Taxane Binding Research Articles

Multiscale Computational Analysis of the Effect of Taxol on Microtubule Mechanics.

Microtubules (MTs) are widely recognized as targets for cancer therapies. They are directly related to unique mechanical properties, closely dependent on MT architecture and tubulin molecular features. Taxol is known to affect tubulin interactions resulting in the stabilization of the MT lattice, and thus the hierarchical organization stability, mechanics, and function. A deeper understanding of the molecular mechanisms through which taxol modulates intertubulin interactions in the MT lattice, and consequently, its stability and mechanical response is crucial to characterize how MT properties are regulated by environmental factors, such as interacting ligands. In this study, a computational analysis of the effect of taxol on the MT was performed at different scales, combining molecular dynamics simulation, dynamical network analysis, and elastic network modeling. The results show that the taxol-induced conformational differences at the M-loop region increase the stability of the lateral interactions and the amount of surface in contact between laterally coupled tubulins. Moreover, the conformational rearrangements in the taxane binding site result in a different structural communication pattern. Finally, the different conformation of the tubulin heterodimers and the stabilized lateral interactions resulted in a tendency toward higher deformation of the vibrating MT in the presence of taxol. Overall, this work provides additional insights into taxol-induced stabilization and relates the conformational changes at the tubulin level to the MT mechanics. Besides providing useful insights into taxol effect on MT mechanics, a methodological framework that could be used to characterize the effects of other MT stabilizing agents is presented.

Abstract 2009: Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance

Abstract Taxanes are microtubule (MT)-stabilizing drugs widely used for the treatment of solid tumors, including the highly prevalent cancers of the breast, the prostate and gastric cancer. However, despite their initial activity, taxane resistance invariably occurs, leaving patients with limited therapeutic options. Currently, the molecular determinants of clinical taxane resistance remain poorly understood. We recently identified CLIP-170S, a novel truncated variant of the MT plus-end binding protein CLIP-170, that confers taxane resistance in vitro and is associated with clinical taxane resistance in patients with gastric cancer. We further showed that CLIP-170S is aberrantly localized along the MT lattice, thus impairing taxane access to its binding site in the MT lumen. However, the mechanism underlying the resistance-inducing extensive lattice localization of CLIP-170S is unknown. The activity and MT binding properties of canonical CLIP-170 are regulated by conformational changes mediated by the interaction of its N- and C-terminal domains. In its inactive, “closed” conformation, CLIP-170 is soluble as the N- and C-terminal domains interact with each other. In the active “open” conformation, CLIP-170’s N- and C-terminal domains are apart, thus exposing its MT-binding domain and allowing MT plus-end binding. As CLIP-170S is missing part of its N-terminal domain, we hypothesized that CLIP-170S is constitutively displayed in an open conformation that promotes its extensive and aberrant localization to the MT lattice. To prove this hypothesis, we used the FKBP-rapalog-FRB system to “force” CLIP-170S to assume a “closed” conformation. We generated a fusion CLIP-170S protein tagged with FKBP and FRB domains in its N- and C-terminal, respectively. The addition of rapalog induced FKBP (N-ter) and FRB (C-ter) domains to interact, promoting CLIP-170S to adopt a “closed” conformation. CLIP-170S in this “closed” conformation resulted in its release from the MT-lattice and restored taxane binding to MTs. We next extended our search to other CLIP-170 clinically prevalent variants with sequence alteration predicted to bind to the MT-lattice and potentially interfere with taxane sensitivity. This search identified CLIP-170IR (an intron retention variant) and several other truncation mutants prevalent in gastric, breast and lung cancer cell lines. Experimentally, we showed that these variants not only phenocopy CLIP-170S binding to the MT- lattice but also confer taxane-resistance. Together, these data demonstrate a novel mechanism of taxane resistance mediated by CLIP-170 mutations that induce CLIP-170 to adopt a constitutively “open” MT-lattice binding conformation, which renders them susceptible to causing taxane resistance. Citation Format: Urko del Castillo, Kiran Kumari Sahu, William G. Stone, Manish A. Shah, Paraskevi Giannakakou. Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2009.

