Doublecortin-like Kinase Research Articles

Expression of Serum DCLK1 in Gastric Cancer Patients and Its Relationship with CEA, CA19-9, and CA72-4

Objective: To investigate the expression of Doublecortin-like kinase-1 (DCLK1) in the serum of gastric cancer patients and its relationship with carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 72-4 (CA72-4). Methods: Fifty patients diagnosed with gastric cancer at the hospital from January to December 2021 were selected as the gastric cancer group, and 50 patients diagnosed with chronic atrophic gastritis during the same period were selected as the control group. The serum concentrations of DCLK1, CEA, CA19-9, and CA72-4 were measured in both groups. Cut-off values and AUC (Area Under the Curve) were determined based on the ROC curve, and the expression of DCLK1 and its relationship with CEA, CA19-9, and CA72-4 were analyzed. Results: The average concentrations of DCLK1, CEA, CA19-9, and CA72-4 in the serum of gastric cancer patients were significantly higher than those in the control group (P < 0.05). CA72-4 had the highest sensitivity (62%), CEA had the highest specificity (98%), and DCLK1 had the largest AUC (0.709). The combined diagnosis of gastric cancer using DCLK1, CEA, and CA19-9 resulted in the largest AUC (0.826), with a sensitivity of 82% and a specificity of 76%. Conclusion: The expression of DCLK1, CEA, CA19-9, and CA72-4 in the serum of gastric cancer patients is significantly higher than that in the control group. The combined detection of DCLK1, CEA, and CA19-9 offers better sensitivity and specificity for the diagnosis of gastric cancer.

DCLK1 induces a pro-tumorigenic phenotype to drive gastric cancer progression.

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell-intrinsic and-extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1D511N). MKN1D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1DCLK1) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

Targeting the PI3K/AKT/mTOR pathway offer a promising therapeutic strategy for cholangiocarcinoma patients with high doublecortin-like kinase 1 expression

BackgroundCholangiocarcinoma (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.MethodsThe 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.ResultsDCLK1 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.ConclusionsDCLK1 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.

Enteric tuft cells coordinate timely expulsion of the tapeworm Hymenolepis diminuta from the murine host by coordinating local but not systemic immunity.

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri.

Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis

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.

From Inflammation to Oncogenesis: Tracing Serum DCLK1 and miRNA Signatures in Chronic Liver Diseases.

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.

Clinical, histopathological, and molecular biomarkers in a multicentric cohort of Mexican women with triple-negative breast cancer.

e13152 Background: Triple negative breast cancer (TNBC) is a heterogeneous entity with a marked variation in biological behavior, treatment responses, and patient outcomes. Thus, the investigation of clinical, histopathological, and molecular biomarkers is essential to understand this variability and potentially inform more effective management approaches across different patient profiles. Methods: We conducted a retrospective multicenter study of women >18 years diagnosed with TNBC between 2006 and 2021 in two Mexican centers. We systematically gathered clinical and histopathological data, including an assessment of H&E-stained slides for histopathological features and tumor-infiltrating lymphocytes (TILs), alongside immunohistochemistry analyses for androgen receptors (AR), CD8, and doublecortin-like kinase 1 (DCLK1). Statistical analyses were conducted using R version 4.3.2. Results: In our cohort, comprising 289 patients, the average age at diagnosis was 49.13 years, with a median overall survival (OS) of 63 months and a 5-year OS rate of 55%. The distribution of clinical stages at diagnosis was as follows: I (n=12, 4.2%), II (n=94, 32.5%), III (n=130, 45.0%), and IV (n=53, 18.3%). CD8 expression was higher in patients with no recurrence compared to patients with stage IV at diagnosis (p<0.001), and it decreased with advancing clinical stages. We also found statistically significant differences in OS between negative and positive CD8 expression, favoring the latter (5-year OS 51.8% vs. 72.2%, p=0.032). Disease-free survival (DFS) and OS also differed significantly between low (≤10%) and high (>10%) stromal TILs, favoring the high expression group (5-year DFS 51.9% vs. 70.8%, p=0.0066; 5-year OS 50.1% vs. 70.3%, p=0.0018). CD8 levels positively correlated with stromal and intratumoral TILs. Positive AR expression (>10%) was seen in 8.9% of cases, with no observed differences in DFS or OS according to negative or positive status (5-year DFS 56.0% vs. 72.2%, p=0.42; 5-year OS 55.0% vs. 60.3%, p=0.68). Finally, DCLK1 positivity (≥10%) was seen in 23% of cases, with no influence on survival (negative vs. positive status, 5-year DFS 58.5% vs. 53.8%, p=0.63; 5-year OS 54.6% vs. 58.1%, p=0.60). Conclusions: Our study underscores the prognostic significance of CD8 and TILs in predicting DFS and OS among TNBC patients. Notably, the expressions of AR and DCLK1 were not independently associated with survival, although their prognostic impact in combination with other biomarkers is still an area of active research. Overall, our findings advocate for a more detailed examination of the molecular characteristics of TNBC within our demographic, encompassing an expanded array of biomarkers and a thorough investigation of the tumor microenvironment.

