Combining bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) improves systemic antitumor CD8+ T cell cytotoxicity over BEMPEG+RT.

BACKGROUND: Tumor cell death caused by radiation therapy (RT) triggers antitumor immunity in part because dying cells release adjuvant factors that amplify and sustain dendritic cell and T cell responses. We previously demonstrated that bempegaldesleukin (BEMPEG: NKTR-214, an immunostimulatory IL-2 cytokine prodrug) significantly enhanced the antitumor efficacy of RT through a T cell-dependent mechanism. Because RT can induce either immunogenic or tolerogenic cell death, depending on various factors (radiation dose, cell cycle phase), we hypothesized that providing a specific immunogenic adjuvant, like intratumoral therapy with a novel toll-like receptor (TLR) 7/8 agonist, NKTR-262, would improve systemic tumor-specific responses through the activation of local innate immunity. Therefore, we evaluated whether intratumoral NKTR-262 combined with systemic BEMPEG treatment would elicit improved tumor-specific immunity and survival compared with RT combined with BEMPEG. METHODS: Tumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; intravenously), RT (12 Gy × 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4+ and CD8+ T cell responses in the blood and tumor 7 days post-treatment. The contribution of specific immune subsets was determined by depletion of CD4+, CD8+, or NK cells. CD8+ T cell cytolytic activity was determined by an in vitro CTL assay. Data are representative of 1-2 independent experiments (n=5-14/group) and statistical significance was determined by 1-way analysis of variance (ANOVA) or repeated measures ANOVA (p value cut-off of 0.05). RESULTS: BEMPEG+NKTR-262 significantly improved survival compared with BEMPEG+RT in a CD8+ T cell-dependent manner. Response to BEMPEG+NKTR-262 was characterized by a significant expansion of activated CD8+ T cells (GzmA+; Ki-67+; ICOS+; PD-1+) in the blood, which correlated with reduced tumor size (p<0.05). In the tumor, BEMPEG+NKTR-262 induced higher frequencies of GzmA+ CD8+ T cells exhibiting reduced expression of suppressive molecules (PD-1+), compared with BEMPEG+RT (p<0.05). Further, BEMPEG+NKTR-262 treatment induced greater tumor-specific CD8+ T cell cytolytic function than BEMPEG+RT. CONCLUSIONS: BEMPEG+NKTR-262 therapy elicited more robust expansion of activated CD8+ T cells compared with BEMPEG+RT, suggesting that intratumoral TLR stimulation provides superior antigen presentation and costimulatory activity compared with RT. A clinical trial of BEMPEG+NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640).

NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy.

BACKGROUND: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines. METHODS: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function. In vivo pharmacokinetic (PK) and pharmacodynamic profile of the cytokines were evaluated in normal mice. Finally, immunomodulatory effect and antitumor activity were assessed in a Daudi lymphoma model. RESULTS: NKTR-255 and rhIL-15 exhibited similar in vitro properties in receptor affinity, signaling and leukocyte degranulation, which collectively differed from precomplexed cytokines. Notably, NKTR-255 and rhIL-15 stimulated greater granzyme B secretion in human peripheral blood mononuclear cells versus precomplexed cytokines. In vivo, NKTR-255 exhibited a PK profile with reduced clearance and a longer half-life relative to rhIL-15 and demonstrated prolonged IL-15R engagement in lymphocytes compared with only transient engagement observed for rhIL-15 and precomplexed rhIL-15 N72D/IL-15Rα Fc. As a consequent, NKTR-255 provided a durable and sustained proliferation and activation of natural killer (NK) and CD8+ T cells. Importantly, NKTR-255 is more effective than the precomplexed cytokine at inducing functionally competent, cytotoxic NK cells in the tumor microenvironment and the properties of NKTR-255 translated into superior antitumor activity in a B-cell lymphoma model versus the precomplexed cytokine. CONCLUSIONS: Our results show that the novel immunotherapeutic, NKTR-255, retains the full spectrum of IL-15 biology, but with improved PK properties, over rhIL-15. These findings support the ongoing phase 1 first-in-human trial (NCT04136756) of NKTR-255 in participants with relapsed or refractory hematologic malignancies, potentially advancing rhIL-15-based immunotherapies for the treatment of cancer.

