RESUMO
ABSTRACT: Epigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.
Assuntos
Epigênese Genética , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Animais , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Camundongos , Interferon Tipo I/metabolismo , Autorrenovação Celular , Regulação Leucêmica da Expressão GênicaRESUMO
Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.
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Biomarcadores Tumorais , Proteômica , Proteína EWS de Ligação a RNA , Sarcoma de Células Pequenas , Translocação Genética , Humanos , Proteína EWS de Ligação a RNA/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Sarcoma de Células Pequenas/genética , Sarcoma de Células Pequenas/patologia , Sarcoma de Células Pequenas/diagnóstico , Feminino , Masculino , Adulto , Adolescente , Adulto Jovem , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/diagnóstico , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/diagnóstico , Criança , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genéticaRESUMO
For more than 20 years gastrointestinal stromal tumors (GIST) have been a paradigm for a targeted treatment with tyrosine kinase inhibitors. A fundamental prerequisite for a neoadjuvant or adjuvant treatment of localized GIST or an additive treatment of metastatic GIST is the molecular typing of tumors, ideally at the initial diagnosis. In addition, the possibility of a hereditary or syndromic predisposition must be considered because this results in consequences for the treatment and a different follow-up strategy.
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Tumores do Estroma Gastrointestinal , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/terapia , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/terapia , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/tratamento farmacológico , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
The detection of disialoganglioside GD2 on tumor biopsies, especially in paraffin-embedded tissues, has been challenging due to the glycolipid structure of GD2 and its membrane anchorage. Here, we present an immunofluorescence protocol for the reliable assessment of GD2 on formalin-fixed paraffin-embedded (FFPE) tissues. We describe steps for antigen retrieval with Tris-EDTA buffer and staining with unconjugated anti-GD2 antibody (clone 14.G2a) and horse radish peroxidase (HRP)-conjugated secondary antibody. We then detail procedures for signal amplification using the tyramide signal amplification technique. For complete details on the use and execution of this protocol, please refer to Fischer-Riepe et al.1.
RESUMO
Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.
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Sarcoma de Ewing , Antígenos Embrionários Estágio-Específicos , Animais , Feminino , Humanos , Camundongos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Gangliosídeos , Glicoesfingolipídeos , Sarcoma de Ewing/patologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/genética , Antígenos Embrionários Estágio-Específicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022- 501725-21-00). See related commentary by Locatelli and Quintarelli, p. 3361.
Assuntos
Gangliosídeos , Imunoterapia Adotiva , Interleucina-18 , Neoplasias , Receptores de Antígenos Quiméricos , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Gangliosídeos/imunologia , Interleucina-18/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/patologia , Linhagem Celular Tumoral , Microambiente Tumoral/imunologia , FemininoRESUMO
Background: Ewing sarcoma (EwS) is a rare and highly malignant bone tumor primarily affecting children, adolescents, and young adults. The pelvis, trunk, and lower extremities are the most common sites, while EwS of the sacrum as a primary site is very rare, and only few studies focusing on this location are published. Due to the anatomical condition, local treatment is challenging in sacral malignancies. We analyzed factors that might influence the outcome of patients suffering from sacral EwS. Methods: We retrospectively analyzed data of the GPOH EURO-E.W.I.N.G 99 trial and the EWING 2008 trial, with a cohort of 124 patients with localized or metastatic sacral EwS. The study endpoints were overall survival (OS) and event-free survival (EFS). OS and EFS were calculated using the Kaplan-Meier method, and univariate comparisons were estimated using the log-rank test. Hazard ratios (HRs) with respective 95% confidence intervals (CIs) were estimated in a multivariable Cox regression model. Results: The presence of metastases (3y-EFS: 0.33 vs. 0.68; P < 0.001; HR = 3.4, 95% CI 1.7 to 6.6; 3y-OS: 0.48 vs. 0.85; P < 0.001; HR = 4.23, 95% CI 1.8 to 9.7), large tumor volume (≥200 ml) (3y-EFS: 0.36 vs. 0.69; P=0.02; HR = 2.1, 95% CI 1.1 to 4.0; 3y-OS: 0.42 vs. 0.73; P=0.04; HR = 2.1, 95% CI 1.03 to 4.5), and age ≥18 years (3y-EFS: 0.41 vs. 0.60; P=0.02; HR = 2.6, 95% CI 1.3 to 5.2; 3y-OS: 0.294 vs. 0.59; P=0.01; HR = 2.92, 95% CI 1.29 to 6.6) were revealed as adverse prognostic factors. Conclusion: Young age seems to positively influence patients` survival, especially in patients with primary metastatic disease. In this context, our results support other studies, stating that older age has a negative impact on survival. Tumor volume, metastases, and the type of local therapy modality have an impact on the outcome of sacral EwS. Level of evidence: Level 2. This trial is registered with NCT00020566 and NCT00987636.
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Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.