RESUMO
Neoantigens derived from somatic mutations in Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), the most frequently mutated oncogene, represent promising targets for cancer immunotherapy. Recent research highlights the potential role of human leukocyte antigen (HLA) allele A*11:01 in presenting these altered KRAS variants to the immune system. In this study, we successfully generate and identify murine T-cell receptors (TCRs) that specifically recognize KRAS8-16G12V from three predicted high affinity peptides. By determining the structure of the tumor-specific 4TCR2 bound to KRASG12V-HLA-A*11:01, we conduct structure-based design to create and evaluate TCR variants with markedly enhanced affinity, up to 15.8-fold. This high-affinity TCR mutant, which involved only two amino acid substitutions, display minimal conformational alterations while maintaining a high degree of specificity for the KRASG12V peptide. Our research unveils the molecular mechanisms governing TCR recognition towards KRASG12V neoantigen and yields a range of affinity-enhanced TCR mutants with significant potential for immunotherapy strategies targeting tumors harboring the KRASG12V mutation.
Assuntos
Antígenos de Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Receptores de Antígenos de Linfócitos T , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/química , Camundongos , Humanos , Neoplasias/imunologia , Neoplasias/genética , Neoplasias/terapia , Mutação , ImunoterapiaRESUMO
In this commentary, we advance the notion that mutant KRAS (mKRAS) is an ideal tumor neoantigen that is amenable for targeting by the adaptive immune system. Recent progress highlights key advances on various fronts that validate mKRAS as a molecular target and support further pursuit as an immunological target. Because mKRAS is an intracellular membrane localized protein and not normally expressed on the cell surface, we surmise that proteasome degradation will generate short peptides that bind to HLA class I (HLA-I) molecules in the endoplasmic reticulum for transport through the Golgi for display on the cell surface. T-cell receptors (TCR)αß and antibodies have been isolated that specifically recognize mKRAS encoded epitope(s) or haptenated-mKRAS peptides in the context of HLA-I on tumor cells. Case reports using adoptive T-cell therapy provide proof of principle that KRAS G12D can be successfully targeted by the immune system in patients with cancer. Among the challenges facing investigators is the requirement of precision medicine to identify and match patients to available mKRAS peptide/HLA therapeutics and to increase the population coverage by targeting additional mKRAS epitopes. Ultimately, we envision mKRAS-directed immunotherapy as an effective treatment option for selected patients that will complement and perhaps synergize with small-molecule mKRAS inhibitors and targeted mKRAS degraders.
Assuntos
Antígenos de Neoplasias , Imunoterapia , Mutação , Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Neoplasias/terapia , Neoplasias/imunologia , Neoplasias/genética , Imunoterapia/métodos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/genética , Terapia de Alvo MolecularRESUMO
PURPOSE: Cell membrane penetrating peptide BR2 can bind with ganglioside and introduce foreign drugs into tumor cells. In this study, we employed BR2 to carry the broad-spectrum anti-p21Ras scFv prepared in our laboratory into ganglioside expressing tumor cells for therapy of ras-driven tumors. METHODS: BR2-p21Ras scFv gene was cloned to prokaryotic expression vector and expressed in E. coli BL21, then the fusion protein was purified with HisPur Ni-NTA. The immunoreactivity of the fusion protein with p21Ras was detected by ELISA and western blotting. The membrane-penetrating and immune co-localization with p21Ras of the fusion protein were determined by immunofluorescence. The antitumor activity was investigated using MTT, wound healing, colone formation, and apoptosis assays in vitro. RESULTS: BR2-p21Ras scFv fusion protein was successfully expressed and purified. We found that the fusion protein could specifically penetrate into human tumor cell lines which express ganglioside including human neuroblastoma cell line SK-N-SH, human colon cancer cell line HCT116 and human glioma cell line U251. After entering tumor cells the fusion protein bonded specifically with p21Ras. In vitro experiments revealed that it could significantly inhibit the proliferation, migration, and colone formation of HCT116, SK-N-SH, and U251 cells and promote the apoptosis of these tumor cells. CONCLUSIONS: BR2-p21Ras scFv can penetrate ganglioside expressing tumor cells and inhibit the growth of ras-driven tumor by binding with p21Ras, and producing an inhibitory effect. It is suggested that BR2-p21Ras scFv is a potential ras-driven tumor therapeutic antibody.
