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This file contains supplementary materials and methods; figures and figure legends and Tables.

Genes differentially expressed between IgG control and anti-PD-L1 treatment.

Supplementary title page. Figure S1: CD38 on anti-PD-L1 resistant tumor cells is up-regulated, which is associated with tumor progression. Figure S2: anti-PD-L1 resistant tumors demonstrate the distinct mRNA and protein profiling for immune signature, reflecting the upregulation of CD38. Figure S3: PD-1/PD-L1 blockade results in CD38 up-regulation and acquired resistance in KP-derived lung and melanoma transplantation tumors. Figure S4: Tumor-associated PD-L1 promotes tumor growth but PD-L1 knockout cancer cells still form tumors. Figure S5: PD-L1 knockout effect on tumor growth is CD8 T cell-dependent. Figure S6: CD38 up-regulation after anti-PD-L1 treatment is associated with all-trans retinoic acid signaling. Figure S7: IFN-b, which is enriched in anti-PD-L1 treated tumors, up-regulates CD38 expression on multiple cancer cell lines. Figure S8: IRF1, which links ATRA and IFN-b, is upregulated after anti-PD-L1 treatment. Figure S9: IFN-g, TNF-a, IL-2, and IL-1b don''t regulate CD38 expression on lung cancer cells. Figure S10: The anti-PD-L1 resistant tumors demonstrate an immune suppressive microenvironment. Figure S11: CD38 substantially changes in vivo tumor formation of PD-L1KO cancer cells, but does not change in vitro cell growth rate and cell cycle. Figure S12: The elimination of PD-L1KOCD38negative cancer cells is CD8+ T cell dependent. Figure S13: CD38 on tumor cells inhibits CD8+ T cell function and protects tumor cells from CD8+ T cell killing. Figure S14: CD38-mediated CD8+TIL dysfunction is not affected by tumor growth rate/tumor size. Figure S15: CD38 expression in cancer cell lines and patient tissues, which is associated with the differentiated immune features. Figure S16: CD38 or PD-L1 expression is not correlated with overall survival in early-stage lung cancer. Figure S17: Pre-treatment levels of CD38 and PD-L1 expression in NSCLC patients who received anti-PD-1/PD-L1 therapy, as divided by clinical outcome. Figure S18: The effect of cancer immunotherapy by anti-CD38 is CD8 T cell dependent. Figure S19: Combined inhibitors of CD38 and PD-L1 inhibits tumor growth and metastases. Figure S20: The co-inhibition of PD-L1 and CD38 leads to a favorable antitumor immune microenvironment. Figure S21: Sequential treatment of anti-PD-L1 and anti-CD38 results in enhanced immune response in tumor microenvironment. Figure S22: anti-mouse CD38 antibody (NIMR-5) does not directly kill tumor cells through ADCC and CDC, but causes CD38 internalization. Table S2: Tumor immune markers with the greatest differential transcriptional levels after anti-PD-L1 treatment. Table S6: The most changed immune-related genes after anti-PD-L1 treatment. Table S9:The correlation between CD38 and suppressive immune markers in LUAD dataset. Table S10: The correlation between CD38 and suppressive immune markers in LUSC dataset. Table S11: The available NSCLC patients with CD38 and PD-L1 tumor cell IHC staining and responses to anti-PD-1 therapy. Table S12: The co-inhibition effect of PD-L1 and CD38 on tumor growth and metastasis. Supplementary Material and Methods: Reagents, Cells and Mice, CRISPR/Cas9 Editing, Antibody-mediated Cell Depletion, CD8+ T Cell Adoptive Transfer, mRNA Profiling of Murine Tumors, Flow Cytometry, Nanostring Analysis, qRT-PCR and Western Blotting, Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis, Histologic Analysis, ELISA and RPPA, ADCC/CDC and Internalization Assays, Human Samples, Statistics. Supplementary references

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

MET supplementary methods

Supplementary Table 1. Patient demographic and clinical characteristics. Supplementary Table 2. Immune-related gene list. Immune related molecules demonstrating roles in tumor microenvironment based on publications were listed in the table and used in this study. Supplementary Table 3. Common mutational events in "epithelial" and "mesenchymal" lung adenocarcinomas versus EMT status. Supplementary Figure 1. Validation of anti-PD-L1 antibody E1L3N (Cell Signaling Technology). Supplementary Figure 2. Validation of anti-PD-L1 antibody ab174838 (Abcam). Supplementary Figure 3. Elevation of multiple immune molecules in "mesenchymal" compared to "epithelial" lung adenocarcinoma. Supplementary Figure 4. B7-H3 is associated with poor OS and RFS in lung adenocarcinoma. Supplementary Figure 5. The association between EMT and inflammatory tumor microenvironment in advanced lung adenocarcinoma.

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 melanoma patients treated with PD-1 blockade predicted poorer overall survival and response. Our study validates a triple combinatorial therapeutic strategy to overcome resistance to combined MEK inhibitor and PD-L1 blockade.

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.

