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Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations., Tumor extracellular matrix has been associated with cancer progression, therapy resistance and immune suppression. Here, the authors show that collagen generates resistance to PD-1/PD-L1 immunotherapy by upregulating LAIR1 expression and downstream signaling, leading to increased CD8+ T cell exhaustion.

CD38 is a transmembrane glycoprotein that is widely expressed in a variety of human tissues and cells, especially those in the immune system. CD38 protein was previously considered as a cell activation marker, and today monoclonal antibodies targeting CD38 have witnessed great achievements in multiple myeloma and promoted researchers to conduct research on other tumors. In this review, we provide a wide-ranging review of the biology and function of the human molecule outside the field of myeloma. We focus mainly on current research findings to summarize and update the findings gathered from diverse areas of study. Based on these findings, we attempt to extend the role of CD38 in the context of therapy of solid tumors and expand the role of the molecule from a simple marker to an immunomodulator.

Understanding resistance mechanisms to targeted therapies and immune checkpoint blockade in mutant KRAS lung cancers is critical to developing novel combination therapies and improving patient survival. Here, we show that MEK inhibition enhanced PD-L1 expression while PD-L1 blockade upregulated MAPK signaling in mutant KRAS lung tumors. Combined MEK inhibition with anti-PD-L1 synergistically reduced lung tumor growth and metastasis, but tumors eventually developed resistance to sustained combinatorial therapy. Multi-platform profiling revealed that resistant lung tumors have increased infiltration of Th17 cells, which secrete IL-17 and IL-22 cytokines to promote lung cancer cell invasiveness and MEK inhibitor resistance. Antibody depletion of IL-17A in combination with MEK inhibition and PD-L1 blockade markedly reduced therapy-resistance in vivo. Clinically, increased expression of Th17-associated genes in patients treated with PD-1 blockade predicted poorer overall survival and response in melanoma and predicated poorer response to anti-PD1 in NSCLC patients. Here we show a triple combinatorial therapeutic strategy to overcome resistance to combined MEK inhibitor and PD-L1 blockade. Recent clinical trials combining MEK inhibitors with anti-PD-L1 in solid tumours show moderate responses. Here, the authors demonstrate that the combination of MEK inhibition and PD-L1 blockade in KRAS mutant lung cancer models leads to a transient tumour regressions and resistance due to increased infiltration of Th17 cells and that the triple therapy targeting MEK, PD-L1 and IL-17 produced better in vivo responses.

Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422

Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8+ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8+ T cells in combined DDR–PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway–mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC.Significance:Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565

Purpose of the Study: Despite recent advances in the use of immunotherapy, only a minority of small cell lung cancer (SCLC) patients respond to immune checkpoint blockade (ICB) with programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) antibodies as monotherapy or combination. Therefore, there is a critical need to develop strategies to enhance the efficacy of ICB in SCLC, an otherwise immunosuppressed disease with dismal 5-year survival rate of 30 years. We have previously shown that prexasertib and olaparib treatment enhanced the protein (but not mRNA) and surface expression of PD-L1 in SCLC cell lines and tumor models. Here we elucidate the previously unexplored mechanism of how DNA damage response targeting enhances antitumor immunity in SCLC. Methods: SCLC cell lines and immune competent murine models were treated with inhibitors targeting DNA damage repair (DDR) proteins, checkpoint kinase 1 (CHK1) (by prexasertib), poly (ADP-ribose) polymerase (PARP) (by olaparib) either as single agents or in combination with anti-PD-L1. End point analyses were done by Western blot, real-time RT-PCR, multicolor flow cytometry and reverse phase protein array (RPPA). Result: We observed increased PD-L1 glycosylation post-prexasertib and olaparib treatment. Prexasertib and/or olaparib +/- anti-PD-L1 combination activates mTOR and inactivates GSK3β pathway thus providing mechanistic insight into DDR-targeting mediated PD-L1 glycosylation and stabilization. We treated tumor-bearing immune-competent B6129F1 mice with either IgG (control), prexasertib (10mg/kg, 2/7), anti-PD-L1 (300ug, 1/7) or combination (n=10/group). At Day 21, anti-PD-L1 did not cause tumor regression and prexasertib treatment delayed tumor growth [T/C=0.31(p Citation Format: Triparna Sen, Bertha Leticia Rodriguez, Limo Chen, Naoto Morikawa, Junya Fujimoto, Lixia Diao, Youhong Fan, Jing Wang, Bonnie S. Glisson, Ignasio Wistuba, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. Targeting DNA damage response upregulates PD-L1 level and promotes antitumor immunity in small-cell lung cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A153.

