RESUMO
Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Craniofaciais/genética , Ciclinas/genética , Deficiências do Desenvolvimento/genética , Atrofia Muscular/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Haploinsuficiência/genética , Heterozigoto , Humanos , Hipertelorismo/genética , Deficiência Intelectual/genética , Masculino , Anormalidades Musculoesqueléticas/genética , Malformações do Sistema Nervoso/genética , Fenótipo , Síndrome , Peixe-ZebraRESUMO
Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteólise , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Dano ao DNA , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteômica , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of a CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that recruits its target protein directly to DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.