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Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2α (eukaryotic translation initiation factor 2 alpha). Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2α undermines the ability of T cells to suppress tumor growth. Reprograming T-cell metabolism was able to alleviate p-eIF2α accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2α to support optimal antitumor T-cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy. Significance: Proteasome function is a necessary cellular component for endowing T cells with tumor killing capacity by mitigating translation attenuation resulting from the unfolded protein response induced by stress in the tumor microenvironment.

The requisites for protein translation in T cells are poorly understood and how translation shapes the antitumor efficacy of T cells is unknown. Here we demonstrated that IL15-conditioned T cells were primed by the metabolic energy sensor AMP-activated protein kinase to undergo diminished translation relative to effector T cells. However, we showed that IL15-conditioned T cells exhibited a remarkable capacity to enhance their protein translation in tumors, which effector T cells were unable to duplicate. Studying the modulation of translation for applications in cancer immunotherapy revealed that direct ex vivo pharmacologic inhibition of translation elongation primed robust T-cell antitumor immunity. Our work elucidates that altering protein translation in CD8+ T cells can shape their antitumor capability.

Proteomics Table 1

Supplementary Figs 1-4

Protein Expression, Shotgun Proteomics

Quantification and viability of T cell groups

mtROS+ CD8 TILs are highly activated

FACS antibodies

Tumor antigen–specific T cells rapidly lose energy and effector function in tumors. The cellular mechanisms by which energy loss and inhibition of effector function occur in tumor-infiltrating lymphocytes (TILs) are ill-defined, and methods to identify tumor antigen–specific TILs that experience such stress are unknown. Processes upstream of the mitochondria guide cell-intrinsic energy depletion. We hypothesized that a mechanism of T-cell–intrinsic energy consumption was the process of oxidative protein folding and disulfide bond formation that takes place in the endoplasmic reticulum (ER) guided by protein kinase R-like endoplasmic reticulum kinase (PERK) and downstream PERK axis target ER oxidoreductase 1 (ERO1α). To test this hypothesis, we created TCR transgenic mice with a T-cell–specific PERK gene deletion (OT1+Lckcre+PERKf/f, PERK KO). We found that PERK KO and T cells that were pharmacologically inhibited by PERK or ERO1α maintained reserve energy and exhibited a protein profile consistent with reduced oxidative stress. These T-cell groups displayed superior tumor control compared with T effectors. We identified a biomarker of ER-induced mitochondrial exhaustion in T cells as mitochondrial reactive oxygen species (mtROS), and found that PD-1+ tumor antigen–specific CD8+ TILs express mtROS. In vivo treatment with a PERK inhibitor abrogated mtROS in PD-1+ CD8+ TILs and bolstered CD8+ TIL viability. Combination therapy enabled 100% survival and 71% tumor clearance in a sarcoma mouse model. Our data identify the ER as a regulator of T-cell energetics and indicate that ER elements are effective targets to improve cancer immunotherapy.

Supplemental Figures S1-S11

Mixed linear modeling results for tumor curves

Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2α (eukaryotic translation initiation factor 2 alpha). Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2α undermines the ability of T cells to suppress tumor growth. Reprograming T-cell metabolism was able to alleviate p-eIF2α accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2α to support optimal antitumor T-cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.Significance:Proteasome function is a necessary cellular component for endowing T cells with tumor killing capacity by mitigating translation attenuation resulting from the unfolded protein response induced by stress in the tumor microenvironment.

Emerging evidence indicates associations between high-fat diet (HFD), metabolic syndrome (MetS), and increased risk of pancreatic cancer. However, individual components of an HFD that increase cancer risk have not been isolated. In addition, a specific pattern of cytokine elevation by which MetS drives pancreatic tumor progression is not well described. We hypothesized that oleic acid (OA), a major component of HFD, would augment pancreatic neoplastic processes.An orthotopic pancreatic cancer model with Panc02 cells was used to compare the effect of low-fat diet to OA-based HFD on cancer progression. Tumors were quantitated, analyzed by immunohistochemistry. In addition, serum cytokine levels were quantitated. Proliferation, migration assays, and expression of epithelial-to-mesenchymal transition factors were evaluated on Panc02 and MiaPaCa-2 pancreatic cancer cells cultured in high concentrations of OA.HFD tumor-bearing mice (n = 8) had an 18% weight increase (P 0.001) and increased tumor burden (P 0.05) compared with the low-fat diet tumor-bearing group (n = 6). HFD tumors had significantly increased angiogenesis (P 0.001) and decreased apoptosis (P 0.05). Serum of HFD mice demonstrated increased levels of glucagon and glucagon-like peptide-1. Two pancreatic cancer cell lines cultured in OA demonstrated significant increases in proliferation (P 0.001) and a2.5-fold increase in cell migration (P 0.001) when treated with OA. Panc02 treated with OA had increased expression of epithelial-to-mesenchymal transition factors SNAI-1 (Snail) and Zeb-1(P 0.01).High-fat conditions in vitro and in vivo resulted in an aggressive pancreatic cancer phenotype. Our data support further investigations elucidating molecular pathways augmented by MetS conditions to identify novel therapeutic strategies for pancreatic cancer.