The single agent or the combination of anti-PD-1, anti-PD-L1, and anti-CTLA-4 is an effective strategy that is being clinically explored to treat a variety of cancer types. Some patients display primary resistance to the treatment, while others relapse after treatment. Resistance is a major issue that needs to be addressed. Using multiple immunocompetent syngeneic and K-rasLA1/+p53R172HΔg/+ spontaneous animal models of lung cancer, we have explored the mechanisms of resistance to treatments by evaluating the molecular and cellular immune profiles of the tumor microenvironment. We observed that tumor-bearing mice treated with PD-1/PD-L1 blocking antibodies developed resistance through the up-regulation of CD38. We also observed this in the combination therapy of anti-PD-1 and anti-CTLA-4, suggesting that CD38 is a major mechanism of resistance to immune checkpoint inhibitors. In vitro and in vivo studies demonstrated that CD38 impacted CD8+ T-cell function via adenosine receptor signaling and dendritic cell-mediated B7 signaling. Antibody-mediated cell depletion assays were conducted to validate the mechanisms. To determine the applicability to patients, we analyzed 793 lung cancer patients’ specimens with immunohistochemistry staining and assessed biomarker relationships in multiple large independent patient databases (~1,900 tumors). Pathologic analysis revealed positive immunohistochemical staining for CD38 on tumor cells in 15-23% of cases, and bioinformatic analyses revealed a strong correlation between CD38 expression and the immune signature. Lastly, targeting CD38 abolished the treatment resistance by modulating the adenosine levels and thereby enhancing the effector immune cell infiltrates into the tumor microenvironment. Based on our study, CD38 blockade improves the efficacy of single-agent anti-PD-1/PD-L1, or with anti-CTLA-4 combination in lung cancer. CD38 could potentially serve as a novel biomarker of resistance for immune checkpoint inhibition. The data from this study provide a unique target, biomarker, and therapeutic strategy that can be translated into the clinical practice. Citation Format: Limo Chen, Lixia Diao, Xiaohui Yi, Bertha Leticia Rodriguez, Yanli Li, Pamela Villalobos, Tina Cascone, Xi Liu, Lin Tan, Philip Lorenzi, Jared Fradette, David Peng, Ferdinandos Skoulidis, Youhong Fan, Jaime Rodriguez-Canales, Vassiliki Papadimitrakopoulou, Ethan Dmitrovsky, Lauren A Byers, Jing Wang, Ignasio Wistuba, Jim Heymach, Don Gibbons. Tackling the tumor microenvironment with CD38 blockade to enhance cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A059.

Cancer cells modulate the recruitment and function of inflammatory cells to create an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the tumor-derived regulatory programs that promote intratumoral immunosuppression remain poorly defined. Here, we show in a KrasLA1/+p53R172HΔg/+-based mouse model that bone morphogenetic protein-4 (BMP4) augments the expression of the T cell co-inhibitory receptor ligand PD-L1 in the mesenchymal subset of lung cancer cells, leading to profound CD8+ T cell-mediated immunosuppression, producing tumor growth and metastasis. We previously reported in this model that BMP4 functions as a pro-tumorigenic factor regulated by miR-200 via GATA4/6. Thus, BMP4-mediated immunosuppression is part of a larger miR-200-directed gene expression program in tumors that promotes tumor progression, which could have important implications for cancer treatment.

