Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a–dependent neurite outgrowth

Centrosomal localization of kinase-active CK1δ is required for neurite outgrowth in response to Wnt-3a

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation

Novel Apoptosis-Inducing Agents for the Treatment of Cancer, a New Arsenal in the Toolbox

Evasion from apoptosis is an important hallmark of cancer cells. Alterations of apoptosis pathways are especially critical as they confer resistance to conventional anti-cancer therapeutics, e.g., chemotherapy, radiotherapy, and targeted therapeutics. Thus, successful induction of apoptosis using novel therapeutics may be a key strategy for preventing recurrence and metastasis. Inhibitors of anti-apoptotic molecules and enhancers of pro-apoptotic molecules are being actively developed for hematologic malignancies and solid tumors in particular over the last decade. However, due to the complicated apoptosis process caused by a multifaceted connection with cross-talk pathways, protein–protein interaction, and diverse resistance mechanisms, drug development within the category has been extremely challenging. Careful design and development of clinical trials incorporating predictive biomarkers along with novel apoptosis-inducing agents based on rational combination strategies are needed to ensure the successful development of these molecules. Here, we review the landscape of currently available direct apoptosis-targeting agents in clinical development for cancer treatment and update the related biomarker advancement to detect and validate the efficacy of apoptosis-targeted therapies, along with strategies to combine them with other agents

Targeting TRAIL Death Receptors in Triple-Negative Breast Cancers: Challenges and Strategies for Cancer Therapy

The tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells via death receptor (DR) activation with little toxicity to normal cells or tissues. The selectivity for activating apoptosis in cancer cells confers an ideal therapeutic characteristic to TRAIL, which has led to the development and clinical testing of many DR agonists. However, TRAIL/DR targeting therapies have been widely ineffective in clinical trials of various malignancies for reasons that remain poorly understood. Triple negative breast cancer (TNBC) has the worst prognosis among breast cancers. Targeting the TRAIL DR pathway has shown notable efficacy in a subset of TNBC in preclinical models but again has not shown appreciable activity in clinical trials. In this review, we will discuss the signaling components and mechanisms governing TRAIL pathway activation and clinical trial findings discussed with a focus on TNBC. Challenges and potential solutions for using DR agonists in the clinic are also discussed, including consideration of the pharmacokinetic and pharmacodynamic properties of DR agonists, patient selection by predictive biomarkers, and potential combination therapies. Moreover, recent findings on the impact of TRAIL treatment on the immune response, as well as novel strategies to address those challenges, are discussed

Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1

<div><p>Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from <i>Csnk1d</i> null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEF^{<i>Csnk1d null</i>}). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEF^{<i>Csnk1d null</i>} cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEF^{<i>Csnk1d null</i>} cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1.</p></div

Analysis of total and phosphorylated Chk1 in MEF^Ctl. cells after HU treatment, following CK1δ knockdown with or without subsequent expression of siRNA-resistant CK1δ constructs.

<p>(A) MEF^<i>Ctl</i>. cells were transfected with control siRNA (siNeg) or siRNA targeting CK1δ (siCK1δ Q2). Twenty-four hours later, cells were transfected with the indicated DNA constructs (pcDNA 3.3 was the empty vector control). Seventy hours later, cells were treated with HU for 2 h and subjected to western blotting. Data are from one representative experiment among 3 independent experiments. (B) Statistical analysis of band intensity shown in (A). Relative band intensity of CK1δ, p-Chk1 and total Chk1 was analyzed in 3 independent experiments and shown as mean +/- SD. *<i>p</i><0.05, **<i>p</i><0.01, NS = not significant.</p

MEF^{<i>Csnk1d null</i>} cells have lower levels of total and phosphorylated Chk1 and Cdc2/CDK1 than MEF^Ctl. cells.

<p>(A) Western blot analysis of total Chk1, total and phosphorylated (Tyr15) Cdc2/CDK1, and CK1δ in MEF^<i>Ctl</i>. and MEF^{<i>Csnk1d null</i>} cells from two sets of cells with different passage number (left, P6; right, P20). Band intensity relative to the HSC70 or tubulin loading control is indicated directly below each band. (B) Time course of Chk1 activation, as indicated by Ser345 phosphorylation, in the same passage of MEF^<i>Ctl</i>. and MEF^{<i>Csnk1d null</i>} cells treated with hydroxyurea (HU). Histogram shows the band intensity of p-Chk1and total Chk1 relative to the HSC70 loading control. Band intensity of MEF^<i>Ctl</i>. cells not exposed to HU was defined as 1.0. (C) MEF^<i>Ctl</i>. (P9) cells were transfected with siRNA, and 46 h later cells were treated with HU for 1.5 h, followed by western blotting. Data in left panel are from one representative experiment. Histograms to the right show relative band intensities of p-Chk1, total Chk1 and CK1δ from two independent experiments. Band intensity of MEF^<i>Ctl</i>. cells transfected with siNeg and not exposed to HU was defined as 1.0.</p