ABSTRACT
Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.
Subject(s)
Cell Cycle Proteins , Enhancer Elements, Genetic , Kinetochores , Protein Serine-Threonine Kinases , Cell Cycle Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Humans , Polo-Like Kinase 1ABSTRACT
To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.
Subject(s)
Antineoplastic Agents/pharmacology , Electron Transport Complex I/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Oxazoles/pharmacology , Oxidative Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Triazoles/pharmacology , Animals , Biomarkers , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Hematopoiesis/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Mice , Models, Biological , Receptor, ErbB-2/antagonists & inhibitorsABSTRACT
High plasma concentrations of free fatty acids (FFA) and insulin are common features in atherosclerotic patients with type 2 diabetes. FFA, according to their nature, can have various effects on vascular smooth muscle cells (VSMC). These cells play important roles throughout atherosclerosis pathogenesis, from plaque development to plaque instability. Thus, this study aims to assess the impact of two FFA combinations and insulin on murine VSMC viability. The two combinations contain the same FFA but at different ratios, one being richer in saturated fatty acids (SFA) and the other having a higher proportion of monounsaturated fatty acids (MUFA). Both combinations inhibited VSMC proliferation due to their pro-apoptotic potential, with SFA being the major inducers of apoptosis. However, the presence of oleic acid (OLA) attenuated this impact in a dose-dependent manner. OLA had also the capacity to reduce apoptosis rates more strongly when combined with a SFA than when used alone in VSMC treatments. This effect was significant only for specific proportions of these FFA and was even more effective in presence of insulin. These results highlight the presence of a competition between pro-apoptotic and anti-apoptotic mechanisms in VSMC that is dependent on FFA ratios (saturated vs. monounsaturated) and on insulinemia. They also underline the importance of the presence of MUFA such as OLA in diets containing high proportions of SFA.
Subject(s)
Fatty Acids, Monounsaturated/pharmacology , Fatty Acids/pharmacology , Muscle, Smooth, Vascular/drug effects , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Structure-Activity RelationshipABSTRACT
The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosan-phosphorylcholine (CH-PC). On CH-PC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CH-PC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH-PC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CH-PC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CH-PC) increased the percentage of progenitor cells (CD117(+)CD34(+)) (7.1 ± 0.8%, FN: 4.1 ± 0.8%) and EPC (CD117(+)CD34(+)VEGFR-2(+)CD31(+)) (2.33 ± 0.6%, FN: 0.25 ± 0.1%), while the mesenchymal stem cell fraction (CD44(+)CD106(+)CD90(+)) was decreased (0.07 ± 0.01%, FN: 0.55 ± 0.22%). Polymeric substrate CH-PC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.
