Down-Regulation of Casein Kinase 1α Contributes to Endometriosis through Phosphatase and Tensin Homolog/Autophagy-Related 7-Mediated Autophagy.

The present study aimed to explore the roles of casein kinase 1α (CK1α) in endometriosis and its underlying mechanisms. Endometrial specimen were collected from the patients and healthy volunteers. The expression patterns of CK1α, phosphatase and tensin homolog (PTEN), and autophagy-related proteins were determined using immunohistochemistry staining, Western blot analysis, and quantitative RT-PCR. Besides, the CK1α-overexpressing cells and PTEN knockdown cells were constructed in the endometrial stromal cells isolated from endometriosis patients. In addition, the cells were transfected with pcDNA3.1-CK1α or pcDNA3.1-CK1α plus siRNA- PTEN. The expressions of CK1α, PTEN, and autophagy-related proteins were determined using Western blot and quantitative RT-PCR. The expressions of CK1α and autophagy-related 7 (Atg7) were significantly decreased in the ectopic endometrium compared with the eutopic endometrium. Spearman rank correlation analysis revealed positive correlations between CK1α and PTEN, CK1α and Atg7, and PTEN and Atg7. In addition, CK1α, PTEN, and autophagy-related proteins were down-regulated in ectopic endometrium. Interestingly, overexpression of CK1α significantly increased the expressions of autophagy-related proteins, whereas the protein expression of autophagy-related proteins was decreased with PTEN knock-down. CK1α regulated PTEN/Atg7-mediated autophagy in endometriosis.

Alternative polyadenylation drives oncogenic gene expression in pancreatic ductal adenocarcinoma.

Alternative polyadenylation (APA) is a gene regulatory process that dictates mRNA 3'-UTR length, resulting in changes in mRNA stability and localization. APA is frequently disrupted in cancer and promotes tumorigenesis through altered expression of oncogenes and tumor suppressors. Pan-cancer analyses have revealed common APA events across the tumor landscape; however, little is known about tumor type-specific alterations that may uncover novel events and vulnerabilities. Here, we integrate RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) to comprehensively analyze APA events in 148 pancreatic ductal adenocarcinomas (PDACs). We report widespread, recurrent, and functionally relevant 3'-UTR alterations associated with gene expression changes of known and newly identified PDAC growth-promoting genes and experimentally validate the effects of these APA events on protein expression. We find enrichment for APA events in genes associated with known PDAC pathways, loss of tumor-suppressive miRNA binding sites, and increased heterogeneity in 3'-UTR forms of metabolic genes. Survival analyses reveal a subset of 3'-UTR alterations that independently characterize a poor prognostic cohort among PDAC patients. Finally, we identify and validate the casein kinase CSNK1A1 (also known as CK1alpha or CK1a) as an APA-regulated therapeutic target in PDAC. Knockdown or pharmacological inhibition of CSNK1A1 attenuates PDAC cell proliferation and clonogenic growth. Our single-cancer analysis reveals APA as an underappreciated driver of protumorigenic gene expression in PDAC via the loss of miRNA regulation.

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Rps14, Csnk1a1 and miRNA145/miRNA146a deficiency cooperate in the clinical phenotype and activation of the innate immune system in the 5q- syndrome.

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

CK1α Collaborates with DOUBLETIME to Regulate PERIOD Function in the Drosophila Circadian Clock.

