Charli Dominguez

The IL-8/IL-8R Axis: A Double Agent in Tumor Immune Resistance

Interleukin-8 (IL-8, CXCL8) is a pro-inflammatory chemokine produced by various cell types to recruit leukocytes to sites of infection or tissue injury. Acquisition of IL-8 and/or its receptors CXCR1 and CXCR2 are known to be a relatively common occurrence during tumor progression. Emerging research now indicates that paracrine signaling by tumor-derived IL-8 promotes the trafficking of neutrophils and myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment, which have the ability to dampen anti-tumor immune responses. Furthermore, recent studies have also shown that IL-8 produced by the tumor mass can induce tumor cells to undergo the transdifferentiation process epithelial-to-mesenchymal transition (EMT) in which tumor cells shed their epithelial characteristics and acquire mesenchymal characteristics. EMT can increase metastatic dissemination, stemness, and intrinsic resistance, including to killing by cytotoxic immune cells. This review highlights the dual potential roles that the inflammatory cytokine IL-8 plays in promoting tumor resistance by enhancing the immunosuppressive microenvironment and activating EMT, and then discusses the potential for targeting the IL-8/IL-8 receptor axis to combat these various resistance mechanisms.

IL-8 signaling is involved in resistance of lung carcinoma cells to erlotinib

A signaling pathway that is frequently deregulated in human carcinomas and has been explored as a therapeutic target involves the activation of the epidermal growth factor receptor (EGFR). Inhibition of EGFR via the small molecule inhibitors erlotinib and gefitinib commonly results in tumor resistance, even in patients with EGFR-mutant tumors that initially show substantial clinical responses. This study was designed to broaden our understanding of the molecular mechanisms of acquired resistance to erlotinib in lung cancer cells bearing wild type or mutated EGFR. We report here that generation of erlotinib-resistant lung cancer cells in vitro resulted in a phenotypic alteration reminiscent of an epithelial-mesenchymal transition (EMT) concomitant with a robust upregulation of the IL-8/IL-8R axis. Our results also demonstrate that upregulation of p38 MAPK signaling is responsible for the enhanced IL-8 secretion in the erlotinib-resistant tumor cells. Blockade of IL-8 signaling effectively reduced mesenchymal features of the resistant cells and also markedly enhanced their susceptibility to erlotinib. These results provide a rationale for the development of new therapeutic approaches involving blockade of IL-8 signaling for the management of acquired resistance to EGFR inhibition in patients with lung cancer.

Epithelial-mesenchymal transition and inflammation at the site of the primary tumor

Tumor growth and progression are the products of complex signaling networks between different cell types within the tumor and its surrounding stroma. In particular, established tumors are known to stimulate an inflammatory reaction via the secretion of cytokines, chemokines, and growth factors that favor the recruitment of a range of infiltrating immune cell populations into the tumor microenvironment. While potentially able to exert tumor control, this inflammatory reaction is typically seized upon by the tumor to promote its own growth and progression towards metastasis. This review focuses on recent advances in understanding how an established tumor can initiate an inflammatory response via the release of pro-inflammatory mediators, such as IL-6 and IL-8, and their roles in cancer metastasis. In particular, the role of the epithelial-mesenchymal transition (EMT), a phenotypic switch observed in carcinomas that promotes progression towards metastasis, is discussed here in relation to cancer inflammation.

A novel bifunctional anti-PD-L1/TGF-β Trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells

ABSTRACT Mesenchymalization is a cellular and molecular program in which epithelial cells progressively lose their well-differentiated phenotype and adopt mesenchymal characteristics. Tumor mesenchymalization occurs during the progression of cancer to metastatic disease, and is also associated with resistance to multiple therapeutics, including killing by cytotoxic immune cells. Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1, and emerging research has found higher PD-L1 expression in mesenchymalized tumors. Here, the association between TGF-β1-mediated mesenchymalization and PD-L1 was investigated in non-small cell lung cancer cells (NSCLC). TGF-β1 was found to upregulate PD-L1 gene transcription in a Smad2-dependent manner, and a positive association between PD-L1 and phosphorylated Smad2 was found in NSCLC tumors. The potential to target these 2 negative immune regulators with a single agent was investigated using M7824, a novel clinical-stage bifunctional agent that targets both PD-L1 and TGF-β. Treatment of NSCLC cells with M7824 in vitro and in vivo attenuated features of TGF-β1-mediated mesenchymalization, including mesenchymal marker expression, proliferation suppression, and chemoresistance. These findings demonstrate that upregulation of tumor cell PD-L1 is a novel mechanism of TGF-β1-induced immunosuppression in NSCLC, and that treatment with M7824 has the potential to simultaneously block both tumor mesenchymalization and PD-L1-dependent immunosuppression.

Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: rationale for combination therapies

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib has been approved for years as a first-line therapy for patients harboring EGFR-sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial–mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Our data shows that short-term exposure of tumor cells to low-dose erlotinib modulates tumor plasticity and immune-mediated cytotoxicity in lung cancer cells harboring a sensitizing EGFR mutation, leading to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as to increase susceptibility to caspase-mediated apoptosis. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, before the establishment of tumor relapse with long-term EGFR inhibition.

Development of Cancer Vaccines Targeting Brachyury, a Transcription Factor Associated with Tumor Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is recognized as a relevant process during the progression of carcinomas towards metastatic disease. Epithelial cancer cells undergoing an EMT program may acquire mesenchymal features, motility, invasiveness, and resistance to a variety of anticancer therapeutics. Preventing or reverting the EMT process in carcinomas has the potential to minimize tumor dissemination and the emergence of therapeutic resistance. One of the strategies currently under investigation to target tumor cells undergoing EMT is the generation of a sustained immune response directed against an essential molecular driver of the process. This review focuses on the current development of immune-mediated anticancer interventions aimed at targeting a transcription factor, brachyury, associated with human tumor EMT. Also presented here is a summary of recent studies demonstrating a role for EMT in tumor resistance to immune effector cytotoxicity, and the study of novel strategies aimed at reverting the EMT to be used in combination with immune-mediated anticancer interventions.

Pharmacological and immunological targeting of tumor mesenchymalization

ABSTRACT Controlling the spread of carcinoma cells to distant organs is the foremost challenge in cancer treatment, as metastatic disease is generally resistant to therapy and is ultimately incurable for the majority of patients. The plasticity of tumor cell phenotype, in which the behaviors and functions of individual tumor cells differ markedly depending upon intrinsic and extrinsic factors, is now known to be a central mechanism in cancer progression. Our expanding knowledge of epithelial and mesenchymal phenotypic states in tumor cells, and the dynamic nature of the transitions between these phenotypes has created new opportunities to intervene to better control the behavior of tumor cells. There are now a variety of innovative pharmacological approaches to preferentially target tumor cells that have acquired mesenchymal features, including cytotoxic agents that directly kill these cells, and inhibitors that block or revert the process of mesenchymalization. Furthermore, novel immunological strategies have been developed to engage the immune system in seeking out and destroying mesenchymalized tumor cells. This review highlights the relevance of phenotypic plasticity in tumor biology, and discusses recently developed pharmacological and immunological means of targeting this phenomenon.

Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer

The complex signaling networks of the tumor microenvironment that facilitate tumor growth and progression toward metastatic disease are becoming a focus of potential therapeutic options. The chemokine IL-8 is overexpressed in multiple cancer types, including triple-negative breast cancer (TNBC), where it promotes the acquisition of mesenchymal features, stemness, resistance to therapies, and the recruitment of immune-suppressive cells to the tumor site. The present study explores the utility of a clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as a potential therapeutic option for TNBC. HuMax-IL8 was shown to revert mesenchymalization in claudin-low TNBC models both in vitro and in vivo as well as to significantly decrease the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) at the tumor site, an effect substantiated when used in combination with docetaxel. In addition, HuMax-IL8 enhanced the susceptibility of claudin-low breast cancer cells to immune-mediated lysis with NK and antigen-specific T cells in vitro. These results demonstrate the multifaceted way in which neutralizing this single chemokine reverts mesenchymalization, decreases recruitment of MDSCs at the tumor site, assists in immune-mediated killing, and forms the rationale for using HuMax-IL8 in combination with chemotherapy or immune-based therapies for the treatment of TNBC.

