RESUMO
BACKGROUND: The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAFV600E+ melanomas and its role in solTNF-driven resistance reprogramming to MAPKi. METHODS: Flow cytometry was used to test TNFR1, TNFR2 and CD271 expression on, as well as NF-kB phosphorylation in human BRAF-mutant melanoma. The ability of melanoma cell lines to acquire MAPKi resistance in response to recombinant or macrophage-derived TNF was evaluated using the MTT cytotoxicity assay. Gene editing was implemented to knock out or knock in TNF receptors in melanoma cell lines. Knockout and knock-in cell line variants were employed to assess the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. Multicolor immunofluorescence microscopy was utilized to test TNFR2 expression by melanoma in patients receiving MAPKi therapy. RESULTS: TNFR1 and TNFR2 are co-expressed at various levels on 4/7 BRAFV600E+ melanoma cell lines evaluated in this study. In vitro treatments with solTNF induce MAPKi resistance solely in TNFR2-expressing BRAFV600E+ melanoma cell lines. TNFR1 and TNFR2 knockout and knock-in studies indicate that solTNF-mediated MAPKi resistance in BRAFV600E+ melanomas is predicated on TNFR1 and TNFR2 co-expression, where TNFR1 is the central mediator of NF-kB signaling, while TNFR2 plays an auxiliary role. solTNF-mediated effects are transient and can be abrogated with biologics. Evaluation of patient specimens indicates that TNFR2 is expressed on 50% of primary BRAFV600E+ melanoma cells and that MAPKi therapy may lead to the enrichment of TNFR2-expressing tumor cells. CONCLUSIONS: Our data suggest that TNFR2 is essential to solTNF-induced MAPKi resistance and a possible biomarker to identify melanoma patients that can benefit from solTNF-targeting therapies.
Assuntos
Melanoma , Receptores Tipo II do Fator de Necrose Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , NF-kappa B , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismoRESUMO
The variant acute promyelocytic leukemia (APL) translocation t(5;17)(q35;q21) fuses the N-terminus of nucleophosmin (NPM1) to the retinoic acid receptor alpha (RARA). We found that ectopic NPM1-RARA expression decreased TP53 protein levels in target cells. NPM1-RARA impaired TP53-dependent transcription. Cells expressing NPM1-RARA were more resistant to apoptotic stimuli. This work identifies the TP53 tumor suppressor as a novel target through which NPM1-RARA impacts leukemogenesis, and confirms the importance of impairment of TP53 in establishment of the APL phenotype.
Assuntos
Leucemia Promielocítica Aguda/patologia , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Células COS , Carcinogênese/genética , Carcinogênese/metabolismo , Chlorocebus aethiops , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 5/genética , Humanos , Leucemia Promielocítica Aguda/genética , Proteínas Nucleares/genética , Nucleofosmina , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Ácido Retinoico/genética , Translocação Genética , Células U937RESUMO
The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the nucleophosmin (NPM) gene at 5q35 with the retinoic acid receptor alpha (RARA) at 17q12-22. We have previously shown that leukemic cells express both NPM-RAR and RAR- NPM reciprocal translocation products. In this study we investigated the potential role of both proteins in modulating myeloid differentiation. Expression of NPM-RAR inhibited vitamin D3/transforming growth factor ß (TGFß)-mediated differentiation of U937 cells by more than 50%. In contrast, RAR-NPM expression did not alter vitamin D3/TGFß-induced differentiation of U937 clones. These results indicate that NPM-RAR, not RAR-NPM, is the prime mediator of myeloid differentiation arrest in t(5;17) APL.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Gradação de TumoresRESUMO
The t(5;17)(q35;q21) APL variant results in expression of a fusion protein linking the N-terminus of nucleophosmin (NPM) to the C-terminus of the retinoic acid receptor alpha (RAR). We have previously shown that NPM-RAR is capable of binding to DNA either as a homodimer or heterodimer with RXR. To determine the biological significance of NPM-RAR/RXR interaction, we developed two mutants of NPM-RAR that showed markedly diminished ability to bind RXR. U937 subclones expressing the NPM-RAR mutants showed significantly less inhibition of vitamin D3/TGFbeta-induced differentiation, compared with NPM-RAR. These results support the hypothesis that RXR interaction is necessary for NPM-RAR-mediated myeloid maturation arrest.
Assuntos
Diferenciação Celular , Células Mieloides/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Receptores X de Retinoides/metabolismo , Animais , Células COS , Chlorocebus aethiops , Regulação para Baixo , Humanos , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Células U937RESUMO
The PML protein localizes to regions of the nucleus known as nuclear bodies or PODs. However, in t(15;17) Acute Promyelocytic Leukemia (APL) blasts, PML is found in a micro-punctate pattern. In order to test the hypothesis that delocalization of PML from PODs is necessary for APL, we investigated the interaction of the t(5;17) APL fusion protein NPM-RAR with PML. NPM-RAR localizes diffusely throughout the nucleoplasm. NPM-RAR does not alter the localization of PML in transfected HeLa cells, and does not associate with PML in vitro. These studies suggest that NPM-RAR does not interact with PML.
