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Supplementary Figures S1-S7; Supplementary Materials and Methods; Supplementary Table S1.

Stimulator of interferon genes (STING) is a critical component of an innate immune pathway that activates robust antiviral and antitumor responses in mouse models. Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) is being explored as a cancer therapy and has shown potent antitumor activity in preclinical models. Here we assessed the benefit of combining immune checkpoint blockade with ADU-S100 (MIW815), a CDN under clinical evaluation, in different syngeneic mouse tumor models. In mice bearing dual flank 4T1 mammary carcinoma tumors resistant to anti-PD-1 treatment, adding a single dose of ADU-S100 with anti-PD-1 induced eradication of both injected and noninjected tumors, leading to near-complete responses, demonstrating that ADU-S100 potentiates the activity of checkpoint blockade. Tumor control was CD8+ T cell-dependent and correlated with an enhanced CD8+ T-cell effector profile in both the periphery and in noninjected tumors. Combining a single injection of ADU-S100 with anti-PD-1 also elicited enhanced tumor control in the dual flank MC-38 colon carcinoma model compared to ADU-S100 or anti-PD-1 treatment alone. Those mice cured by combination treatment were also protected from tumor rechallenge. Moreover, in the poorly immunogenic B16.F10 model, adding ADU-S100 to the ineffective combination therapy of anti-PD-1 and anti-CTLA-4 induced tumor-specific CD8+ T-cell responses and tumor control, leading to multiple complete responses and durable immunity in surviving animals. Together, these results highlight the immune correlates of STING-mediated antitumor efficacy and illustrate the potential of combining ADU-S100 with checkpoint inhibitors for the treatment of human cancer. Clinical trials of ADU-S100 in combination with anti-PD-1 or with anti-CTLA-4 are ongoing and could further elucidate the immunologic mechanism of action and therapeutic effect in humans. This abstract is also being presented as Poster B47. Citation Format: Weiwen Deng, Anthony L. Desbien, Kelsey Sivick Gauthier, Gabrielle Reiner, Leticia Corrales, Tamara Schroeder, Laura Hix Glickman, Natalie H. Surh, Brian Francica, Justin J. Leong, Ken Metchette, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, Steven L. Bender, Chudi Ndubaku, Meredith L. Leong, Andrea van Elsas, Sarah M. McWhirter. ADU-S100 (MIW815) synergizes with checkpoint blockade to elicit an antitumor CD8+ T-cell response to control distal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR09.

Summary: Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFα is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity. : Intratumoral STING pathway activation is a promising therapeutic approach to treat cancer. While high doses of STING agonist are effective at clearing injected tumors, Sivick et al. find that lower doses of STING agonist are optimal for generating robust systemic tumor-specific T cell responses and durable anti-tumor immunity. Keywords: STING, cyclic dinucleotide, intratumoral, ImmunoOncology, anti-tumor immunity, CD8+ T cell, checkpoint inhibitor, ADU-S100, type I interferon, abscopal immunity

Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with and an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.

Osteoarthritis and Kartogenin Osteoarthritis is characterized by progressive breakdown of articular cartilage and affects over 25 million people in the United States. Mesenchymal stem cells (MSCs), which reside in healthy and diseased joints, are multipotent adult stem cells that are able to differentiate into a variety of cell types. Johnson et al. (p. 717 , published online 5 April) identified a small molecule, kartogenin, which was able to induce MSCs to differentiate into chondrocytes in vitro. When administered locally, kartogenin was efficacious in two animal models of osteoarthritis.

Background: Tamoxifen is commonly used for breast cancer therapy. However, tamoxifen resistance is an important clinical problem. Continuous treatment with conventional therapy may contribute to cancer progression in recurring cancers through the accumulation of drug-resistant cancer progenitors. Methods: To investigate signalling mechanisms important for the maintenance and viability of drug-resistant cancer progenitors, we used microarray analysis, PCR array for genes involved in cancer drug resistance and metabolism, flow cytometry, soft agar colony formation assay, in vivo tumourigenicity assay and immunohistochemical analysis using tamoxifen-sensitive and tamoxifen-resistant breast cancer MCF7 cells. Results: Downregulation of CXCR4 signalling by small molecule antagonist AMD3100 specifically inhibits growth of progenitor cell population in MCF7(TAM-R) cells both in vitro and in vivo. Microarray analysis revealed aryl hydrocarbon receptor (AhR) signalling as one of the top networks that is differentially regulated in MCF7(TAM-R) and MCF7 xenograft tumours treated with AMD3100. Further, small molecule antagonists of AhR signalling specifically inhibit the progenitor population in MCF7(TAM-R) cells and growth of MCF7(TAM-R) xenografts in vivo. Conclusion: The chemokine receptor CXCR4 maintains a cancer progenitor population in tamoxifen-resistant MCF7 cells through AhR signalling and could be a putative target for the treatment of tamoxifen-resistant breast cancers.

