Anti-allergic drug azelastine suppresses colon tumorigenesis by directly targeting ARF 1 to 1 inhibit IQGAP 1-ERK-Drp 1-mediated mitochondrial fission 2 Running title : Azelastine targets ARF 1 to suppress colon tumorigenesis 3 4

24 This study aimed to screen novel anticancer strategies from FDA-approved non-cancer drugs and 25 identify potential biomarkers and therapeutic targets for colorectal cancer (CRC). 26 Methods: A library consisting of 1056 FDA-approved drugs was screened for anticancer agents. 27 WST-1, colony-formation, flow cytometry, and tumor xenograft assays were used to determine the 28 anticancer effect of azelastine. Quantitative proteomics, confocal imaging, Western blotting and 29 JC-1 assays were performed to examine the effects on mitochondrial pathways. The target protein 30 of azelastine was analyzed and confirmed by DARTS, WST-1, Biacore and tumor xenograft assays. 31 Immunohistochemistry, gainand loss-of-function experiments, WST-1, colony-formation, 32 immunoprecipitation, and tumor xenograft assays were used to examine the functional and clinical 33 significance of ARF1 in colon tumorigenesis. 34 Results: Azelastine, a current anti-allergic drug, was found to exert a significant inhibitory effect 35 on CRC cell proliferation in vitro and in vivo, but not on ARF1-deficient or ARF1-T48S mutant 36 cells. ARF1 was identified as a direct target of azelastine. High ARF1 expression was associated 37 with advanced stages and poor survival of CRC. ARF1 promoted colon tumorigenesis through its 38 interaction with IQGAP1 and subsequent activation of ERK signaling and mitochondrial fission 39 by enhancing the interaction of IQGAP1 with MEK and ERK. Mechanistically, azelastine bound 40 to Thr-48 in ARF1 and repressed its activity, decreasing Drp1 phosphorylation. This, in turn, 41 inhibited mitochondrial fission and suppressed colon tumorigenesis by blocking IQGAP1-ERK 42 signaling. 43 Conclusions: This study provides the first evidence that azelastine may be novel therapeutics for 44 CRC treatment. ARF1 promotes colon tumorigenesis, representing a promising biomarker and 45 therapeutic target in CRC. 46 47