Opposing Wnt pathways orient cell polarity during organogenesis.

The orientation of asymmetric cell division contributes to the organization of cells within a tissue or organ. For example, mirror-image symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. We characterized the molecular mechanisms contributing to this division pattern. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs toward the center. These Wnts act via Fz/LIN-17 and Ryk/LIN-18, which control beta-catenin localization and activate gene transcription. In addition, VPCs on both sides of the axis of symmetry possess a uniform underlying "ground" polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving the Ror receptor tyrosine kinase CAM-1 and the planar cell polarity component Van Gogh/VANG-1. Thus, tissue polarity is determined by the integration of multiple Wnt pathways.

Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation.

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.

Paracrine Wnt signaling both promotes and inhibits human breast tumor growth.

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

Examination of cocaine dose in a preclinical model of natural reward devaluation by cocaine.

In a preclinical model of natural reward devaluation by cocaine, taste cues elicit aversive taste reactivity when they predict impending but delayed cocaine self-administration. Here, we investigated this negative affective state as a function of cocaine dose. Male, Sprague-Dawley rats were given 45 brief intraoral infusions of a 0.15% saccharin solution before 2 h cocaine self-administration for 14 days. Rats were video recorded; taste reactivity and patterns of self-administration were quantified on the first and last days. On day 14, a significant decrease in appetitive taste reactivity and increase in aversive taste reactivity was observed (compared with day 1) that did not vary as a function of cocaine dose. In contrast, patterns of cocaine self-administration (i.e. the total number of lever presses and load-up behavior) varied as a function of dose across days. Further, load-up behavior was positively correlated with aversive taste reactivity (i.e. gapes) on day 14 across all doses tested. Collectively, these findings indicate that the emergence of negative affect in this preclinical model is not dependent on cocaine dose.

Cocaine-induced endocannabinoid release modulates behavioral and neurochemical sensitization in mice.

The endocannabinoid system has been implicated in the development of synaptic plasticity induced by several drugs abused by humans, including cocaine. However, there remains some debate about the involvement of cannabinoid receptors/ligands in cocaine-induced plasticity and corresponding behavioral actions. Here, we show that a single cocaine injection in Swiss-Webster mice produces behavioral and neurochemical alterations that are under the control of the endocannabinoid system. This plasticity may be the initial basis for changes in brain processes leading from recreational use of cocaine to its abuse and ultimately to dependence. Locomotor activity was monitored with photobeam cell detectors, and accumbens shell/core microdialysate dopamine levels were monitored by high-performance liquid chromatography with electrochemical detection. Development of single-trial cocaine-induced behavioral sensitization, measured as increased distance traveled in sensitized mice compared to control mice, was paralleled by a larger stimulation of extracellular dopamine levels in the core but not the shell of the nucleus accumbens. Both the behavioral and neurochemical effects were reversed by CB1 receptor blockade produced by rimonabant pre-treatments. Further, both behavioral and neurochemical cocaine sensitization were facilitated by pharmacological blockade of endocannabinoid metabolism, achieved by inhibiting the fatty acid amide hydrolase enzyme. In conclusion, our results suggest that a single unconditioned exposure to cocaine produces sensitization through neuronal alterations that require regionally specific release of endocannabinoids. Further, the present results suggest that endocannabinoids play a primary role from the earliest stage of cocaine use, mediating the inception of long-term brain-adaptive responses, shaping central pathways and likely increasing vulnerability to stimulant abuse disorders.

Molecular characterization of type I IFN-induced cytotoxicity in bladder cancer cells reveals biomarkers of resistance.

Although anti-tumor activities of type I interferons (IFNs) have been recognized for decades, the molecular mechanisms contributing to clinical response remain poorly understood. The complex functions of these pleiotropic cytokines include stimulation of innate and adaptive immune responses against tumors as well as direct inhibition of tumor cells. In high-grade, Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer, nadofaragene firadenovec, a non-replicating adenovirus administered locally to express the IFNα2b transgene, embodies a novel approach to deploy the therapeutic activity of type I IFNs while minimizing systemic toxicities. Deciphering which functions of type I IFN are required for clinical activity will bolster efforts to maximize the efficacy of nadofaragene firadenovec and other type I IFN-based therapies, and inform strategies to address resistance. As such, we characterized the phenotypic and molecular response of human bladder cancer cell lines to IFNα delivered in multiple contexts, including adenoviral delivery. We found that constitutive activation of the type I IFN signaling pathway is a biomarker for resistance to both transcriptional response and direct cytotoxic effects of IFNα. We present several genes that discriminate between sensitive and resistant tumor cells, suggesting they should be explored for utility as biomarkers in future clinical trials of type I IFN-based anti-tumor therapies.