Time shift A high-throughput cell-based screen identified a benzothiazole analogue, LH846, which induces period lengthening of the circadian rhythm. Affinity chromatography coupled with mass spectrometry and genomic analysis identified protein kinase CKIδ as the biological target of LH846 (see picture).

The EBI2 receptor (Epstein–Barr virus-induced gene 2, also known as GPR183) was recently shown to be linked to autoimmune disease, and is a critical regulator of the humoral immune response. It is a G-protein-coupled receptor, and its natural ligand has been unknown. Two groups now bring an end to the 'orphan' status of this receptor with identification of specific oxysterols as its natural ligands. The most potent ligand and activator is 7a,25-dihydroxycholesterol, and the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response. Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1,2,3. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H)4. Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.

Small molecules do the job: Somatic cells are reprogrammed into iPS cells upon ectopic expression of Oct4, Sox2, Klf4 and c-Myc. Application of a cell-based, high-throughput chemical screen led to the identification of Src family kinase (SFK) inhibitors as chemical replacements for retroviral Sox2 delivery. These compounds are used to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application., National Institutes of Health (U.S.) (Grant HD 045022), National Institutes of Health (U.S.) (Grant 5 R37A084198)

Members of the nuclear receptor superfamily are thought to activate transcription by recruitment of one or more recently identified coactivator complexes. Here we demonstrate that both peroxisome proliferator-activated receptor binding protein (PBP) and steroid receptor coactivator-1 (SRC-1) are required for ligand-dependent transcription of transiently transfected and chromosomally integrated reporter genes by the estrogen receptor (ER) and retinoic acid receptor (RAR). To examine ligand-dependent interactions between nuclear receptors and specific coactivators in living cells, these proteins were tagged with cyan (CFP) and yellow (YFP) mutants of the green fluorescent protein. Fluorescence resonance energy transfer (FRET) from the CFP to the YFP indicated interaction between the receptor and coactivator. CFP fusions to RAR or its ligand-binding domain exhibited rapid ligand-dependent FRET to YFP-tagged nuclear receptor interaction domains of the coactivators SRC-1 and PBP. The ER-ligand-binding domain, unlike RAR, also exhibited some basal interaction with coactivators in unstimulated cells that was abolished by the receptor antagonists tamoxifen or ICI182,780. Inhibition of FRET by tamoxifen but not ICI182,780 could be reversed by estradiol, whereas estradiol-enhanced FRET could not be inhibited by either antagonist, indicating that ligand effects can show varying degrees of hysteresis. These findings suggest that ligand-dependent transcriptional activities of the RAR and ER require concurrent or sequential recruitment of SRC-1 and PBP-containing coactivator complexes.

Synthetic methodology has been developed for the generation of large, diverse libraries of “unnatural” carbamate oligomers using the “one bead, one peptide” method. Using a pool of 27 structurally and functionally diverse monomers, one acyclic and two cyclic libraries were synthesized and screened for binding to the integrin GPIIb/IIIa. Several classes of oligocarbamate ligands for GPIIb/IIIa were discovered, and two cyclic ligands have activities that are within a factor of 3 of kistrin, a snake venom protein that effectively inhibits platelet aggregation. Preliminary pharmacokinetic characterization was performed on a linear oligocarbamate ligand, which was cleared from plasma with a half-life of 3.6 min.

An efficient method for the solid phase synthesis of N -alkylcarbamate oligomers from alternating carboxylic acid and N α -Fmoc protected chiral p -nitrophenylcarbonate monomers has been developed. The general synthetic scheme involves four steps per coupling cycle: deprotection of the terminal amino group of the growing oligomer, acylation of the free amine with a carboxylic acid monomer, reduction of the resulting amide bond with borane and coupling of the secondary amine to a N α -Fmoc protected p -nitrophenyl carbonate monomer. This novel biopolymer which has two side chain residues per backbone carbamate linkage and no backbone hydrogen bond donors may provide new frameworks for drug design as well as folded domains with novel physical and biological properties.

