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Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R–MyD88–IRAK2–PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity. Obesity is often accompanied by chronic inflammation. Li and colleagues show that, in mice fed high-fat diets, IL-1 signaling in adipocytes induces an unconventional IRAK2 translocation to mitochondria and suppresses respiratory super-complex formation to alter mitochondrial function, and exacerbates obesity.

Background Prostate cancer (PCa) is primarily driven by androgen receptor (AR) signaling and has a highly immunosuppressive microenvironment. Although genomic and histopathological differences between low- and high-grade primary PCa (lgPCa and hgPCa) have been reported, an integrative assessment of multiple molecular features in the context of disease grade and metastatic outcome is lacking. We propose that a subset of hgPCa patients who relapse under SOC may benefit from adjuvant immune-checkpoint blockade (ICB) added to SOC to overcome immune suppression. Methods We analyzed treatment naive prostatectomy tissue from a cohort of 124 primary PCa patients (n= 58, Gleason score ≤6; n= 66, Gleason score ≥ 8). We performed RNAseq expression profiling, whole-exome sequencing (WES) and immunohistochemistry. We employed digital spatial analysis in tumor vs. stromal regions to characterize differences in CD8+ T-cell topology between lgPCa and hgPCa Results 1.Comparisons in lg vs. hgPCA: Digital spatial analysis assessing the proximity of CD8+ T-cells to tumor cells revealed a T-cell exclusion phenotype that is more prominent in hgPCa, whereas evaluation of overall CD8+ T-cell density in tumor and stromal regions did not differentiate disease grades. HgPCa had a higher frequency of at least one functional mutation in either TP53, RHPN2, or KMT2D genes compared to lgPCa. Assessment of MHC-I deficiency by IHC and mRNA revealed that hgPCa has significantly lower MHC-I protein expression compared to lgPCa. Interestingly, MHC-I loss in hgPCa associated with a T-cell exclusion phenotype. Moreover, RNAseq gene expression signatures revealed higher expression of tumor-associated macrophage (TAMs), T-regs, Cancer-Associated Fibroblasts (CAFs), DNA damage repair (DDR) genes and lower Interferon-γ (IFN- γ) expression in hgPCa compared to lgPCa. Overall, hgPCa is characterized by a combined phenotype of ‘MHCIloss/IFN- γ low/CAFhigh/TAMhigh/T-reghigh/DDRhigh’. 2.Comparisons within hgPCA that develop metastasis: Unsupervised analysis of molecular features in hgPCa patients that developed metastases identified a subset of patients that exhibit a less immunosuppressive phenotype with lower tumor AR expression, retained tumor MHC-I expression, moderate CD8+ T-cell infiltration and a high IFN-γ RNA signature (figure 1), suggesting potential benefit from ICB therapy Conclusions Our analysis suggests that hgPCa is characterized by low antigenicity as assessed by loss of MHC-I protein expression and an immunosuppressive microenvironment rich in CAFs, macrophages, T-regs and T-cell exclusion phenotypes. Unlike lgPCa, hgPCA can have a poor prognosis (within 5 years relapse). However, a subset of hgPCa patients that metastasized while on SOC exhibited a biomarker profile that might benefit from combination of SOC with ICB Ethics Approval This study was approved by BMS Cambridge Massachusetts Institutional Biosafety Committee, approval number CAM_2020_12050_6 Consent ‘Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.’

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren’s-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1–STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren’s-like diseases in Act1−/− mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T–B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren’s-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren’s-like syndrome in patients containing Act1 mutation., Adaptor for IL-17 receptors (Act1) is known to be crucial for IL-17-mediated immune responses. Here the authors show that Act1 also functions as a negative regulator of T and B cells by direct inhibition of STAT3.

