Antitumor Immune Responses最新文献

{"title":"Abstract A73: Optogenetic regulation of T cell metabolism in the tumor microenvironment","authors":"Andrea M. Amitrano, Brandon L. Walling, Kyun-Do Kim, Brandon J. Berry, A. Trewin, Andrew P. Wojtovich, Minsoo Kim","doi":"10.1158/2326-6074.TUMIMM17-A73","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A73","url":null,"abstract":"The tumor microenvironment presents significant metabolic challenges to T cells by depleting oxygen and glucose, as well as limiting the uptake of key nutrients. Therefore, T cells and tumor cells engage in fierce metabolic competition, as the demand for both oxygen and glucose in the niche is extremely high. The transition from a resting naive T cell into an activated and highly proliferative effector T cell requires substantial metabolic reprogramming from relying primarily on oxidative phosphorylation (OxPhos) to the rapid induction of aerobic glycolysis. However, evidence suggests that tumor infiltrating CD8 + T cells show defects in glycolytic functions. In addition, our data indicates that actively migrating effector CD8 + T cells have greater levels of OxPhos activity than stationary cells, imposing an increasing demand for oxygen during T cell migration to the tumor site. To overcome the glycolytic deficiency of the tumor-targeting T cells and boost anti-tumor effector functions at the tumor microenvironment, we developed a genetically encoded light-activated proton pump (fungal proton pump, “Mac”), namely photoactivatable OxPhos (PA-OxPhos) that is expressed in the inner mitochondrial membrane. During OxPhos, electrons enter the electron transport chain (ETC), causing protons to be pumped across the inner mitochondrial membrane to establish a proton gradient. The gradient is then used to generate ATP through complex V (CxV). Therefore, the outward proton pumping through the inner mitochondrial membrane by light stimulation of PA-OxPhos mimics the ETC function and boosts ATP generation in T cells, even in the presence of low levels of oxygen and substrates, giving T cells a metabolic competitive advantage in the tumor microenvironment. PA-OxPhos is tagged with GFP and is expressed in the mitochondria of transfected 293T cells and in activated mouse CD8 + T cells. When cells were treated with Rotenone (an inhibitor of complex I of the ETC), ATP production was decreased after 90 minutes. Importantly, light stimulation of 293T cells expressing PA-OxPhos successfully increased ATP production even in the presence of Rotenone. Our data suggests that PA-OxPhos can remotely provide a competitive metabolic advantage and boost T cell functions in the tumor microenvironment. The utilization of an alternative mechanism for ATP production in T cells could potentially dissipate the failures of T-cell-based cancer immunotherapies in destroying malignant cells of solid tumors. Citation Format: Andrea Amitrano, Brandon Walling, Kyun Do Kim, Brandon Berry, Adam Trewin, Andrew Wojtovich, Minsoo Kim. Optogenetic regulation of T cell metabolism in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A73.","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"13 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115203515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A69: Mutagenicity of urea cycle dysregulation and its implications for cancer immunotherapy","authors":"J. Lee, N. Carmel, H. Karathia, Noam Auslander, S. Rabinovich, R. Keshet, Noa Stettner, Alon Silberman, L. Agemy, D. Helbling, R. Eilam, Qin Sun, A. Brandis, H. Weiss, D. Dimmock, Noam Stern-Ginossar, A. Scherz, I. Ulitsky, S. Nagamani, R. Elhasid, S. Hannenhalli, E. Ruppin, A. Erez","doi":"10.1158/2326-6074.TUMIMM17-A69","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A69","url":null,"abstract":"Immune checkpoint therapy leads to durable clinical responses in many cancer patients, but fails in others. To improve the clinical response to immunotherapy, it is highly important to identify predictive biomarkers. While checkpoint genes’ expression levels, tumor neo-antigen load and microsatellite instability (MSI) have been associated with enhanced response to checkpoint immunotherapies, they yet provide only a modest predictive signal and hence there is a need to identify additional predictive factors. Specifically, while there is growing evidence that metabolic alterations can affect the tumor and modulate the immune response, the potential effects of altered cancer metabolism on tumor mutagenesis and immunotherapy remain unexplored. The urea cycle (UC) converts excess nitrogen derived from the breakdown of nitrogen-containing molecules (e.g., ammonia) to urea, a relatively non-toxic and disposable nitrogenous compound. We and others have shown that silencing of the UC enzyme ASS1 promotes cancer proliferation by diverting its substrate aspartate toward CAD enzyme, which mediates the first three reactions in the pyrimidine synthesis pathway. We now demonstrate, by analysis of the TCGA data, tumor samples and cancer cell line experiments, that UC dysregulation (UCD) is a much wider common metabolic phenomenon that maximizes nitrogen utilization in cancer, favoring pyrimidine synthesis over urea disposal. Of note, while UCD is significantly associated with decreased cancer patient survival, the overall mutational load is not. Remarkably, we find that the UCD changes the 1:1 purine (R)-to-pyrimidine (Y) ratio in favor of pyrimidine in cancer cells. Moreover, in