Stimulator of Interferon Genes (STING) is a critical component of the cytosolic DNA sensing pathway of the innate immune system. STING is activated by cyclic dinucleotides (CDNs), a product of the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), in response to presence of cytosolic DNA, including tumor-derived DNA. Production of type I interferon within the tumor microenvironment (TME), mediated by the STING pathway, leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. Therapeutic activation of STING through intratumoral (IT) administration of CDNs results in anti-tumor efficacy and long-lived survival in several mouse syngeneic tumor models. Rational design of synthetic CDN derivatives has shown that certain modifications alter STING binding, increase cellular potency, enhance maturation of human dendritic cells to promote in vitro T cell expansion, and are able to broadly activate all human STING haplotypes. Mechanistic studies in mouse tumor models demonstrate that CDNs mediate anti-tumor immunity by inducing an acute innate immune response, leading to collapse of the injected tumor, and promoting a tumor-specific CD8+ T cell response that protects against tumor re-challenge. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, informing future clinical development. The ability to elicit innate and adaptive anti-tumor immunity via activation of STING in the TME demonstrates that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. The design of an ongoing Phase 1 first-in-human clinical study to evaluate the safety, tolerability and possible antitumor activity of ADU-S100 in subjects with cutaneously accessible tumors and lymphomas will also be presented. Citation Format: Sarah M. McWhirter, Laura Hix Glickman, Tony Desbien, Kelsey Sivick Gauthier, David Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, George Katibah, Ed Lemmens, Leticia Corrales, Meredith Leong, Chudi Ndubaku, Justin Leong, Leonard Sung, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Jr.. STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B020.

Recent reports have provided the mechanistic insight of how innate immune activation promotes priming of anti-tumor immunity and inform the development of clinical approaches to facilitate this process. Spontaneous T cell infiltration of melanoma lesions in humans is correlated with a type I interferon (IFN) transcriptional profile in the tumor microenvironment (TME) and infiltration of lymphocytes, indicative of ongoing innate immune recognition within the tumor. Substantial evidence indicates that tumor infiltrating lymphocytes (TILs), including activated CD8+ T cells, is predictive of a positive clinical outcome in response to several immunotherapy strategies. Similarly, in mice bearing melanoma, there is a correlation between expression of IFN-β by tumor-resident dendritic cells (DCs), and spontaneous priming of tumor-specific immunity. Induction of IFN-β expression and co-regulated IFN-responsive genes and pro-inflammatory chemokines is dependent upon activation of the STING (Stimulator of Interferon Genes) pathway, mediated through sensing of tumor dsDNA in TME-resident CD8α+ DCs by cyclic GMP-AMP (cGAMP) synthase (cGAS), which in turn synthesizes cGAMP. The cyclic dinucleotide (CDN) cGAMP produced by cGAS is the natural STING agonist ligand. Thus, the cGAS-STING signaling axis has emerged as a central node for sensing damage in the host. We hypothesized that direct activation of the STING pathway in the TME by intratumoral (IT) injection of specific CDNs would be an effective therapeutic strategy to promote broad tumor-initiated T cell priming against an individual's tumor antigen repertoire. There are five variant human STING alleles that exist at varying frequencies. While the natural STING ligand cGAMP activates signaling in all variants, other natural CDNs, including those produced by bacteria, have structural differences and are unable to activate particular STING variants, such as the REF (R232H) allele, informing the development of compounds that activate all human STING alleles. We sought to develop synthetic CDN compounds with increased activity in human cells as well as the ability to engage all known polymorphic human STING molecules. Using human 293T cell lines engineered to express the various STING proteins, we screened a large panel of CDN derivatives that varied in purine nucleotide base, structure of the phosphate bridge linkage, and substitution of the non-bridging oxygen atoms at the phosphate bridge with sulfur atoms. ADU-S100 is composed of two adenosine monophosphate (AMP) analogues cyclized via a 2’-5’ (non-canonical) and a 3’-5’ (canonical) phosphodiester bond, and was selected for clinical translation based on properties of enhanced cellular uptake, human STING activation, stability and anti-tumor efficacy, as compared to bacterial and mammalian derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING haplotypes, including donors with a homozygous haplotype for the refractory human REF allele, indicates that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through IT administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. A bell-shaped ADU-S100 dose response curve (which varied based on tumor model) delineated regression of injected tumor, induction of tumor-specific CD8+ T cell immunity, and regression of distal non-injected tumors (abscopal effect), and/or protection against autologous tumor challenge. At low dose levels, regression of the treated tumor was suboptimal. At optimal doses, regression of the treated and distal untreated tumors, or protection against tumor re-challenge was observed, and correlated with induction of a robust tumor Ag-specific CD8+ T cell response. At higher dose levels there was a loss of protection against tumor re-challenge which correlated with increases in acute systemic cytokines and reduction in CD8+ T cell responses even though growth of the treated tumor was inhibited. These results suggest that the mechanism of ADU-S100-induced tumor regression is due to both an acute pro-inflammatory cytokine response and also tumor-specific CD8+ T cell immunity. The local anti-tumor effect without systemic immunity is consistent with well-established data in which excessive innate immune stimulation and induction of pro-inflammatory cytokines such as TNF-α are known to inhibit both priming of CD8+ T cell immunity and establishment of a stable and self-renewing memory CD8+ T cell population. In addition, anti-tumor efficacy was enhanced by combination with immune checkpoint inhibitors, for example α-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. A Phase 1 clinical study to evaluate the safety and tolerability and possible anti-tumor effects in subjects with cutaneously accessible non UV-induced and UV-induced malignancies or lymphomas given repeated IT doses of ADU-S100 is planned. Citation Format: Laura Hix Glickman, Leticia Corrales, David B. Kanne, Shailaja Kasibhatla, Jie Li, Anne Marie Culazzo Pferdekamper, Kelsey Sivick Gauthier, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Weiwen Deng, Anthony L. Desbien, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Sarah M. McWhirter, Thomas F. Gajewski, Thomas W. Dubensky. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY39-02.

