Chemical Biology Toolkit for DCLK1 Reveals Connection to RNA Processing

Liu, Yan; Ferguson, Fleur M.; Li, Lianbo; Kuljanin, Miljan; Mills, Caitlin E.; Subramanian, Kartik; Harshbarger, Wayne; Gondi, Sudershan; Wang, Jinhua; Sorger, Peter K.; Mancias, Joseph D.; Gray, Nathanael S.; Westover, Kenneth D.

doi:10.1016/j.chembiol.2020.07.011

Cited by 21 publications

(41 citation statements)

References 79 publications

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“…In this study, we establish a new functional role for the DCLK1 in supporting sEV biogenesis, secretion and reprogramming sEV cargo towards a pro-migratory phenotype, in vitro. This is in line with previous reports linking DCLK1 expression to the induction of signaling pathways effecting cancer cell motility, invasion and EMT [7,8,10,13,19,20,22,23,115]. Our results also align and extend mechanistic models of DCLK1 as a polymerizer and stabilizer of microtubules and therefore facilitator of vesicular trafficking [31,32,39,41,45,116].…”

Section: Discussionsupporting

confidence: 92%

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells

et al. 2021

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Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1 OE -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1 PAR ). Quantitative proteome analysis of MKN1 OE -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1 PAR -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.

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Section: Discussionsupporting

confidence: 92%

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells

et al. 2021

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“…So far, DCLK1-IN-1 has been shown to be efficient in only a subset of patient derived organoid samples that expressed DCLK1-S 37 . The use of DCLK1-IN-1 in colorectal cancer cell line model has also recently allowed a connection to be established between DCLK1 and RNA processing pathways, although it is not clear in this study which isoform was overexpressed 49 . Regardless, DCLK1-IN-1 represents an invaluable tool to specifically dissect the biological kinase function of the DCLK1-short isoforms and their contribution to tumorigenesis.…”

Section: Discussionmentioning

confidence: 90%

Structural basis for small molecule targeting of Doublecortin Like Kinase 1 DCLK1

Patel¹,

Roy²,

Kropp³

et al. 2021

Preprint

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Doublecortin-like kinase 1 (DCLK1) is a bi-functional protein classified as a Microtubule-Associated Protein (MAP) and as a serine/threonine kinase that plays a critical role in regulating microtubule assembly. This understudied kinase is upregulated or mutated in a wide range of cancers. Knockdown studies have shown that DCLK1 is functionally important for tumour growth. However, the presence of tissue and development specific spliced DCLK1 isoforms and the lack of systematic evaluation of their biological function have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a potent and selective DCLK1 kinase inhibitor, a powerful new tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with two DCLK1-IN-1 precursors and DCLK-IN-1. Combined, our structural data analysis illuminates and rationalises the structure-activity relationship that informed development of DCLK1-IN-1 and provides the basis for DCLK1-IN-1 increased selectivity. We show that DCLK1-IN-1 induces a drastic conformational change of the N-lobe, which uncovered a new allosteric site. In addition, we demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms with high affinity but does not prevent DCLK1 MAP function. Together, our work outlines the need for in-depth studies to rationally design of isoform-specific modulators and provides an invaluable structural platform to further the design of selective DCLK1 therapeutic agents.

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“…Additional potential substrates in nucleic acid processing such as CDK11, MATR3, and DNA topoisomerase 2-beta (TOP2B) were also identified and phosphopeptides including TOP2B, CDK11B, and MATR3 were significantly decreased after treatment with DCLK1-IN-1. Pathway analysis suggested substrate involvement in RNA processing, insulin signaling, ErbB signaling, proteoglycan synthesis, and maintenance of focal adhesion and tight junction pathways [72]. Finally, Koizumi et al experimentally identified MAP7D1 (microtubule-associated protein 7 domain containing 1) as a substrate of DCLK1 in cortical neurons and the phosphomimetic MAP7D1 fully rescued the impaired callosal axon elongation in neurons after DCLK1 knockdown [73].…”

Section: Interactions Between Dclk1 and The Tumor Microenvironmentmentioning

confidence: 99%

“…Importantly, this inhibitor showed significant activity against clinically relevant DCLK1+ patient-derived pancreatic ductal adenocarcinoma organoids [49]. Additionally, DCLK1-IN-1 was shown to be effective in CRC using kinase-modified engineered DCLK1 in the DLD-1 cell line [72]. Further studies are needed using DCLK1-IN-1 and other specific DCLK1 kinase inhibitors, and an assessment of its ability to influence anti-tumor immunity is especially desirable as the clinical use of kinase inhibitors in conjunction with immune checkpoint therapies is emerging [82].…”

Section: Development Of Dclk1-targeted Therapeutic Agents and Biologicsmentioning

confidence: 99%

Tuft and Cancer Stem Cell Marker DCLK1: A New Target to Enhance Anti-Tumor Immunity in the Tumor Microenvironment

Cao

Weygant

Chandrakesan

et al. 2020

Cancers

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Microtubule-associated doublecortin-like kinase 1 (DCLK1) is an accepted marker of tuft cells (TCs) and several kinds of cancer stem cells (CSCs), and emerging evidence suggests that DCLK1-positive TCs participate in the initiation and formation of inflammation-associated cancer. DCLK1-expressing CSCs regulate multiple biological processes in cancer, promote resistance to therapy, and are associated with metastasis. In solid tumor cancers, tumor epithelia, immune cells, cancer-associated fibroblasts, endothelial cells and blood vessels, extracellular matrix, and hypoxia all support a CSC phenotype characterized by drug resistance, recurrence, and metastasis. Recently, studies have shown that DCLK1-positive CSCs are associated with epithelial-mesenchymal transition, angiogenesis, and immune checkpoint. Emerging data concerning targeting DCLK1 with small molecular inhibitors, monoclonal antibodies, and chimeric antigen receptor T-cells shows promising effects on inhibiting tumor growth and regulating the tumor immune microenvironment. Overall, DCLK1 is reaching maturity as an anti-cancer target and therapies directed against it may have potential against CSCs directly, in remodeling the tumor microenvironment, and as immunotherapies.

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Chemical Biology Toolkit for DCLK1 Reveals Connection to RNA Processing

Cited by 21 publications

References 79 publications

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells

Structural basis for small molecule targeting of Doublecortin Like Kinase 1 DCLK1

Tuft and Cancer Stem Cell Marker DCLK1: A New Target to Enhance Anti-Tumor Immunity in the Tumor Microenvironment

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