Epidermal growth factor receptor and Janus Kinase 2 regulation of programmed death ligand 1 and immunoescape in head and neck cancer

Co-inhibitory immune checkpoint receptors (ICR) are novel targets for cancer immunotherapy. Programmed death ligand 1 (PD-L1), expressed in many cancers, including head and neck cancers (HNC), interacts with its receptor, programmed death 1 (PD-1), resulting in an exhausted phenotype. As yet, the stimuli and pathways that induce PD-L1 expression in tumor cells are not fully understood. Interferon gamma (IFNγ) and the epidermal growth factor receptor (EGFR) utilize Janus kinase 2 (JAK2) as a common signaling node transmitting tumor cell-mediated extrinsic or intrinsic signals, respectively. We investigated the mechanisms by which these factors upregulate PD-L1 and immunosuppressive cytokine expression in HNC cells in the context of EGFR/JAK/STAT pathway activation. We found that wild type overexpressed EGFR significantly correlated with JAK2 and PD-L1 expression. Furthermore, PD-L1 expression was induced in an EGFR- and JAK2-dependent manner, and specific JAK2 inhibition prevented PD-L1 upregulation in HNC, enhancing their immunogenicity. HNC tumors have higher expression of immunosuppressive cytokines including TGFβ, IL-10, VEGF-A and IDO and lower expression of inflammatory cytokines such as IL-12A and IL-17A than controls. EGFR/JAK2 inhibition downregulated secretion of these STAT3-dependent cytokines in vitro, suggesting that targeting the EGFR/JAK2/STAT3 suppressive pathway may reverse tumor immunoescape. This view is supported by in vivo findings where HNC patients unresponsive to cetuximab therapy had significantly higher concentrations of immunosuppressive cytokines. NK cells are crucial for promoting T cell responses against cancer. However, NK cell PD-1 expression remains largely undefined. Cetuximab-activated NK cells constitute the major effector cell subset that lyses tumor targets via antibody dependent cellular cytotoxicity (ADCC). We demonstrate that expression of PD-1 in HNC tumors correlates with NK cell activation markers. HNC patients exhibit higher levels of circulating PD-1+ NK cells, which are further enriched in the tumor. Interestingly, cetuximab treatment increased this frequency in vitro and in vivo. Inhibition of the PD-L1/PD-1 axis increased cetuximab-mediated NK cell activation and cytotoxicity. Collectively, our findings suggest a novel role for JAK2 in EGFR-mediated PD-L1 upregulation and immunosuppressive cytokine secretion. Importantly, combined inhibition of the EGFR and PD-L1/PD-1 axis presents a potential strategy to reverse cetuximab-resistant immune evasion of HNC by enhancing NK cell cytotoxicity

IFNγ-induced PD-L1 expression is JAK2 but not JAK1 dependent and its inhibition enhances NK-cetuximab mediated ADCC of HNSCC cells

Programmed death ligand 1 (PD-L1) is an immunosuppressive molecule expressed by many cancer types, including a large proportion of head and neck cancers (HNC), and ligation of its receptor, programmed death 1 (PD-1), induces exhaustion of effector T cells. It has been shown that interferon gamma (IFNγ) induces PD-L1 expression in many cancer types including glioblastoma, melanoma, lung and kidney cancer. Importantly, the stimuli and mechanism for PD-L1 upregulation in HNC cells are not well characterized. IFNγ signals through Janus Kinase 1/2 (JAK1/2) heterodimer complex and mediates signal transducer and activator of transcription 1 (STAT1) phosphorylation, leading to type I cytokine expression, upregulation of antigen presentation, and tumor cell recognition by cytolytic T lymphocytes (CTL). We investigated basal PD-L1 expression and the mechanism by which IFNγ signaling upregulates PD-L1 in HNC cells including dependence on JAK/STAT pathway. We observed that IFNγ signaling increased PD-L1 expression in a JAK2 but not JAK1 dependent fashion. In addition, interferon alpha (IFNα), which signals via JAK1/TYK2 did not upregulate PD-L1 expression while still upregulated HLA class I. Specific JAK2 inhibition downregulated NK cell-derived IFNγ induced PD-L1 expression and enhanced cetuximab mediated ADCC. Our data suggest a crucial role for JAK2/STAT1 in IFNγ mediated PD-L1 upregulation. JAK2 inhibition provides a promising strategy to increase tumor cell lysis through maintaining HLA class I while suppressing tumor cell expressed PD-L1 in combination with anti-EGFR cetuximab therapy