Bempegaldesleukin (BEMPEG; NKTR-214) efficacy as a single agent and in combination with checkpoint-inhibitor therapy in mouse models of osteosarcoma.

Survival of patients with relapsed/refractory osteosarcoma has not improved in the last 30 years. Several immunotherapeutic approaches have shown benefit in murine osteosarcoma models, including the anti-programmed death-1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) immune checkpoint inhibitors. Treatment with the T-cell growth factor interleukin-2 (IL-2) has shown some clinical benefit but has limitations due to poor tolerability. Therefore, we evaluated the efficacy of bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, alone and in combination with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors in metastatic and orthotopic murine models of osteosarcoma. Treatment with BEMPEG delayed tumor growth and increased overall survival of mice with K7M2-WT osteosarcoma pulmonary metastases. BEMPEG also inhibited primary tumor growth and metastatic relapse in lungs and bone in the K7M3 orthotopic osteosarcoma mouse model. In addition, it enhanced therapeutic activity of anti-CTLA-4 and anti-PD-1 checkpoint blockade in the DLM8 subcutaneous murine osteosarcoma model. Finally, BEMPEG strongly increased accumulation of intratumoral effector T cells and natural killer cells, but not T-regulatory cells, resulting in improved effector:inhibitory cell ratios. Collectively, these data in multiple murine models of osteosarcoma provide a path toward clinical evaluation of BEMPEG-based regimens in human osteosarcoma.

Clodronate Improves Survival of Transplanted Hoxb8 Myeloid Progenitors with Constitutively Active GMCSFR in Immunocompetent Mice.

New methods to produce large numbers of myeloid progenitor cells, precursors to macrophages (MΦs), by maintaining Hoxb8 transcription factor activity1 has reinvigorated interest in MΦ cell therapies. We generated Hoxb8-dependent myeloid progenitors (HDPs) by transducing lineage-negative bone marrow cells with a constitutively expressed Hoxb8 flanked by loxP. HDPs proliferate indefinitely and differentiate into MΦ when Hoxb8 is removed by a tamoxifen-inducible Cre. We genetically modified HDPs with a constitutively active GMCSF receptor and the tamoxifen-induced transcription factor IRF8, which we have termed "HDP-on." The HDP-on proliferates without GMCSF and differentiates into the MΦ upon exposure to tamoxifen and ruxolitinib (GMCSF inhibitor via JAK1/2 blockade). We quantified the biodistribution of HDPs transplanted via intraperitoneal injection into immunodeficient NCG mice with a luciferase reporter; HDPs are detected for 14 days in the peritoneal cavity, liver, spleen, kidney, bone marrow, brain, lung, heart, and blood. In immunocompetent BALB/c mice, HDP-on cells, but not HDPs, are detected 1 day post-transplantation in the peritoneal cavity. Pretreatment of BALB/c mice with liposomal clodronate significantly enhances survival at day 7 for HDPs and HDP-on cells in the peritoneal cavity, spleen, and liver, but cells are undetectable at day 14. Short-term post-transplantation survival of HDPs is significantly improved using HDP-on and liposomal clodronate, opening a path for MΦ-based therapeutics.

Macrophage-based cell therapies: The long and winding road.

