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Aims/hypothesisDiabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets.MethodsWe performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets.ResultsThe meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells1. Although the thymic medulla consists mostly of developing and mature AIRE+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought2. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers3. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

Recent research has shown that Doublecortin-like kinase 1 (DCLK1) is overexpressed in different types of cancer. It has recently been described as a cancer stem cells (CSCs) marker, is associated with carcinogenesis, and positively correlates with infiltration of multiple immune cell types in some cancers. However, studies focused on assessing DCLK1 expression in HCC are limited, and the role of DCLK1 in HCC tumor immunity remains to be determined. In this study, we used a modified model of the resistant hepatocyte (MRHM) to evaluate DCLK1 expression in HCC. Furthermore, DCLK1 expression in HCC was analyzed using TIMER 2.0, UALCAN, GEPIA, GEO, and HPA web-based tools. Correlations between DCLK1 expression and clinicopathological factors in patients were analyzed using the UALCAN web-based tool. Finally, correlations between DCLK1 and immune infiltrates were investigated using the TIMER 2.0 and TISIDB web-based tools. The results showed that DCLK1 is significantly overexpressed during progression of the HCC carcinogenic process in the MRHM. DCLK1 is overexpressed in HCC according to multiple publics web-based tools, and its overexpression is associated with cancer stage. Furthermore, DCLK1 expression was correlated with infiltration levels of multiple immune cells, immunomodulatory factors, immunoinhibitors, MHC molecules, chemokines, receptors, and immune cell-specific markers. These results suggest that DCLK1 is a potential prognostic biomarker that determines cancer progression and correlates with immune cell infiltration in HCC., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflicts of interest. Ethical approval: All experimental animal studies were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of CINVESTAV-IPN, protocol 0168 − 15. Informed Consent: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Co-infection with oncogenic retrovirus and herpesvirus significantly facilitates tumor metastasis in human and animals. Co-infection with avian leukosis virus subgroup J (ALV-J) and Marek's disease virus (MDV), which are typical oncogenic retrovirus and herpesvirus, respectively, leads to enhanced oncogenicity and accelerated tumor formation, resulting in increased mortality of affected chickens. Previously, we found that ALV-J and MDV cooperatively promoted tumor metastasis. However, the molecular mechanism remains elusive. Here, we found that doublecortin-like kinase 1 (DCLK1) mediated cooperative acceleration of epithelial-mesenchymal transition (EMT) by ALV-J and MDV promoted tumor metastasis. Mechanistically, DCLK1 induced EMT via activating Wnt/β-catenin pathway by interacting with β-catenin, thereby cooperatively promoting tumor metastasis. Initially, we screened and found that DCLK1 was a potential mediator for the cooperative activation of EMT by ALV-J and MDV, and enhanced cell proliferation, migration, and invasion. Subsequently, we revealed that DCLK1 physically interacted with β-catenin to promote the formation of the β-catenin-TCF4 complex, inducing transcription of the Wnt target gene, c-Myc, promoting EMT by increasing the expression of N-cadherin, Vimentin, and Snail, and decreasing the expression of E-cadherin. Taken together, we discovered that jointly activated DCLK1 by ALV-J and MDV accelerated cell proliferation, migration and invasion, and ultimately activated EMT, paving the way for tumor metastasis. This study elucidated the molecular mechanism underlying cooperative metastasis induced by co-infection with retrovirus and herpesvirus., Importance: Tumor metastasis, a complex phenomenon in which tumor cells spread to new organs, is one of the greatest challenges in cancer research and is the leading cause of cancer-induced death. Numerous studies have shown that oncoviruses and their encoded proteins significantly affect metastasis, especially the EMT process. ALV-J and MDV are classic tumorigenic retrovirus and herpesvirus, respectively. We found that ALV-J and MDV synergistically promoted EMT. Further, we identified the tumor stem cell marker DCLK1 in ALV-J and MDV co-infected cells. DCLK1 directly interacted with β-catenin, promoting the formation of the β-catenin-TCF4 complex. This interaction activated the Wnt/β-catenin pathway, thereby inducing EMT and paving the way for synergistic tumor metastasis. Exploring the molecular mechanisms by which ALV-J and MDV cooperate during EMT will contribute to our understanding of tumor progression and metastasis. This study provides new insights into the cooperative induced tumor metastasis by retroviruses and herpesviruses., Competing Interests: The authors declare no conflict of interest.

