ABSTRACT
Overexpression of MYC oncogene is highly prevalent in many malignancies such as aggressive triple-negative breast cancers (TNBCs) and it is associated with very poor outcome. Despite decades of research, attempts to effectively inhibit MYC, particularly with small molecules, still remain challenging due to the featureless nature of its protein structure. Herein, we describe the engineering of the dominant-negative MYC peptide (OmoMYC) linked to a functional penetrating 'Phylomer' peptide (FPPa) as a therapeutic strategy to inhibit MYC in TNBC. We found FPPa-OmoMYC to be a potent inducer of apoptosis (with IC50 from 1-2 µM) in TNBC cells with negligible effects in non-tumorigenic cells. Transcriptome analysis of FPPa-OmoMYC-treated cells indicated that the fusion protein inhibited MYC-dependent networks, inducing dynamic changes in transcriptional, metabolic, and apoptotic processes. We demonstrated the efficacy of FPPa-OmoMYC in inhibiting breast cancer growth when injected orthotopically in TNBC allografts. Lastly, we identified strong pharmacological synergisms between FPPa-OmoMYC and chemotherapeutic agents. This study highlights a novel therapeutic approach to target highly aggressive and chemoresistant MYC-activated cancers.
Subject(s)
Cell-Penetrating Peptides/pharmacology , Molecular Targeted Therapy/methods , Neoplasm Proteins/antagonists & inhibitors , Peptide Fragments/therapeutic use , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Triple Negative Breast Neoplasms/drug therapy , Amino Acid Sequence , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/therapeutic use , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Drug Synergism , Female , Genes, myc , Humans , Inhibitory Concentration 50 , Leucine Zippers/genetics , Mice , Models, Molecular , Mutation , Peptide Fragments/administration & dosage , Peptide Fragments/genetics , Peptide Fragments/pharmacokinetics , Peptide Library , Protein Conformation , Protein Engineering , Proto-Oncogene Proteins c-myc/administration & dosage , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/pharmacokinetics , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacokineticsABSTRACT
Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.
Subject(s)
Cell-Penetrating Peptides/pharmacology , Drug Delivery Systems/methods , Drug Evaluation, Preclinical/methods , Animals , Bacteriophage T7 , Biotinylation , CHO Cells , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/toxicity , Circular Dichroism , Cricetulus , Disease Models, Animal , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Microscopy, Fluorescence , Muscular Dystrophy, Duchenne/drug therapy , Peptide Library , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.