analysis of both TCGA data and UC perturbed cancer cells we find that: (a) UCD is significantly associated with a novel and unique pattern of purine-to-pyrimidine transversion mutational bias across many cancer types at the DNA coding (sense) strand, and (b) this trend becomes stronger and more significant at both the mRNA and protein levels, testifying to its functional implications. Notably, the overall mutational load in cancer is negatively correlated with UCD, testifying to their independence. To test whether the mutational bias is associated with better immunotherapy response, we analyze published data of three large melanoma cohorts. We find that responders of both anti-PD1 and anti-CTLA4 therapy exhibit significantly higher UCD and R->Y mutational bias than non-responders. We further observe that the peptides carrying transverse R->Y mutations are preferentially presented as neoantigens in responders independent of mutational load, and this trend becomes significant for more clonal neoantigens, promoting UCD as a potential biomarker for the success of immunotherapy. Finally, as nitrogen metabolites are excreted in the urine, we hypothesize that these changes may be detectable in urine of UCD-cancers. We observe increased levels of pyrimidine derived metabolites in the urine of m","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132034293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract PR08: Metabolic adaptations establish immunotherapy resistance in melanoma","authors":"A. Jaiswal, Shivanand Pudakalakatti, Prasanta Dutta, Arthur Liu, T. Bartkowiak, C. Ager, C. Ivan, R. Davis, M. Davies, J. Wargo, J. Allison, P. Bhattacharya, D. Hong, M. Curran","doi":"10.1158/2326-6074.TUMIMM17-PR08","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-PR08","url":null,"abstract":"Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~80% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF analysis, flow cytometry, confocal microscopy, and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by coordinate upregulation of the glycolytic and aldose reductase pathways to create a metabolically hostile microenvironment in which T-cell function is profoundly suppressed. When reintroduced into the parental tumor, the genes most closely associated with these metabolic adaptations confer enhanced immunotherapy resistance. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed MRI imaging to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patients. Conclusion: Upregulation of glycolytic metabolism and the aldose reductase pathway by melanoma tumor cells cripples T cells in the microenvironment and confers resistance to checkpoint blockade. This abstract is also being presented as PosterA71. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Prasanta Dutta, Arthur Liu, Todd Bartkowiak, Casey Ager, Cristina Ivan, Richard Eric Davis, Michael A. Davies, Jennifer Wargo, James P. Allison, Pratip K. Bhattacharya, David Hong, Michael A. Curran. Metabolic adaptations establish immunotherapy resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR08.","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"17 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123703259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A74: T cell activation standardization for therapeutic assay development","authors":"Shilan Dong, Jason Cahoon, Tasnim Khalifa, R. Ohri","doi":"10.1158/2326-6074.TUMIMM17-A74","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A74","url":null,"abstract":"Introduction: Several cancer immunotherapy strategies rely on T-cell activation (1). While T-cell activation mechanisms are well established, there is a dearth of quantified standardization of these activation pathways. Standardization with quantified end-points [induced by known activators and pathways] allows the development of useful assays to evaluate emerging immunotherapies. Our initial work has focused on T-cell activation. Materials and Methods: Activation of the Jurkat T-cell line was induced by the combined action of ionomycin and PMA (phorbol 12-myristate 13-acetate) (2). Jurkat cells were cultured in 24 well plates in RPMI 1640 media (with 10% FBS and 1% Penicillin/Streptomycin) at a density of 106 cells/ml. We looked at a wide concentration range for both activators (500 - 1500 ng/ml for ionomycin and 10 - 100 ng/ml for PMA), in order to identify their most synergistic combination for T-cell activation as quantified by biomarker expression (i.e. IL-2 ELISA). The negative controls were 1% DMSO (used for solubilizing the activators), and PBS (phosphate buffered saline). For a specific combination of 1000 ng/ml ionomycin and 100 ng/ml PMA, the designed time points were 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The cells and supernatant were preserved at the end of these time-points. The viability of the cells was tested using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and the IL-2 production was detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: In addition to identifying the optimal activating concentrations of ionomycin and PMA, the obtained results characterized the time-course of maximal Jurkat cell activation over a 24 h period. During this 24 h period, the IL-2 levels and the cell proliferation rates progressively increased before starting to plateau. The production of IL-2 started increasing at 4 h from 157.6 pg/ml and continued to go up until 24 h to 687.6 pg/ml. Cell