Malignant pleural mesothelioma (MPM) is an aggressive cancer for which more effective treatments are needed. In this study, strong to moderate staining of MET and ERK5 was detected in 67.1 and 48% of the analyzed 73 human mesothelioma tumors, and significant correlation of MET and ERK5 expression was identified (P

Chronic cerebral ischemia (CCI) is a clinical syndrome characterised by brain dysfunction due to decreased chronic cerebral perfusion. CCI initiates several inflammatory pathways, including pyroptosis. RNA-binding proteins (RBPs) play important roles in CCI. This study aimed to explore whether the interaction between RBP-Cpeb4 and Dclk2 affected Ehf phosphorylation to regulate neuronal pyroptosis. HT22 cells and mice were used to construct oxygen glucose deprivation (OGD)/CCI models. We found that Cpeb4 and Dclk2 were upregulated in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. Cpeb4 upregulated Dclk2 expression by increasing Dclk2 mRNA stability. Knockdown of Cpeb4 or Dclk2 inhibited neuronal pyroptosis in OGD-treated HT22 cells and CCI-induced hippocampal CA1 tissues. By binding to the promoter regions of Caspase1 and Caspase3, the transcription factor Ehf reduced their promoter activities and inhibited the transcription. Dclk2 phosphorylated Ehf and changed its nucleoplasmic distribution, resulting in the exit of p-Ehf from the nucleus and decreased Ehf levels. It promoted the expression of Caspase1 and Caspase3 and stimulated neuronal pyroptosis of HT22 cells induced by OGD. Cpeb4/Dclk2/Ehf pathway plays an important role in the regulation of cerebral ischemia-induced neuronal pyroptosis., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Cholangiocarcinoma (CCA), characterized by high heterogeneity and extreme malignancy, has a poor prognosis. Doublecortin-like kinase 1 (DCLK1) promotes a variety of malignant cancers in their progression. Targeting DCLK1 or its associated regulatory pathways can prevent the generation and deterioration of several malignancies. However, the role of DCLK1 in CCA progression and its molecular mechanisms remain unknown. Therefore, we aimed to investigate whether and how DCLK1 contributes to CCA progression., Methods: The expression of DCLK1 in CCA patients was detected using Immunohistochemistry (IHC). We established DCLK1 knockout and DCLK1 overexpression cell lines for Colony Formation Assay and Transwell experiments to explore the tumor-promoting role of DCLK1. RT-PCR, Western blot and multiple fluorescent staining were used to assess the association between DCLK1 and epithelial-mesenchymal transition (EMT) markers. RNA sequencing and bioinformatics analysis were performed to identify the underlying mechanisms by which DCLK1 regulates CCA progression and the EMT program., Results: DCLK1 was overexpressed in CCA tissues and was associated with poor prognosis. DCLK1 overexpression facilitated CCA cell invasion, migration, and proliferation, whereas DCLK1 knockdown reversed the malignant tendencies of CCA cells, which had been confirmed both in vivo and in vitro. Furthermore, we demonstrated that DCLK1 was substantially linked to the advancement of the EMT program, which included the overexpression of mesenchymal markers and the downregulation of epithelial markers. For the underlying mechanism, we proposed that the PI3K/AKT/mTOR pathway is the key process for the role of DCLK1 in tumor progression and the occurrence of the EMT program. When administered with LY294002, an inhibitor of the PI3K/AKT/mTOR pathway, the tumor's ability to proliferate, migrate, and invade was greatly suppressed, and the EMT process was generally reversed., Conclusions: DCLK1 facilitates the malignant biological behavior of CCA cells through the PI3K/AKT/mTOR pathway. In individuals with cholangiocarcinoma who express DCLK1 at high levels, inhibitors of the PI3K/AKT/mTOR signaling pathway may be an effective therapeutic approach., (© 2024. The Author(s).)

