ABSTRACT
The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Cyclin-Dependent Kinases , Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Sulfonamides , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Drug Synergism , Hedgehog Proteins/metabolism , Hedgehog Proteins/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/therapeutic use , Sulfonamides/pharmacology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.
Subject(s)
Centrioles/enzymology , Cilia/enzymology , Epithelial Cells/enzymology , Mitosis , NIMA-Related Kinases/metabolism , Retinal Pigment Epithelium/enzymology , Animals , Binding Sites , Cell Line , Centrioles/genetics , Cilia/genetics , Female , G2 Phase Cell Cycle Checkpoints , Hematopoietic Stem Cells/enzymology , Humans , Mammary Glands, Animal/cytology , Mammary Glands, Animal/enzymology , Mice , Microtubule Proteins/genetics , Microtubule Proteins/metabolism , NIMA-Related Kinases/genetics , Protein Binding , Retinal Pigment Epithelium/cytology , Signal Transduction , Time FactorsABSTRACT
Ciliogenesis initiates at the mother centriole through a series of events that include membrane docking, displacement of cilia-inhibitory proteins and axoneme elongation. Centriolar proteins, in particular at distal and subdistal appendages, carry out these functions. Recently, cytoplasmic complexes named centriolar satellites have also been shown to promote ciliogenesis. Little is known about the functional and molecular relationship between appendage proteins, satellites and cilia biogenesis. Here, we identified the WD-repeat protein 8 (WDR8, also known as WRAP73) as a satellite and centriolar component. We show that WDR8 interacts with the satellite proteins SSX2IP and PCM1 as well as the centriolar proximal end component Cep135. Cep135 is required for the recruitment of WDR8 to centrioles. Depletion experiments revealed that WDR8 and Cep135 have strongly overlapping functions in ciliogenesis. Both are indispensable for ciliary vesicle docking to the mother centriole and for unlocking the distal end of the mother centriole from the ciliary inhibitory complex CP110-Cep97. Our data thus point to an important function of centriolar proximal end proteins in ciliary membrane biogenesis, and establish WDR8 and Cep135 as two factors that are essential for the initial steps of ciliation.