ABSTRACT
The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.
Subject(s)
Aurora Kinase A/physiology , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Multienzyme Complexes/metabolism , Allosteric Regulation , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Cell Line , G1 Phase Cell Cycle Checkpoints , HeLa Cells , Humans , Interphase/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Replication OriginABSTRACT
Mutations activating KRAS underlie many forms of cancer, but are refractory to therapeutic targeting. Here, we develop Poloppin, an inhibitor of protein-protein interactions via the Polo-box domain (PBD) of the mitotic Polo-like kinases (PLKs), in monotherapeutic and combination strategies to target mutant KRAS. Poloppin engages its targets in biochemical and cellular assays, triggering mitotic arrest with defective chromosome congression. Poloppin kills cells expressing mutant KRAS, selectively enhancing death in mitosis. PLK1 or PLK4 depletion recapitulates these cellular effects, as does PBD overexpression, corroborating Poloppin's mechanism of action. An optimized analog with favorable pharmacokinetics, Poloppin-II, is effective against KRAS-expressing cancer xenografts. Poloppin resistance develops less readily than to an ATP-competitive PLK1 inhibitor; moreover, cross-sensitivity persists. Poloppin sensitizes mutant KRAS-expressing cells to clinical inhibitors of c-MET, opening opportunities for combination therapy. Our findings exemplify the utility of small molecules modulating the protein-protein interactions of PLKs to therapeutically target mutant KRAS-expressing cancers.
Subject(s)
Cell Cycle Proteins/metabolism , Mutation , Protein Interaction Domains and Motifs/drug effects , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Mitosis , Molecular Structure , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/chemistry , Structure-Activity Relationship , Substrate Specificity , Polo-Like Kinase 1ABSTRACT
Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases É and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.