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems., (© 2024. The Author(s).)

Chronic liver diseases, fibrosis, cirrhosis, and HCC are often a consequence of persistent inflammation. However, the transition mechanisms from a normal liver to fibrosis, then cirrhosis, and further to HCC are not well understood. This study focused on the role of the tumor stem cell protein doublecortin-like kinase 1 (DCLK1) in the modulation of molecular factors in fibrosis, cirrhosis, or HCC. Serum samples from patients with hepatic fibrosis, cirrhosis, and HCC were analyzed via ELISA or NextGen sequencing and were compared with control samples. Differentially expressed (DE) microRNAs (miRNA) identified from these patient sera were correlated with DCLK1 expression. We observed elevated serum DCLK1 levels in fibrosis, cirrhosis, and HCC patients; however, TGF-β levels were only elevated in fibrosis and cirrhosis. While DE miRNAs were identified for all three disease states, miR-12136 was elevated in fibrosis but was significantly increased further in cirrhosis. Additionally, miR-1246 and miR-184 were upregulated when DCLK1 was high, while miR-206 was downregulated. This work distinguishes DCLK1 and miRNAs' potential role in different axes promoting inflammation to tumor progression and may serve to identify biomarkers for tracking the progression from pre-neoplastic states to HCC in chronic liver disease patients as well as provide targets for treatment.

Background/aim: Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown., Materials and Methods: DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1∼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors., Results: The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1∼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner., Conclusion: DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein kinase involved in neurogenesis and human cancer. Recent studies have revealed a novel functional role for DCLK1 in inflammatory signaling, thus positioning it as a novel target kinase for respiratory inflammatory disease treatment. In this study, we designed and synthesized a series of NVP-TAE684-based derivatives as novel anti-inflammatory agents targeting DCLK1. Bio-layer interferometry binding screening and kinase assays of the NVP-TAE684 derivatives led to the discovery of an effective DCLK1 inhibitor (a24), with an IC 50 of 179.7 nM. Compound a24 effectively inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages with higher potency than the lead compound. Mechanistically, compound a24 inhibited LPS-induced inflammation by inhibiting DCLK1-mediated IKKβ phosphorylation. Furthermore, compound a24 showed in vivo anti-inflammatory activity in an LPS-challenged acute lung injury model. These findings suggest that compound a24 may serve as a novel candidate for the development of DCLK1 inhibitors and a potential therapeutic agent for the treatment of inflammatory diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lingfeng Chen reports financial support was provided by National Natural Science Foundation of China. Lingfeng Chen reports financial support was provided by Natural Science Funding of Zhejiang Province. Guang Liang reports financial support was provided by Zhejiang Provincial Key Scientific Project. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Double cortin-like kinase 1 (DCLK1) exhibits high expression levels across various cancers, notably in human colorectal cancer (CRC). Diacerein, a clinically approved interleukin (IL)-1β inhibitor for osteoarthritis treatment, was evaluated for its impact on CRC proliferation and migration, alongside its underlying mechanisms, through both in vitro and in vivo analyses. The study employed MTT assay, colony formation, wound healing, transwell assays, flow cytometry, and Hoechst 33342 staining to assess cell proliferation, migration, and apoptosis. Additionally, proteome microarray assay and western blotting analyses were conducted to elucidate diacerein's specific mechanism of action. Our findings indicate that diacerein significantly inhibits DCLK1-dependent CRC growth in vitro and in vivo. Through high-throughput proteomics microarray and molecular docking studies, we identified that diacerein directly interacts with DCLK1. Mechanistically, the suppression of p-STAT3 expression following DCLK1 inhibition by diacerein or specific DCLK1 siRNA was observed. Furthermore, diacerein effectively disrupted the DCLK1/STAT3 signaling pathway and its downstream targets, including MCL-1, VEGF, and survivin, thereby inhibiting CRC progression in a mouse model, thereby inhibiting CRC progression in a mouse model., (Copyright © 2024 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2 203 , a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2 203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The olfactory neuroepithelium serves as a sensory organ for odors and forms part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. Among them, microvillous cells (MVCs) are strategically positioned at the apical surface, but their specific functions are enigmatic, and their relationship to the other specialized epithelial cells is unclear. Here, we establish that the family of MVCs comprises tuft cells and ionocytes in both mice and humans. Integrating analysis of the respiratory and olfactory epithelia, we define the distinct receptor expression of TRPM5 + tuft-MVCs compared with Gɑ-gustducin high respiratory tuft cells and characterize a previously undescribed population of glandular DCLK1 + tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we used an integrated single-cell transcriptional and protein analysis. Inhalation of Alternaria induced mucosal epithelial effector molecules including Chil4 and a distinct pathway leading to proliferation of the quiescent olfactory horizontal basal stem cell (HBC) pool, both triggered in the absence of olfactory apoptosis. Alternaria- and ATP-elicited HBC proliferation was dependent on TRPM5 + tuft-MVCs, identifying these specialized epithelial cells as regulators of olfactory stem cell responses. Together, our data provide high-resolution characterization of nasal tuft cell heterogeneity and identify a function of TRPM5 + tuft-MVCs in directing the olfactory mucosal response to allergens.

