Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

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.

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.

OBJECTIVE: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a first-line treatment for lung adenocarcinoma with EGFR-sensitive mutations, but acquired resistance to EGFR-TKIs remains a problem in clinical practice. The development of epithelial-mesenchymal transition (EMT) is a critical mechanism that induces acquired resistance to TKIs. Reversing acquired resistance to EGFR-TKIs through targeting the key molecules driving EMT provides an alternative choice for patients. We, therefore, aimed to explore the role of doublecortin-like kinase 1 (DCLK1) as an EMT driver gene in the acquired resistance of lung adenocarcinoma to EGFR-TKIs.
METHODS: The IC50 of Gefitinib or Osimertinib in PC9/HCC827 cells was measured using a cell counting kit-8 (CCK8) assay. The expression levels of EMT-related genes in PC9 and HCC827 cells were detected using RT-PCR and Western blot. Cell migration and invasion abilities were assessed via a transwell assay. For the in vivo experiments, PC9 cells were subcutaneously injected into BALB/c nude mice to form tumors. Upon harvesting, tumor tissues were retained for RT-PCR, Western blot, and polychromatic fluorescence staining to detect biomarker changes in the EMT process.
RESULTS: Gefitinib-resistant PC9 (PC9/GR) and Osimertinib-resistant HCC827 (HCC827/OR) cells showed remarkable activation of EMT and enhanced migration and invasion abilities compared to TKI-sensitive cells. In addition, DCLK1 expression was markedly increased in EGFR-TKI-resistant lung adenocarcinoma cells. The targeted knockout of DCLK1 effectively reversed the EMT phenotype in TKI-resistant cells and improved EGFR-TKI sensitivity, which was further validated by the in vivo experiments.
CONCLUSIONS: DCLK1 facilitates acquired resistance to EGFR-TKI in lung adenocarcinoma by inducting EMT and accelerating the migration and invasion abilities of TKI-resistant cells.

Rationale: Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that selectively marks cancer stem-like cells (CSCs) and promotes malignant progression in colorectal cancer (CRC). However, the exact molecular mechanism by which DCLK1 drives the aggressive phenotype of cancer cells is incompletely determined. Methods: Here, we performed comprehensive genomics and proteomics analyses to identify binding proteins of DCLK1 and discovered X-ray repair cross-complementing 5 (XRCC5). Thus, we explored the biological role and downstream events of the DCLK1/XRCC5 axis in human CRC cells and CRC mouse models. Results: The results of comprehensive bioinformatics analyses suggested that DCLK1-driven CRC aggressiveness is linked to inflammation. Mechanistically, DCLK1 bound and phosphorylated XRCC5, which in turn transcriptionally activated cyclooxygenase-2 expression and enhanced prostaglandin E2 production; these events collectively generated the inflammatory tumor microenvironment and enhanced the aggressive behavior of CRC cells. Consistent with the discovered mechanism, inhibition of DCLK1 kinase activity strongly impaired the tumor seeding and growth capabilities in CRC mouse models. Conclusion: Our study illuminates a novel mechanism that mediates the pro-inflammatory function of CSCs in driving the aggressive phenotype of CRC, broadening the biological function of DCLK1 in CRC.

