Abstract CC03-01: Exosome pathway inhibition as therapeutic strategy in colorectal cancer

Exosomes are small extracellular vesicles (~100 nm) that are secreted by all cells and participate in cellular communication. Tumor-derived exosomes play a role in cancer progression by affecting the tumor microenvironment and establishing the pro-metastatic niche. Cancer cells are reported to have an increased release of exosomes, in addition to modulating their cargo. Therefore, blocking exosome secretion by cancer cells could halt cancer progression and be a promising novel drug-target for therapy. Global insight into the functional protein players of this process is key for development of a successful therapeutic strategy. To identify novel drug targets, Mass Spectrometry (MS)-based proteomics was performed on a unique sample set collected from 22 patients, comprising multiple fractions (tissue lysate, soluble secretome and extracellular vesicles) of matched normal and colorectal cancer (CRC) tissues (n=18) and adenomas (n=4). Available CPTAC phospho-proteomic CRC dataset was used to identify phospho-proteins that regulate the potential drug targets. Network clustering was performed using ClusterOne and gene ontology using BinGO (cytoscape). CD63 was measured using confocal microscopy. Exosome release using Coomassie stained gels and TSG101 expression using Western blot. Gene Ontology revealed deregulated pathways in normal versus cancer comparison that were linked to vesicle trafficking. Mainly, the ESCRT-pathway of the exosome biogenesis machinery was down-regulated in cancer while alternative proteins related to endocytosis and exosome release were up-regulated. Targeted data mining to the exosome biogenesis pathway revealed 12 up-regulated proteins (FC<1.5, p<0.05) and 14 proteins with increased cellular phosphorylation levels (p<0.05). Three upstream kinases with increased overall activity in CRC context, assessed by the INKA algorithm, were selected for further functional analysis using drugs in clinical trials or FDA-approved. To this end, two CRC cell lines were treated at IC50-correspondent dose for 16h. All three drugs caused a decrease in released exosomes after kinase inhibition, as evaluated by Coomassie staining and TSG101 levels. Intracellular immunofluorescent staining of multivesicular bodies (MVBs) by CD63, revealed increased intensity after treatment. This increase in CD63 intensity suggests an intracellular accumulation of MVBs which indicates an inability of the cell to release the exosomes. We aim to further validate the mechanisms of action underlying the inhibition of kinase-mediated exosome production and release after treatment using (phospho-)proteomics. Furthermore, we will assess its effect on exosome-mediated migration/invasion and angiogenesis. In conclusion, we describe a unique clinical dataset to identify drug targets in the exosome-release pathway, which presents a novel therapeutic strategy against CRC. Citation Format: Madalena N. Monteiro, Catarina Almeida-Marques, Meike de Wit, Valerie Dusseldorp, Logan Bishop-Currey, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Hanieh Sadeghi, Gerrit Meijer, Remond J. A. Fijneman, Angelika Hausser, Irene V. Bijnsdorp, Connie R. Jimenez. Exosome pathway inhibition as therapeutic strategy in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC03-01.