Members of the Protein kinase D (PKD) kinase family each play important cell-specific roles in the regulation of normal pancreas functions. In pancreatic diseases PKD1 is the most widely characterized isoform with roles in pancreatitis and in induction of pancreatic cancer and its progression. PKD1 expression and activation increases in pancreatic acinar cells through macrophage secreted factors, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling, and reactive oxygen species (ROS), driving the formation of precancerous lesions. In precancerous lesions PKD1 regulates cell survival, growth, senescence, and generation of doublecortin like kinase 1 (DCLK1)-positive cancer stem cells (CSCs). Within tumors, regulation by PKD1 includes chemoresistance, apoptosis, proliferation, CSC features, and the Warburg effect. Thus, PKD1 plays a critical role throughout pancreatic disease initiation and progression., Competing Interests: Declaration of competing interest The authors declare they have no potential conflicts of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Diabetic cardiomyopathy (DCM) is a pathophysiological condition triggered by diabetes mellitus and can lead to heart failure. Doublecortin-like kinase protein 1 (DCLK1) is a multifunctional protein kinase involved in the regulation of cell proliferation, differentiation, survival, and migration. Current studies on DCLK1 mainly focus on cancer development; however, its role in non-tumor diseases such as DCM is yet to be deciphered. Our analysis revealed that DCLK1 was upregulated in cardiomyocytes of streptozotocin (STZ)-induced type 1 diabetic mouse, suggesting a correlation between DCLK1 and DCM progression. It was further demonstrated that either cardiomyocyte-specific DCLK1 knockout or pharmacological DCLK1 inhibitor DCLK1-IN-1 significantly alleviated cardiac hypertrophy and fibrosis in STZ-induced diabetic mice. RNA-seq analysis of heart tissues revealed that DCLK1 regulated the nuclear factor kappa B (NF-κB)-mediated inflammatory response in DCM. In vitro, DCLK1 activated NF-κB and the inflammatory response by inducing the IKKβ phosphorylation in high-concentration glucose (HG)-challenged cardiomyocytes. DCLK1-IN-1 also prevented HG-induced IKKβ/NF-κB activation and inflammatory injuries in cardiomyocytes. In conclusion, this study highlights the novel role of cardiomyocyte DCLK1 in regulating IKKβ/NF-κB, which aggravates inflammation to promote the pathogenesis of DCM. DCLK1 may serve as a new target for DCM treatment., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Ovarian cancer (OvCa) has a dismal prognosis because of its late-stage diagnosis and the emergence of chemoresistance. Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase known to regulate cancer cell "stemness", epithelial-mesenchymal transition (EMT), and drug resistance. Here we show that DCLK1 is a druggable target that promotes chemoresistance and tumor progression of high-grade serous OvCa (HGSOC). Importantly, high DCLK1 expression significantly correlates with poor overall and progression-free survival in OvCa patients treated with platinum chemotherapy. DCLK1 expression was elevated in a subset of HGSOC cell lines in adherent (2D) and spheroid (3D) cultures, and the expression was further increased in cisplatin-resistant (CPR) spheroids relative to their sensitive controls. Using cisplatin-sensitive and resistant isogenic cell lines, pharmacologic inhibition (DCLK1-IN-1), and genetic manipulation, we demonstrate that DCLK1 inhibition was effective at re-sensitizing cells to cisplatin, reducing cell proliferation, migration, and invasion. Using kinase domain mutants, we demonstrate that DCLK1 kinase activity is critical for mediating CPR. The combination of cisplatin and DCLK1-IN-1 showed a synergistic cytotoxic effect against OvCa cells in 3D conditions. Targeted gene expression profiling revealed that DCLK1 inhibition in CPR OvCa spheroids significantly reduced TGFβ signaling, and EMT. We show in vivo efficacy of combined DCLK1 inhibition and cisplatin in significantly reducing tumor metastases. Our study shows that DCLK1 is a relevant target in OvCa and combined targeting of DCLK1 in combination with existing chemotherapy could be a novel therapeutic approach to overcome resistance and prevent OvCa recurrence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C·W.H. is an inventor on a patent for nanoparticle-encapsulated DCLK1-targeted siRNAs. C.W.H. is the co-founder of COARE Holdings, Inc. The remaining authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Delay in type II alveolar epithelial cell (AECII) regeneration has been linked to higher mortality in patients with acute respiratory distress syndrome (ARDS). However, the interaction between Doublecortin-like kinase 1 (DCLK1) and the Hippo signaling pathway in ARDS-associated AECII differentiation remains unclear. Therefore, the objective of this study was to understand the role of the DCLK1/Hippo pathway in mediating AECII differentiation in ARDS., Materials and Methods: AECII MLE-12 cells were exposed to 0, 0.1, or 1 μg/mL of lipopolysaccharide (LPS) for 6 and 12 h. In the mouse model, C57BL/6JNarl mice were intratracheally (i.t.) injected with 0 (control) or 5 mg/kg LPS and were euthanized for lung collection on days 3 and 7., Results: We found that LPS induced AECII markers of differentiation by reducing surfactant protein C (SPC) and p53 while increasing T1α (podoplanin) and E-cadherin at 12 h. Concurrently, nuclear YAP dynamic regulation and increased TAZ levels were observed in LPS-exposed AECII within 12 h. Inhibition of YAP consistently decreased cell levels of SPC, claudin 4 (CLDN-4), galectin 3 (LGALS-3), and p53 while increasing transepithelial electrical resistance (TEER) at 6 h. Furthermore, DCLK1 expression was reduced in isolated human AECII of ARDS, consistent with the results in LPS-exposed AECII at 6 h and mouse SPC-positive (SPC + ) cells after 3-day LPS exposure. We observed that downregulated DCLK1 increased p-YAP/YAP, while DCLK1 overexpression slightly reduced p-YAP/YAP, indicating an association between DCLK1 and Hippo-YAP pathway., Conclusions: We conclude that DCLK1-mediated Hippo signaling components of YAP/TAZ regulated markers of AECII-to-AECI differentiation in an LPS-induced ARDS model., (© 2023. The Author(s).)