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

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

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. 1066. This article is highlighted in the In This Issue feature, p. 1047

The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of translation initiation in vitro and in vivo and an efficacious anticancer agent in animal models of human cancers. We report on the design, synthesis, and in vitro characterization of a series of rigidified mimetic of this prototypic inhibitor in which the phenyl in the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety was bridged into a tricyclic system. The bridge consisted one of the following: ethylene, methylene oxide, methylenesulfide, methylenesulfoxide, and methylenesulfone. Numerous analogues in this series were found to be markedly more potent than the parent prototypic inhibitor in the inhibition of eIF4E/eIF4G interaction, thus preventing the eIF4F complex formation, a rate limiting step in the translation initiation cascade in eukaryotes, and in inhibition of human cancer cell proliferation.

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.

Symmetrical N,N′-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP· tRNA i Met ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N′-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.

CXCL14 is a relatively new chemokine with unidentified receptor and undefined function. Recently, we found that CXCL14 is upregulated in arthritic joints in a mouse model of autoimmune arthritis, collagen-induced arthritis. To examine the role of CXCL14 in the development and pathogenesis of autoimmune arthritis, we have generated transgenic (Tg) mice that overexpress CXCL14 under control of phosphoglycerate kinase promoter. The results showed that CXCL14-Tg mice developed more severe arthritis compared with wild-type controls. The draining lymph nodes of CXCL14-Tg mice were significantly enlarged and contained an increased number of activated T cells, particularly the CD44+CD62Llow effector memory cells. In addition, T cells from CXCL14-Tg mice exhibited an enhanced proliferative response against collagen II and produced higher levels of IFN-γ but not IL-4 or IL-17. CXCL14-Tg mice also had elevated levels of IgG2a autoantibodies. These findings indicated that CXCL14 plays an important role in the autoimmune arthritis, which may have an implication in understanding the pathogenic mechanisms of rheumatoid arthritis in humans and, ultimately, therapeutic interference.

SummaryActivation-induced deaminase (AID) is a prerequisite for immunoglobulin (Ig) class-switch recombination and somatic hypermutation, which is critical for antibody affinity maturation. IgM and IgG autoantibodies are characteristic of the systemic autoimmune disorders such as lupus. However, the relative contributions of hypermutated high-affinity IgG antibodies and germline-encoded IgM antibodies to systemic autoimmunity are not defined fully. The role of AID in autoimmunity is unclear. The current study used AID-deficient mice to investigate the role of AID in the development and pathogenesis of murine lupus. C57BL/6 mice deficient in both Fas and AID were generated. Compared to their AID-competent littermates, AID−/− lymphoproliferative (lpr) mice produced significantly elevated levels of IgM autoreactive antibodies with enhanced germinal centre (GC) response, developed more advanced splenomegaly and exhibited more severe glomerulonephritis. Thus, AID may play an important role in the negative regulation of systemic autoimmune manifestations in murine lupus. The results also indicate that hypermutated high-affinity IgG antibodies are not necessary for the development of autoimmune syndrome in lpr mice on a C57BL/6 background.

Epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P

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.

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

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.

4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.

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.

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.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR01) of the Conference Proceedings. 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 A05.

Translation initiation factors are over-expressed and/or activated in many human cancers and may contribute to their genesis and/or progression. Removal of physiologic restraints on translation initiation causes malignant transformation. Conversely, restoration of physiological restrains on translation initiation reverts malignant phenotypes. Here, we extensively characterize the anti-cancer activity of two small molecule inhibitors of translation initiation: #1181, which targets the eIF2∙GTP∙Met-tRNA i ternary complex, and 4EGI-1, which targets the eIF4F complex. In vitro, both molecules inhibit translation initiation, abrogate preferentially translation of mRNAs coding for oncogenic proteins, and inhibit proliferation of human cancer cells. In vivo, both #1181 and 4EGI-1 strongly inhibit growth of human breast and melanoma cancer xenografts without any apparent macroscopic- or microscopictoxicity. Mechanistically, #1181 phosphorylates eIF2α while 4EGI-1 disrupts eIF4G/ eIF4E interaction in the tumors excised from mice treated with these agents. These data indicate that inhibition of translation initiation is a new paradigm in cancer therapy.

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2–GTP–tRNAi Met translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2–GTP–tRNAi Met ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N9-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N9-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2a and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N9-diarylureas are potent and specific tools for studying the role of eIF2–GTP–tRNAi Met ternary complex in the pathobiology of human disorders.

Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with immunomodulatory agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune-based approaches, such as immune checkpoint blockade, is critical to successful translation of these agents. By using integrated gene array analysis in three large independent NSCLC patient datasets along with immunohistochemistry study (IHC), reverse phase protein array (RPPA) and in vivo animal study, we demonstrated that epithelial-mesenchymal transition (EMT) is highly associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. Our data revealed immune activation and simultaneous development of multiple immune suppressive mechanisms, including elevation of immune checkpoints such as PD-L1, PD-L2, PD-1, TIM-3, BTLA and CTLA-4, as well as an increase in tumor infiltrating CD4+Foxp3+ regulatory T cells, IL-6 and indoleamine 2,3-dioxygenase (IDO) in lung adenocarcinomas with a mesenchymal phenotype. Our data suggested that EMT might represent a potential biomarker to select the patients who will benefit from immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly other cancers. Note: This abstract was not presented at the meeting. Citation Format: Yanyan Lou, Lixia Diao, Parra Cuentas Edwin Roger, Warren L. Denning, Limo Chen, Youhong Fan, Jaime Rodriguez, Lauren Byers, Jing Wang, Vassiliki Papadimitrakopoulou, Behrens Carmen, Ignacio I. Wistuba, Patrick Hwu, John V. Heymach, Don L. Gibbons. Epithelial-mesenchymal transition is associated with a profound inflammatory tumor microenvironment in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2015-3364

Mucinl (MUC1) promoter has been cloned from the 5' flanking region of the MUC1 gene in breast carcinoma, functionally characterized and applied in gene therapy of breast and esophageal carcinoma. In the present study, we amplified a 786 base pair (bp) MUC1 promoter by two-step nest PCR, and identified the activity and tumor-specificity using an enhanced green fluorescent protein (EGFP) gene as a reporter gene by fluorescence microscopy and flow cytometry analysis in Panc-1, primary normal pancreatic (PNPC), and cervical cancer HeLa cell lines. Subsequently, the human somatostatin receptor subtype 2 (hSSTR2) gene driven by MUC1 promoter was cloned into the pAdTrack to produce recombinant adenovirus AdMUC1-hSSTR2. The anticancer effect of AdMUC1-hSSTR2 was determined in Panc-1. The results demonstrated that there was no AdMUC1-hSSTR2-induced apoptosis, but a significant cell proliferation inhibition even without somatostatin (SST) analogue Octreotide, involved in the up-regulation of the cyclin-dependent kinase (CDK) inhibitors p21 and p27. Moreover, the anticancer effect could not be augmented by the addition of Octreotide, revealing a mechanism that was independent from induction of Octreotide. Therefore, this adenovirus system can be used as a novel, potent and specific tool for gene-targeting therapy in the MUC 1 positive pancreatic carcinoma as shown in Panc-1.

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.

Background: Tumor cell regulation of the recruitment and activity of diverse inflammatory cells creates an immunosuppressive microenvironment that favors both tumor growth and metastasis. However, the mechanistic basis of how tumor cells trigger intra-tumoral immunosuppression have not been fully defined. Here we show that metastasis-prone lung adenocarcinoma cells suppress the proliferation and activity of intra-tumoral CD8+ T cells through a PD-L1 dependent mechanism and that PD-L1 expression is regulated by microRNA-200 (miR-200), a known mediator of tumor cell EMT. Results: Analysis of patient tumors from The Cancer Genome Atlas (TCGA) (230 adenocarcinomas) and a large MDACC cohort of resected tumors (N>150) demonstrated that tumors with a mesenchymal gene expression signature had repression of miR-200 family expression and higher levels of PD-L1 expression. These findings were confirmed experimentally in human and murine NSCLC cell lines. In vivo experiments with a Kras/p53 mutant mouse model of lung adenocarcinoma showed that decreased miR-200 led to increased expression of PD-L1 levels on tumor cells, causing profound CD8+ T cell suppression. Upon suppression of CD8+ T cell activity tumors acquired a growth advantage and metastatic capability. Confirmation of these results with the LLC model in PD-L1 knockout mice also demonstrated that PD-L1 expression on the tumor cells, rather than other cells in the tumor microenvironment, was critical to defining tumor immunity and metastastic ability. The metastases could be suppressed by genetic and pharmacologic blockade of PD-L1. Conclusion: Our findings demonstrate a novel biological role for miR-200 in cancer cells by simultaneous control of the cell-intrinsic EMT program and the cell-non-autonomous PD-L1-mediated immune evasion. These findings support further investigation into the mechanistic effects of miR-200 on metastasis and the use of specific EMT-related biomarkers to select immunomodulatory therapies. Citation Format: Don L. Gibbons, Limo Chen, Sangeeta Goswami, Young-Ho Ahn, Lauren A. Byers, Lixia Diao, Jing Wang, Angelica Cortez, Jonathon Roybal, James Welsh, Xuejun Zhang, David Dwyer, Xiaohui Yi, Chen Lieping, Ignacio Wistuba, John V. Heymach, Jonathan M. Kurie, F. Xiao-Feng Qin. microRNA-200 regulates tumor cell PD-L1 expression to control lung cancer metastasis. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A21.

To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism.A 63-nucleotide (nt) oligonucleotide encoding K-ras(val12) and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-ras(val12). After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-ras(val12) was obtained. Stable suppression of K-ras(val12) was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry.We obtained adenovirus AdH1-K-ras(val12) carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-ras(val12), the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-ras(val12)-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdH1-p53, respectively) (P0.05).These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-ras(val12) infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.