RESUMEN
Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).
Asunto(s)
Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacologíaRESUMEN
OBJECTIVE: Cyclin-dependent kinase 9 (CDK-9) controls the activation of primary inflammatory response genes. The purpose of this study was to determine whether CDK-9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines. METHODS: Human chondrocytes were challenged with different proinflammatory stimuli (interleukin-1ß [IL-1ß], lipopolysaccharides, and tumor necrosis factor α) in the presence or absence of either the CDK-9 inhibitor flavopiridol or small interfering RNA (siRNA). The expression of messenger RNA (mRNA) for inflammatory mediator genes, catabolic genes, and anabolic genes were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Cartilage explants were incubated for 6 days with IL-1ß in the presence or absence of flavopiridol. Cartilage matrix degradation was assessed by the release of glycosaminoglycan (GAG) and cleaved type II collagen (COL2A) peptides. RESULTS: CDK-9 inhibition by flavopiridol or knockdown by siRNA effectively suppressed the induction of mRNA for inducible nitric oxide synthase by all 3 proinflammatory stimuli. Results from NF-κB-targeted PCR array analysis showed that flavopiridol suppressed IL-1ß induction of a broad range of inflammatory mediator genes (59 of 67 tested). CDK-9 inhibition also suppressed the induction of catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5), but did not affect the basal expression of anabolic genes (COL2A, aggrecan, and cartilage oligomeric matrix protein) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by G6PDH activity. Finally, in IL-1ß-treated cartilage explants, flavopiridol reduced the release of the matrix degradation product GAG and cleaved COL2A peptides, but did not affect long-term chondrocyte viability. CONCLUSION: CDK-9 activity is required for the primary inflammatory response in chondrocytes. Flavopiridol suppresses the induction of inflammatory mediator genes and catabolic genes to protect cartilage from the deleterious effects of proinflammatory cytokines, without affecting cell viability and functions.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Citocinas/farmacología , Flavonoides/farmacología , Inflamación/prevención & control , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Adulto , Anciano , Anciano de 80 o más Años , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Colágeno Tipo II/metabolismo , Quinasa 9 Dependiente de la Ciclina/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Humanos , Técnicas In Vitro , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/farmacología , Lipopolisacáridos/farmacología , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , Procolágeno N-Endopeptidasa/metabolismo , ARN Interferente Pequeño/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
EBNA 2 is one of only five viral genes essential for the infection and immortalization of human B cells by the cancer-associated virus Epstein-Barr virus (EBV). EBNA 2 activates cellular and viral transcription and associates with components of the basal transcription apparatus and a number of coactivators. We provide the first evidence to show that the mechanism of transcriptional activation by EBNA 2 also involves phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II). We found that transcriptional activation by EBNA 2 was inhibited by a dominant-negative mutant of the pol II CTD kinase, CDK9, and by low concentrations of the CDK9 inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Moreover, using chromatin immunoprecipitation assays we demonstrated that EBNA 2 stimulates both pol II recruitment and pol II phosphorylation on serine 5 of the CTD in vivo. These results identify a new step in the transcription cycle that is subject to regulation by a key EBV-encoded transcription factor and highlight CDK9 inhibitors as potential anti-EBV agents.