Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition.

Introduction: Approximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy. Methods: To compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition. Results: RNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells. Conclusions: The findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple-Negative Breast Cancer.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein-coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. IMPLICATIONS: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

A molecular basis for neurofibroma-associated skeletal manifestations in NF1.

PURPOSE: Plexiform neurofibromas (pNF) develop in children with neurofibromatosis type 1 (NF1) and can be associated with several skeletal comorbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. METHODS: We used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used dual-energy X-ray absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. RESULTS: We detected increased nonmineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. CONCLUSION: Results indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.

Nanobody-enabled monitoring of kappa opioid receptor states.

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.

GSK2801, a BAZ2/BRD9 Bromodomain Inhibitor, Synergizes with BET Inhibitors to Induce Apoptosis in Triple-Negative Breast Cancer.

Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triple-negative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFb-mediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. IMPLICATIONS: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.

Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2.

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.

Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer.

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

New Mechanisms of Resistance to MEK Inhibitors in Melanoma Revealed by Intravital Imaging.

Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors in situ, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance.Significance: A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. Cancer Res; 78(2); 542-57. ©2017 AACR.

Kinome and Transcriptome Profiling Reveal Broad and Distinct Activities of Erlotinib, Sunitinib, and Sorafenib in the Mouse Heart and Suggest Cardiotoxicity From Combined Signal Transducer and Activator of Transcription and Epidermal Growth Factor Receptor Inhibition.

BACKGROUND: Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. METHODS AND RESULTS: We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. CONCLUSIONS: Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment.

Enhancer Remodeling during Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacologic Targeting of the P-TEFb Complex.

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11-q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642-two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)-activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS-U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS-U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.

Inhibition of Lapatinib-Induced Kinome Reprogramming in ERBB2-Positive Breast Cancer by Targeting BET Family Bromodomains.

Therapeutics that target ERBB2, such as lapatinib, often provide initial clinical benefit, but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that, to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance, including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib, and overcoming the dilemma of heterogeneity in the adaptive response.

The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells.

The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.

SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c controls epithelial-mesenchymal transition by inducing Wnt5a signaling.

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM-) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM- breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons.

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

The presynaptic component of the serotonergic system is required for clozapine's efficacy.

Clozapine, by virtue of its absence of extrapyramidal side effects and greater efficacy, revolutionized the treatment of schizophrenia, although the mechanisms underlying this exceptional activity remain controversial. Combining an unbiased cheminformatics and physical screening approach, we evaluated clozapine's activity at >2350 distinct molecular targets. Clozapine, and the closely related atypical antipsychotic drug olanzapine, interacted potently with a unique spectrum of molecular targets. This distinct pattern, which was not shared with the typical antipsychotic drug haloperidol, suggested that the serotonergic neuronal system was a key determinant of clozapine's actions. To test this hypothesis, we used pet1(-/-) mice, which are deficient in serotonergic presynaptic markers. We discovered that the antipsychotic-like properties of the atypical antipsychotic drugs clozapine and olanzapine were abolished in a pharmacological model that mimics NMDA-receptor hypofunction in pet1(-/-) mice, whereas haloperidol's efficacy was unaffected. These results show that clozapine's ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.

Symplekin specifies mitotic fidelity by supporting microtubule dynamics.

Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly.

Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype.

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.

Genetic deletion of p90 ribosomal S6 kinase 2 alters patterns of 5-hydroxytryptamine 2A serotonin receptor functional selectivity.

The concept of functional selectivity has now thoroughly supplanted the previously entrenched notion of intrinsic efficacy by explaining how agonists and antagonists exhibit a range of efficacies for distinct receptor-mediated responses. It is noteworthy that functional selectivity accommodates significant changes in efficacy resulting from differential expression of G protein-coupled receptor modifying proteins (i.e., "conditional efficacy")-a phenomenon with profound implications for drug discovery. We have uncovered a novel regulatory mechanism whereby p90 ribosomal S6 kinase 2 (RSK2) interacts with 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin receptors and attenuates receptor signaling via direct receptor phosphorylation (Proc Natl Acad Sci U S A 103:4717-4722, 2006; J Biol Chem 284:5557-5573, 2009). This discovery, together with the mounting evidence for conditional efficacy, suggested to us that 5-HT(2A) agonist signaling might be disproportionately affected by alterations in RSK2 expression. To test this hypothesis, we evaluated a chemically diverse set of 5-HT(2A) agonists at three readouts of 5-HT(2A) receptor activation in both wild-type (WT) and RSK2 knock-out (KO) mouse embryonic fibroblasts (MEFs). Here we report that 5-HT(2A) receptor agonist efficacies were significantly and variably augmented in RSK2 KO MEFs compared with WT MEFs. As a result, relative agonist efficacies were significantly altered, and even reversed, between WT and RSK2 KO MEFs for a single effector readout. This study provides the first evidence that deletion of a single kinase can elicit profound changes in patterns of agonist functional selectivity.