Modulation of taxane binding to tubulin curved and straight conformations by systematic 3′N modification provides for improved microtubule binding, persistent cytotoxicity and in vivo potency

The taxane class of microtubule stabilizers are some of the most effective and widely used chemotherapeutics. The anticancer activity of taxanes arises from their ability to induce tubulin assembly by selectively recognizing the curved (c-) conformation in unassembled tubulin as compared to the straight (s-) conformation in assembled tubulin. We first designed and synthesized a series of 3′N-modified taxanes bearing covalent groups. Instead of discovering covalent taxanes, we found a series of non-covalent taxanes 2, in which the 3′N side chain was found to be essential for cytotoxicity due to its role in locking tubulin in the s-conformation. A representative compound bearing an acrylamide moiety (2h) exhibited increased binding affinity to the unassembled tubulin c-conformation and less cytotoxicity than paclitaxel. Further exploration of chemical space around 2h afforded a new series 3, in which derivatives such as 3l bind more tightly to both the s- and c-conformations of tubulin compared to paclitaxel, leading to more efficient promotion of tubulin polymerization and a greater persistence of in vitro efficacy against breast cancer cells after drug washout. Although 3l also had improved in vivo potency as compared to paclitaxel, it was also associated with increased systemic toxicity that required localized, intratumoral injection to observe potent and prolonged antitumor efficacy.

Structural insight into the stabilization of microtubules by taxanes.

Paclitaxel (Taxol®) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drug's mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 Å resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the bM loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, the longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is, however, not related to the microtubule stabilization mechanism of taxanes, as all analogs tested expand the microtubule lattice, despite the fact that one of them, Baccatin III, is biochemically inactive. In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.

Discovery of novel microtubule stabilizers targeting taxane binding site by applying molecular docking, molecular dynamics simulation, and anticancer activity testing

Disruption of the dynamic equilibrium of microtubules can induce cell cycle arrest in G2/M phase and apoptosis. Hence, discovery of novel tubulin polymerization inhibitors is very necessary and an important task in drug research and development for treatment of various tumors. In this investigation, 50 compounds were screened as microtubule stabilizers targeting the taxane site by combination of molecular docking methods. Among these hits, hits 19 and 38 with novel scaffolds exhibited the highest anti-proliferative activity with IC50 ranging from 9.50 to 13.81 μM in four cancer cell lines. The molecular dynamics simulations confirmed that tubulin and two hits could form stable systems. Meanwhile, the mechanism of the interactions between tubulin and two hits at simulated physiological conditions were probed. The in vitro tubulin polymerization assay revealed hits 19 and 38 were able to promote tubulin polymerization in a dose-dependent manner. Further, the immunofluorescence assay suggested that hits 19 and 38 could accelerate microtubule assembly in A549 and HeLa cells. Finally, studies on antitumor activity indicated that hits 19 and 38 induced G2/M phase cell cycle arrest and apoptosis, and inhibited cancer cell motility and migration in A549 and HeLa cells. Importantly, hit38 exhibited better anti-tubulin and anti-cancer activity than hit19 in A549 and HeLa cells. Therefore, these results suggest that hit38 represents a promising microtubule stabilizer for treating cancer and deserves further investigation.

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 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.

Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 μM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M−1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.

EVALUATION OF [11C]MPC-6827 AS A MICROTUBULE TARGETING PET RADIOTRACER IN CANCER CELL LINES

Objective: The objective of this study was to evaluate the uptake and specificity of [11C]MPC-6827, a MT targeted PET ligand in prostate, glioblastoma and breast cancer cells.
 Methods: [11C]MPC-6827 was synthesized by reacting corresponding desmethyl precursors with [11C]CH3I in a GE-FX2MeI/FX2M radiochemistry module. In vitro binding of [11C]MPC-6827 was performed in breast cancer MDA-MB-231, glioblastoma (GBM) patient-derived tumor (GBM-PDX), GBM U251 and prostate cancer 3 (PC3) cell lines at 37 °C in quadruplicate at 5, 15, 30, 60, and 90 minute incubation time. The nonspecific bindings were determined by incubation with unlabeled microtubule targeting agents MPC-6827, HD-800, colchicine, paclitaxel and docetaxel (5.0 mM).
 Results: [11C]MPC-6827 provided the highest binding in the breast cancer cell, MDA-MB-231, among all the cells studied, with 90% specific binding. [11C]MPC-6827 binds to glioblastoma PDX and U251 cells with ~50% and 40% specific binding, whereas, prostate cancer cell line, PC3 cells showed 40% specific binding. [11C]MPC-6827 also exhibits binding to the taxane and colchicine binding sites of MTs, in MDA-MB-231 cells.
 Conclusion: These data indicate that [11C]MPC-6827 can be a promising PET radiotracer for preclinical imaging of the brain and peripheral cancers.