Combinations of cocaine with other dopamine uptake inhibitors: assessment of additivity.

Drugs that inhibit dopamine (DA) reuptake through actions at the dopamine transporter (DAT) have been proposed as candidates for development as pharmacotherapies for cocaine abuse. Accordingly, it is important to understand the potential pharmacological interactions of cocaine with other drugs acting at the DAT. Effects of combinations of cocaine with a cocaine analog, 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (WIN 35,428), were compared quantitatively with the combinations of cocaine with the N-butyl,4',4''-diF benztropine analog, 3-(bis(4-fluorophenyl)methoxy)-8-butyl-8-azabicyclo[3.2.1]octane (JHW 007), to determine whether their effects on DA levels in the shell of the nucleus accumbens (NAC) in mice differed. Each of the drugs alone produced dose-related elevations in NAC DA levels. In contrast to the other drugs, JHW 007 was less effective, producing maximal effects that approached 400% of control versus approximately 700% with the other drugs. In addition, the JHW 007 dose-effect curve was not as steep as those for cocaine and WIN 35,428. Combinations of cocaine with its analog, WIN 35,428, were most often greater than those predicted based on dose additivity. In contrast, combinations of cocaine with JHW 007 were most often subadditive. This outcome is consistent with recent studies suggesting that structurally divergent DA uptake inhibitors bind to different domains of the DAT, which can result in different DAT conformations. The conformational changes occurring with JHW 007 binding may result in functional outcomes that alter its abuse liability and its effects in combination with cocaine.

Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs.

The interaction of a drug with its target is critical to achieve drug efficacy. In cases where cellular environment influences target engagement, differences between individuals and cell types present a challenge for a priori prediction of drug efficacy. As such, characterization of environments conducive to achieving the desired pharmacologic outcome is warranted. We recently reported that the clinical CDK4/6 inhibitor palbociclib displays cell type-specific target engagement: Palbociclib engaged CDK4 in cells biologically sensitive to the drug, but not in biologically insensitive cells. Here, we report a molecular explanation for this phenomenon. Palbociclib target engagement is determined by the interaction of CDK4 with CDKN2A, a physiologically relevant protein inhibitor of CDK4. Because both the drug and CDKN2A prevent CDK4 kinase activity, discrimination between these modes of inhibition is not possible by traditional kinase assays. Here, we describe a chemo-proteomics approach that demonstrates high CDK4 target engagement by palbociclib in cells without functional CDKN2A and attenuated target engagement when CDKN2A (or related CDKN2/INK4 family proteins) is abundant. Analysis of biological sensitivity in engineered isogenic cells with low or absent CDKN2A and of a panel of previously characterized cell lines indicates that high levels of CDKN2A predict insensitivity to palbociclib, whereas low levels do not correlate with sensitivity. Therefore, high CDKN2A may provide a useful biomarker to exclude patients from CDK4/6 inhibitor therapy. This work exemplifies modulation of kinase target engagement by endogenous proteinaceous regulators and highlights the importance of cellular context in predicting inhibitor efficacy.

HER-2 overexpression differentially alters transforming growth factor-beta responses in luminal versus mesenchymal human breast cancer cells.

INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-beta) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-beta1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-beta on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-beta1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-beta signaling pathway was assessed using TGF-beta1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-beta1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-beta in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-beta induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-beta. In contrast, HER-2 and TGF-beta signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.

Use of a molecular genetic platform technology to produce human Wnt proteins reveals distinct local and distal signaling abilities.

Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.

Relations between stimulation of mesolimbic dopamine and place conditioning in rats produced by cocaine or drugs that are tolerant to dopamine transporter conformational change.