DCLK1 Expression and its Functional Significance in Intrahepatic Cholangiocarcinoma and Bil-IN Stage.

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. 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. 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. 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.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P&amp;lt;0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p&amp;lt;0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p&amp;lt;0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.

Repurposing diacerein to suppress colorectal cancer growth by inhibiting the DCLK1/STAT3 signaling pathway

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.

Abstract 6885: Investigating a novel role of DCLK1 isoforms in colitis and colitis associated colon cancer

Abstract Chronic intestinal inflammation in patients diagnosed with inflammatory bowel disease (IBD) significantly increases their risk of colorectal cancer (CRC). Despite the rising global frequency of IBD, the precise mechanisms that regulate the transition from IBD to colon cancer are poorly understood. Doublecortin-like kinase 1 (DCLK1) is a protein with two major isoforms, DCLK1-L and DCLK1-S. Previous studies have shown that hypermethylation of the DCLK-1α promoter encoding DCLK1-L, particularly in colorectal cancer, allows switching to the DCLK1-S isoform that confers an invasive tumor phenotype. The DCLK1-L is a tuft cell marker that protects against colitis by sensing and eliminating gut pathogenic infection through various mechanisms. On the contrary, the role of DCLK1-S in colitis and colitis-associated cancer has not been thoroughly investigated. Studies from our lab have identified a marked elevation of DCLK1-S in tissue samples of IBD patients. We also recently discovered that DCLK1-S is predominantly expressed in Ly6G+; MHCII- neutrophils, which coincide with elevated levels of inflammation and tissue damage in the colon. To further understand the role of DCLK1 isoforms in driving the progression of colitis to colon cancer, we bred Dclk1fl/fl mice with MRP8-Cre-ires/GFP mice to generate Dclk1fl/fl;MRP8-Cre+/- that eliminates DCLK1-L upon tamoxifen injection resulting in sustained expression of DCLK1-S in the granulocytes especially neutrophils. When infected with Citrobacter rodentium (CR), MRP8;Dclk1−/− mice compared to MRP8;Dclk1+/− or WT mice developed more severe colitis. We have also discovered a novel interaction between DCLK1-S and MMP13 in an unbiased docking study that correlated with significant co-localization of DCLK1-S with MMP13 in the colons of CR-infected mice. Employing a knock-in mice over-expressing the DCLK1-S +19 under the CAG promoter, the DCLK1-MMP13 co-localization correlated with severe ulceration and colitis with loss of crypts and immune cell infiltration including Ly6G+ neutrophils. Studies are underway to see if the severity of colitis in MRP8;Dclk1−/− mice translates into enhanced tumorigenesis and to see if potential interaction between DCLK1-S and MMP13 could provide a plausible mechanism of colitis progression to colon cancer through ECM remodeling and tissue damage. Citation Format: Kafayat Aderonke Yusuf, Badal C. Roy, Shrikant Anant, Shahid Umar. Investigating a novel role of DCLK1 isoforms in colitis and colitis associated colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6885.