Abstract 3817: Systems biology identifies that Gleevec reverses taxane resistance in solid tumors by selective inhibition of a novel +Tip microtubule-binding variant

Abstract 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. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Neel S. Madhukar, Kyle Cleveland, Isabel Barasoain, Jose Fernando Diaz, Olivier Elemento, Manish A. Shah, Paraskevi Giannakakou. Systems biology identifies that Gleevec reverses taxane resistance in solid tumors by selective inhibition of a novel +Tip microtubule-binding variant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3817.

Pocket similarity identifies selective estrogen receptor modulators as microtubule modulators at the taxane site

Taxanes are a family of natural products with a broad spectrum of anticancer activity. This activity is mediated by interaction with the taxane site of beta-tubulin, leading to microtubule stabilization and cell death. Although widely used in the treatment of breast cancer and other malignancies, existing taxane-based therapies including paclitaxel and the second-generation docetaxel are currently limited by severe adverse effects and dose-limiting toxicity. To discover taxane site modulators, we employ a computational binding site similarity screen of > 14,000 drug-like pockets from PDB, revealing an unexpected similarity between the estrogen receptor and the beta-tubulin taxane binding pocket. Evaluation of nine selective estrogen receptor modulators (SERMs) via cellular and biochemical assays confirms taxane site interaction, microtubule stabilization, and cell proliferation inhibition. Our study demonstrates that SERMs can modulate microtubule assembly and raises the possibility of an estrogen receptor-independent mechanism for inhibiting cell proliferation.

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

A brain-penetrant triazolopyrimidine enhances microtubule-stability, reduces axonal dysfunction and decreases tau pathology in a mouse tauopathy model

BackgroundAlzheimer’s disease (AD) and related tauopathies are neurodegenerative diseases that are characterized by the presence of insoluble inclusions of the protein tau within brain neurons and often glia. Tau is normally found associated with axonal microtubules (MTs) in the brain, and in tauopathies this MT binding is diminished due to tau hyperphosphorylation. As MTs play a critical role in the movement of cellular constituents within neurons via axonal transport, it is likely that the dissociation of tau from MTs alters MT structure and axonal transport, and there is evidence of this in tauopathy mouse models as well as in AD brain. We previously demonstrated that different natural products which stabilize MTs by interacting with β-tubulin at the taxane binding site provide significant benefit in transgenic mouse models of tauopathy. More recently, we have reported on a series of MT-stabilizing triazolopyrimidines (TPDs), which interact with β-tubulin at the vinblastine binding site, that exhibit favorable properties including brain penetration and oral bioavailability. Here, we have examined a prototype TPD example, CNDR-51657, in a secondary prevention study utilizing aged tau transgenic mice.Methods9-Month old female PS19 mice with a low amount of existing tau pathology received twice-weekly administration of vehicle, or 3 or 10 mg/kg of CNDR-51657, for 3 months. Mice were examined in the Barnes maze at the end of the dosing period, and brain tissue and optic nerves were examined immunohistochemically or biochemically for changes in MT density, axonal dystrophy, and tau pathology. Mice were also assessed for changes in organ weights and blood cell numbers.ResultsCNDR-51657 caused a significant amelioration of the MT deficit and axonal dystrophy observed in vehicle-treated aged PS19 mice. Moreover, PS19 mice receiving CNDR-51657 had significantly lower tau pathology, with a trend toward improved Barnes maze performance. Importantly, no adverse effects were observed in the compound-treated mice, including no change in white blood cell counts as is often observed in cancer patients receiving high doses of MT-stabilizing drugs.ConclusionsA brain-penetrant MT-stabilizing TPD can safely correct MT and axonal deficits in an established mouse model of tauopathy, resulting in reduced tau pathology.