In the quest for better medicines, attention is increasingly turning to cell-based therapies. The rationale is that infused cells can provide a targeted therapy to precisely correct a complex disease phenotype. Between 1987 and 2010, autologous macrophages (MΦs) were used in clinical trials to treat a variety of human tumors; this approach provided a modest therapeutic benefit in some patients but no lasting remissions. These trials were initiated prior to an understanding of: the complexity of MΦ phenotypes, their ability to alter their phenotype in response to various cytokines and/or the environment, and the extent of survival of the re-infused MΦs. It is now known that while inflammatory MΦs can kill tumor cells, the tumor environment is able to reprogram MΦs into a tumorigenic phenotype; inducing blood vessel formation and contributing to a cancer cell growth-promoting milieu. We review how new information enables the development of large numbers of ex vivo generated MΦs, and how conditioning and gene engineering strategies are used to restrict the MΦ to an appropriate phenotype or to enable production of therapeutic proteins. We survey applications in which the MΦ is loaded with nanomedicines, such as liposomes ex vivo, so when the drug-loaded MΦs are infused into an animal, the drug is released at the disease site. Finally, we also review the current status of MΦ biodistribution and survival after transplantation into an animal. The combination of these recent advances opens the way for improved MΦ cell therapies.

The effect of polymer backbone chemistry on the induction of the accelerated blood clearance in polymer modified liposomes.

A variety of water-soluble polymers, when attached to a liposome, substantially increase liposome circulation half-life in animals. However, in certain conditions, liposomes modified with the most widely used polymer, polyethylene glycol (PEG), induce an IgM response resulting in an accelerated blood clearance (ABC) of the liposome upon the second injection. Modification of liposomes with other water-soluble polymers: HPMA (poly[N-(2-hydroxypropyl) methacrylamide]), PVP (poly(vinylpyrrolidone)), PMOX (poly(2-methyl-2-oxazoline)), PDMA (poly(N,N-dimethyl acrylamide)), and PAcM (poly(N-acryloyl morpholine)), increases circulation times of liposomes; but a precise comparison of their ability to promote long circulation or induce the ABC effect has not been reported. To obtain a more nuanced understanding of the role of polymer structure/MW to promote long circulation, we synthesized a library of polymer diacyl chain lipids with low polydispersity (1.04-1.09), similar polymer molecular weights (2.1-2.5kDa) and incorporated them into 100nm liposomes of a narrow polydispersity (0.25-1.3) composed of polymer-lipid/hydrogenated soy phosphatidylcholine/cholesterol/diD: 5.0/54.5/40/0.5. We confirm that HPMA, PVP, PMOX, PDMA and PAcM modified liposome have increased circulation times in rodents and that PVP, PDMA, and PAcM do not induce the ABC effect. We demonstrate for the first time, that HPMA does not cause an ABC effect whereas PMOX induces a pronounced ABC effect in rats. We find that a single dose of liposomes coated with PEG and PMOX generates an IgM response in rats towards the respective polymer. Finally, in this homologous polymer series, we observe a positive correlation (R=0.84 in rats, R=0.92 in mice) between the circulation time of polymer-modified liposomes and polymer viscosity; PEG and PMOX, the polymers that can initiate an ABC response were the two most viscous polymers. Our findings suggest that polymers that do not cause an ABC effect such as, HPMA or PVP, deserve further consideration as polymer coatings to improve the circulation of liposomes and other nanoparticles.

Delivery of lipid micelles into infarcted myocardium using a lipid-linked matrix metalloproteinase targeting peptide.

There is a great need for delivery strategies capable of efficiently localizing drugs to the damaged myocardium that do not require direct intramyocardial injection of therapeutic molecules. In the work discussed here, we exploited the myocardium-specific upregulation of matrix metalloproteinases (MMPs) that occurs during myocardium remodeling by designing a micellar vehicle containing an MMP-targeting peptide (MMP-TP). The binding of MMP-TP to MMP was evaluated with purified MMP-2 protein and U-937 cells induced to overexpress MMP. Inhibition of MMP-2 activity was not observed in the presence of unmodified micelles but was pronounced at a 5 mol % MMP-TP ligand density. In a FACS analysis, MMP-TP micelles containing 5 mol % of the MMP-targeting peptide showed ∼10-fold higher binding to activated U937 cells than plain micelles and micelles containing a control peptide with two amino acid replacements. MMP-TP-micelles and plain micelles were injected intravenously into C57BL/6 mice 1, 3, and 7 days after the induction of a myocardial infarction (MI). Immunohistochemistry performed on heart tissue sections revealed that MMP-TP-micelles colocalize with both MMP and infiltrating macrophages. MMP-TP micelles showed significantly enhanced accumulation to the necrotic area of the heart after MI on days 3 and 7 when compared to plain micelles and negative control peptide micelles. This is coincident with the measured temporal profile of MMP gene expression in the heart after MI. These results suggest that MMP-TP micelles are candidates for the development of targeted regenerative heart therapeutics because of their ability to target the infarcted myocardium in a MMP dependent manner.

Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice.

We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287:1279-1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼ 40 nM and ∼ 20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼ 8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.

Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan.

Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.

Chemoenzymatic synthesis of oligohyaluronan-lipid conjugates.

Herein, we describe an efficient and high-yielding method to synthesize hyaluronan oligosaccharide-lipid conjugates. This strategy is based on first covalently attaching diphytanoyl glycerophosphatidylethanolamine (DiPhPE) to commercially available high molecular weight hyaluronic acid (HA), via the carboxylate group of the glucuronic acid using carbodiimide chemistry. The HA-lipid conjugate mixture is then digested with bovine testicular hyaluronidase to yield HA-DiPhPE conjugates that have a narrow distribution of moderately sized HA oligosaccharides. These HA-lipid conjugates can be incorporated into liposomes or micelles to selectively target CD44 that is overexpressed on many cancer or cancer initiating cells.

Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock.

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3ß-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3ß. Interestingly, OGT activity is regulated by GSK3ß; hence, OGT and GSK3ß exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

All Dact (Dapper/Frodo) scaffold proteins dimerize and exhibit conserved interactions with Vangl, Dvl, and serine/threonine kinases.

BACKGROUND: The Dact family of scaffold proteins was discovered by virtue of binding to Dvl proteins central to Wnt and Planar Cell Polarity (PCP) signaling. Subsequently Dact proteins have been linked to a growing list of potential partners implicated in ß-catenin-dependent and ß-catenin-independent forms of Wnt and other signaling. To clarify conserved and non-conserved roles for this protein family, we systematically compared molecular interactions of all three murine Dact paralogs by co-immunoprecipitation of proteins recombinantly expressed in cultured human embryonic kidney cells. RESULTS: Every Dact paralog readily formed complexes with the Vangl, Dvl, and CK1δ/ε proteins of species ranging from fruit flies to humans, as well as with PKA and PKC. Dact proteins also formed complexes with themselves and with each other; their conserved N-terminal leucine-zipper domains, which have no known binding partners, were necessary and sufficient for this interaction, suggesting that it reflects leucine-zipper-mediated homo- and hetero-dimerization. We also found weaker, though conserved, interactions of all three Dact paralogs with the catenin superfamily member p120ctn. Complex formation with other previously proposed partners including most other catenins, GSK3, LEF/TCF, HDAC1, and TGFß receptors was paralog-specific, comparatively weak, and/or more sensitive to empirical conditions. CONCLUSIONS: Combined with published functional evidence from targeted knock-out mice, these data support a conserved role for Dact proteins in kinase-regulated biochemistry involving Vangl and Dvl. This strongly suggests that a principal role for all Dact family members is in the PCP pathway or a molecularly related signaling cascade in vertebrates.

Dact1 is a postsynaptic protein required for dendrite, spine, and excitatory synapse development in the mouse forebrain.

Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian CNS, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knock-out mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild-type littermates. This correlated with reductions in excitatory synapses and miniature EPSCs, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that, during neuronal differentiation, Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.

Dact1-3 mRNAs exhibit distinct expression domains during tooth development.

