RESUMEN
Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C-C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).
Asunto(s)
Receptores CCR8 , Linfocitos T Reguladores , Microambiente Tumoral , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Receptores CCR8/inmunología , Receptores CCR8/antagonistas & inhibidores , Animales , Ratones , Humanos , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Femenino , Citotoxicidad Celular Dependiente de Anticuerpos , Depleción Linfocítica , Línea Celular Tumoral , Fagocitosis/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.
Asunto(s)
Linfocitos B , Leucemia Linfocítica Crónica de Células B , Inhibidores de Proteínas Quinasas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Dasatinib/farmacología , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/fisiopatología , Monoéster Fosfórico Hidrolasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de Antígenos de Linfocitos B , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL.
Asunto(s)
Metilación de ADN , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Factores de Transcripción NFATC/genética , Recurrencia Local de Neoplasia/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Adenina/análogos & derivados , Anciano , Biomarcadores de Tumor , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Piperidinas , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Células Tumorales CultivadasAsunto(s)
ADN de Neoplasias , Factores de Transcripción de Tipo Kruppel , Leucemia Linfocítica Crónica de Células B , Proteínas de Neoplasias , Regiones Promotoras Genéticas , Receptores Notch , Transducción de Señal , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Femenino , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
AIM: The synthesis of rRNA is a key determinant of normal and malignant cell growth and subject to epigenetic regulation. Yet, the epigenomic features of rDNA arrays clustered in nucleolar organizer regions are largely unknown. We set out to explore for the first time how DNA methylation is distributed on individual rDNA arrays. MATERIALS & METHODS: Here we combined immunofluorescence detection of DNA modifications with fluorescence hybridization of single DNA fibers, metaphase immuno-FISH and methylation-sensitive restriction enzyme digestions followed by Southern blot. RESULTS: We found clustering of both hypomethylated and hypermethylated repeat units and hypermethylation of noncanonical rDNA in IMR90 fibroblasts and HCT116 colorectal carcinoma cells. Surprisingly, we also found transitions between hypo- and hypermethylated rDNA repeat clusters on single DNA fibers. CONCLUSION: Collectively, our analyses revealed co-existence of different epialleles on individual nucleolar organizer regions and showed that epi-combing is a valuable approach to analyze epigenomic patterns of repetitive DNA.
Asunto(s)
Metilación de ADN , ADN Ribosómico/metabolismo , Epigénesis Genética , Región Organizadora del Nucléolo , ADN Ribosómico/química , Femenino , Genes de ARNr , Genoma Humano , Humanos , Masculino , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.