In the present study, total membrane proteins from tumor cell lines including HepG2, Hep3B2, H226, Ovcar3 and N87 were extracted and digested with γLysC and trypsin. The resulting peptide lysate were pre-fractionated and subjected to untargeted quantitative proteomics analysis using a high resolution mass spectrometer. The mass spectra were processed by the MaxQuant and the protein abundances were estimated using total peak area (TPA) method. A total of 6037 proteins were identified, and the analysis resulted in the identification of 2647 membrane proteins. Of those, tumor antigens and absorption, metabolism, disposition and elimination (ADME) proteins including UDP-glucuronosyltransferase, cytochrome P450, solute carriers and ATP-binding cassette transporters were detected and disclosed significant variations among the cell lines. The principal component analysis was performed for the cluster of cell lines. The results demonstrated that H226 is closely related with N87, while Hep3B2 aligned with HepG2. The protein cluster of Ovcar3 was apart from that of other cell lines investigated. By providing for the first time quantitative untargeted proteomics analysis, the results delineated the expression profiles of membrane proteins. These findings provided a useful resource for selecting targets of choice for anticancer therapy through advancing data obtained from preclinical tumor cell line models to clinical outcomes., Graphical abstract Comparative untargeted proteomics analysis was conducted to identify and quantify membrane proteins in tumor cell lines through database searching with MaxQuant software. The identified proteins were annotated by gene ontology (GO) and assigned into four distinct subcellular categories. The proteomics analysis demonstrated significant variations among the tumor cell lines and lead to a greater appreciation for the role of membrane proteins in tumor development and drug resistance, and present targets of choice as tumor marker proteins.fx1

Background Patients with primary Sjogren’s syndrome (pSS) present with heterogeneous clinical symptoms and immune dysregulation. How immune dysregulation in SS arises is poorly understood, which hampers development of effective therapies. Identification of key immune pathways contributing to pSS pathogenesis is essential for successful drug development. Systemic treatment of patients early in the disease process will limit irreversible damage to salivary and lacrimal glands, as well as to other affected tissues. Objectives To identify early systemic biomarkers and dysregulated immune pathways in newly diagnosed pSS patients by multidimensional immuno-profiling. Methods We included 40 newly diagnosed pSS patients (39 female; mean age 51±14) and 20 age- and sex-matched non-SS sicca patients (19 female; mean age 50±13). All pSS patients fulfilled ACR-EULAR criteria. Serum and peripheral blood mononuclear cells (PBMC) were collected and cryopreserved. PBMCs were thawed for immunophenotyping by flow cytometry and RNA isolation. RNA sequencing was performed using TruSeq Stranded Total RNA Library Prep Gold (Illumina), following manufacturer’s recommendations, and RNAseq libraries were sequenced on a HiSeq2500 system. Additionally, serum proteomics and immunoassays for pro-inflammatory cytokines in serum were performed. Results Interferon (IFN) type I signaling pathways were at the top of enriched pathways in PBMCs from pSS patients, compared with non-SS sicca controls (adjusted p Conclusion Newly diagnosed patients with pSS show co-activation of the IFN type I and B cell activation pathways, compared to non-SS sicca controls. At the same time, CD4+ T cell subsets critical for B cell function are activated, which can enhance B cell activation and plasma cell formation. Personalized treatment based on the activity of each pathway in individual patients potentially increases the efficacy of such treatments. Acknowledgement Part of this work was funded by NIH grant RFA-DE-06-004 Disclosure of Interests Gwenny M. Verstappen: None declared, Lu Gao Employee of: Bristol-Myers Squibb, Sarah A. Pringle: None declared, Janneke H. Terpstra: None declared, Silvia C. Liefers: None declared, Jolien F. van Nimwegen Speakers bureau: Bristol-Myers Squibb, Arjan Vissink: None declared, Vishal Patel Employee of: Bristol-Myers Squibb, Aiqing He Employee of: Bristol-Myers Squibb, Amy Truong Employee of: Bristol-Myers Squibb, Sarah Hu Employee of: Bristol-Myers Squibb, Ashok Dongre Employee of: Bristol-Myers Squibb, Arathi Krishnakumar Employee of: Bristol-Myers Squibb, Stefan Kirov Employee of: Bristol-Myers Squibb, Julie Carman Employee of: Bristol-Myers Squibb, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant for: Roche, Bristol-Myers Squibb, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Bristol-Myers Squibb, Novartis, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Speakers bureau: Bristol-Myers Squibb, Roche, Janssen-Cilag