WNT/β-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair β-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of β-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as β-catenin activity and its target gene VEGFA . Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed β-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since β-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and β-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients.

While significant strides have been made in understanding cancer biology, the enhancement in patient survival is limited, underscoring the urgency for innovative strategies. Epigenetic modifications characterized by hereditary shifts in gene expression without changes to the DNA sequence play a critical role in producing alternative gene isoforms. When these processes go awry, they influence cancer onset, growth, spread, and cancer stemness. In this review, we delve into the epigenetic and isoform nuances of the protein kinase, doublecortin-like kinase 1 (DCLK1). Recognized as a hallmark of tumor stemness, DCLK1 plays a pivotal role in tumorigenesis, and DCLK1 isoforms, shaped by alternative promoter usage and splicing, can reveal potential therapeutic touchpoints. Our discussion centers on recent findings pertaining to the specific functions of DCLK1 isoforms and the prevailing understanding of its epigenetic regulation via its two distinct promoters. It is noteworthy that all DCLK1 isoforms retain their kinase domain, suggesting that their unique functionalities arise from non-kinase mechanisms. Consequently, our research has pivoted to drugs that specifically influence the epigenetic generation of these DCLK1 isoforms. We posit that a combined therapeutic approach, harnessing both the epigenetic regulators of specific DCLK1 isoforms and DCLK1-targeted drugs, may prove more effective than therapies that solely target DCLK1.