The requisites for protein translation in T cells are poorly understood and how translation shapes the antitumor efficacy of T cells is unknown. Here we demonstrated that IL15-conditioned T cells were primed by the metabolic energy sensor AMP-activated protein kinase to undergo diminished translation relative to effector T cells. However, we showed that IL15-conditioned T cells exhibited a remarkable capacity to enhance their protein translation in tumors, which effector T cells were unable to duplicate. Studying the modulation of translation for applications in cancer immunotherapy revealed that direct ex vivo pharmacologic inhibition of translation elongation primed robust T-cell antitumor immunity. Our work elucidates that altering protein translation in CD8+ T cells can shape their antitumor capability.

Protein synthesis enables cell growth and survival, but the molecular mechanisms through which T cells suppress or maintain protein translation in the stress of solid tumors are unknown. Using mouse models and human tumors we demonstrate that protein translation in T cells is repressed by the solid tumor microenvironment (TME) due to activation of the unfolded protein response (UPR) via phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (p-eIF2α). Given that acute glucose deprivation in T cells exacerbated p-eIF2α, we show that metabolic reprogramming toward glycolytic independence allays the UPR and p-eIF2α, enabling sustained protein translation in T cells in TME stress. UPR mitigation was associated with enhanced degradation of proteins in antitumor T cells, as proteasome inhibition resulted in eIF2α phosphorylation, attenuation of translation, and loss of antitumor efficacy. In contrast, proteasome stimulation relieved translation inhibition, inducing robust T cell tumor control, offering a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.

Background T cells are a secretory immune subset with the capacity to control solid tumors. Protein translation is of paramount importance in CD8 T cells, controlling proliferation, stimulation and lineage fate. Methods Herein, we used both the fluorescent analogue of methionine homopropargylglycine (HPG) incorporation assay and O-propargyl-puromycin (OPP) method which enters the A-site of the ribosome and effectively labels and terminates nascent polypeptide chains to monitor protein synthesis in mouse and human tumors. Moreover, we employed label free quantitative proteomics (LFQ), lipidomics, metabolic analysis, and in vivo animal modeling to elucidate mechanisms of protein translation in antitumor immunity. Results We found that canonical protein synthesis is restricted in endogenous CD8 tumor infiltrating lymphocytes (TILs) by the tumor microenvironment (TME). Proteomic analysis revealed that gluconeogenesis and B-oxidation of fatty acids (FAO) were upregulated in CD8 T cells under tumor stress but these metabolic sources were unable to support translation in the TME. Further, we discovered that glucose metabolism and mammalian target of rapamycin complex 1 (mTORC1) preferentially hinder protein synthesis in CD8 TILs. These data enabled the discovery that proteasomal protein degradation is the optimal source to fuel protein translation in T cells in the stress of solid tumors. We demonstrate that Rapamycin-primed T cells are preferentially powered by proteasomal proteolysis and are able to sustain protein translation in tumors and control tumor growth. Conclusions Our data establish that canonical protein translation governed by mTORC1 and glucose metabolism is subject to inhibition in the TME and promotion of protein catabolism is a new strategy to support antitumor immunity. Ethics Approval All animal experiments were in accordance with the MUSC Institutional Animal Care and Use Committee (IACUC), protocol # IACUC-2018-00422 and # IACUC-2018-00347

Background Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Methods Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Results Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Conclusions Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Epithelial-to-mesenchymal transition (EMT) has been closely linked with therapy resistance and cancer stem cells (CSCs). However, EMT pathways have proven challenging to therapeutically target. MicroRNA 145 (miR-145) targets multiple stem cell transcription factors and its expression is inversely correlated with EMT. Therefore, we hypothesized that miR-145 represents a therapeutic target to reverse snail family transcriptional repressor 1 (SNAI1)-mediated stemness and radiation resistance (RT). Stable expression of SNAI1 in DLD1 and HCT116 cells (DLD1-SNAI1; HCT116-SNAI1) increased expression of Nanog and decreased miR-145 expression compared to control cells. Using a miR-145 luciferase reporter assay, we determined that ectopic SNAI1 expression significantly repressed the miR-145 promoter. DLD1-SNAI1 and HCT116-SNAI1 cells demonstrated decreased RT sensitivity and, conversely, miR-145 replacement significantly enhanced RT sensitivity. Of the five parental colon cancer cell lines, SW620 cells demonstrated relatively high endogenous SNAI1 and low miR-145 levels. In the SW620 cells, miR-145 replacement decreased CSC-related transcription factor expression, spheroid formation, and radiation resistance. In rectal cancer patient-derived xenografts, CSC identified by EpCAM+/aldehyde dehydrogenase (ALDH)+ demonstrated high expression of SNAI1, c-Myc, and Nanog compared with non-CSCs (EpCAM+/ALDH-). Conversely, patient-derived CSCs demonstrated low miR-145 expression levels relative to non-CSCs. These results suggest that the SNAI1:miR-145 pathway represents a novel therapeutic target in colorectal cancer to overcome RT resistance.

