RESUMEN
BACKGROUND: Cancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies. METHODS: To identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs. RESULTS: The screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB) nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF-NF-κB axis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis. CONCLUSION: Our data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.
Asunto(s)
FN-kappa B , Factor de Necrosis Tumoral alfa , Apoptosis , Humanos , FN-kappa B/metabolismo , Fosforilación , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.
Asunto(s)
Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Linfocitos T Reguladores/inmunología , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Células Clonales , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Activación de Linfocitos/inmunología , Estadificación de Neoplasias , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de la Célula Individual , Células TH1/inmunología , Transcriptoma/genéticaRESUMEN
The success of cancer immunotherapy is limited by resistance to immune checkpoint blockade. We therefore conducted a genetic screen to identify genes that mediated resistance against CTLs in anti-PD-L1 treatment-refractory human tumors. Using PD-L1-positive multiple myeloma cells cocultured with tumor-reactive bone marrow-infiltrating CTL as a model, we identified calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key modulator of tumor-intrinsic immune resistance. CAMK1D was coexpressed with PD-L1 in anti-PD-L1/PD-1 treatment-refractory cancer types and correlated with poor prognosis in these tumors. CAMK1D was activated by CTL through Fas-receptor stimulation, which led to CAMK1D binding to and phosphorylating caspase-3, -6, and -7, inhibiting their activation and function. Consistently, CAMK1D mediated immune resistance of murine colorectal cancer cells in vivo The pharmacologic inhibition of CAMK1D, on the other hand, restored the sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro Thus, rapid inhibition of the terminal apoptotic cascade by CAMK1D expressed in anti-PD-L1-refractory tumors via T-cell recognition may have contributed to tumor immune resistance.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/inmunología , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Animales , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/inmunología , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/biosíntesis , Resistencia a Antineoplásicos , Humanos , Ratones , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapiaRESUMEN
The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the 'immune modulatome' of cancer.
Asunto(s)
Linfocitos T CD8-positivos , Inmunidad Celular , Inmunoterapia/métodos , Neoplasias Experimentales , Interferencia de ARN , Células TH1 , Animales , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Femenino , Humanos , Células MCF-7 , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Receptores CCR/inmunología , Células TH1/inmunología , Células TH1/patologíaRESUMEN
The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Inflamación/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Células Mieloides/efectos de los fármacos , Paclitaxel/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/farmacología , Calgranulina B/genética , Calgranulina B/inmunología , Enfermedad Crónica , Relación Dosis-Respuesta Inmunológica , Humanos , Terapia de Inmunosupresión , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/patología , Melanoma/complicaciones , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/inmunología , Células Mieloides/inmunología , Células Mieloides/patología , Paclitaxel/farmacología , Cultivo Primario de Células , Neoplasias Cutáneas/complicaciones , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Low-dose cyclophosphamide (CP) therapy induces immunogenic tumor cell death and decreases regulatory T cell (Treg) numbers in mice with transplantable tumors. Using the ret transgenic murine melanoma model that resembles human melanoma, we detected no beneficial antitumor effects with such treatment, despite a decrease in Tregs. On the contrary, low-dose CP enhanced the production of chronic inflammatory mediators in melanoma lesions associated with increased accumulation of Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), which exhibit elevated suppressive activity and nitric oxide (NO) production as well as inhibition of T-cell proliferation. Moreover, the frequencies of CD8(+) T cells in the tumors and their ability to produce perforin were decreased. To study whether the observed CP-induced MDSC expansion and activation also occurs under chronic inflammatory tumor-free conditions, mice exhibiting chronic inflammation were treated with CP. Similar to tumor-bearing mice, CP-treated inflamed mice displayed elevated levels of MDSCs with enhanced production of NO, reactive oxygen species, and a suppressed in vivo natural killer (NK) cell cytotoxic activity indicating CP effects on the host immune system independent of the tumor. We suggest that melanoma therapy with low-dose CP could be efficient only when combined with the neutralization of MDSC immunosuppressive function and chronic inflammatory microenvironment.
Asunto(s)
Ciclofosfamida/farmacología , Terapia de Inmunosupresión/métodos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Inmunosupresores/farmacología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Melanoma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias Cutáneas/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Aspartimide formation is one of the major obstacles that impedes the solid phase synthesis of large peptides and proteins. Until now, no cost-effective strategy to suppress this side reaction has been developed. Here it is demonstrated that addition of small amounts of organic acids to the standard Fmoc cleavage agent piperidine efficiently prevents formation of aspartimide side products. This effect is shown to be virtually independent of the acid strength.
Asunto(s)
Ácidos/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntesis química , Concentración de Iones de Hidrógeno , Estructura Molecular , Técnicas de Síntesis en Fase SólidaRESUMEN
Myeloid cells play a key role in the outcome of anti-tumor immunity and response to anti-cancer therapy, since in the tumor microenvironment they may exert both stimulatory and inhibitory pressures on the proliferative, angiogenic, metastatic, and immunomodulating potential of tumor cells. Therefore, understanding the mechanisms of myeloid regulatory cell differentiation is critical for developing strategies for the therapeutic reversal of myeloid derived suppressor cell (MDSC) accumulation in the tumor-bearing hosts. Here, using an in vitro model system, several potential mechanisms of the direct effect of paclitaxel on MDSC were tested, which might be responsible for the anti-tumor potential of low-dose paclitaxel therapy in mice. It was hypothesized that a decreased level of MDSC in vivo after paclitaxel administration might be due to (i) the blockage of MDSC generation, (ii) an induction of MDSC apoptosis, or (iii) the stimulation of MDSC differentiation. The results revealed that paclitaxel in ultra-low concentrations neither increased MDSC apoptosis nor blocked MDSC generation, but stimulated MDSC differentiation towards dendritic cells. This effect of paclitaxel was TLR4-independent since it was not diminished in cell cultures originated from TLR4-/- mice. These results support a new concept that certain chemotherapeutic agents in ultra-low non-cytotoxic doses may suppress tumor progression by targeting several cell populations in the tumor microenvironment, including MDSC.