RESUMO
Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.
Assuntos
Anemia de Diamond-Blackfan/patologia , Dano ao DNA , Modelos Biológicos , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Anemia de Diamond-Blackfan/embriologia , Anemia de Diamond-Blackfan/genética , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Feto/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Nucleosídeos/farmacologia , Ribonucleotídeo Redutases/metabolismo , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Measuring target coverage of small molecule inhibitors is paramount-first, for selection of molecules to progress through the drug development process and second, once a candidate drug moves to clinical testing, for guiding dose/schedule selection. Single cell network profiling (SCNP) using multiparameter flow cytometry can measure compound effects on multiple signaling cascades in a cell-type-specific manner. We applied SCNP to a panel of compounds with reported inhibitory effects on Jak/Stat signaling using a novel system where modulation of multiple signaling cascades are simultaneously measured in discrete cell subsets in whole (ie, unfractionated) blood. Jak2 vs. Jak3 selectivity as well as "off-target" effects on other cell signaling pathways were measured using a combination of cytokines that target different white blood cell subsets, namely GM-CSF (monocytes/granulocytes), IL-2 (T cells), and CD40L (B cells). The compounds were then rank-ordered by potency and selectivity against the different pathways tested. Notably, SCNP performed in whole unfractionated blood compared to fractionated peripheral blood mononuclear cells (PBMC) from the same donors revealed potency loss for all compounds, with one exception. These studies show that SCNP can be used to efficiently measure a drug candidate's potency and selectivity in a physiologically relevant environment (eg, whole blood) and that robust IC(50) are attainable from rare subpopulations (<100 cells). The ability to generate in vitro IC(50) measurements in whole blood can be used not only for the preclinical selection of lead molecules, but also to determine the target plasma concentration for clinical studies and to measure target coverage after drug administration in early phase clinical trials. Knowledge of the compound plasma concentration necessary to achieve biochemical coverage permits rational design of clinical trials based on biologically active dose vs. the traditional maximum tolerated dose (MTD) design, which is better suited for cytotoxic, nontargeted drugs.