Abstract 4169: A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer

Abstract The microtubule (MT) cytoskeleton is one of the most validated therapeutic targets in clinical oncology, as evidenced by the wide clinical activity of taxanes and other MT inhibitors. However, patients with intrinsic drug resistance do not benefit from taxane chemotherapy, such as patients with diffuse gastric cancer (GC). Currently, the molecular mechanisms underlying clinical taxane resistance remain poorly elucidated. Using a panel of GC cell lines with intrinsic sensitivity or resistance to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular accumulation of the drug and in the absence of tubulin mutations or altered tubulin isotype expression. Herein, we show for the first time that the resistant cells expressed a novel, short isoform of the MT plus-end binding protein, CLIP170 (CLIP1). CLIP1 binds to the plus-ends of microtubules (+TIPs) and regulates dynein-mediated MT-based trafficking and the tubulin tyrosination cycle. We showed by both mass-spec proteomics and 5’-RACE that the short CLIP1 isoform (CLIP1S) lacks the first 150 amino acids, thus, missing the first Cap-Gly (Cytoskeleton-Associated Protein-Glycine) domain, which is required for proper +TIP localization. Indeed, confocal microscopy experiments showed that CLIP1S was mislocalized to the microtubule lattice in contrast to the canonical comet-like pattern of CLIP1 seen in taxane sensitive cell lines. CLIP1S expression was specifically correlated with taxane resistance (docetaxel, cabazitaxel) as no correlation was observed with other DNA-damaging agents. Since, CLIP1S expression has never been reported before, to establish causation, we stably knocked down CLIP1 and CLIP1S in taxane-sensitive and resistant cells, respectively. CLIP1SKD entirely reversed taxane-resistance (~300 fold) while CLIP1KD had no effect in the taxane-sensitive cell lines. These data suggest a gain-of-function of CLIP1S that leads to taxane resistance. To study the binding kinetics of taxanes to MTs in the presence or absence of CLIP1S we performed live cell imaging of native cytoskeletons using fluorescently-labeled paclitaxel (Flutax). Our data revealed significantly faster dissociation rates of Flutax from MTs in the resistant cells, indicating transient interaction with MTs. Taxane binding to MTs is a two-step process. First taxanes bind to the MT-pore low affinity surface site, which then facilitates access to the high affinity luminal site. Using a small molecule that binds only to MT-pore site, we showed that we showed that taxol binding to the pore is significantly slower in resistant cells expressing CLIP1S, which together with mislocalization of this variant along the MT lattice suggests that it obstructs access to the MT-pore thus restricting entry of taxane into the lumen of microtubules. This finding will have profound implications for taxane resistance as well as microtubule biology broadly. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Isabel Barasoain, Jose Fernando Diaz, Manish A. Shah, Paraskevi Giannakakou. A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2017-4169

Abstract 2932: Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer

Abstract Successful clinical application of taxanes (microtubule-stabilizers) is limited due to intrinsic or acquired drug resistance. Thus, it is critical to unveil the molecular mechanisms of taxane resistance to significantly improve clinical outcomes. Retrospective analysis of the TAX-325 gastric cancer (GC) trial revealed that the addition of docetaxel (DTX) to cisplatin/fluorouracil increased progression-free plus overall survival, in intestinal (INT) but not in diffuse (DIF) GC subtypes. Our preclinical data confirmed that DIF GC cell lines are intrinsically resistant to taxanes. The incidence of DTX resistance in DIF GC cell lines was 2.5 times higher than INT GC cell lines. Drug efflux, tubulin posttranslational modification and differential β-tubulin isotype expression were ruled out as potential mechanisms of intrinsic taxane resistance. Thus, a new molecular mechanism must underlie the intrinsic taxane resistance in DIF GC. To quantify the kinetics of taxol binding to cellular MTs, we treated both DIF and INT groups of GC lines with the fluorescein-conjugated paclitaxel analog, Flutax-2. The Flutax-2 staining intensity of cellular MTs was assessed by live-cell confocal microscopy at different time points. Following a 3h incubation, there was less than 20% decrease in Flutax-2 intensity in the sensitive cell lines, compared to 59∼89% decrease in the resistant cell lines. These data suggested different binding kinetics between sensitive and resistant cells. We added 1μM Flutax-2 to sensitive/resistant cells’ native cytoskeletons for 0∼60 seconds to determine the association rate (kon) of Flutax-2 binding to MTs. The kon of Flutax-2 in the sensitive cell lines was significantly higher in sensitive (5.8×104M−1s−1) versus resistant cells (0.3∼2.6×104M−1s−1). Next, we measured the dissociation rates (koff) by competing the pre-bound Flutax-2 with DTX (0∼600 seconds). Although 20∼40% faster koff was observed in resistant cells, it appeared that the association rate of Flutax-2 was the dominant factor of differential taxane binding to MTs in GC cells. The binding mode of taxanes to MTs involves 1) binding to MT pores and 2) internalization to the high-affinity binding site at the MT lumen. To differentiate between the two, we used hexaflutax, which binds exclusively to the MT pores. We observed hexaflutax decorating radial MT arrays in sensitive but not in the resistant cells. As tubulin mutations around the high-affinity taxane binding site or tubulin posttranslational modifications/differential expression of β-tubulin isotype is not the case in DIF GC cell lines, our data suggest that modifications of the MT pore conformation or occlusion of the pore site is responsible for the intrinsic taxane resistance in DIF GC. Determining the origin of the defect at the pores will help design better MT-stabilizing drugs to overcome chemo-resistance, the major obstacle hindering overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2932.