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The doublecortin-like kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory 'tail' segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations, and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to 'supercharge' the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other calcium calmodulin kinases (CAMKs), and a 'Swiss Army' assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for autoregulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically divergent DCLK1 modulators, stabilizers, or degraders., Competing Interests: AV, GW, DB, BO, SS, NG, EF, LD, CB, WY, IA, SK, CE, PE, NK No competing interests declared, (© 2023, Venkat, Watterson, Byrne et al.)

Doublecortin-like kinase 1 (DCLK1) is a prominent kinase involved in carcinogenesis, serving as a diagnostic marker for early cancer detection and prevention, as well as a target for cancer therapy. Extensive research efforts have been dedicated to understanding its role in cancer development and designing selective inhibitors. In our previous work, we successfully determined the crystal structure of DCLK1 while it was bound to its autoinhibitory domain (AID) at the active site. By analyzing this structure, we were able to uncover the intricate molecular mechanisms behind specific cancer-causing mutations in DCLK1. Utilizing molecular dynamics simulations, we discovered that these mutations disrupt the smooth assembly of the AID, particularly affecting the R2 helix, into the kinase domain (KD). This disruption leads to the exposure of the D533 residue of the DFG (Asp-Phe-Gly) motif in the KD, either through steric hindrance, the rearrangement of electrostatic interactions, or the disruption of local structures in the AID. With these molecular insights, we conducted a screening process to identify potential small-molecule inhibitors that could bind to DCLK1 through an alternative binding mode. To assess the binding affinity of these inhibitors to the KD of DCLK1, we performed calculations on their binding energy and conducted SPR experiments. We anticipate that our study will contribute novel perspectives to the field of drug screening and optimization, particularly in targeting DCLK1.

Background: Osteopenia has been well documented in adolescent idiopathic scoliosis (AIS). Bone marrow stem cells (BMSCs) are a crucial regulator of bone homeostasis. Our previous study revealed a decreased osteogenic ability of BMSCs in AIS-related osteopenia, but the underlying mechanism of this phenomenon remains unclear., Methods: A total of 22 AIS patients and 18 age-matched controls were recruited for this study. Anthropometry and bone mass were measured in all participants. Bone marrow blood was collected for BMSC isolation and culture. Osteogenic and adipogenic induction were performed to observe the differences in the differentiation of BMSCs between the AIS-related osteopenia group and the control group. Furthermore, a total RNA was extracted from isolated BMSCs to perform RNA sequencing and subsequent analysis., Results: A lower osteogenic capacity and increased adipogenic capacity of BMSCs in AIS-related osteopenia were revealed. Differences in mRNA expression levels between the AIS-related osteopenia group and the control group were identified, including differences in the expression of LRRC17 , DCLK1 , PCDH7 , TSPAN5 , NHSL2 , and CPT1B . Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed several biological processes involved in the regulation of autophagy and mitophagy. The Western blotting results of autophagy markers in BMSCs suggested impaired autophagic activity in BMSCs in the AIS-related osteopenia group., Conclusion: Our study revealed that BMSCs from AIS-related osteopenia patients have lower autophagic activity, which may be related to the lower osteogenic capacity and higher adipogenic capacity of BMSCs and consequently lead to the lower bone mass in AIS patients., (Copyright © 2022 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652., (© 2023. The Author(s).)

