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Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4(CRBN)) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4(CRBN) substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clinical efficacy in the treatment of multiple myeloma and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS), respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4(CRBN) is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.

Necroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation.

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis. Osteosarcoma is an aggressive tumour and little is known the mechanisms underpinning its highly metastatic nature. Here, the authors highlight a role for the CK1α/CBX4 axis in driving metastasis, suggesting that this pathway might be targeted for therapeutic benefit.

The contribution of autophagy to cancer development remains controversial, largely owing to the fact that autophagy can be tumour suppressive or oncogenic in different biological contexts. Here, we show that in non-small-cell lung cancer (NSCLC), casein kinase 1 alpha 1 (CK1α) suppresses tumour growth by functioning as an autophagy inducer to activate an autophagy-regulating, tumour-suppressive PTEN/AKT/FOXO3a/Atg7 axis. Specifically, CK1α bound the C-terminal tail of PTEN and enhanced both PTEN stability and activity by competitively antagonizing NEDD4-1-induced PTEN polyubiquitination and abrogating PTEN phosphorylation, thereby inhibiting AKT activity and activating FOXO3a-induced transcription of Atg7. Notably, blocking CK1α-induced Atg7-dependent autophagy cooperates with oncogenic HRas V12 to initiate tumorigenesis of lung epithelial cells. An association of a CK1α-modulated autophagic program with the anti-neoplastic activities of the CK1α/PTEN/FOXO3a/Atg7 axis was demonstrated in xenografted tumour models and human NSCLC specimens. This provides insights into the biological and potentially clinical significance of autophagy in NSCLC. Cai et al. show that CK1α suppresses lung cancer growth by inducing autophagy through a PTEN–AKT–FOXO3a pathway that ultimately leads to ATG7 expression.

WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

Eryptosis is a coordinated non-lytic cell death of erythrocytes characterized by cell shrinkage, cell membrane scrambling, Ca2+ influx, ceramide accumulation, oxidative stress, activation of calpain and caspases. Physiologically, it aims at removing damaged or aged erythrocytes from circulation. A plethora of diseases are associated with enhanced eryptosis, including metabolic diseases, cardiovascular pathology, renal and hepatic diseases, hematological disorders, systemic autoimmune pathology, and cancer. This makes eryptosis and eryptosis-regulating signaling pathways a target for therapeutic interventions. This review highlights the eryptotic signaling machinery containing several protein kinases and its small molecular inhibitors with a special emphasis on casein kinase 1α (CK1α), a serine/threonine protein kinase with a broad spectrum of activity. In this review article, we provide a critical analysis of the regulatory role of CK1α in eryptosis, highlight triggers of CK1α-mediated suicidal death of red blood cells, cover the knowledge gaps in understanding CK1α-driven eryptosis and discover the opportunity of CK1α-targeted pharmacological modulation of eryptosis. Moreover, we discuss the directions of future research focusing on uncovering crosstalks between CK1α and other eryptosis-regulating kinases and pathways.

A Xenopus laevis two-reporter screen identifies the antihelminthic drug pyrvinium as an inhibitor of the Wnt/β-catenin signaling pathway that works by activating CK1α, which is likely working at the level of Pygopus, a core transcriptional component of the Wnt pathway. Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC 50 of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore is subject to regulation by cell signalling; however, the kinase(s) responsible for this event have not been definitively identified. Here using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson’s disease. The kinase LRRK2 is implicated in Parkinson’s disease progression and is known to be phosphorylated. Chia et al. show that this phosphorylation is mediated by the kinase CK1a, and is required for the recruitment of LRRK2 to Golgi-derived vesicles, suggesting a role for this protein in Golgi maintenance.

The role of p53 in metastasis In a mouse model of intestinal cancer, Yinon Ben-Neriah and colleagues show that in the absence of CK1-α, the loss of p53 dramatically enhances tumour progression and metastasis. The tumour repressor p53 normally limits cancer cell invasion through the regulation of p21 and a set of invasion genes that include Prox1. This study adds more detail to the emerging picture that during tumour development, the p53 gene not only controls cell death and proliferation but also metastasis. This study shows, via a mouse model of intestinal cancer, that in the absence of CKIα, the loss of p53 dramatically enhances tumour progression and metastasis. p53 is shown to normally limit cancer cell invasion via the regulation of p21 and a set of invasion genes that include Prox1 . This study adds important insights to the emerging picture that during tumour development the p53 tumour suppressor gene not only controls cell death and proliferation but also metastasis. The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer 1 . Here we show that casein kinase Iα (CKIα), a component of the β-catenin-destruction complex 1 , is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation 2 . The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression 3 ; however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1 , Cip1 , Sdi1 and Cdkn1a ) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53.

Disturbance of protein kinase activity may result in dramatic consequences that often lead to cancer development and progression. In tumors of blood origin, both tyrosine kinases and serine/threonine kinases are altered by different types of mutations, critically regulating cancer hallmarks. CK1α and CK2 are highly conserved, ubiquitously expressed and constitutively active pleiotropic kinases, which participate in multiple biological processes. The involvement of these kinases in solid and blood cancers is well documented. CK1α and CK2 are overactive in multiple myeloma, leukemias and lymphomas. Intriguingly, they are not required to the same degree for the viability of normal cells, corroborating the idea of “druggable” kinases. Different to other kinases, mutations on the gene encoding CK1α and CK2 are rare or not reported. Actually, these two kinases are outside the paradigm of oncogene addiction, since cancer cells’ dependency on these proteins resembles the phenomenon of “non-oncogene” addiction. In this review, we will summarize the general features of CK1α and CK2 and the most relevant oncogenic and stress-related signaling nodes, regulated by kinase phosphorylation, that may lead to tumor progression. Finally, we will report the current data, which support the positioning of these two kinases in the therapeutic scene of hematological cancers.