Abstract 2896: 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity

Abstract Taxane based chemotherapy is commonly used to treat a wide variety of solid tumors including breast, prostate, lung and ovarian. Taxanes bind to and stabilize microtubules inhibiting their function in interphase and mitosis, and leading to cancer cell apoptosis. Although taxanes are widely used clinically and their target, tubulin, is ubiquitously expressed, patient response is heterogeneous and the molecular mechanism of clinical drug resistance remains elusive. In addition to their antimitotic effects, taxanes affect several signaling pathways whose perturbation by microtubule stabilization may determine cell sensitivity or resistance. Our recent work has identified one such pathway by showing that Taxol treatment inhibits the translation of HIF-1α mRNA by inducing its sequestration to P-bodies where it is silenced. However, the signaling cascade that is initiated in response to microtubule damage leading to translation suppression of HIF-1α is not understood. Using polysome analysis we showed that Taxol treatment inhibited translation at the initiation step of protein synthesis, evidenced by the increase in the 80S peak. Post-translational modifications of various translation initiation factors are implicated in the regulation of translation initiation. Hence, we analyzed phosphorylation states of several initiation factors, including eIF4E, eIF2α, 4E-BP1, 4E-BP2 and rpS6. We didn't observe any change in the phosphorylation state of these initiation factors except for 4E-BP1. We observed that Taxol treatment led to 4E-BP1 hyper-phosphorylation, at a unique previously unrecognized, site. Using isogenic cell lines with acquired mutations at the taxane binding site we conclusively showed that microtubule disruption was required for the hyper-phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 is known to activate cap-dependent translation. Instead, our data using polysome profiling revealed that Taxol-induced 4E-BP1 hyper-phosphorylation was associated with translation suppression. We confirmed these results by performing the cap-binding assay, which demonstrated that Taxol treatment had no effect on cap-dependent translation. Taken together these results suggest a novel biological function for 4E-BP1 hyper-phosporylation. Further, treatment with the mTOR inhibitor, Torin-1, had no effect on Taxol-induced 4E-BP1 hyper-phosphorylation, suggesting mTOR pathway independence. Interestingly, treatment with the CDK inhibitor Roscovitine, prevented both Taxol-induced 4E-BP1 hyper-phosphorylation and Taxol-induced cell death. Although we do not yet know which of the three CDKs exerts the inhibitory effect on taxanes, our data suggest that there is a functional link between 4E-BP1 hyper-phosphorylation and taxane activity. We are currently investigating the role of CDK1, CDK2 and CDK5 in this pathway to decipher the novel biological function of 4E-BP1 hyper-phosphorylation. Citation Format: Prashant Khade, Paraskevi Giannakakou. 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2896.