Stimulator of Interferon Genes (STING) is a critical signaling sensor of the innate immune system. STING binds cyclic dinucleotides (CDN) produced by an intracellular enzyme in response to presence of intracellular DNA, including tumor-derived DNA. STING-mediated production of host type I interferon within the tumor microenvironment (TME) leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. A novel synthetic CDN derivative (ADU-S100), with superior STING-activating and anti-tumor properties, was developed for clinical translation. ADU-S100 has enhanced cellular uptake properties and stability, as compared to bacterial- and mammalian-derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING alleles, including a homozygous haplotype for the most refractory human allele (R232H), indicate that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through intratumoral (IT) administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provides long-lived immunologic memory. Mechanistic studies demonstrate that STING-mediated anti-tumor immunity is due in part to an acute pro-inflammatory cytokine response as well as a tumor-specific CD8+ T cell response. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, for example anti-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. Citation Format: Laura Hix Glickman, David B. Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, Kelsey Sivick Gauthier, Weiwen Deng, Anthony L. Desbien, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffrey M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Sarah M. McWhirter. STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1445.

Purpose: The cancer stem cell hypothesis predicts that standard prostate cancer monotherapy eliminates bulk tumor cells but not a tumor-initiating cell population, eventually leading to relapse. Many studies have sought to determine the underlying differences between bulk tumor and cancer stem cells. Experimental Design: Our previous data suggest that the PTEN/PI3K/AKT pathway is critical for the in vitromaintenance of CD133+/CD44+ prostate cancer progenitors and, consequently, that targeting PI3K signaling may be beneficial in treatment of prostate cancer. Results: Here, we show that inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 leads to a decrease in the population of CD133+/CD44+ prostate cancer progenitor cells in vivo. Moreover, the combination of the PI3K/mTOR modulator NVP-BEZ235, which eliminates prostate cancer progenitor populations, and the chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors in a prostate cancer xenograft model than monotherapy. Conclusion: This combination treatment ultimately leads to the expansion of cancer progenitors with a PTEN E91D mutation, suggesting that the analysis of PTEN mutations could predict therapeutic response to the dual therapy. Clin Cancer Res; 16(23); 5692–702. ©2010 AACR.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Crystal structures are known for aspartate carbamoyltransferase (ATCase) in the T and R states, with and without the allosteric activator adenosine triphosphate (ATP) or inhibitor cytidine triphosphate (CTP). Visual inspection of X-ray crystal structures does not provide all of the information necessary for the determination of structure--function relationships in protein molecules. This problem is compounded because the crystalline states of the molecule may introduce effects due to crystal packing, restricted flexibility and less than optimum enzymatic conditions. Therefore, alternative techniques are required to test mechanisms conjectured from three-dimensional crystal structures of proteins. The technique of site-specific mutagenesis allows the researcher to test structure--function models based on three-dimensional structures and to obtain further insight into characteristics of the enzyme. Site-specific mutagenesis has been used to probe residues believed to be critical in the structure and function of ATCase. Selection of residues to be mutated has depended extensively on three-dimensional crystal structures of the enzyme. To date, 48 site-specific mutations at 37 different amino acid sites have been published. Although a total of 118 mutants at 58 different sites has been communicated to our laboratory, only published mutants will be considered in this review. In this paper, we compile for the first time, review, and analyze the site-specific mutants of ATCase. Site-specific mutagenesis of proteins has become a powerful technique in modern-day molecular biology, especially in studying a molecule as large as aspartate carbamoyltransferase. In this review, the role of site-specific mutagenesis of ATCase is discussed and improvements in the analysis are suggested.

