Abstract 641: Systems biology identifies Gleevec as a specific inhibitor of CLIP-170S, a novel +TIP isoform, which causes taxane resistance in cancer cells and patients by obstructing the Microtubule pore

Abstract

Abstract Taxanes are widely used in the treatment of solid tumor patients including gastric cancer (GC). Post-hoc analysis of the clinical trial that led to docetaxel approval in GC, revealed that patients with diffuse histological subtype were intrinsically resistant to taxanes. As yet, the molecular basis of clinical drug resistance remains poorly elucidated. Using a panel of GC cell lines, we identified a subset with intrinsic taxane resistance due to impaired drug-target engagement, in the absence of tubulin mutations or decreased drug accumulation. We discovered a novel, short variant of the microtubule (MT) +TIP binding protein CLIP-170, hereafter CLIP-170S, which was preferentially expressed in resistant cells. Mass-spec proteomics and 5'RACE showed that CLIP-170S lacked the first 150 amino acids, thus, missing the Cap-Gly domain required for +TIP localization. Microscopy of endogenous or exogenous proteins revealed that CLIP-170S was mislocalized from +TIP to the MT lattice in contrast to the canonical CLIP-170. Stable CLIP-170S knock down (KD) entirely reversed taxane-resistance (300 fold), directly establishing CLIP-170S as the cause of taxane resistance. Quantitation of Flutax-2 (fluorescently labeled taxane) binding kinetics by live-cell imaging of native cytoskeletons in sensitive and resistant cells, showed that Flutax-2 dissociated faster from MTs in CLIP-170S-expressing resistant cells due to slower association rate. CLIP-170S-KD fully restored Flutax-2 binding to MTs, indicating that CLIP-170S impedes taxane-MT interaction. As taxane binding to MT lumen requires entry via the MT pore, we used chemical probes binding at the outer-only (hexaflutax) or luminal (cyclostreptin) pore sites and showed reduced binding of both compounds to resistant cell cytoskeletons. In contrast, CLIP-170S had no effect on peluroside whose MT binding does not require access through the pore. Together, these data indicate that CLIP-170S obstructs the MT pore, preventing drug access to the MT lumen and causing taxane resistance. Clinically, we found CLIP-170S to be expressed in ~60% of GC patient tumors and that its expression was significantly associated with resistance to cabazitaxel monotherapy. Computational analyses of RNAseq data from sensitive and resistant cells predicted Gleevec (Imatinib) as a drug that could overcome taxane resistance. Indeed, we showed that Gleevec reversed taxane resistance by specific depletion of CLIP-170S protein. Taken together, these data reveal an entirely novel mechanism of taxane resistance via obstruction of the MT pore by the previously unrecognized CLIP-170S. We further found CLIP-170S to be highly prevalent in patient tumors and identified Gleevec as the first specific inhibitor of CLIP-170S. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Neel Madhukar, Elena Vila Navarro, Kyle Cleveland, Isabel Barasoain, Holly V. Goodson, Dan Sackett, Jose Fernando Diaz, Olivier Elemento, Manish A. Shah, Paraskevi Giannakakou. Systems biology identifies Gleevec as a specific inhibitor of CLIP-170S, a novel +TIP isoform, which causes taxane resistance in cancer cells and patients by obstructing the Microtubule pore [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 641.