Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

Identification of the Doublecortin like kinase 1 (DCLK1) interactome reveals novel kinase dependent processes involved in gastric cancer progression.   (#58)

Annalisa L.E. Carli 1 2 , Shoukat Afshar-Sterle 1 2 , Najma Adnan 1 , Ryan N. O'Keefe 1 2 , Onisha Patel 3 , Isabelle Lucet 3 , Fleur M. Ferguson 4 5 , Nathaneal S. Gray 4 5 , Pierre Faou 6 , Harinda Rajapaksha 6 , Joel Steel 7 , Ralf Schittenhelm 7 , Matthias Ernst 1 2 , Michael Buchert 1 2
  1. Cancer and Inflammation Program, Olivia Newton-John Cancer Research Institute, HEIDELBERG, VIC, Australia
  2. School of Cancer medicine, La Trobe University, Bundoora, VIC, Australia
  3. The Walter and Eliza Hall Institute of Medical Research, Parkville , VIC, Australia
  4. Department of Cancer Biology, Dana-Faber Cancer Institute, Boston, MA, USA
  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
  6. Proteomics and Metabolomics Platform, La Trobe University, Bundoora, VIC, Australia
  7. Monash Proteomics & Metabolomics Facility, Monash University, Clayton, VIC, Australia

Introduction: Gastric cancer (GC) is one of the most aggressive and 3rd in cancer related deaths worldwide. Since the cancer genome atlas molecularly profiled GCs many new promising targets have emerged for Eppstein-Barr virus positive, microsatellite-, and chromosomal instable GCs. Unfortunately, no new therapeutic targets have emerged for the genomic stable (GS) subtype, which has the lowest overall survival. However, Ser/Thr-protein kinase Doublecortin like kinase 1 (DCLK1) is significantly upregulated in GS GCs. DCLK1 is a microtubule associated protein family member, hence important for cellular shape, polarity, migration, mitosis, and vesicular transport. DCLK1 has been shown to promote epithelial-to-mesenchymal transition and inducing migration and invasion in many different solid cancers. Therefore, we aimed to investigate whether DCLK1 could be a potential therapeutic target for GS GCs and determine how molecularly DCLK1 contributes to gastric cancer progression and identifying kinase substrates.

Methods/Results: DCLK1 overexpression resulted in increased cellular protrusions and cell migration in vitro, which was reversed upon kinase inhibition with the highly specific DCLK1-IN-1 inhibitor. Subsequent label-free quantitative (phospho)proteomics revealed significant changes in RNA-processing, cell-cell adhesion, cell cycle processes, cellular matrix organization, chromatin organization, and vesicular transport upon either DCLK1 overexpression or inhibition. 91 DCLK1 interactors were confidently identified with the Significance Analysis of INTeractome (SAINT) software on the quantitative affinity purification coupled to mass spectrometry (AP-MS) experiment on FLAG-tagged DCLK1, DCLK1-IN-1 treated, and kinase dead mutant. Next we compared the interactome and phospho-proteome data and identified 22 overlapping potential DCLK1 kinase substrates. These 22 proteins are again involved in membrane trafficking, RNA pol II transcription, RNA processing, ribosome biogenesis, cytoskeletal-, mitotic spindle-, and chromosome organization.

Conclusion: Our comprehensive proteomics approach revealed novel reversible processes for DCLK1’s contribution to cancer progression and identified potential kinase substrates. Together, this study establishes DCLK1 as a promising targetable regulator of GC.