RATIONALE: Dopamine transporter (DAT) conformation plays a role in the effectiveness of cocaine-like and other DAT inhibitors. Cocaine-like stimulants are intolerant to DAT conformation changes having decreased potency in cells transfected with DAT constructs that face the cytosol compared to wild-type DAT. In contrast, analogs of benztropine (BZT) are among compounds that are less affected by DAT conformational change. METHODS: We compared the displacement of radioligand binding to various mammalian CNS sites, acute stimulation of accumbens shell dopamine levels, and place conditioning in rats among cocaine and four BZT analogs with Cl substitutions on the diphenyl-ether system including two with carboalkoxy substitutions at the 2-position of the tropane ring. RESULTS: Binding assays confirmed high-affinity and selectivity for the DAT with the BZT analogs which also produced significant stimulation of mesolimbic dopamine efflux. Because BZT analogs produced temporal patterns of extracellular dopamine levels different from those by cocaine (3-10 mg/kg, i.p.), the place conditioning produced by BZT analogs and cocaine was compared at doses and times at which both the increase in dopamine levels and rates of increase were similar to those produced by an effective dose of cocaine. Despite this equilibration, none of the BZT analogs tested produced significant place conditioning. CONCLUSIONS: The present results extend previous findings suggesting that cocaine-like actions are dependent on a binding equilibrium that favors the outward conformational state of the DAT. In contrast, BZT analogs with reduced dependence on DAT conformation have reduced cocaine-like behavioral effects and may prove useful in development of medications for stimulant abuse.

Sigma receptor agonists: receptor binding and effects on mesolimbic dopamine neurotransmission assessed by microdialysis.

BACKGROUND: Subtypes of sigma (σ) receptors, σ1 and σ2, can be pharmacologically distinguished, and each may be involved in substance-abuse disorders. σ-Receptor antagonists block cocaine place conditioning and σ-receptor agonists are self-administered in rats that previously self-administered cocaine. Self-administration of abused drugs has been related to increased dopamine (DA) neurotransmission, however, σ-receptor agonist effects on mesolimbic DA are not fully characterized. METHODS: Receptor-binding studies assessed affinities of σ-receptor ligands for σ-receptor subtypes and the DA transporter; effects on DA transmission in the rat nucleus accumbens shell were assessed using in vivo microdialysis. RESULTS: Cocaine (.1-1.0 mg/kg intravenous [IV]), the nonselective σ(½)-receptor agonist DTG (1.0-5.6 mg/kg IV), and the selective σ1-receptor agonist PRE-084 (.32-10 mg/kg IV) dose-dependently increased DA to ∼275%, ∼150%, and ∼160% maxima, respectively. DTG-induced stimulation of DA was antagonized by the nonselective σ(½)-receptor antagonist BD 1008 (10 mg/kg intraperitoneal [IP]) and the preferential σ2-receptor antagonist SN 79 (1-3 mg/kg IP), but not by the preferential σ1-receptor antagonist, BD 1063 (10-30 mg/kg IP). Neither PRE-084 nor cocaine was antagonized by BD 1063 or BD 1008. CONCLUSIONS: σ-Receptor agonists stimulated DA in a brain area critical for reinforcing effects of cocaine. DTG effects on DA appear to be mediated by σ2-receptors rather than σ1-receptors. However, DA stimulation by cocaine or PRE-084 does not likely involve σ-receptors. The relatively low potency on DA transmission of the selective σ1-receptor agonist, PRE-084, and its previously reported potent reinforcing effects, suggest a dopamine-independent reinforcing pathway that may contribute to substance-abuse disorders.

Design, Synthesis, and Preclinical Characterization of the Selective Androgen Receptor Modulator (SARM) RAD140.

This report describes the discovery of RAD140, a potent, orally bioavailable, nonsteroidal selective androgen receptor modulator (SARM). The characterization of RAD140 in several preclinical models of anabolic androgen action is also described.

Ror receptor tyrosine kinases: orphans no more.

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway.

Inhibitors of Wnt signaling promote normal development and prevent cancer by restraining when and where the Wnt pathway is activated. ROR proteins, a class of Wnt-binding receptor tyrosine kinases, inhibit Wnt signaling by an unknown mechanism. To clarify how RORs inhibit the Wnt pathway, we examined the relationship between Wnts and the sole C. elegans ROR homolog, cam-1, during C. elegans vulval development, a Wnt-regulated process. We found that loss and overexpression of cam-1 causes reciprocal defects in Wnt-mediated cell-fate specification. Our molecular and genetic analyses revealed that the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, suggesting that the CAM-1/ROR ECD sequesters Wnts. A sequestration model is supported by our findings that the CAM-1 ECD binds to several Wnts in vitro. These results demonstrate how ROR proteins help to refine the spatial pattern of Wnt activity in a complex multicellular environment.