Background: Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, are urgently needed. SCLC has a relatively immunosuppressed phenotype with relatively low levels of infiltrating T-cells and reduced antigen presentation. Only a minority of SCLC patients responds to programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors as monotherapy. Therefore, there is a critical need to develop strategies to enhance the efficacy of immunotherapy in SCLC. We previously discovered that DNA damage repair (DDR) protein, checkpoint kinase 1 (CHK1), is overexpressed in SCLC; CHK1 inhibitor, LY2606368, has activity in preclinical models of SCLC, a finding that supported the phase 2 clinical trial of LY2606368 for SCLC patients (NCT02735980). We hypothesize that targeting CHK1 by inducing DNA damage can enhance immunogenicity, antitumor immunity, and response to immune checkpoint targeting. Methods: SCLC cell lines and immune competent murine models were treated with single agent LY2606368, anti-PD-L1, or combination. End point analyses were done by Western blot, real-time RT-PCR, multicolor flow cytometry, and reverse phase protein array (RPPA). Result: SCLC tumors with higher DDR mutation burden have a higher expression of PD-L1 (p30 years. Citation Format: Triparna Sen, Limo Chen, B. Leticia Rodriguez, Carl M. Gay, Lerong Li, Yanli Li, Youhong Fan, Bonnie Glisson, Jing Wang, Helen Piwnica-Worms, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. DNA damage repair targeting upregulates PD-L1 level and potentiates the effect of PD-L1 blockade in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A108.

79 Background: Although immune checkpoint inhibitors including PD-L1 blockade provide significant clinical benefit for patients with lung cancer, barriers to immunotherapy clinical successes have been due to a high rate of resistance. The therapeutic improvement requires a thorough understanding of the biological process of resistance. Until recently, there have been only a few studies reporting the mechanisms of resistance to PD-L1 blockade. The mechanistic basis remains poorly defined. Methods: In multiple immunocompetent syngeneic and K-rasLA1/+p53R172H?g/+ spontaneous animal models of lung cancer, we have explored the resistance mechanisms using pharmacological and genetic approaches (monoclonal antibody treatment and CRISPR/Cas9-mediated editing). The molecular and immune profiles of the tumor microenvironment were evaluated. More importantly, to determine the applicability to patients with lung cancer, we analyzed 259 patients’ specimens with IHC staining and mined many immune markers in TCGA adeno and squamous datasets. Results: We identified the up-regulation of CD38 on tumor cells as well as enrichment of CD38highTregs and CD38highMDSCs in tumor as the markers of treatment resistance. We observed the same resistance mechanism caused by CD38 in PD-L1 KO mice bearing PD-L1 KO Lewis lung tumors edited with the CRISPR/Cas9 system. Furthermore, by manipulating CD38 on a panel of lung cancer cell lines, in vitro and in vivo data demonstrates that CD38 inhibits CD8+ T cell proliferation, antitumor cytokine secretion, and tumor cell killing capability. To test whether CD38 blockade might be therapeutically efficacious to anti-PD-L1 resistance, we applied the combination therapy of anti-CD38 and anti-PD-L1 and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Additionally, in 259 lung patients, 18.5% of cases exhibited positive staining for CD38 on tumor cells, showing a great potential benefit for treating lung patients. Conclusions: CD38 is defined as a novel immune checkpoint and acts as a mechanism of resistance in the context of PD-L1 therapy. Targeting this novel immune checkpoint may broaden the benefit of PD-L1/PD-1 axis blockade for lung cancer treatment.