A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.

An essential function of the human epidermis is the maintenance of a protective barrier against the environment. As a consequence, keratinocytes, which make up this layer of the skin, undergo an elaborate process of self-renewal, terminal differentiation, and cell death. Misregulation of these processes can lead to several human diseases, including psoriasis and basal cell and squamous cell carcinomas. To identify novel regulators of keratinocyte differentiation, a cell-based screen of small-molecule libraries was carried out for molecules that induce terminal differentiation of normal human epidermal keratinocytes. One class of molecules was identified, the 2-(3,4,5-trimethoxyphenylamino)-pyrrolo[2,3-d]pyrimidines, which were shown to induce differentiation of epidermal progenitor cells to terminally differentiated keratinocytes. These molecules serve as useful mechanistic probes of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis and may lead to novel therapeutic approaches for the treatment of skin hyperproliferative disorders.

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs). Analysis of >2 million images, acquired by quantitative fluorescence microscopy, showed that depletion of 1,152 genes strongly affected cell-cycle progression. These genes clustered into eight distinct phenotypic categories based on phase of arrest, nuclear area, and nuclear morphology. Phase-specific networks were built by interrogating knowledge-based and physical interaction databases with identified genes. Genome-wide analysis of cell-cycle regulators revealed a number of kinase, phosphatase, and proteolytic proteins and also suggests that processes thought to regulate G 1 -S phase progression like receptor-mediated signaling, nutrient status, and translation also play important roles in the regulation of G 2 /M phase transition. Moreover, 15 genes that are integral to TNF/NF-κB signaling were found to regulate G 2 /M, a previously unanticipated role for this pathway. These analyses provide systems-level insight into both known and novel genes as well as pathways that regulate cell-cycle progression, a number of which may provide new therapeutic approaches for the treatment of cancer.

The epidermal growth factor receptor (EGFR) tyrosine kinase was one of the first receptor tyrosine kinases to be targeted for drug development by the pharmaceutical industry due to its ubiquitous overexpression in a variety of tumors. Despite the validation of several quinazoline-based scaffolds in the clinic, there is a dearth of alternative chemical structure classes that are capable of inhibiting EGFR kinase activity selectively. Here we describe the discovery of potent and highly selective 4,6-disubstituted pyrimidine inhibitors of enzymatic and cellular EGFR activity and provide an explanation for their exceptional degree of kinase selectivity.

SummaryInsulin resistance is a primary defect in type 2 diabetes characterized by impaired peripheral glucose uptake and insufficient suppression of hepatic glucose output. Insulin signaling inhibits liver glucose production by inducing nuclear exclusion of the gluconeogenic transcription factor FOXO1 in an Akt-dependent manner. Through the concomitant application of genome-scale functional screening and quantitative image analysis, we have identified PTP-MEG2 as a modulator of insulin-dependent FOXO1 subcellular localization. Ectopic expression of PTP-MEG2 in cells inhibited insulin-induced phosphorylation of the insulin receptor, while RNAi-mediated reduction of PTP-MEG2 transcript levels enhanced insulin action. Additionally, adenoviral-mediated depletion of PTP-MEG2 in livers of diabetic (db/db) mice resulted in insulin sensitization and normalization of hyperglycemia. These data implicate PTP-MEG2 as a mediator of blood glucose homeostasis through antagonism of insulin signaling, and suggest that modulation of PTP-MEG2 activity may be an effective strategy in the treatment of type 2 diabetes.