Reversing EGFR Mediated Immunoescape by Targeted Monoclonal Antibody Therapy

Uncontrolled growth is a signature of carcinogenesis, in part mediated by overexpression or overstimulation of growth factor receptors. The epidermal growth factor receptor (EGFR) mediates activation of multiple oncogenic signaling pathways and escape from recognition by the host immune system. We discuss how EGFR signaling downregulates tumor antigen presentation, upregulates suppressive checkpoint receptor ligand programmed death ligand (PD-L1), induces secretion of inhibitory molecules such as transforming growth factor beta (TGFβ) and reprograms the metabolic pathways in cancer cells to upregulate aerobic glycolysis and lactate secretion that ultimately lead to impaired cellular immunity mediated by natural killer (NK) cell and cytotoxic T lymphocytes (CTL). Ultimately, our understanding of EGFR-mediated escape mechanisms has led us to design EGFR-specific monoclonal antibody therapies that not only inhibit tumor cell metabolic changes and intrinsic oncogenic signaling but also activates immune cells that mediate tumor clearance. Importantly, targeted immunotherapy may also benefit from combination with antibodies that target other immunosuppressive pathways such PD-L1 or TGFβ and ultimately enhance clinical efficacy

EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3

The epidermal growth factor receptor (EGFR) supports the escape of malignant cells from immunosurveillance by inhibiting the activation of signal transducer and activator of transcription 1 (STAT1) while promoting that of STAT3. We have recently demonstrated that protein tyrosine phosphatase, non-receptor type 11 (PTNP11, best known as SHP2), a phosphatase that operates downstream of EGFR, is responsible for the dephosphorylation of active STAT1 and for the inhibition of the antigen-processing machinery (APM), hence favoring tumor immunoescape. Thus, EGFR signaling may skew the tumor microenvironment to suppress cellular immune responses

PD-1 is a marker of activation on tumor infiltrating NK cells in head and neck cancer

Co-inhibitory immune checkpoint receptors have become important targets for cancer immunotherapy. Programmed death 1 (PD-1) has been well-characterized on T cells in many cancer types, including head and neck cancer (HNC), for its ability to mediate activation and eventually T cell exhaustion in the tumor microenvironment. However, PD-1 expression on NK cells, which are crucial innate immune effector cells against cancer, remains largely undefined. In the setting of HNC, NK cells mediate lysis of EGFR-overexpressing tumor targets via cetuximab-mediated antibody dependent cytotoxicity (ADCC). Indeed, cetuximab has shown to be clinically effective but only to a modest extent. Therefore, it is necessary to investigate how cetuximab modulates activation of immune effector cell infiltrates in the tumor microenvironment in order to improve or extend its therapeutic efficacy. We hypothesized that expression of PD-1 per se on NK cells may constitute a marker of a chronically activated phenotype, which is suppressed only after ligation by its cognate ligand programmed death ligand-1 (PD-L1). Thus, tumor cell-expressing PD-L1 may present as a crucial mediator of immunosuppression in the tumor microenvironment decreasing cytotoxicity of cetuximab activated PD-1 expressing NK cells. Herein, using The Cancer Genome Atlas (TCGA) data for 500 HNC patients' tumors, we found that PD-1 expression correlates with NK activation markers. Indeed, HNC patients also exhibit higher levels of circulating and tumor infiltrating PD-1+ NK cells, and neoadjuvant cetuximab treatment increased this frequency in vitro and in vivo in a prospective Phase II trial. In addition, anti-PD-1 mAb nivolumab enhanced cetuximab mediated NK cell activation and HNC cell lysis. Therefore, blocking PD-L1/PD-1 axis may be a useful approach to reverse immune evasion of HNC tumors to cetuximab therapy by reversing NK cell dysfunction

Anti-EGFR targeted monoclonal antibody isotype influences anti-tumor immunity in head and neck cancer patients