Wnt signaling is essential for tooth formation and Dact proteins modulate Wnt signaling by binding to the intracellular protein Dishevelled (Dvl). Comparison of the three known mouse Dact genes, Dact1-3, from the morphological initiation of mandibular first molar development through the onset of root formation using section in situ hybridization showed distinct, complementary and overlapping expression patterns for these genes. Whereas Dact2 expression was restricted to the dental epithelium, including the enamel knot signaling centers and pre-ameloblasts, Dact1 and Dact3 showed developmentally regulated expression in the dental mesenchyme. Both Dact1 and Dact3 mRNAs were first detected in the presumptive dental mesenchyme. After being downregulated from the condensing dental mesenchyme of the bud stage tooth germ, Dact1 was upregulated in the dental follicle mesenchyme at the cap stage and subsequently also in the dental papilla at the bell stage, where the expression persisted to the postnatal stages. In contrast, Dact3 transcripts persisted throughout the dental mesenchyme, including the preodontoblasts, during embryogenesis before transcripts were largely downregulated from the tooth germ postnatally. Collectively, these results suggest that Dact1 and -3 may contribute to early tooth formation by modulation of Wnt signaling pathways in the mesenchyme, including preodontoblasts, whereas Dact2 may play important signal-modulating roles in the adjacent epithelial cells including the enamel knot signaling centers and pre-ameloblasts. Future loss-of-function studies will help elucidate whether any of these functions are redundant, particularly for Dact1 and Dact3.

Posterior malformations in Dact1 mutant mice arise through misregulated Vangl2 at the primitive streak.

Mice homozygous for mutations in Dact1 (also called Dapper or Frodo) phenocopy human malformations involving the spine, genitourinary system and distal digestive tract. We traced this phenotype to disrupted germ-layer morphogenesis at the primitive streak. Notably, heterozygous mutation of Vangl2, a transmembrane component of the planar cell polarity (PCP) pathway, rescued recessive Dact1 phenotypes, whereas loss of Dact1 reciprocally rescued semidominant Vangl2 phenotypes. We show that Dact1, an intracellular protein, forms a complex with Vangl2. In Dact1 mutants, Vangl2 was increased at the primitive streak, where cells ordinarily undergo an epithelial-mesenchymal transition. This is associated with abnormal E-cadherin distribution and changes in biochemical measures of the PCP pathway. We conclude that Dact1 contributes to morphogenesis at the primitive streak by regulating Vangl2 upstream of cell adhesion and the PCP pathway.

Activating PER repressor through a DBT-directed phosphorylation switch.

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)epsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per(S) mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.

DACT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modifications.

Genetic and epigenetic defects in Wnt/beta-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/beta-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly derepressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/beta-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/beta-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/beta-catenin signaling in colorectal cancer.

Three Dact gene family members are expressed during embryonic development and in the adult brains of mice.

Members of the Dact protein family initially were identified through binding to Dishevelled (Dvl), a cytoplasmic protein central to Wnt signaling. During mouse development, Dact1 is detected in the presomitic mesoderm and somites during segmentation, in the limb bud mesenchyme and other mesoderm-derived tissues, and in the central nervous system (CNS). Dact2 expression is most prominent during organogenesis of the thymus, kidneys, and salivary glands, with much lower levels in the somites and in the developing CNS. Dact3, not previously described in any organism, is expressed in the ventral region of maturing somites, limb bud and branchial arch mesenchyme, and in the embryonic CNS; of the three paralogs, it is the most highly expressed in the adult cerebral cortex. These data are consistent with studies in other vertebrates showing that Dact paralogs have distinct signaling and developmental roles and suggest they may differentially contribute to postnatal brain physiology.

Posttranscriptional and posttranslational regulation of clock genes.

Circadian rhythms have been observed in diverse organisms, including plants, animals, bacteria, and fungi. In such organisms, the circadian clock is primarily composed of a cell-autonomous transcriptional feedback loop. In addition to transcriptional regulation, the modification of core clock transcripts and proteins can dramatically affect the circadian clock. In this review, the authors discuss some of the posttranscriptional and posttranslational modifications and their effects on the circadian clock. The combined outcome of these modifications is to adjust the timing of the clock to produce a circadian oscillator that takes approximately 24 h.