ABSTRACT
The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, with both functions being crucial for HPV-induced oncogenesis. A chimeric protein was built by fusing a PDZ domain and an LxxLL motif, both known to bind E6. NMR spectroscopy, calorimetry and a mammalian protein complementation assay converged to show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all of the hrm-HPV E6â proteins tested but not to low-risk mucosal or cutaneous HPVâ E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspaseâ 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers.
Subject(s)
DNA-Binding Proteins/metabolism , Human papillomavirus 16/metabolism , Human papillomavirus 18/metabolism , Neoplasms/virology , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/virology , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Adenoviridae/genetics , Amino Acid Motifs , Binding Sites , Cell Death , Cell Line , DNA-Binding Proteins/chemistry , Gene Expression , HeLa Cells , Human papillomavirus 16/chemistry , Human papillomavirus 18/chemistry , Humans , Ligands , Models, Molecular , Neoplasms/metabolism , Neoplasms/therapy , Oncogene Proteins, Viral/chemistry , PDZ Domains , Papillomavirus Infections/metabolism , Papillomavirus Infections/therapy , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Repressor Proteins/chemistry , Tumor Suppressor Protein p53/metabolismABSTRACT
BET bromodomain inhibitors hold promise as therapeutic agents in diverse indications, but their clinical progression has been challenging and none have received regulatory approval. Early clinical trials in cancer have shown heterogeneous clinical responses, development of resistance, and adverse events. Increased understanding of their mechanism(s) of action and identification of biomarkers are needed to identify appropriate indication(s) and achieve efficacious dosing. Using genome-wide CRISPR-Cas9 screens at different concentrations, we report molecular mechanisms defining cellular responses to BET inhibitors, some of which appear specific to a single compound concentration. We identify multiple transcriptional regulators and mTOR pathway members as key determinants of JQ1 sensitivity and two Ca2+/Mn2+ transporters, ATP2C1 and TMEM165, as key determinants of JQ1 resistance. Our study reveals new molecular mediators of BET bromodomain inhibitor effects, suggests the involvement of manganese, and provides a rich resource for discovery of biomarkers and targets for combination therapies.