proliferation decreased a little during the first 2 h and increased from 57% to 114% (of the PBS control) between 8 to 24 h. Conclusions and Discussion: Our work establishes a standardized basis to determine the extent and the time-course of maximal T-cell activation through a given mechanism - in this case through the mechanism of calcium ionophore induced (ionomycin) and phorbol ester induced (PMA) T-cell activation (3). Such quantified standardization and maximization of T-cell activation in turn presents a cell-culture model (or assay) to evaluate candidate therapeutics designed to work through the same activation pathway. In addition to T-cells, we are working on the quantified standardization of the activation of NK-cells (4) and dendritic cells (5), with activation measured by the end-points of biomarker expression profiling (ELISA/Luminex), proliferation (MTT assay), microscopy and flow cytometry (CD receptor expression profiling). References: 1. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: Moving beyon","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"30 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125010679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A67: Bi-phasic metabolic responses to in situ macrophage activation","authors":"Y. Kam, Pamela M. Swain, B. Dranka","doi":"10.1158/2326-6074.TUMIMM17-A67","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A67","url":null,"abstract":"The cellular metabolism of macrophages is an emerging element regulating inflammatory macrophages which are a critical component of tumor microenvironment. The inflammatory macrophage with highly glycolytic phenotype is also known to elevate the glycolytic activity upon pathogenic stimulation such as lipopolysaccharide (LPS). In this study, the dynamic changes in glycolysis were traced in a real-time manner by measuring proton efflux rates (PERs) and oxygen consumption rates (OCR) after an in-situ activation using Seahorse XFe96 analyzer. The PER of human peripheral blood monocyte (PBMC) derived M1 macrophages was increased within an hour after injection of LPS, which corresponding to cytokine release, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In contrast to PBMC-derived M1 macrophage activation, macrophage cell lines of RAW264.7 and J774.A1 required co-stimulation with interferon γ (IFNγ) for the full activation. Interestingly, the LPS and IFNγ co-stimulation modulates glycolytic rates in a bi-phasic manner which was identified only in long term (> 6 hr) monitoring. A series of long term XF analysis using in situ activation revealed that the immediate early glycolytic response fully relies on LPS stimulation while the secondary elevation in PER depends on IFNγ stimulus, which turns on inducible nitric oxide synthase (iNOS) signaling and in turn suppresses mitochondrial respiration. The TNFα production is closely related to the immediate early glycolytic elevation, but independent from IFNγ-induced second elevation. The IFNγ-dependent second glycolysis increase was totally abolished by iNOS inhibitors whereas the immediate early glycolysis elevation was not affected at all. These data imply a temporal orchestration mechanism of LPS and IFNγ signaling in the metabolic regulation and activation of inflammatory macrophages. Citation Format: Yoonseok Kam, Pamela M. Swain, Brian P. Dranka. Bi-phasic metabolic responses to in situ macrophage activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A67.","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"62 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125025942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A68: Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade","authors":"Sun Hye Kim, Man Li, S. Trousil, B. Zheng","doi":"10.1158/2326-6074.TUMIMM17-A68","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A68","url":null,"abstract":"Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that differentiate from bone marrow precursors and expand in cancer-bearing hosts. MDSCs significantly contribute to tumor-induced immune suppression and represent a promising target for cancer immune therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin, a mitochondrial complex I inhibitor, selectively inhibits granulocytic myeloid-derived suppressor cells (G-MDSCs) in spleens of tumor bearing mice and ex vivo. Metabolomics analysis reveal that, compared to CD11b+Ly6G- cells, CD11b+Ly6G+ MDSCs have lower levels of glutathione and NADPH/NADP+ ratio, suggesting these cells have higher level of endogenous ROS. Interestingly, phenformin further induces production of ROS in G-MDSC, whereas co-treatment of the antioxidant N-acetylcysteine dampens ROS levels in these cells and rescues the effect of phenformin on reducing the numbers of G-MDSCs. Importantly, co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8+ T cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of MDSCs. Our findings demonstrate a selective, inhibitory effect of phenformin on G-MDSCs-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma. Citation Format: Sun Hye Kim, Man Li, Sebastian Trousil, bin zheng. Targeting metabolic vulnerabilities of MDSCs to enhance the antitumor activity of PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A68.","PeriodicalId":414218,"journal":{"name":"Antitumor Immune Responses","volume":"38 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122298491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}