A highly efficient method has been developed for the solid-phase synthesis of an "unnatural biopolymer" consisting of chiral aminocarbonate monomers linked via a carbamate backbone. Oligocarbamates were synthesized from N-protected p-nitrophenyl carbonate monomers, substituted with a variety of side chains, with greater than 99 percent overall coupling efficiencies per step. A spatially defined library of oligocarbamates was generated by using photochemical methods and screened for binding affinity to a monoclonal antibody. A number of high-affinity ligands were then synthesized and analyzed in solution with respect to their inhibition concentration values, water/octanol partitioning coefficients, and proteolytic stability. These and other unnatural polymers may provide new frameworks for drug development and for testing theories of protein and peptide folding and structure.

Several classes of compounds have been tested as potential inhibitors of the serine protease thrombin, an important regulator of blood coagulation cascades. We describe here the discovery of a new class of thrombin inhibitors based on an unnatural carbamate biopolymer. Oligocarbamate thrombin inhibitors were identified through the screening of diverse cyclic trimer, cyclic tetramer, and linear tetramer libraries using the one bead, one peptide method. Whereas the cyclic trimer oligocarbamate ligands bound thrombin with modest affinity, a cyclic tetramer oligocarbamate inhibited thrombin with an apparent K i of 31 nM. Linear oligocarbamate tetramers bound thrombin with inhibition constants in the 100-nM range. These nonpeptidic, oligomeric molecules may provide the basis for further drug development and studies of thrombin–ligand interactions.

Page 1672. Eric C. Peters was mistakenly listed in the Acknowledgment of the published Communication rather than in the author list. Eric C. Peters should be included in the author list between Jonathan G. Swoboda and Charles Y. Cho, along with indication (‡) of his affiliation with The Genomics Institute of the Novartis Research Foundation. The first sentence of the Acknowledgment should now read as follows: The authors acknowledge the efforts of John Walker for experimental support.

The solid phase synthesis and generation of libraries of "unnatural biopolymers" is described. These polymers are characterized by novel backbones and building blocks, the properties of which may modify their pharmacological and folding properties.

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.

Hymenialdisine (HMD) is a sponge-derived natural product kinase inhibitor with nanomolar activity against CDKs, Mek1, GSK3β, and CK1 and micromolar activity against Chk1. In order to explore the broader application of the pyrrolo[2,3-c]azepine skeleton of HMD as a general kinase inhibitory scaffold, we searched for additional protein targets using affinity chromatography in conjunction with the synthesis of diverse HMD analogs and profiled HMD against a panel of 60 recombinant enzymes. This effort has led to nanomolar to micromolar inhibitors of 11 new targets including p90RSK, KDR, c-Kit, Fes, MAPK1, PAK2, PDK1, PKCθ, PKD2, Rsk1, and SGK. The synthesis of HMD analogs has resulted in the identification of compounds with enhanced and/or dramatically altered selectivities relative to HMD (28n) and in molecules with antiproliferative activities 30-fold higher than HMD (28p).

SummaryEmbryonic stem cells (ESCs) are an attractive source of cells for disease modeling in vitro and may eventually provide access to cells/tissues for the treatment of many degenerative diseases. However, applications of ESC-derived cell types are largely hindered by the lack of highly efficient methods for lineage-specific differentiation. Using a high-content screen, we have identified a small molecule, named stauprimide, that increases the efficiency of the directed differentiation of mouse and human ESCs in synergy with defined extracellular signaling cues. Affinity-based methods revealed that stauprimide interacts with NME2 and inhibits its nuclear localization. This, in turn, leads to downregulation of c-Myc, a key regulator of the pluripotent state. Thus, our findings identify a chemical tool that primes ESCs for efficient differentiation through a mechanism that affects c-Myc expression, and this study points to an important role for NME2 in ESC self-renewal.