BACKGROUND: Low-grade duodenal inflammation has recently been identified in patients with functional dyspepsia (FD). Chemosensory tuft cells were reported to be associated with gastrointestinal diseases. We therefore assessed duodenal tuft cell density and microinflammation in patients with FD to determine whether these measures could serve as useful biomarkers, and also correlated tuft cell density and microinflammation in FD patients.
METHODS: Duodenal biopsy specimens were obtained from patients with FD and from controls. Tuft cells, eosinophils, and mast cells were immunochemically stained with specific antibodies. Tuft cells were identified by immunostaining for doublecortin-like kinase 1 (DCLK1); cholinergic tuft cells were assessed by double staining for choline acetyltransferase (ChAT) and DCLK1. Immune-type tuft cells were assessed by IL-25 mRNA expression using real-time PCR.
RESULTS: The density of intramucosal eosinophils and mast cells was significantly higher in the duodenum of FD patients than in controls. The density of tuft cells was significantly higher in the duodenum of FD patients compared with controls, and significantly correlated with eosinophil density in the duodenum of FD patients and controls. Moreover, a fraction of ChAT-positive cells was DCLK1 positive; all duodenal DCLK1+ tuft cells were ChAT-immunoreactive in FD and in control subjects.
CONCLUSIONS: Cholinergic tuft cell density was higher in the duodenum of patients with FD and significantly correlated with eosinophil density. Further studies are needed to investigate the pathophysiological significance of tuft cells in FD and may provide valuable clues to the pathophysiology of FD.

Avian leukosis virus subgroup J (ALV-J) induces myelocytomas, which can metastasize to multiple organs in diseased chickens. Although metastasis is the primary cause of death in such cases, the mechanism for it remains unclear. Here, we found that interaction between ALV-J surface protein (SU) and doublecortin-like kinase 1 (DCLK1) promotes epithelial-mesenchymal transition (EMT) and cell proliferation. We found that ALV-J can activate EMT in infected cells. Subsequently, proteomics analysis revealed that DCLK1, a well-established putative tumor stem cell marker, which is highly expressed in ALV-J-infected DF-1 cells and chickens, might be a potential factor mediating EMT. Furthermore, using immunofluorescence and immunoprecipitation, we verified that SU interacts with DCLK1. Functional studies suggested that overexpression of DCLK1 increased viral replication and promoted cell proliferation by accelerating the progression of cells from the G0/G1 phase to the S phase of cell cycle, whereas RNA interference of DCLK1 reduced viral replication and arrested cell proliferation by retarding cell cycle progression from the late G1 phase into the S phase in ALV-J-infected cells. Moreover, we demonstrate that the increased accumulation of DCLK1 promotes EMT by increasing the expression of N-cadherin, vimentin, MMP2, and transcription factor Snail1 and decreasing the expression of epithelial marker E-cadherin. These results suggest that ALV-J SU interacts with DCLK1, and accelerates cell proliferation, leading to increased viral replication and ultimately activating EMT, which paves the way for tumor metastasis. IMPORTANCE Tumor metastasis is a major challenge in cancer research, because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. It is estimated that >90% of mortality from cancer is attributable to metastases. We found that ALV-J can activate EMT, which plays a critical role in cancer metastasis. Subsequently, we identified a tumor stem cell marker, DCLK1, in ALV-J infected cells, which interacts with surface protein (SU) of ALV-J to promote virus replication, activate EMT, and accelerate cell proliferation enabling ALV-J to obtain metastatic ability. Understanding the process of participation of ALV-J in EMT and the route of metastasis will help elucidate the mechanism of virus-induced tumor metastasis and help identify promising molecular targets and key obstacles for ALV-J control and clinical technology development.

Given the functional attributes of Doublecortin-like kinase 1 (DCLK1) in tumor growth, invasion, metastasis, cell motility, and tumor stemness, it is emerging as a therapeutic target in gastrointestinal cancers. Although a series of specific or nonspecific ATP-competitive inhibitors were identified against DCLK1, different types of scaffolds that can be utilized for the development of highly selective inhibitors or structural understanding of binding specificities of the compounds remain limited. Here, we present our work to repurpose a Janus kinase 1 inhibitor, ruxolitinib as a DCLK1 inhibitor, showing micromolar binding affinity and inhibitory activity. Furthermore, to gain an insight into its interaction mode with DCLK1, a crystal structure of the ruxolitinib-complexed DCLK1 has been determined and analyzed. Ruxolitinib as a nonspecific DCLK1 inhibitor characterized in this work is anticipated to provide a starting point for the structure-guided discovery of selective DCLK1 inhibitors.

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.

Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.