Background: Small cell lung cancer (SCLC) is a highly aggressive disease for which standard treatment remains virtually unchanged since the 1980s. SCLC has a relatively immunosuppressed phenotype with low levels of infiltrating T-cells and evidence of reduced antigen presentation. Only a minority of SCLC patients responds to programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors as monotherapy. Therefore, though the clinical data is promising, there is a strong need to develop strategies to enhance the efficacy of immunotherapy in SCLC. Our group previously discovered that the DNA damage repair (DDR) protein, checkpoint kinase 1 (CHK1), is overexpressed in SCLC and that CHK1 inhibitors have activity in preclinical models of SCLC. Based on data from others and our group, we hypothesize that tumor associated neoantigen (TAA) expression is suppressed in SCLC by several mechanisms, including DDR machinery, and that targeting CHK1 may enhance antitumor immunity and response to immune checkpoint targeting. Results: In SCLC models, inhibition of CHK1 by genetic knockdown and small molecule inhibition (LY2606368) induces DNA damage as demonstrated by increased γ-H2AX levels. We also observed increased protein levels of immune checkpoint ligand, PD-L1, following pharmacologic inhibition with LY2606368. We next tested whether co-targeting CHK1+PD-L1 enhances the anti-tumor effect in an immune-competent SCLC model. B6129F1 mice were injected in the flank with TKO.mTmG cells harboring conditional deletion of Trp53, Rb1 and p130. When the tumor volume reached 120mm3, mice were treated with either IgG (control), LY2606368 (10mg/kg, 2/7), anti-PD-L1 (300ug, 1/7) or combination of LY2606368 and anti-PD-L1 antibody. Single agent treatment with anti-PD-L1 antibody did not cause tumor regression in these models with T/C ratio=0.93 (p Discussion: SCLC has an immunosuppressed phenotype (despite a high mutational burden); however, only a minority of tumors expresses PD-L1, suggesting that immunosuppressive mechanisms other than the PD-1/PD-L1 pathway are likely to contribute. This study shows that targeting CHK1 (by genetic knockdown and pharmacological inhibition) leads to increased DNA damage and increased expression of immune checkpoint ligand, PD-L1. Combining CHK1 inhibition and PD-L1 targeting significantly enhanced the effect of PD-L1 antibody leading to tumor regression in an immune competent SCLC model. Biomarker analyses from these models are ongoing to confirm the expression of immune markers. PD-L1 inhibitors as monotherapy have led to objective responses in only a minority of SCLC patients. The CHK1 inhibitor LY2606368 is currently in clinical trial for SCLC patients. The complementary modes of action of the two promising modalities, immune checkpoint targeting and CHK1 inhibition, suggest intriguing possibilities for therapeutic synergy with combination treatment and warrants further clinical investigation. Citation Format: Triparna Sen, Limo Chen, Bertha Leticia Rodriguez, Yongbin Yang, You Hong Fan, Catherine Allison Stewart, Bonnie Glisson, Helen Piwnica-Worms, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. Combining immune checkpoint inhibition and DNA damage repair (DDR) targeted therapy in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B72.

Immunosuppression of tumour-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8(+) TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumour cells, leading to CD8(+) T-cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T-cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

The UVB (290–320 nm) radiation in sunlight is responsible for inducing skin cancer. Exposure to UV radiation is also immunosuppressive, and the systemic immune suppression induced by UV is a well-recognized risk factor for cancer induction. As UVB radiation is absorbed within the upper layers of the skin, indirect mechanisms must play a role in activating systemic immune suppression. One prominent example is mast cell migration, which from the skin to the draining LN is an essential step in the cascade of events leading to immune suppression. What triggers mast cell migration is not entirely clear. Here, we tested the hypothesis that PAF, a lipid mediator of inflammation produced by the skin in response to UV exposure, is involved. Mast cell-deficient mice (KitW-sh/W-sh) are resistant to the suppressive effect of UV radiation, and reconstituting mast cell-deficient mice with normal bone marrow-derived mast cells restores susceptibility to immunosuppression. However, when mast cells from PAFR−/− mice were used, the reconstituted mice were not susceptible to the suppressive effects of UV. Furthermore, PAFR−/− mice showed impaired UV-induced mast cell migration when compared with WT mice. Finally, injecting PAF into WT mice mimicked the effect of UV irradiation and induced mast cell migration but not in PAFR−/− mice. Our findings indicate that PAFR binding induces mast cells to migrate from the skin to the LNs, where they mediate immune suppression.