The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day-dependent manner. Normal progression of circadian rhythms is dependent on the daily cycling of core transcriptional factors that make up cell-autonomous molecular oscillators. In Drosophila, PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional-translational feedback mechanism to regulate the circadian transcriptome. Posttranslational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, casein kinase 1α (CK1α) has been identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. Using Drosophila as a model, we show that CK1α has an overall function of speeding up PER metabolism and is required to maintain the 24 h period of circadian rhythms. Our results indicate that CK1α collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, we observe that CK1α promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1α enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus.SIGNIFICANCE STATEMENT Circadian clocks are endogenous timers that integrate environmental signals to impose temporal control over organismal physiology over the 24 h day/night cycle. To maintain the 24 h period length of circadian clocks and to ensure that circadian rhythms are in synchrony with the external environment, key proteins that make up the molecular oscillator are extensively regulated by phosphorylation to ensure that they perform proper time-of-day-specific functions. Casein kinase 1α (CK1α) has previously been identified as a kinase that phosphorylates mammalian PERIOD (PER) proteins to promote their degradation, but the mechanism by which it modulates PER stability is unclear. In this study, we characterize the mechanisms by which CK1α interacts with DOUBLETIME (DBT) to achieve the overall function of speeding up PER metabolism and to ensure proper time-keeping.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

CK1α and IRF4 are essential and independent effectors of immunomodulatory drugs in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive cancer with few treatment options. The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been shown to kill PEL cell lines, and lenalidomide is in clinical trials against PEL. IMiDs bind to the CRL4CRBN E3 ubiquitin ligase complex, leading to the acquisition of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), casein kinase 1 α (CK1α), and zinc finger protein 91 (ZFP91) as neosubstrates. IMiDs are effective against multiple myeloma because of degradation of IKZF1 and IKZF3 and the consequent loss of interferon regulatory factor 4 (IRF4) and MYC expression. Lenalidomide is also effective in chromosome 5q deletion-associated myelodysplastic syndrome as a result of degradation of CK1α. An essential IKZF1-IRF4-MYC axis has recently been proposed to underlie the toxicity of IMiDs in PEL. Here, we further investigate IMiD effectors in PEL cell lines, based on genome-wide CRISPR/Cas9 screens for essential human genes. These screens and extensive validation experiments show that, of the 4 neosubstrates, only CK1α is essential for the survival of PEL cell lines. In contrast, IKZF1 and IKZF3 are dispensable, individually or in combination. IRF4 was critical in all 8 PEL cell lines tested, and surprisingly, IMiDs triggered downregulation of IRF4 expression independently of both IKZF1 and IKZF3. Reexpression of CK1α and/or IRF4 partially rescued PEL cell lines from IMiD-mediated toxicity. In conclusion, IMiD toxicity in PEL cell lines is independent of IKZF1 and IKZF3 but proceeds through degradation of the neosubstrate CK1α and downregulation of IRF4.

CSNK1α1 mediates malignant plasma cell survival.

Here we report that targeting casein kinase 1-α1 (CSNK1α1) is a potential novel treatment strategy in multiple myeloma (MM) therapy distinct from proteasome inhibition. CSNK1α1 is expressed in all the tested MM cell lines and patient MM cells, and is not altered during bortezomib-triggered cytotoxicity. Inhibition of CSNK1α1 kinase activity in MM cells with targeted therapy D4476 or small hairpin RNAs triggers cell G0/G1-phase arrest, prolonged G2/M phase and apoptosis. D4476 also induced cytotoxicity in bortezomib-resistant MM cells and enhanced bortezomib-triggered cytotoxicity. CSNK1α1 signaling pathways include CDKN1B, P53 and FADD; gene signatures involved included interferon-α, tumor necrosis factor-α and LIN9. In addition, reduction of Csnk1α1 prevents cMYC/KRAS12V transformation of BaF3 cells independent of interleukin-3. Impartially, reducing Csnk1α1 prevented development of cMYC/KRAS12V-induced plasmacytomas in mice, suggesting that CSNK1α1 may be involved in MM initiation and progression. Our data suggest that targeting CSNK1α1, alone or combined with bortezomib, is a potential novel therapeutic strategy in MM. Moreover, inhibition of CSNK1α1 may prevent the progression of monoclonal gammopathy of undetermined significance to MM.

Casein kinase 1 α phosphorylates the Wnt regulator Jade-1 and modulates its activity.

Tight regulation of Wnt/ß-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting ß-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit ß-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing ß-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates ß-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that ß-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.