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Anticorpos de Cadeia Única , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Gangliosídeos , Humanos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.
Assuntos
Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma de Pulmão , Linfócitos T CD8-Positivos , Glutaminase , Neoplasias Pulmonares , Quinases Proteína-Quinases Ativadas por AMP/deficiência , Quinases Proteína-Quinases Ativadas por AMP/imunologia , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Glutaminase/antagonistas & inibidores , Glutaminase/imunologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Ativação Linfocitária , Camundongos , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente TumoralRESUMO
Background: Pancreatic cancer comprises not only cancer cells but also a collection of cross-talking noncancerous cells within tumor. Therefore, selective delivery of cytotoxic agents towards cancer cells and limiting the collateral damage to tumor suppressive benign cells, such as effector lymphocytes in the tumor microenvironment, is of great value. Methods: Pancreatic cancer cells harbor oncogenic KRAS which induces a constitutively high level of macropinocytosis. Inspired by such uniquity, we sought to explore the targeting potential of dextran, a biomaterial presumed to be endocytosed in the macropinocytosis dependent manner. Cell entry preference, mechanism and subcellular sorting of dextran with different molecular weights were firstly examined. Triptolide (TP), a potent cytotoxin was then set as the model payload for dextran conjugation. KRAS selectivity and the therapeutic effects of dextran-conjugated TP were investigated via both in vitro cellular studies and in vivo tumor model assessment. Results: Dextran, with a specific molecular weight of 70 kDa rather than other weights, was identified as a robust KRAS-responsive intracellular delivery carrier with enhanced entry upon KRAS mutation. The 70 kDa dextran-conjugated TP (DEX-TP) displayed greater efficacy and cellular deposition efficiency towards KRAS mutant cells than KRAS wild-type cells. Treatment with DEX-TP suppressed tumor progression in KRAS mutant pancreatic cancer orthotopic mouse models with reduced toxicity and significantly extended mouse survival time. Furthermore, the conjugate attained a more favorable therapeutic outcome in the tumor immune microenvironment than the free drug, preserving the fraction of T cells and their effector cytokines. Conclusions: In summary, macropinocytic dextran was able to provide drug delivery selectivity towards KRAS mutant cancer cells and reduce tumor immunity depletion caused by the cytotoxic drug in pancreatic cancer.
Assuntos
Dextranos , Diterpenos , Neoplasias Pancreáticas , Fenantrenos , Proteínas Proto-Oncogênicas p21(ras) , Animais , Linhagem Celular Tumoral , Dextranos/administração & dosagem , Diterpenos/administração & dosagem , Diterpenos/farmacologia , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacologia , Camundongos , Terapia de Alvo Molecular , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenantrenos/administração & dosagem , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
KRAS/LKB1 (STK11) NSCLC metastatic tumors are intrinsically resistant to anti-PD-1 or PD-L1 immunotherapy. In this study, we use a humanized mouse model to show that while carboplatin plus pembrolizumab reduce tumor growth moderately and transiently, the addition of the tumor suppressor gene TUSC2, delivered systemically in nanovesicles, to this combination, eradicates tumors in the majority of animals. Immunoprofiling of the tumor microenvironment shows the addition of TUSC2 mediates: (a) significant infiltration of reconstituted human functional cytotoxic T cells, natural killer cells, and dendritic cells; (b) induction of antigen-specific T cell responses; (c) enrichment of functional central and memory effector T cells; and (d) decreased levels of PD-1+ T cells, myeloid-derived suppressor cells, Tregs, and M2 tumor associated macrophages. Depletion studies show the presence of functional central and memory effector T cells are required for the efficacy. TUSC2 sensitizes KRAS/LKB1 tumors to carboplatin plus pembrolizumab through modulation of the immune contexture towards a pro-immune tumor microenvironment.
