RESUMEN
A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , Mutación , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/inmunología , Reparación de la Incompatibilidad de ADN/genética , Frecuencia de los Genes , Genoma Humano/efectos de los fármacos , Genoma Humano/genética , Glioma/inmunología , Humanos , Masculino , Ratones , Repeticiones de Microsatélite/efectos de los fármacos , Repeticiones de Microsatélite/genética , Mutagénesis/efectos de los fármacos , Mutación/efectos de los fármacos , Fenotipo , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Análisis de Secuencia de ADN , Temozolomida/farmacología , Temozolomida/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 106 M-1, similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatchrepair-deficient phenotype.
Asunto(s)
Disparidad de Par Base , Carbocianinas/química , ADN/química , Colorantes Fluorescentes/química , Rodio/química , Línea Celular , Reparación de la Incompatibilidad de ADN , Fluorescencia , HumanosRESUMEN
[Ru(bpy)2(BNIQ)]2+ (BNIQ = Benzo[c][1,7]naphthyridine-1-isoquinoline), which incorporates the sterically expansive BNIQ ligand, is a highly selective luminescent probe for DNA mismatches and abasic sites, possessing a 500-fold higher binding affinity toward these destabilized regions relative to well-matched base pairs. As a result of this higher binding affinity, the complex exhibits an enhanced steady-state emission in the presence of DNA duplexes containing a single base mismatch or abasic site compared to fully well-matched DNA. Luminescence quenching experiments with Cu(phen)22+ and [Fe(CN)6]3- implicate binding of the complex to a mismatch from the minor groove via metalloinsertion. The emission response of the complex to different single base mismatches, binding preferentially to the more destabilized mismatches, is also consistent with binding by metalloinsertion. This work shows that high selectivity toward destabilized regions in duplex DNA can be achieved through the rational design of a complex with a sterically expansive aromatic ligand.
Asunto(s)
Complejos de Coordinación/química , ADN/química , Colorantes Fluorescentes/química , Rutenio/química , Disparidad de Par Base , Secuencia de Bases , Complejos de Coordinación/síntesis química , Colorantes Fluorescentes/síntesis química , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Ligandos , Luminiscencia , Naftiridinas/síntesis químicaRESUMEN
[Ru(Me4phen)2dppz](2+) serves as a luminescent "light switch" for single base mismatches in DNA. The preferential luminescence enhancement observed with mismatches results from two factors: (i) the complex possesses a 26-fold higher binding affinity toward the mismatch compared to well-matched base pairs, and (ii) the excited state emission lifetime of the ruthenium bound to the DNA mismatch is 160 ns versus 35 ns when bound to a matched site. Results indicate that the complex binds to the mismatch through a metalloinsertion binding mode. Cu(phen)2(2+) quenching experiments show that the complex binds to the mismatch from the minor groove, characteristic of metalloinsertion. Additionally, the luminescence intensity of the complex with DNA containing single base mismatches correlates with the thermodynamic destabilization of the mismatch, also consistent with binding through metalloinsertion. This complex represents a potentially new early cancer diagnostic for detecting deficiencies in mismatch repair.
