Virtual Pre-recorded Oral Presentation 34th Lorne Cancer Conference 2022

The kinase activity of the cancer stem cell marker DCLK1 orchestrates pro-tumorigenic stromal changes (#30)

Michael Buchert 1 , Shoukat Asfahar-Sterle 1 , Annalisa L, E Carli 1 , Ryan O'Keefe 1 , Moritz Eissmann 1 , Ashwini Chand 1 , Rita Busuttil 2 , Isabel Lucet 3 , David Greening 4 , Nathanael S Gray 5 , Matthias Ernst 1
  1. Olivia Newton-John Cancer Research Institute, Heidelberg, VIC, Australia
  2. Peter MacCallum Cancer Centre, Parkville, VIC, Australia
  3. Walter and Eliza Hall Institute of Medical Research, Parkville
  4. Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne
  5. Chemical and Systems Biology, Stanford University, Stanford, CA, USA

BACKGROUND & AIMS: Gastric cancer (GC) is the 3rd leading cause of cancer mortality worldwide, therefore providing novel diagnostic and treatment options is crucial for at risk groups. The serine/threonine kinase doublecortin-like kinase 1 (DCLK1) is putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumours and a proposed driver of 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. DCLK1 gene amplification and somatic missense mutations are frequent feature (>10%) of human primary GC yet the underlying molecular mechanism(s) how DCLK1 mediates tumourigenesis are poorly understood. METHODS: RNA sequencing data was used to profile DCLK1 expression in 37 GC cell lines and more than 450 human GCs. We engineered human gastric cancer cells stably expressing DCLK1 to assess their oncogenic potential and to evaluate the contribution of the DCLK1 kinase domain in preclinical orthotopic gastric cancer models. Tumour specimen were analyzed by histology, immunohistochemistry, immunoblotting, flow cytometry and quantitative PCR. RESULTS: We report that the kinase domain of DCLK1 induces an epithelial to mesenchymal transition, promoting an invasive and metastatic behaviour both in vitro and in vivo. Moreover, DCLK1 kinase activity promotes extensive changes to the tumour microenvironment by amplifying the tumor secretome including the biogenesis and cargo selection of small extracellular vesicles (sEVs). Mechanistically, we identify the chemokine CXCL12 as a key mediator responsible for the majority of phenotypes induced by DCLK1 expression and quantitative proteome analysis of sEVs isolated from DCLK1 expressing cells revealed enrichment of migratory and adhesion regulators. Importantly, treatment with DCLK1-IN-1, a specific and selective small molecule kinase inhibitor of DCLK1, reverses the sEV biogenesis and cargo selection phenotypes as well as the pro-tumourigenic and pro-metastatic phenotypes. CONCLUSIONS: Together, this study establishes DCLK1 as a targetable master regulator of the tumour cell secretome and a promising target across all GC cancer subtypes.