Assuntos
Proteínas Quinases Ativadas por AMP , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Supressoras de Tumor , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/imunologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Modelos Animais de Doenças , Genes Supressores de Tumor , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Microambiente Tumoral , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologiaRESUMO
Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinogênese/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Alelos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Peptídeos/genética , Peptídeos/imunologia , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-small cell lung cancer (NSCLC) patients harboring a KRAS mutation have unfavorable therapeutic outcomes with chemotherapies, and the mutation also renders tolerance to immunotherapies. There is an unmet need for a new strategy for overcoming immunosuppression in KRAS-mutant NSCLC. The recently discovered role of melatonin demonstrates a wide spectrum of anticancer impacts; however, the effect of melatonin on modulating tumor immunity is largely unknown. In the present study, melatonin treatment significantly reduced cell viability accompanied by inducing cell apoptosis in KRAS-mutant NSCLC cell lines including A549, H460, and LLC1 cells. Mechanistically, we found that lung cancer cells harboring the KRAS mutation exhibited a higher level of programmed death ligand 1 (PD-L1). However, treatment with melatonin substantially downregulated PD-L1 expressions in both the presence and absence of interferon (IFN)-γ stimulation. Moreover, KRAS-mutant lung cancer cells exhibited higher Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) levels, and PD-L1 expression was positively correlated with YAP and TAZ in lung cancer cells. Treatment with melatonin effectively suppressed YAP and TAZ, which was accompanied by downregulation of YAP/TAZ downstream gene expressions. The combination of melatonin and an inhibitor of YAP/TAZ robustly decreased YAP and PD-L1 expressions. Clinical analysis using public databases revealed that PD-L1 expression was positively correlated with YAP and TAZ in patients with lung cancer, and PD-L1 overexpression suggested poor survival probability. An animal study further revealed that administration of melatonin significantly inhibited tumor growth and modulated tumor immunity in a syngeneic mouse model. Together, our data revealed a novel antitumor mechanism of melatonin in modulating the immunosuppressive tumor microenvironment by suppressing the YAP/PD-L1 axis and suggest the therapeutic potential of melatonin for treating NSCLC.
Assuntos
Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Regulação para Baixo/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias Pulmonares/imunologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Células A549 , Animais , Antígeno B7-H1/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Mucinous adenocarcinoma (MAC) is conventionally diagnosed by WHO definition when the extracellular mucin is >50% of the tumor area, while tumors with <50% mucin are designated as having a mucinous component. The study is aimed at analyzing the clinicopathologic characteristics, mutation spectrum, and prognosis of colorectal adenocarcinoma with mucinous component (CAWMC). Mutation analyses for exon 2 to 4 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing. Expression of DNA mismatch repairs and P53 proteins were evaluated by immunohistochemistry. Density of tumor-infiltrating lymphocyte (TIL) status was scored. We also evaluated the percentage of glands producing mucin and the morphology of the different tumor cell types in mucin pools. We retrospectively analyzed the prognosis of 43 patients with stage II/III. The overall frequencies of KRAS and BRAF mutations were 36% and 8%, respectively. Patients with MAC exhibiting high levels of mucin were related to the increase of tumor diameter (P=0.038) but were not associated with any of the other clinicopathologic parameters. The proportion or variable morphology of mucinous component did not stratify progression-free survival in stage II/III cases. TIL was the most significant predictor of progression-free survival among stage II/III CAWMC. It is interesting to note that signet ring cell carcinoma does not portend a worse prognosis for patients with high TIL levels. Combining use the grade of TIL status with the WHO grade of the entire tumor can help identify patients with a high risk of recurrence more accurately.
Assuntos
Adenocarcinoma , Neoplasias Colorretais , Mucinas , Mutação , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas p21(ras) , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Mucinas/genética , Mucinas/imunologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.
Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Mutação , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Camundongos Nus , Proteínas Mutantes/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.
