ABSTRACT
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Subject(s)
Neoplasms/drug therapy , Nucleosides/pharmacology , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Animals , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , Humans , Imides/pharmacology , Mice , Neoplasms/genetics , Neoplasms/pathology , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/genetics , Protein Binding , Pyrazoles , Pyrimidines , Sulfides , Ubiquitin/antagonists & inhibitors , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Xenograft Model Antitumor AssaysABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/administration & dosage , Antineoplastic Agents/administration & dosage , Benzazepines/administration & dosage , Cell Cycle Proteins/antagonists & inhibitors , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiones/administration & dosage , Administration, Oral , Animals , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzazepines/pharmacokinetics , Benzazepines/pharmacology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Synergism , Female , Gene Knockdown Techniques , Histones/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Rituximab , Thiones/pharmacokinetics , Thiones/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEß as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEß mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEß mutations.
Subject(s)
Cyclopentanes/pharmacology , Enzyme Inhibitors/pharmacology , Mutation , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/genetics , Animals , Binding Sites , Cell Line, Tumor , Clinical Trials as Topic , Drug Resistance, Neoplasm/genetics , Female , Humans , Mice , Mice, Nude , Rats , Rats, Nude , Tumor Cells, Cultured , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/physiology , Xenograft Model Antitumor AssaysABSTRACT
Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.