Although recent advances in targeting immune checkpoints, such as PD-L1, PD-1, and CTLA-4, are producing durable long-term control of cancer, only a fraction of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Epithelial-mesenchymal transition (EMT) plays an important role in driving tumor metastasis and intra-tumoral immunosuppression, being predictive for high risk of cancer recurrence and poor survival prognosis in many major categories of cancers. We previously developed a robust EMT gene signature, highlighting differential patterns for epithelial and mesenchymal tumor cells. To test whether EMT can be used as a potential biomarker for selecting patients more likely to benefit from immune checkpoint blockade agents, we conducted an integrated analysis of gene expression profiling from three independent large datasets (TCGA, MD Anderson's PROSPECT and BATTLE datasets) of lung adenocarcinoma patients. Comprehensive analysis of mRNA gene expression, reverse phase protein array, immunohistochemistry, and correlation with clinical data were performed. Our study demonstrates that EMT is highly associated with an immunosuppressive, inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation co-existent with elevation of immune checkpoints such as PD-L1, PD-L2, PD-1, TIM-3, BTLA, CTLA-4 and B7-H3, along with increases in tumor-infiltrating Foxp3+ regulatory T cells and immunosuppressive cytokines such as IL-6, CCL2, CXCL12, and CCL18 in lung adenocarcinomas that displayed the mesenchymal phenotype. Furthermore, we have used an immune competent syngeneic mouse model of lung adenocarcinoma with mutant Kras and p53 to perform tumor immune cell profiling and functional analyses. The data demonstrates that the ZEB1-mediated EMT represses microRNA-200 expression and de-represses PD-L1 on tumor cells, leading to immunosuppression and metastasis by CD8+ T cell dysfunction. Additionally, EMT produces marked accumulation of Foxp3+ regulatory T cells and myeloid-derived suppressor cells in mesenchymal tumors compared with epithelial tumors. More importantly, when the tumor-bearing mice were treated with anti-PD-L1 antibody alone or in combination with anti-CTLA-4 antibody, the mesenchymal tumors demonstrated greater sensitivity to the immunotherapy. Both human and animal data demonstrate that EMT is highly associated with distinct tumor microenvironment changes, including elevation of multiple targetable immune checkpoints that are regulated at least in part by the microRNA-200/ZEB1 axis. These findings warrant further investigation of the mechanisms by which EMT regulates the immune microenvironment and using EMT as a potential predictive biomarker to guide selection of patients who are likely to benefit from immune checkpoint blockade agents in non-small cell lung cancer. Citation Format: Limo Chen, Yanyan Lou, Ignacio Wistuba, Stephen Ullrich, Xiao-Feng Qin, Lauren Byers, John Heymach, Don Gibbons. EMT produces an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A086.