Abstract 1229: The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources

Abstract Next-generation taxanes are known to be active against taxane-resistant cell lines that overexpress P-glycoprotein (P-gp) and/or βIII-tubulin, both of which are associated with drug resistance. In this study, we examined the effect of two potent next-generation taxanes, SB-T-1214 and SB-CST-10202 (C-seco-taxane), on growth of the ovarian cancer cell line Hey and its drug-resistant daughter cell lines that overexpress different levels of P-gp and express increased levels of βIII-tubulin. We found that SB-T-1214 was ∼6-9-fold more potent than SB-CST-10202 against these drug resistant cells. Since the taxanes bind to both tubulin and P-gp, the relative binding affinity of the taxanes to these two cellular targets may influence the effectiveness of the taxanes in resistant cells. To determine the effects of these two taxanes on binding of Taxol® to P-gp and tubulin, a tritium-labeled Taxol® analog, 2-(m-azidobenzoyl)taxol (2-m-AzTax), was used. We have previously demonstrated that 2-m-AzTax photolabels a peptide (amino acids 217-231) in β-tubulin. To study the relative binding affinities of SB-T-1214 and SB-CST-10202, tubulins from bovine brain (BBT) and chicken erythrocytes (CET) were specifically photolabeled with [3H]2-m-AzTax, in the presence and absence of either Taxol®, Taxotere®, SB-T-1214 or SB-CST-10202. β-tubulin isotype content from BBT and CET is different, the former contains βI-, βII-, βIII- and βIV-tubulins, but the latter has only βVI. Taxol® had a minimal inhibitory effect (∼3%) on BBT, but a strong inhibitory effect (99%) on CET. Taxotere® appeared to have a stronger inhibitory effect than Taxol® on photolabeling (31% and 99% for BBT and CET, respectively). The inhibitory effects elicited by the two next-generation taxanes on photolabeling were distinct for β-tubulins from these two sources. SB-T-1214 and SB-CST-10202 inhibited BBT photolabeling by 38% and 32%, respectively. Interestingly, these two drugs caused a 41% and only 6% inhibition, respectively, on photolabeling of CET that does not contain βIII-tubulin. We are currently analyzing the effect of SB-T-1214 on binding of [3H]2-m-AzTax to different tubulin isotypes resolved by isoelectrofocusing. To study photolabeling of P-gp, plasma membranes from a multidrug resistant (MDR) cell line SKVLB1 that expresses very high levels of P-gp were prepared. SB-T-1214 and SB-CST-10202 inhibited photolabeling of P-gp markedly (> 80% inhibition), suggesting that these two taxanes are good substrates for P-gp. They also caused an increase in steady state [3H]2-m-AzTax accumulation in the MDR cell line SKVLB1. Since drug binding to tubulin and P-gp is the primary event that occurs in the cell following drug administration, our data on taxane binding affinity to the two targets will help in understanding the effectiveness of the next-generation taxanes in taxane-resistance. Citation Format: Chia-Ping H. Yang, Changwei Wang, Iwao Ojima, Susan Band Horwitz. The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1229.