Abstract B45: Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: Rationale for combination therapies

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been approved for years as a first-line therapy for patients harboring EGFR sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial-mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Utilizing a panel of lung carcinoma cell lines with mutated EGFR, we have evaluated the ability of erlotinib to modulate tumor phenotype and to modulate cytotoxic responses to effector NK cells and T cells specific for brachyury and MUC-1. The effect of erlotinib treatment was also evaluated with single cell clonal populations of lung cancer cells isolated based on their expression of epithelial and mesenchymal markers. Our data shows that short-term exposure of tumor cells to low dose erlotinib leads to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as altered apoptotic signaling through enhanced caspase cleavage. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Additionally, with the utilization of single cell clones, it was evident that there was a selection of mesenchymal populations during erlotinib treatment, which could explain the acquired resistance to immune attack seen at longer time points. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, prior to the establishment of tumor relapse with long-term EGFR inhibition. Citation Format: Charli Dominguez, Kwong-Yok Tsang, Claudia Palena. Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: Rationale for combination therapies. [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 B45.

Abstract 4032: Modulation of tumor PD-L1 expression by epithelial-mesenchymal phenotypic plasticity

Epithelial-mesenchymal transition (EMT) is a molecular and cellular program in which epithelial cells lose their well-differentiated phenotype and adopt mesenchymal traits. This process occurs during the progression of cancer to metastatic disease; tumor cells undergoing EMT acquire invasive characteristics, and mesenchymal features can be highly enriched among circulating tumor cells. EMT has also been associated with the resistance to multiple therapeutic agents, and may ultimately lead to relapse. Emerging research indicates that tumor cells undergoing EMT may also exhibit resistance to killing by innate natural killer (NK) cells and antigen-specific cytotoxic T lymphocytes (CTLs). Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1. Given the contribution of tumor cell phenotypic plasticity to the susceptibility of tumor cells to immune attack, we have evaluated the role of EMT in the expression of PD-L1. Using several different cell line model systems, we devised an mRNA-based EMT scoring method to rank tumor cells according to their phenotypic characteristics. Our model systems included groups of isogenic cell lines in which phenotype modulation was achieved via 1) TGF-β1 treatment; 2) manipulation of EMT genes by transfection; 3) isolation of single cell clones; and 4) selection with chemotherapy. Scoring of the cell lines revealed the existence of distinct epithelial and mesenchymal phenotypes, as well as an intermediate (also termed ‘hybrid’ or ‘metastable’) phenotype in which markers of both differentiation states are co-expressed within the same cell line. Analysis of PD-L1 at the mRNA and protein level (antibody clone MIH1) demonstrated that significant changes in PD-L1 expression take place in response to tumor phenotypic modulation. Using multiple systems, it was observed that acquisition of a distinct mesenchymal phenotype associated with increased levels of PD-L1 expression, compared to isogenic tumor cells exhibiting an epithelial phenotype (for example, with tumor cells induced into the mesenchymal phenotype via TGF-β1 treatment or E-cadherin knockdown). In contrast, acquisition of an intermediate tumor phenotype characterized by co-expression of epithelial and mesenchymal features resulted in significant loss of PD-L1 expression. This phenomenon was observed in tumor cells exposed to chemotherapy in which acquisition of mesenchymal markers took place in the presence of sustained levels of epithelial markers (including E-cadherin). Altogether, our results highlight the importance of tumor phenotypic plasticity on PD-L1 expression. We plan to utilize these various models to understand the mechanism(s) of PD-L1 regulation by phenotype modulation and also to evaluate the potential predictive value of tumor cell phenotype, in conjunction with PD-L1 levels, on the susceptibility of tumor cells to killing by immune cells. Citation Format: Justin M. David, Charli L. Dominguez, Jeffrey Schlom, Claudia Palena. Modulation of tumor PD-L1 expression by epithelial-mesenchymal phenotypic plasticity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4032.