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues ( P <0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 ( r =0.53, P <0.001), LATS2 ( r =0.34, P <0.001), MOB1B ( r =0.40, P <0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin ( P <0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group ( P <0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells ( P <0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin ( P <0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P <0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

Obesity increases the risk for cardiovascular diseases and induces cardiomyopathy. Chronic inflammation plays a significant role in obesity-induced cardiomyopathy and may provide new therapeutic targets for this disease. Doublecortin-like kinase 1 (DCLK1) is an important target for cancer therapy and the role of DCLK1 in obesity and cardiovascular diseases is unclear. Herein, we showed that DCLK1 was overexpressed in the cardiac tissue of obese mice and investigated the role of DCLK1 in obesity-induced cardiomyopathy. We generated DCLK1-deleted mice and showed that macrophage-specific DCLK1 knockout, rather than cardiomyocyte-specific DCLK1 knockout, prevented high-fat diet (HFD)-induced heart dysfunction, cardiac hypertrophy, and fibrosis. RNA sequencing analysis showed that DCLK1 deficiency exerted cardioprotective effects by suppressing RIP2/TAK1 activation and inflammatory responses in macrophages. Upon HFD/palmitate (PA) challenge, macrophage DCLK1 mediates RIP2/TAK1 phosphorylation and subsequent inflammatory cytokine release, which further promotes hypertrophy in cardiomyocytes and fibrogenesis in fibroblasts. Finally, a pharmacological inhibitor of DCLK1 significantly protects hearts in HFD-fed mice. Our study demonstrates a novel role and a pro-inflammatory mechanism of macrophage DCLK1 in obesity-induced cardiomyopathy and identifies DCLK1 as a new therapeutic target for the treatment of this disease. Upon HFD/PA challenge, DCLK1 induces RIP2/TAK1-mediated inflammatory response in macrophages, which subsequently promotes cardiac hypertrophy and fibrosis. Macrophage-specific DCLK1 deletion or pharmacological inhibition of DCLK1 protects hearts in HFD-fed mice., (© 2023. The Author(s).)

Tuft cells are chemosensory epithelial cells that increase in number following infection or injury to robustly activate the innate immune response to alleviate or promote disease. Recent studies of castration resistant prostate cancer and its subtype, neuroendocrine prostate cancer, revealed Pou2f3+ populations in mouse models. The transcription factor Pou2f3 is a master regulator of the tuft cell lineage. We show that tuft cells are upregulated early during prostate cancer development, and their numbers increase with progression. Cancer-associated tuft cells in the mouse prostate express DCLK1, COX1, COX2, while human tuft cells express COX1. Mouse and human tuft cells exhibit strong activation of signaling pathways including EGFR and SRC-family kinases. While DCLK1 is a mouse tuft cell marker, it is not present in human prostate tuft cells. Tuft cells that appear in mouse models of prostate cancer display genotype-specific tuft cell gene expression signatures. Using bioinformatic analysis tools and publicly available datasets, we characterized prostate tuft cells in aggressive disease and highlighted differences between tuft cell populations. Our findings indicate that tuft cells contribute to the prostate cancer microenvironment and may promote development of more advanced disease. Further research is needed to understand contributions of tuft cells to prostate cancer progression., (© 2023. The Author(s).)

Tuft cells are solitary chemosensory cells distributed mainly in hollow organs and detected in human and mouse pancreas precursor lesions of pancreatic cancer. Induced by inflammation and KRAS mutation, pancreatic acinar cell-derived tuft cells play a protective role in epithelium injury. The tumour suppression of tuft cells has been indicated in some studies. However, the function of tuft cells in pancreatic cancer remains unclear. In this review, we first introduce the definition of tuft cells and then review the relationship between tuft cells and pancreatic inflammation. In addition, we emphasized the role of tuft cells in the genesis and development of pancreatic cancers, especially the part of markers for tuft cell's doublecortin-like kinase 1 (DCLK1). Finally, we turn to the microscopic perspective and review the interactions between tuft cells and the microbiome in the pancreatic microenvironment. Overall, we describe the role of tuft cells in response to tissue damage and tumour progression in the pancreas. Nevertheless, the specific formation principle and the more detailed mechanism of action of tuft cells in the pancreas remain to be further explored., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)