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Supplementary Figures 1-8 from The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

Supplementary Tables 1-4 from The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

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.’

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

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type–specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A–induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A–induced stress fiber formation in ASMCs and attenuated IL-17A–enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

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.

Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first responder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as well as other reactive radical species. A high throughput screen and triaging protocol was developed to identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor, 7-(benzyloxy)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine is described.

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.

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.

SummaryInterleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3β, we found that GSK3α but not GSK3β formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.

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.

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.

A set of guidelines has been developed for using the peptide hits technique (PHT) as a semi-quantitative screening tool for the identification of proteins that change in abundance in a complex mixture. The dataset that formed the basis for these experiments was created using a cell lysate derived from the yeast Saccharomyces cerevisiae, spiked at various levels with serum albumin (BSA), and analyzed by LC/MS/MS and SEQUEST. Knowing that the level of only one protein (BSA) actually changed in the mixture allowed for the development and refinement of the necessary bioinformatics and statistical analyses, e. g., principal component analysis (PCA), normalization, and analysis of variation (ANOVA). As expected, the number of BSA peptide hits changed in proportion to the amount of BSA added to the sample. PCA was able to clearly distinguish between the spiked samples and the untreated sample, indicating that PCA may be able to classify samples, e. g., healthy versus diseased, in future experiments. The use of an endogenous “housekeeping” protein was found to be superior to the use of total hits for data normalization prior to analysis. An ANOVA based model readily identified BSA as a protein of interest, that is, one likely to be changing from amongst the background proteins, indicating that an ANOVA model may be able to identify individual proteins in target or biomarker discovery experiments. General guidelines based on these combined observations are set forth for future analyses and the rapid screening for candidate proteins of interest.