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1β release in microglia. Noncanonical inflammasome-derived IL-1β produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1β via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation., eLife digest The innate immune system is the body’s first line of defense against invading microbes. Some immune cells carry specific receptor proteins called Toll-like receptors that can identify microbes and the signals they emit. As soon as the receptors have detected a threat – for example through sensing oily molecules that make up the cell membranes of microbes – they produce signaling proteins called cytokines and chemokines to alert other immune cells. The DNA in the cell’s nucleus carries the instructions needed to make proteins. To produce proteins, including cytokines and chemokines, the information first has to be transferred into mRNA templates, which carry the instructions to the sites in the cell where the proteins are made. Cytokine and chemokine mRNAs are generally short-lived, but previous studies in 2009 and 2011 have shown that an enzyme called IRAK2 can stabilize them to make them last longer. IRAK enzymes are activated by the Toll-like receptors after a threat has been detected. However, until now it was not known whether IRAK2 also helps to transport the mRNAs of cytokines and chemokines out of the cell nucleus. Using immune cells of mice, Zhou et al. – including some of the researchers involved in the previous studies – discovered that IRAK2 helped to export the mRNAs of cytokines and chemokines from the immune cell nucleus into the surrounding cell fluid. The Toll-like receptors recognized the oily molecules of the microbes and consequently activated IRAK2, which lead to IRAK2 being moved into the cell nucleus. Once activated, IRAK2 helped to assemble the export machinery that moved selected mRNAs out of the nucleus to build the proteins. To do so, IRAK2 stopped a destabilizing protein from binding to the mRNA, so that instead the export machinery could transport the mRNA of the cytokines and chemokines out of the cell nucleus. A next step will be to test whether IRAK2 is required to guide exported mRNA tothe sites in the cell where the proteins are made. This new insight could help to develop new treatments for various diseases. For example, diseases in which the immune system attacks the cells of the body, rather than invaders, can be caused by too many cytokines and chemokines. Since IRAK2 helps to control the availability of cytokines and chemokines it may in future be used as a new drug target.

NOTCH1 signalling contributes to defective remyelination by impairing differentiation of oligodendrocyte progenitor cells (OPCs). Here we report that IL-17 stimulation induces NOTCH1 activation in OPCs, contributing to Th17-mediated demyelinating disease. Mechanistically, IL-17R interacts with NOTCH1 via the extracellular domain, which facilitates the cleavage of NOTHC1 intracellular domain (NICD1). IL-17-induced NOTCH1 activation results in the interaction of IL-17R adaptor Act1 with NICD1, followed by the translocation of the Act1–NICD1 complex into the nucleus. Act1–NICD1 are recruited to the promoters of several NOTCH1 target genes (including STEAP4, a metalloreductase important for inflammation and cell proliferation) that are specifically induced in the spinal cord by Th17 cells. A decoy peptide disrupting the IL-17RA–NOTCH1 interaction inhibits IL-17-induced NOTCH1 activation and attenuates Th17-mediated experimental autoimmune encephalitis (EAE). Taken together, these findings demonstrate critical crosstalk between the IL-17 and NOTCH1 pathway, regulating Th17-induced inflammatory and proliferative genes to promote demyelinating disease., NOTCH signalling stimulates oligodendrocyte progenitor cell proliferation but how this regulates demyelinating disease is unclear. Here, the authors show that an IL-17 adaptor protein, Act1, interacts with the C-terminal fragment of NOTCH1 (NICD) to activate cell proliferation and an inflammatory response.

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.

