Therapeutic targeting of SLC6A8 creatine transporter suppresses colon cancer progression and modulates human creatine levels.

Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.

LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

Although homologous recombination is considered an accurate form of DNA repair, genetics suggest that the Escherichia coli translesion DNA polymerase IV (Pol IV, also known as DinB) promotes error-prone recombination during stress, which allows cells to overcome adverse conditions. However, how Pol IV functions and is regulated during recombination under stress is unknown. We show that Pol IV is highly proficient in error-prone recombination and is preferentially recruited to displacement loops (D loops) at stress-induced concentrations in vitro. We also found that high-fidelity Pol II switches to exonuclease mode at D loops, which is stimulated by topological stress and reduced deoxyribonucleotide pool concentration during stationary phase. The exonuclease activity of Pol II enables it to compete with Pol IV, which probably suppresses error-prone recombination. These findings indicate that preferential D-loop extension by Pol IV facilitates error-prone recombination and explain how Pol II reduces such errors in vivo.

A solution to release twisted DNA during chromosome replication by coupled DNA polymerases.

Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as 'signal release', had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.

New insights into replisome fluidity during chromosome replication.

Several paradigm shifting advances have recently been made on the composition and function of the chromosomal DNA replication machinery. Replisomes appear to be more fluid and dynamic than ever imagined, enabling rapid and efficient bypass of roadblocks and template lesions while faithfully replicating chromosomal DNA. This fluidity is determined by many layers of regulation, which reach beyond the role of replisome components themselves. In fact, recent studies show that additional polymerases, post-transcriptional modifications, and chromatin structure are required for complete chromosome duplication. Many of these factors are involved with the more complex events that take place during lagging-strand synthesis. These, and other recent discoveries, are the focus of this review.

Single-molecule studies reveal the function of a third polymerase in the replisome.

The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes.

Origin-dependent initiation of DNA replication within telomeric sequences.

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.

Mechanism of polymerase collision release from sliding clamps on the lagging strand.

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the 'collision release' model, the lagging strand polymerase collides with the 5' terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5' terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the tau subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The tau subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.

Replisome Dynamics during Chromosome Duplication.

This review describes the components of the Escherichia coli replisome and the dynamic process in which they function and interact under normal conditions. It also briefly describes the behavior of the replisome during situations in which normal replication fork movement is disturbed, such as when the replication fork collides with sites of DNA damage. E. coli DNA Pol III was isolated first from a polA mutant E. coli strain that lacked the relatively abundant DNA Pol I activity. Further biochemical studies, and the use of double mutant strains, revealed Pol III to be the replicative DNA polymerase essential to cell viability. In a replisome, DnaG primase must interact with DnaB for activity, and this constraint ensures that new RNA primers localize to the replication fork. The leading strand polymerase continually synthesizes DNA in the direction of the replication fork, whereas the lagging-strand polymerase synthesizes short, discontinuous Okazaki fragments in the opposite direction. Discontinuous lagging-strand synthesis requires that the polymerase rapidly dissociate from each new completed Okazaki fragment in order to begin the extension of a new RNA primer. Lesion bypass can be thought of as a two-step reaction that starts with the incorporation of a nucleotide opposite the lesion, followed by the extension of the resulting distorted primer terminus. A remarkable property of E. coli, and many other eubacterial organisms, is the speed at which it propagates. Rapid cell division requires the presence of an extremely efficient replication machinery for the rapid and faithful duplication of the genome.

An affinity oligonucleotide displacement strategy to purify ribonucleoprotein complexes applied to human telomerase.

Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of catalytically active human telomerase and the purification of native and recombinant telomerase using antisense affinity oligonucleotides. The purification procedure involves binding of the RNP complex to NeutrAvidin beads via a biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotide complementary to the RNA subunit. The complex is eluted from the beads through competition with a displacement oligonucleotide. Thus, the purified RNP is eluted under mild conditions, retaining its catalytic activity.

Four functionally distinct populations of human effector-memory CD8+ T lymphocytes.

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro.

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.

Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7-27+28+ and RA+CCR7-27+28-, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon gamma, and tumor necrosis factor alpha. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.