Systems biology identifies that Gleevec reverses taxane resistance in solid tumors by selective inhibition of a novel plus Tip microtubule-binding variant
| Autor: | Thakkar, Prashant V., Kita, Katsuhiro, Galletti, Giuseppe, Madhukar, Neel, Cleveland, Kyle, Barasoain, Isabel, Díaz, José Fernando, Elemento, Olivier, Shah, Manish A., Giannakakou, Paraskevi |
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| Rok vydání: | 2019 |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| Popis: | Trabajo presentado en el AACR Annual Meeting 2019, celebrado en Atlanta, GA (Estados Unidos), del 29 de marzo al 3 de abril de 2019 The microtubule (MT) cytoskeleton is a validated therapeutic target in oncology, evidenced by the wide use of taxanes in solid tumors 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 taxane chemotherapy. Using a panel of GC cell lines intrinsically sensitive or resistant to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular drug accumulation and absence of tubulin mutations. We discovered a novel, truncated variant of the MT +TIP binding protein CLIP1, hereafter CLIP1S, which was significantly enriched in the resistant cells. Mass-spec proteomics and 5¿RACE showed that CLIP1S lacked the first 150 amino acids, thus, missing the Cap-Gly domain required for MT +TIP localization. Confocal microscopy of endogenous or exogenous tagged proteins revealed that CLIP1S was indeed mislocalized from the +TIP to the MT lattice in contrast to +TIP localization of canonical CLIP1. Stable CLIP1S-Knock Down (KD) entirely reversed taxane-resistance (~300 fold), establishing causation between CLIP1S and taxane resistance. Quantitation of drug-binding kinetics using live-cell imaging of Flutax-2 (fluorescently-labeled taxane) in native cytoskeletons, showed that CLIP1S caused Flutax-2 to have significantly reduced affinity and increased dissociation rates from MTs, as compared with cells expressing only the canonical CLIP1. CLIP1S-KD, fully restored Flutax-2 binding, implicating CLIP1S in impeding taxane-MT interaction. Co-administration of chemical probes specific for the low affinity taxane binding site on MT surface further implicated CLIP1S in partially obstructing the MT pore thereby restricting taxane access in the MT lumen where the high affinity taxane binding site is located. Computational analyses of RNA-seq data from untreated or taxane-treated sensitive and resistant GC cells using a novel bayesian drug-target identification algorithm predicted Imatinib (Gleevec¿) as a drug that could overcome CLIP1S mediated taxane resistance. Indeed, imatinib completely reversed taxane resistance, phenocopying the sensitization observed with the CLIP1S-KD. Most importantly, we showed that imatinib reversed taxane resistance by specific inhibition of CLIP1S in a dose-dependent manner as early as 3 h post-treatment. Taken together, these data identify an entirely novel mechanism of taxane resistance that involves obstruction of the MT pore in the presence of a previously unknown +TIP variant. Through systems biology we identified imatinib as the first specific CLIP1S inhibitor, thereby repurposing imatinib as a novel therapeutic to overcome clinical taxane resistance in GC and beyond. |
| Databáze: | OpenAIRE |
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