Background: Integrated analysis of multi-omic data is important in understanding oncogenesis, pathology, and drug mechanism of action. Formalin-fixed, paraffin-embedded (FFPE) tissues comprise the bulk of archival specimens in hospitals and clinical trials. Commercial kits are commonly used to extract RNA, DNA, or protein for biomarker studies; however, high-quality extractions are challenging due to crosslinking. We explored the feasibility of performing proteomic, transcriptomic, and genetic analysis from limited FFPE samples collected in clinical trials through a pilot study in colorectal cancer (CRC) and normal tissue. Methods: Qiagen kits were employed, with some modifications, to extract nucleic acid and protein from FFPE sample lysates that would be amenable to next-generation sequencing and liquid chromatography/mass-spectrometry (LC/MS) proteomic profiling. Commercially sourced FFPE slides from CRC biopsies and normal colon, heart, and skin samples were processed (n = 10 each; ~600-mm2 by 5-μm/slice). RNA-seq library preparation was performed using the Illumina® TruSeq Access kit. Label-free LC/MS proteomic analysis was carried out using trypsin/Lys-C-digested protein fragments on an EASY-nLC™ 1200 coupled to a Q Exactive™ Plus (Thermo Fisher). MS data were processed with MaxQuant to estimate protein abundance (Cox and Mann, Nat Biotech 2008). Results: Of 40 samples tested, 38 yielded quantifiable nucleic acid and protein of sufficient quality for transcriptional and proteomic analyses. Mean RNA yield was ~300 ng (150 ng-1.2 µg). RNA degradation was significant, with a mean DV>200 of 45% (Agilent Bioanalyzer). Mean DNA yield was 530 ng, with a mean fragment size of 2 kb. Mean protein yield was 50 µg (1 µg-300 µg). RNA libraries had a mapping rate range of 85%-90% and a coding rate range of 70%-80%; ~16,000 genes were reliable detected (log2 TPM > 1). The number of unique proteins detected ranged from 3,000 to 4,000 for CRC, normal colon, and heart samples; skin samples averaged 1,000 proteins and had the lowest overall yield. Correlation of RNA and protein expression for the same genes was weak (Spearman's rho ~0.3-0.4) at the per-sample level but statistically significant. Using linear models, we identified differentially expressed transcripts and proteins between the CRC and normal colon samples. Pathway enrichment analysis of both modalities indicated changes in cell cycle, which is consistent with rapid growth of tumor cells. Changes in nucleoside metabolism, extracellular matrix remodeling, and innate immune response were more apparent at the protein level. Conclusion: We found that multi-omic analysis was feasible with FFPE samples, and proteomics can be used to validate RNA results. Additionally, proteomics reveal post-translational events, such as extracellular matrix remodeling, that provide unique insights into cancer pathology. Citation Format: Vishal Patel, Ji Gao, Mingyi Liu, Aiqing He, Xi-Tao Wang, Kathryn Vanderlaag, Ariella Sasson, Stefan Kirov, Sunil Kuppasani, Omar J. Jabado, Kandasamy Ravi, Ashok Dongre, Julie Carman, Heidi LeBlanc. Integrated analysis of colorectal carcinoma by co-extraction of RNA, DNA and protein from FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2707.

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.

A hypothesis was formed that it would be possible to isolate an adequate amount of protein from a patient, having normal renal function, to identify biological markers of a particular disease state using a variety of proteomics techniques. To support this hypothesis, three samples of urine were collected from a volunteer: first when healthy, later when experiencing acute inflammation due to a pilonidal abcess, and again later still after successful recovery from the condition. The urine from these samples was processed by solid-phase extraction to concentrate and desalt the endogenous proteins and peptides. The proteins and peptides from these urine samples were analyzed in three different experiments: (1) traditional two-dimensional gel electrophoresis followed by proteolysis and mass spectrometric identification of various protein spots, (2) whole mixture proteolysis followed by one-dimensional packed capillary liquid chromatography and tandem mass spectrometry, (3) whole mixture proteolysis followed by two-dimensional capillary liquid chromatography and tandem mass spectrometry. In all three cases, a set of proteins was identified representing putative biomarkers. Each of these proteins was then found to have been previously linked in the scientific literature to inflammation. One acute phase reactant in particular, orosomucoid, was readily observed in all three experiments to dramatically increase in abundance, thereby supporting the hypothesis.

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Abin activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptidesderived from cytosolic proteins exists in vivo.

Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II–like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii−/−M−/−). Antigen presenting cells (APCs) from Ii−/−M−/− mice, as compared with APCs from Ii−/− mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab–restricted T cells. As a consequence of this defect in the loading of self peptides, CD4+ thymocyte development is profoundly impaired in Ii−/−M−/− mice, resulting in a peripheral CD4+ T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.

Overexpression and enhanced activity of insulin-like growth factor-I receptor (IGF-IR) in diverse tumor types make it an attractive target for cancer therapy. BMS-536924 is a potent small molecule inhibitor of IGF-IR, which shows antitumor activity in multiple tumor models, including sarcoma. To facilitate the development of IGF-IR inhibitors as cancer therapy, identification of biomarkers for selecting patients most likely to derive clinical benefit is needed. To do so, 28 sarcoma and neuroblastoma cell lines were screened for in vitro response to BMS-536924 to identify sensitive and resistant cell lines. Notably, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma are more responsive to BMS-536924, suggesting these specific subtypes may represent potential targeted patient subpopulations for the IGF-IR inhibitor. Gene expression and protein profiling were performed on these cell lines, and candidate biomarkers correlating with intrinsic and/or acquired resistance to BMS-536924 were identified. IGF-I, IGF-II, and IGF-IR were highly expressed in sensitive cell lines, whereas IGFBP-3 and IGFBP-6 were highly expressed in resistant lines. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in resistant cell lines may represent one possible resistance mechanism by the adaptation of IGF-IR–independent growth using alternative signaling pathways. Based on cross-talk between IGF-IR and EGFR pathways, combination studies to target both pathways were performed, and enhanced inhibitory activities were observed. These results provide a strategy for testing combinations of IGF-IR inhibitors with other targeted therapies in clinical studies to achieve improved patient outcomes. Further exploration of mechanisms for intrinsic and acquired drug resistance by these preclinical studies may lead to more rationally designed drugs that target multiple pathways for enhanced antitumor efficacy. [Cancer Res 2009;69(1):161–70]

The CK2 protein kinases are a small family of two highly related serine/threonine kinases composed of two catalytic subunits, α and α’, and a single β subunit. Numerous substrates have been reported for CK2 and these proteins are known to participate in diverse cellular processes, including cell signaling, transcription, DNA repair, apoptosis regulation and tumor suppression. Elevated CK2 expression and kinase activity has been observed in many cancer types. Further, mRNA knockdown and enzyme inhibition studies have demonstrated that many cancer cell lines are dependent on CK2 for growth and survival. To further evaluate CK2 kinases as targets for therapeutic intervention in cancer, we identified BMS-595, a potent and selective, ATP-competitive CK2 inhibitor. BMS-595 inhibits the in vitro proliferation of human colorectal and lung cancer cell lines with IC50s ranging from less than 10 nM to greater than 1 μM. In sensitive cell lines, anti-proliferative effects of BMS-595 and structurally related analogs strongly correlated with cellular CK2 kinase inhibition. Oral administration of BMS-595 to mice bearing colorectal cancer and lung cancer xenografts demonstrated pharmacodynamic effects and robust efficacy at tolerated doses. These studies confirm the dependence of a subset of human colon and lung cancer cell lines on CK2 activity for growth and demonstrate that pharmacologic inhibition of CK2 can produce anti-tumor efficacy at tolerated doses. Citation Format: Brent A. Rupnow, Chiang Yu, Jonathan G. Pabalan, Urvashi V. Roongta, Jonathan S. Lippy, Ashok R. Dongre, Mary T. Obermeier, Aberra Fura, Paul A. Elzinga, Benjamin J. Henley, Joseph Fargnoli, Francis Y. Lee, William R. Foster, Christine M. Tarby, Brian E. Fink, John S. Tokarski, Ashvinikumar V. Gavai, Tai W. Wong, John T. Hunt, Gregory D. Vite, Ashok V. Purandare. Anti-tumor activity of BMS-595, a novel CK2 kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5395. doi:10.1158/1538-7445.AM2015-5395

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a ‘client’ protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the TH17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.