Effective cancer therapies simultaneously restrict tumor cell growth and improve anti-tumor immune responses. Targeting redox-dependent protein folding enzymes within the endoplasmic reticulum (ER) is an alternative approach to activation of the unfolded protein response (UPR) and a novel therapeutic platform to induce malignant cell death. E64FC26 is a recently identified protein disulfide isomerase (PDI) inhibitor that activates the UPR, oxidative stress, and apoptosis in tumor cells, but not normal cell types. Given that targeting cellular redox homeostasis is a strategy to augment T cell tumor control, we tested the effect of E64FC26 on healthy and oncogenic T cells. In stark contrast to the pro-UPR and pro-death effects we observed in malignant T cells, we found that E64FC26 improved viability and limited the UPR in healthy T cells. E64FC26 treatment also diminished oxidative stress and decreased global PDI expression in normal T cells. Oxidative stress and cell death are limited in memory T cells and we found that PDI inhibition promoted memory traits and reshaped T cell metabolism. Using adoptive transfer of tumor antigen-specific CD8 T cells, we demonstrate that T cells activated and expanded in the presence of E64FC26 control tumor growth better than vehicle-matched controls. Our data indicate that PDI inhibitors are a new class of drug that may dually inhibit tumor cell growth and improve T cell tumor control.

Background: Osteoarthritis (OA) is a leading cause of morbidity affecting 54 million Americans per year at an annual cost of 304 billion dollars to the US healthcare system. OA is characterized as a disease of joint wear and tear, but mounting evidence suggests a role for aberrant activation of immunity to contribute to disease progression. Methods: We hypothesized that OA patients have differential profiles of immunity between peripheral and synovial compartments as an indication that in situ immunity is affected by OA or vice versa. We used FACS analysis to phenotype T cell and myeloid populations between peripheral and synovial compartments from 16 patients undergoing total joint replacement due to advanced OA. Results: Our finding that Foxp3[Formula: see text] T regulatory (Tregs) increasingly comprise SF immunity of OA patients is novel ([Formula: see text]). Though the periphery harbored greater numbers of lymphocyte and myeloid cells compared to synovial fluid ([Formula: see text]), the synovial fluid revealed elevated percentages of myeloid (Cd11b[Formula: see text]) cells that comprised the CD45[Formula: see text] population ([Formula: see text]). Further, characterization elucidated that CD45/CD11b/CD14[Formula: see text]/CD15- cells upregulated HLADR in the affected synovial fluid ([Formula: see text]) and that these cells increase expression of CD68 ([Formula: see text]). Conclusions: Our data indicate that the affected joint space in OA patients harbors phenotypically distinct T-cell and myeloid populations compared to autologous-matched peripheral immunity. The contribution of aberrant immune populations to development and progression of OA is of interest for novel immunotherapies.

Tumor antigen–specific T cells rapidly lose energy and effector function in tumors. The cellular mechanisms by which energy loss and inhibition of effector function occur in tumor-infiltrating lymphocytes (TILs) are ill-defined, and methods to identify tumor antigen–specific TILs that experience such stress are unknown. Processes upstream of the mitochondria guide cell-intrinsic energy depletion. We hypothesized that a mechanism of T-cell–intrinsic energy consumption was the process of oxidative protein folding and disulfide bond formation that takes place in the endoplasmic reticulum (ER) guided by protein kinase R-like endoplasmic reticulum kinase (PERK) and downstream PERK axis target ER oxidoreductase 1 (ERO1α). To test this hypothesis, we created TCR transgenic mice with a T-cell–specific PERK gene deletion (OT1+Lckcre+PERKf/f, PERK KO). We found that PERK KO and T cells that were pharmacologically inhibited by PERK or ERO1α maintained reserve energy and exhibited a protein profile consistent with reduced oxidative stress. These T-cell groups displayed superior tumor control compared with T effectors. We identified a biomarker of ER-induced mitochondrial exhaustion in T cells as mitochondrial reactive oxygen species (mtROS), and found that PD-1+ tumor antigen–specific CD8+ TILs express mtROS. In vivo treatment with a PERK inhibitor abrogated mtROS in PD-1+ CD8+ TILs and bolstered CD8+ TIL viability. Combination therapy enabled 100% survival and 71% tumor clearance in a sarcoma mouse model. Our data identify the ER as a regulator of T-cell energetics and indicate that ER elements are effective targets to improve cancer immunotherapy.