Abstract 1403: The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression

Abstract Background: Tuft cells, marked by Doublecortin-like kinase-1 (Dclk1), are rare in the healthy pancreas but increase markedly during pancreatic tumorigenesis. While Group 2 innate lymphoid cells (ILC2s) are known to modulate inflammation and immunity, their specific roles in cancer-related immunity and immunotherapy interactions with pancreatic tuft cells remain unknown. Methods: To investigate tuft cells during early pancreatic neoplasia, we used a Dclk1 reporter mouse model and single-cell RNA-seq to study Dclk1-expressing cells in normal pancreas and neoplasia using Mist1/Kras mouse. We treated mice with IL-13 peptides and anti-IL-13 antibodies to explore the IL-13-ILC2 axis in tuft cell regulation. Results: Within the normal pancreas, Dclk1 expression marked several specialized epithelial cell types, including tuft-like cells, neuroendocrine cells, and a small number of acinar progenitor cells. A notable increase in Dclk1-expressing cells was observed at the onset of early PanIN lesions, with a subsequent decline in advanced PanIN stages and PDAC. Using flow cytometric analysis, we observed an increase in ILC2 cells in the Mist1/Kras pancreas in response to IL33 produced by pancreatic cancer-associated fibroblasts (CAFs). Blocking ILC2 activation with anti-IL33 antibodies reduced Dclk1 cell hyperplasia during PanIN progression. Given the known association between tuft cells, ILC2 cells, and the IL13/IL4 signaling pathways in the intestine, we discovered that Dclk1-expressing cells highly expressed the IL4 receptor (IL4ra). IL-13 levels increased during PanIN progression, and we found that ILC2s comprised the primary source of IL-13 within pancreatic tumors. In vitro, studies showed that the presence of IL-13 maintained the expansion of Dclk1-expressing tuft-like cells in pancreatic organoids derived from Dclk1-DTR-ZsGreen mice. Correspondingly, the in vivo administration of anti-IL-13 resulted in reduced Dclk1 expression. Conclusion: Previous studies have shown that pancreatic tuft cells inhibit pancreatic cancer progression. Our study reveals a novel regulatory mechanism wherein IL-13 secreted by ILC2s modulates Dclk1 tuft cell expression during pancreatic tumorigenesis. The IL13-ILC2 axis appears to be a potential therapeutic target to regulate Dclk1 cell activity, which could influence the course of pancreatic tumorigenesis. Future investigations will further explore the interaction between the IL13/ILC2 axis and Dclk1 in pancreatic cancer development. Citation Format: Giovanni Valenti, Pasquale Laise, Feijing Wu, Hiroki Kobayashi, Quin T. Waterbury, Ryota Takahashi, Tuo Ruan, Zhengyu Jiang, Yosuke Ochiai, Leah B. Zamechek, Timothy C. Wang. The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1403.

Establishment of a chicken intestinal organoid culture system to assess deoxynivalenol-induced damage of the intestinal barrier function

BackgroundDeoxynivalenol (DON) is a mycotoxin that has received recognition worldwide because of its ability to cause growth delay, nutrient malabsorption, weight loss, emesis, and a reduction of feed intake in livestock. Since DON-contaminated feedstuff is absorbed in the gastrointestinal tract, we used chicken organoids to assess the DON-induced dysfunction of the small intestine.ResultsWe established a culture system using chicken organoids and characterized the organoids at passages 1 and 10. We confirmed the mRNA expression levels of various cell markers in the organoids, such as KI67, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), mucin 2 (MUC2), chromogranin A (CHGA), cytokeratin 19 (CK19), lysozyme (LYZ), and microtubule-associated doublecortin-like kinase 1 (DCLK1), and compared the results to those of the small intestine. Our results showed that the organoids displayed functional similarities in permeability compared to the small intestine. DON damaged the tight junctions of the organoids, which resulted in increased permeability.ConclusionsOur organoid culture displayed topological, genetic, and functional similarities with the small intestine cells. Based on these similarities, we confirmed that DON causes small intestine dysfunction. Chicken organoids offer a practical model for the research of harmful substances.

Discovery of a doublecortin-like kinase 1 inhibitor to prevent inflammatory responses in acute lung injury

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 IC50 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.

DCLK1 is Overexpressed and Associated with Immune Cell Infiltration in Hepatocellular Carcinoma.

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.

Tuft cells are required for a rhinovirus-induced asthma phenotype in immature mice.

Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation, and airway hyperresponsiveness that is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase 1-positive (DCLK1+) tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57BL/6J mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ tuft cells in the large airways. Compared with wild-type mice, RV-infected Pou2f3-/- mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils, and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early-life viral infections could potentiate type 2 inflammatory responses to future infections.

Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma

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 DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis invivo. Mechanistically, DCLK2203 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.

Identification of small molecule inhibitors against doublecortin-like kinase 1 for targeting colon cancer stem cells

Identification of small molecule inhibitors against doublecortin-like kinase 1 for targeting colon cancer stem cells

E74-like ETS transcription factor 1 promotes the progression of pancreatic cancer by regulating doublecortin-like kinase 1/Janus kinase/signal transducer and activator of transcription pathway

E74-like ETS transcription factor 1 promotes the progression of pancreatic cancer by regulating doublecortin-like kinase 1/Janus kinase/signal transducer and activator of transcription pathway

Doublecortin-like kinase 1 (DCLK1) Promotes Platinum-Resistance and is a Novel Therapeutic Target for Ovarian Cancer

Doublecortin-like kinase 1 (DCLK1) Promotes Platinum-Resistance and is a Novel Therapeutic Target for Ovarian Cancer