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.Lung cancer is the leading cause of cancer related deaths in the world, with the majority of cases (~85%) corresponding to the non-small cell lung cancer (NSCLC) type.¹ Frequently, proliferation and survival of NSCLC is driven by the oncogene epidermal growth factor receptor (EGFR), which results deregulated in tumors by means of mutation or gene amplification.² The frequency of EGFR deregulation in NSCLC has led to the development of several EGFR-targeted therapies, including erlotinib, an approved EGFR tyrosine kinase inhibitor (EGFR-TKIs) that is widely used for the treatment of NSCLC.^{3, 4} Presence of EGFR-sensitizing mutations, mainly deletions in exon 19 or the L858R substitution in exon 21 of EGFR, has shown to best predict responses to erlotinib and other EGFR-TKIs in patients;⁵ however, despite a remarkable tumor debulking caused by EGFR signaling blockade, tumor relapse is seen in almost 100% of treated patients, resulting in very low long-term survival rates.⁶Given the high mortality rate of NSCLC, there is a pressing call for novel therapeutic approaches that could circumvent the therapeutic resistance seen in the clinic thus far.⁷ In recent years, immunotherapeutic strategies have become recognized means to stimulate the destruction of tumor cells by manipulating or enhancing anti-tumor immune responses. However, there is still a need for further improvement of immune-mediated approaches for the treatment of NSCLC.⁸ One aspect for consideration in this regard is the ability of tumor cells to evade immune responses by an array of strategies,^{9, 10} including their ability to undergo profound phenotypic plasticity via a process designated as the epithelial–mesenchymal transition (EMT).^{11, 12} This phenomenon is now recognized as a mechanism of progression in carcinomas,^{13, 14, 15} where it promotes tumor cell migration, invasion and metastasis, as well as the acquisition of resistance to a variety of anti-cancer therapeutics, including resistance to immune-mediated cytotoxic lysis.^{16, 17, 18, 19}Accumulating evidence links the EGF/EGFR axis to the phenotypic plasticity of solid tumors. In multiple reports in different cancer types, signaling through this axis has been described as a driver of tumor EMT^{20, 21} and given these observations, blockade of EGFR signaling has been explored and shown to revert the phenotype of tumor cells from a mesenchymal-like to an epithelial one.^{20, 22} Interestingly, long exposure to EGFR-TKIs, resulting in the acquisition of tumor resistance and relapse, has been linked to the acquisition of mesenchymal properties in tumor cells, as mesenchymal-like cells are less sensitive to the cytotoxic effects of EGFR inhibition.^{23, 24, 25, 26}Although the effect of EGFR-TKIs in cancer signaling and clinical outcome are well established, the potential of blocking EGFR signaling as a means to facilitate tumor targeting by anti-tumor immune effector cells has not been thoroughly investigated. In this study, EGFR inhibition has been explored both in vitro and in vivo with xenografts of EGFR-mutated NSCLC cells, in terms of its ability to modulate epithelial versus mesenchymal features and to improve tumor sensitivity to immune-mediated attack. Our data demonstrate that short-term, low-dose erlotinib modulates immune-mediated cytotoxicity of NSCLC cells, leading to a remarkable enhancement of tumor cell lysis. This effect positively correlated with the ability of short-term blockade of EGFR signaling to modulate tumor phenotype towards a more epithelial one. The effect, however, was lost when erlotinib was utilized for long periods of time (⩾72 h both in vitro or in vivo), which resulted in tumor mesenchymalization and decreased tumor lysis in response to immune effector mechanisms. The data presented here thus provide 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 treatment with an EGFR-TKI.