Epithelial-to-mesenchymal transition (EMT) has been associated with poor treatment outcomes in various malignancies and is inversely associated with miRNA145 expression. Therefore, we hypothesized that SNAI2 (Slug) may mediate 5-fluorouracil (5FU) chemotherapy resistance through inhibition of miR145 in colorectal cancer and thus represents a novel therapeutic target to enhance current colorectal cancer treatment strategies. Compared with parental DLD1 colon cancer cells, 5FU-resistant (5FUr) DLD1 cells demonstrated features of EMT, including >2-fold enhanced invasion (P < 0.001) and migration, suppressed E-cadherin expression, and 2-fold increased SNAI2 expression. DLD1 and HCT116 cells with stable expression of SNAI2 (DLD1/SNAI2; HCT116/SNAI2) also demonstrated EMT features such as the decreased E-cadherin as well as significantly decreased miR145 expression, as compared with control empty vector cells. On the basis of an miR145 luciferase promoter assay, we demonstrated that SNAI2 repressed activity of the miR145 promoter in the DLD1 and HCT116 cells. In addition, the ectopic expressing SNAI2 cell lines demonstrated decreased 5FU sensitivity, and, conversely, miR145 replacement significantly enhanced 5FU sensitivity. In the parental SW620 colon cancer cell line with high SNAI2 and low miR145 levels, inhibition of SNAI2 directly with short hairpin sequence for SNAI2 and miR145 replacement therapy both decreased vimentin expression and increased in vitro 5FU sensitivity. In pretreatment rectal cancer patient biopsy samples, low miR145 expression levels correlated with poor response to neoadjuvant 5FU-based chemoradiation. These results suggested that the SNAI2:miR145 pathway may represent a novel clinical therapeutic target in colorectal cancer and may serve as a response predictor to chemoradiation therapy. Mol Cancer Ther; 13(11); 2713–26. ©2014 AACR.

Mechanisms of locally advanced rectal cancer (LARC) neoadjuvant chemoradiation resistance are poorly understood. Current research models to study this issue are incomplete and restricted by the limited supply of untreated tissue samples. Patient-derived xenografts (PDX) are commonly developed from surgical specimens and can serve as effective tools to investigate therapy. We previously demonstrated that the SNAI2/ miR-145 signaling axis enhanced the tumor-initiating cell (TIC) and chemotherapy resistance. We developed a unique PDX model from pre-neoadjuvant therapy LARC biopsy samples to study chemoradiation resistance. Further, we hypothesized that TICs isolated from LARC patient-derived xenografts would demonstrated increased SNAI2/ miR-145 signaling. Methods: Fresh LARC endoscopic biopsy samples were digested using Liberase DH to yield a dissociated single-cell solution. All in vivo experiments consisted of injecting cells with Matrigel subcutaneously into NSG NODSCID/IL2Rgnull immunocompromised mice. Xenograft (Passage 1, P1) single cells were resuspended in ALDEFLUOR assay buffer. ALDEFLUOR and EPCAM positive cells (ALDH+/EPCAM+) were then detected using a flow cytometer to identify the TICs. Using the negative control ALDH inhibitor, DEAB (1.5 μM), identify the ALDH+ cells compared with background. For limited dilution (LD) in vitro spheroid assay, 5000 EPCAM+/ALDH+ and ALDH- cells were cultured in ultra-low attachment plates in cancer stem-cell medium (DMEM-F12; 1X B-27, βEGF, βFGF, 1% penicillin/streptomycin, BSA, heparin, β-mercaptoethanol). For LD in vivo study, EPCAM+ ALDH sorted cells were implanted subcutaneously with Matrigel and closely observed. Droplet Digital PCR was conducted using the QX200 Droplet Digital PCR System. miR-145 PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU6B. Results: Our LARC PDX model had high outgrowth and engraftment rates of 70 and 85% respectively and maintained critical histologic features. Both LD in vitro and in vivo experiments demonstrated that EPCAM+/ALDH+ cells were highly tumorigenic suggestive of TICs (p Conclusions: The lack of novel strategies plays a central role in the inability to overcome therapeutic resistance in LARC. Our results demonstrate a novel model to investigate LARC and suggest that a novel miR-145 therapeutic strategy may be able to target the TIC in LARC patients. Citation Format: Ernest R. Camp, Cindy Wang, Katie Hurst, Lourdes M. Nogueira, Victoria J. Findlay. Improving rectal cancer therapy using a patient-derived tumor xenograft model. [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 LB-043.