Either tumor cell epithelial-mesenchymal transition (EMT) or immunosuppression plays the very important role in metastasis during cancer progression. How these distinct processes are co-opted has remained obscure. To address this question, we have generated a cohort of epithelial and mesenchymal cell lines with manipulation of microRNA-200/ZEB1 (an EMT regulatory axis) in KrasLA1/+p53R172HΔg/+ spontaneous mouse model of lung cancer and performed tumor immune cell profiling and functional analysis. The data demonstrates that the microRNA-200 repression and ZEB1 activation target PD-L1 on tumor cells, leading to immunosuppression and metastasis by CD8+ T cell dysfunction, and that marked accumulation of regulatory T cells and myeloid-derived suppressor cells in mesenchymal tumors compared with epithelial tumors. Furthermore, by analyzing two large independent datasets (TCGA and PROSPECT) from lung cancer patients, our study demonstrates that EMT is highly associated with a distinct inflammatory tumor microenvironment, showing the elevation of multiple immune checkpoints such as PD-L1, PD-1, TIM-3, BTLA, CTLA-4 and B7-H3, high levels of tumor-infiltrating Foxp3+ regulatory T cells and immunosuppressive cytokines such as CXCL12, CCL2 and CCL18. The data discovered a link between EMT and immunosuppression, indicating that EMT causes immunosuppression, thereby metastasis. On the other hand, the immunosuppressive microenvironment transforms and/or maintains cancer cells in a mesenchymal state, therefore, cancer cells get the potential/power to metastasize. Immune suppressive cells are able to produce a diverse array of EMT inducers. For instance, T regulatory cells produce TGF-β;, IL-6, IL-10, and TNF-α;. Other immune suppressive cells such as myeloid-derived suppressor cells, tumor-associated macrophages, and tumor-associated neutrophils can also produce strong EMT inducers. Both human and mouse data convincingly established that EMT and immunosuppression form a double positive feed-forward loop, synergizing to promote tumor development. However, when the tumor-bearing mice were treated with anti-PD-L1 antibody, the mesenchymal tumors demonstrated greater sensitivity to the immunotherapy. This is a striking result because we have reported that the mesenchymal subpopulation accounts for aggressive growth, invasion, and metastasis. Since previous studies suggested that some chemotherapeutic agents can induce EMT, we likely find a way, targeting the loop of EMT and immunosuppression, to turn cancer's strengths into targetable weaknesses by combining EMT-induced chemotherapeutics and immunotherapeutics to reduce/prevent tumor metastasis. Citation Format: Limo Chen, Yanyan Lou, John Heymach, Ignacio Wistuba, Stephen Ullrich, Xiao-Feng Qin, Don Gibbons. Targeting the EMT-immunosuppression loop in lung cancer as a strategy to prevent metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR01.

Immunosuppression of tumor-infiltrating lymphocytes (TIL) is a common feature of advanced epithelial tumors, but its biological basis has remained obscure. In addition, anti-PD-1/PD-L1 treatment has produced encouraging clinical responses, but it is still unclear which patients are likely to benefit. Thus, successful therapeutic translation will require a thorough understanding of mechanisms of treatment resistance and predictive biomarkers to select patients. Using multiple lung cancer mouse models (spontaneous and transplant models), we demonstrate a molecular link between epithelial-to-mesenchymal transition (EMT) and intra-tumoral CD8+ T cell immunosuppression, two key drivers of malignant tumor progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1, a checkpoint inhibitor of CD8+ T cell immunity. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumor cells, leading to CD8+TIL dysfunction and metastasis. These findings were further supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets: The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset and the Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (PROSPECT) project at MD Anderson Cancer Center. In addition to revealing a link between EMT and T cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and they suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists. Our data also suggest that EMT represents an important biomarker to select the patients who may benefit from immune checkpoint blockade agents and other immunotherapies in lung cancer and possibly a broader range of other cancers. Citation Format: Limo Chen, Sangeeta Goswami, Xiaohui Yi, Lauren Byers, Lixia Diao, Jonathon Roybal, Christin Ungewiss, David Peng, Jing Wang, Ignacio Wistuba, Lieping Chen, Stephen Ullrich, John Heymach, Jonathan Kurie, Xiao-Feng Qin, Don Gibbons. A big role in tumor immune microenvironment of a small non-coding RNA: microRNA-200. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B67.

Epithelial-mesenchymal transition and immunosuppression are crucial for cancer metastasis and treatment resistance. The mechanism by which these distinct processes are co-opted remains incompletely understood. Our recent work has exposed the "dirty affairs" of the 2 at the tumor site, thus calling for a combined therapy to break such a dangerous liaison.

10035 Background: Major barriers to durable remission in cancer are drug-resistant clones and tumor stem cells. We sought to harness and enhance endogenous antitumor immunity by combining imatinib ...