various degrees of chromosome instability and/or cell arrest or death, whereas knock out of TRF1 is embryonic lethal (12). On the other hand, overexpressing these proteins alters telomere length (13-16). As access of telomerase, the enzyme that elongates the G-strand overhang of telomeres, to telomere ends is mediated by proteins in lower eukaryotes as a means of regulating telomere length; human telomere-binding proteins may also serve in this capacity (17). Thus, telomere-binding proteins can function in telomere stability and/or telomerase-mediated replication of telomeres.Accumulating evidence argues that the ssDNA-and dsDNAte...