EGFR is frequently overexpressed on several cancers, and two targeted antibodies are FDA approved but differ by isotype. Cetuximab (IgG1 isotype) has been shown to be effective at both inhibiting downstream signaling of EGFR and activating anti-tumor, cellular immune mechanisms. While panitumumab (IgG2 isotype) can inhibit downstream EGFR signaling similar to cetuximab, panitumumab might also induce antibody-dependent cell cytotoxicity (ADCC) or adaptive immunity. We sought to investigate the cellular immunity specifically activated by cetuximab or panitumumab showing that both mAb primarily activate NK cells, although cetuximab was significantly more potent than panitumumab. We also observed that although panitumumab may activate monocytes through the CD32 (FcγRIIa) receptor, neither mAb activated monocytes sufficiently to mediate ADCC. Cetuximab enhanced DC maturation to a greater extent than panitumumab, corresponding with improved cross presentation of tumor antigen by cetuximab compared with panitumumab. Indeed, improved adaptive immune responses with increased EGFR-specific cytotoxic CD8+ T cells were present in patients treated with cetuximab compared to those treated with panitumumab. These results suggest that although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at mediating anti-tumor, cellular immune mechanisms which may be crucial for effective therapy for HNSCC

Evidencias de paleosismos en la estratigrafía de la cuenca cuaternaria Cusco

El Perú se encuentra en el borde occidental del continente sudamericano, donde la Cordillera de los Andes se hace más ancha y alta. Esta cadena montañosa se ubica sobre el límite de placas convergentes en el cual la placa oceánica de Nazca subduce la placa Sudamericana o Continental. El borde occidental de América del Sur se caracteriza por ser una de las zonas sísmicamente más activas en el mundo, donde la sismicidad está asociada al proceso de subducción y a las reactivaciones de fallas. En el Perú, se ha avanzado hasta el punto de haber identificado, cartografiado y reconocido las características de los sistemas de fallas activas mayores del país. Uno de los sistemas de fallas más importantes del Perú se encuentra en la Cordillera Oriental, exactamente en el departamento del Cusco, ubicado en el sur del Perú (Fig. 1 ). La información sobre sismos en el Cusco es insuficiente para realizar trabajos sobre peligro sísmico. Crónicas históricas (Esquivel & Navia, 1775; Silgado, 1978) mencionan la ocurrencia de sismos muy devastadores para la ciudad del Cusco, estas remontándose hasta la época Inca. La falta de datos y limitaciones de la sismicidad histórica nos conduce a realizar trabajos de paleosismología, que permitirán identificar las estructuras geológicas asociadas a sismos ocurridos antes de las primeras crónicas históricas, mediante un análisis integral de disciplinas como son: La Geología Estructural, Estratigrafía y Sedimentología. La paleosismología estudia la estructura sísmica directa (la falla) realizando una trinchera o zanja siempre y cuando ocurra ruptura superficial durante el sismo, pero esto no siempre es así, ya que se pueden dar terremotos muy importantes sin rupturas superficiales. Por este motivo el estudio de las estructuras de origen sísmico desarrollado en sedimentos no consolidados cobra especial importancia, siendo el tema central del presente trabajo

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Efecto in vitro del látex de Ficus insipida sobre la cascada de la coagulación sanguínea

Es necesario investigar drogas naturales nuevas que aporten principios farmacológicos activos para ser utilizadas como una alternativa terapéutica. Por este motivo nos propusimos estudiar a Ficus insipida, cuyo látex ha sido usado como antihelmíntico durante muchos años en la amazonía, pero se investigó solo superficialmente su efecto anticoagulante in vitro. Objetivo: Comprobar el efecto anticoagulante in vitro y determinar la vía de la coagulación sobre la que actúa el látex de Ficus insípida. Material y métodos: Se obtuvo el látex de Ficus insipida y se prepararon diferentes concentraciones del mismo. Se obtuvieron muestras de sangre venosa periférica de 5 donantes voluntarios, anticoagulándolas con citrato de sodio. Luego, éstas se mezclaron con las diluciones del látex, se centrifugaron y se extrajo el plasma. Posteriormente, se obtuvo un pool de plasma para cada concentración del látex y se procedió a determinar el Tiempo de Protrombina (TP) y el Tiempo de Tromboplastina Parcial activada (TTPa), respectivamente. Resultados: Se encontró que el látex de Ficus insipida prolongó el TP a una concentración mayor o igual a 0,03125% (V/V), y ambos, el TP y TTPa a una concentración mayor o igual a 0,15% (V/V). Conclusiones: Los resultados obtenidos nos permiten afirmar que el látex de Ficus insipida posee un efecto anticoagulante in vitro dosis dependiente sobre la vía extrínseca de la coagulación sanguínea a una concentración igual o mayor a 0,03125% (V/V) y que a una concentración igual o mayor a 0,15% (V/V) posee un potente efecto anticoagulante sobre ambas vías de la coagulación.(Rev Med Hered 2010;21:146-152)