Limo Chen

Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression

Immunosuppression of tumor-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 tumor 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.

Epithelial-Mesenchymal Transition Is Associated with a Distinct Tumor Microenvironment Including Elevation of Inflammatory Signals and Multiple Immune Checkpoints in Lung Adenocarcinoma.

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

An essential role for platelet-activating factor in activating mast cell migration following ultraviolet irradiation

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.

The mutually regulatory loop of epithelial–mesenchymal transition and immunosuppression in cancer progression

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.

Growth and metastasis of lung adenocarcinoma is potentiated by BMP4-mediated immunosuppression

ABSTRACT 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.

CD38-mediated immunosuppression as a mechanism of tumor cell escape from PD-1/PD-L1 blockade

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNβ in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.

Abstract B72: Combining immune checkpoint inhibition and DNA damage repair (DDR) targeted therapy in small cell lung cancer (SCLC)

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.

Abstract 567: CD38 blockade overcomes the immune resistance to anti-PD-L1 therapy by boosting CD8 T cell response

Although strategies incorporating immune checkpoint inhibition, e.g. PD-1/PD-L1 blockade, are achieving unprecedented successes and increasingly becoming incorporated into standard of care regimens for cancer patients, high rates of resistance still limit the potential efficacy. Therapeutic improvement requires a thorough understanding of the biological process of resistance. To date there have been few studies reporting mechanisms of resistance to PD-L1 blockade. We have explored the resistance mechanisms to functional PD-L1 loss in preclinical lung cancer models by using pharmacological and genetic approaches (PD-L1 blocking antibody treatment or CRISPR/Cas9-mediated deletion of PD-L1 on tumor cells). The molecular and immune profiles of the tumor microenvironment were evaluated in mutant K-ras/p53 (KP) GEM lung cancer models and multiple immunocompetent syngeneic models (both KP and Lewis lung cancer). Additionally, to determine the applicability of the results to patients with lung cancer, we analyzed 259 patient tumor specimens with IHC staining and evaluated the immune markers in TCGA datasets (adenocarcinoma and squamous) and the MD Anderson PROSPECT dataset. We observed that lung tumors gained resistance to anti-PD-L1 antibody treatment over time, and that the up-regulation of CD38 on tumor cells accounted for the treatment resistance. We also observed the same resistance mechanism caused by CD38 up-regulation in PD-L1 KO mice bearing PD-L1 KO Lewis lung tumors generated with the CRISPR/Cas9 system. Manipulation of CD38 on a panel of lung cancer cell lines, demonstrated that CD38 inhibits CD8+ T cell proliferation, antitumor cytokine secretion, and tumor cell killing capability in vitro and in vivo. Furthermore, to test whether CD38 blockade might be therapeutically efficacious to counter anti-PD-L1 resistance, we applied the combination therapy of anti-CD38 and anti-PD-L1 in lung cancer animal models and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Bioinformatic analyses of the patient tumor databases revealed a strong correlation between CD38 expression and an immune suppressive inflammatory signature. Finally, in 259 lung cancer specimens, 18.5% of cases exhibited positive staining for CD38 on tumor cells. Based upon our studies, we conclude that the up-regulation of CD38 on tumor cells is a major mechanism of resistance to anti-PD-L1 therapy, and that CD38 is a novel immune checkpoint that inhibits CD8+ T cell function. The blockade of CD38 and PD-L1 is a rational combination to prevent immune resistance and increase the response rate for lung cancer patients. Citation Format: Limo Chen, Lixia Diao, Yongbin Yang, Xiaohui Yi, Jaime Rodriguez, Youhong Fan, Leticia Rodriguez, Jared Fradette, Christin Ungewiss, Jonothan Roybal, Jingfen Zhu, Jing Wang, Lauren Byers, Stephen Ullrich, Ignacio Wistuba, John Heymach, Xiao-Feng Qin, Don Gibbons. CD38 blockade overcomes the immune resistance to anti-PD-L1 therapy by boosting CD8 T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 567. doi:10.1158/1538-7445.AM2017-567

Abstract A086: EMT produces an immunosuppressive tumor microenvironment

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 Anderson9s 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.

Abstract PR01: Targeting the EMT-immunosuppression loop in lung cancer as a strategy to prevent metastasis

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 cancer9s 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.