Assuntos
Carcinoma Ductal Pancreático/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Neoplasias Pancreáticas/genética , Células Estreladas do Pâncreas/metabolismo , Evasão Tumoral/efeitos dos fármacos , Aloenxertos , Animais , Antineoplásicos/farmacologia , Carbono/imunologia , Carbono/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Formiatos/imunologia , Formiatos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Interferon gama/genética , Interferon gama/imunologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Oximas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Serina/imunologia , Serina/metabolismo , Serina/farmacologia , Transdução de Sinais , Sulfonamidas/farmacologia , Triptofano/imunologia , Triptofano/metabolismo , Triptofano/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRASG12V- and RAC2P29L-derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.
Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Epitopos , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Células HEK293 , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/genética , Neoplasias/imunologia , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Proteína RAC2 de Ligação ao GTPRESUMO
Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.
Assuntos
Evasão da Resposta Imune/fisiologia , Modelos Genéticos , Neoplasias Pancreáticas/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Transcriptoma , Neoplasias PancreáticasRESUMO
Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. Both genetically engineered mouse models (GEMMs) and syngeneic cell transplant approaches use immunocompetent mice to define immune-dependent events in tumor development and progression. Due to their rapid and reproducible nature, there is expanded interest in developing new syngeneic tools from established primary tumor models. However, few studies have examined the extent that syngeneic tumors reflect the immune profile of their originating primary models. Here, we describe comprehensive immunophenotyping of two well-established GEMMs and four new syngeneic models derived from these parental primary tumors. To our knowledge, this is the first systematic analysis comparing immune landscapes between primary and orthotopic syngeneic tumors. These models all use the same well-defined human-relevant driver mutations, arise at identical orthotopic locations, and are generated in mice of the same background strain. This allows for a direct and focused comparison of tumor immune landscapes in carefully controlled mouse models. We identify key differences between the immune infiltrate of GEMM models and their corresponding syngeneic tumors. Most notable is the divergence of T cell populations, with different proportions of CD8+ T cells and regulatory T cells across several models. We also observe immune variation across syngeneic tumors derived from the same primary model. These findings highlight the importance of immune variance across mouse modeling approaches, which has strong implications for the design of rigorous and reproducible translational studies.
Assuntos
Neoplasias/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Modelos Animais de Doenças , Humanos , Imunidade , Camundongos , Linfócitos T/imunologiaRESUMO
High ERß/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.
Assuntos
Receptor beta de Estrogênio/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor ErbB-2/genética , Microambiente Tumoral/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinogênese/genética , Carcinogênese/imunologia , Linhagem Celular Tumoral , Receptor beta de Estrogênio/imunologia , Feminino , Humanos , Imunoterapia/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Oncogenes/genética , Oncogenes/imunologia , Receptor de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Quinazolinonas/farmacologia , Receptor ErbB-2/imunologia , Microambiente Tumoral/imunologiaRESUMO
KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Ductal Pancreático/imunologia , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/imunologia , Mutação de Sentido Incorreto , Neoplasias Pancreáticas/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Microambiente Tumoral/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Células Th17/imunologia , Aloenxertos , Animais , Linhagem Celular Tumoral , Doença Enxerto-Hospedeiro/genética , Efeito Enxerto vs Leucemia/genética , Interleucina-6/genética , Interleucina-6/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/imunologiaRESUMO
BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.
Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer , Neoplasias Gastrointestinais , Imunidade Celular , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro , Linfócitos T/imunologia , Substituição de Aminoácidos , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/terapia , Humanos , Masculino , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Oncogenic KRAS mutations are the most frequent mutations in human cancer, but most difficult to target. While sustained proliferation caused by oncogenic KRAS-downstream signalling is a main driver of carcinogenesis, there is increasing evidence that it also mediates autocrine effects and crosstalk with the tumour microenvironment (TME). Here, we discuss recent reports connecting KRAS mutations with tumour-promoting inflammation and immune modulation caused by KRAS that leads to immune escape in the TME. We discuss the preclinical work on KRAS-induced inflammation and immune modulation in the context of currently ongoing clinical trials targeting cancer entities that carry KRAS mutations and strategies to overcome the oncogene-induced effects on the immune system.
Assuntos
Carcinogênese/genética , Carcinogênese/imunologia , Imunomodulação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Modelos Imunológicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pesquisa Translacional BiomédicaRESUMO
KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.