RESUMO
PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.
Assuntos
Carcinoma de Células Renais/patologia , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Túbulos Renais Proximais/metabolismo , Fator de Transcrição PAX8/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Camundongos Nus , Mutagênese , Fator de Transcrição PAX8/genética , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Ativação Transcricional , Transplante HeterólogoRESUMO
Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.