Abstract 317: Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy

Abstract Ellagic acid (EA) is a polyphenolic compound found in berries, pomegranates and other foods, which has cancer preventive and therapeutic properties. EA inhibits proliferation, induces apoptosis, blocks cell cycle progression and inhibits ABC drug transporters. Based on these anti-cancer activities of EA, we hypothesized that EA could enhance the efficacy of taxane chemotherapy on prostate cancer cells and inhibit drug resistance. We used castration-resistant 22Rv1, VCaP, and C4-2 prostate cancer cells and androgen-independent PC-3 cells to study effects of EA on efficacy of the taxane drug cabazitaxel (CBZ). Cells were treated with EA (0.1 μM - 30 μM) and after 72 hr proliferation was measured using the SRB assay. EA inhibited proliferation at levels greater than 10 μM. At 30 μM the greatest inhibition was observed in the most proliferative cell lines (22Rv1: 66.7%, PC-3: 82.7%), with a more modest effect on the slower growing cells (VCaP: 25.2%, C4-2: 31.7%). We next tested if growth inhibitory concentrations of EA could enhance the microtubule stabilizing effects of CBZ. In a cell-free assay, CBZ (1 nM - 1 μM) dose dependently increased tubulin polymerization, and EA (10 μM) increased polymerization by 61.0%. However, we observed an antagonistic response with the combination of CBZ and EA, reducing polymerized tubulin by 13.9% compared to CBZ alone. To confirm these results in cell culture, we treated 22Rv1 cells with EA (10 μM) and CBZ (10 nM) and determined levels of polymerized tubulin by western blotting. We observed a 1.6 fold increase in polymerized tubulin by EA, a 5.5 fold increase by CBZ, which was reduced to a 4.6 fold increase by combination of CBZ with EA. Contrary to our hypothesis, these results suggest that EA may interfere with taxanes, leading to a reduction in drug effectiveness. Additionally, the ability of EA alone to induce polymerization suggests the possibility that the growth inhibitory activity of EA is a consequence of its effects on microtubule dynamics, potentially through an interaction with taxane binding site of tubulin. This is consistent with our observation that EA has the greatest growth inhibitory effect on very proliferative cell lines, since microtubule targeting agents are most effective at killing rapidly dividing cells. To our knowledge, this is the first evidence of microtubule targeting as an anticancer mechanism of EA. We also studied the effects of EA on drug efflux to determine if EA could inhibit development of taxane drug resistance. We treated C4-2 cells with EA (0.01 μM - 30 μM) and measured calcein retention as marker of the activity of the P-glycoprotein (P-gp) pump. There was a dose dependent increase in calcein retention by EA, with a 1.9 fold increase at the highest concentration (30 μM), suggesting an inhibition of P-gp pump activity with increasing amounts of EA. Thus, EA may increase intracellular taxane levels, but at the same time reduce their microtubule-polymerization effects. Citation Format: Jillian N. Eskra, Alaina Dodge, Maarten C. Bosland. Microtubule polymerizing effects of ellagic acid reduce efficacy of taxane chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 317.

Abstract P6-13-07: The taccalonolides are novel microtubule stabilizers that covalently bind tubulin and have in vivo efficacy in drug resistant tumors

Abstract Some of the most effective drugs used in the treatment of breast cancer are microtubule stabilizers. However, there are limitations to their clinical efficacy, including inherent and acquired drug resistance. All microtubule stabilizers that are currently approved for clinical use bind within the taxane pocket on β-tubulin in a reversible manner. The taccalonolides are a novel class of microtubule stabilizers that have a similar profile of microtubule stabilization as the taxanes, but circumvent drug resistance mediated by expression of drug efflux pumps, mutations in the taxane binding site, or overexpression of the βIII isotype of tubulin. We have shown that one important difference between the taccalonolides and clinically approved microtubule stabilizers is that the taccalonolides form a covalent bond to β-tubulin. This distinct interaction allows for irreversible binding, which explains their ability to avoid drug efflux mechanisms and likely belies their exquisite potency in in vivo antitumor models which allows for delivery in aqueous solvents. Serum stability and binding studies, microsomal clearance and pharmacokinetic analysis were performed with both taccalonolides AF and AJ to more fully understand the properties of this class of compounds. We found that both taccalonolides had low microsomal intrinsic clearance rates with no evidence of serum binding and had half-lives similar to paclitaxel in vivo. Like other microtubule targeted agents, taccalonolide AF has a narrow therapeutic window with antitumor effects accompanied by body weight loss. Interestingly, direct injection of taccalonolide AF into a xenograft tumor was highly effective with no associated toxicities at low doses, indicating that targeted delivery to the tumor would greatly increase the efficacy and decrease toxicities. To this end, efforts to promote the targeted delivery of taccalonolide AF to the tumor are being evaluated. Citation Format: Risinger AL, Li J, Benavides R, Kuhn JG, Mooberry SL. The taccalonolides are novel microtubule stabilizers that covalently bind tubulin and have in vivo efficacy in drug resistant tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-07.

Abstract 2121: Microtubule-regulated mRNA translation signaling and therapeutic implications in cancer

Abstract Drugs that affect microtubule dynamics and function, both those that stabilize microtubules (e.g. taxanes) and those that depolymerize them (e.g. vincas, Eribulin) are widely used in cancer chemotherapy. However, their mechanism of action at the molecular level and the signaling pathways regulated by microtubule dynamics are not fully understood, precluding chemotherapy customization to the individuals. Previously, we have reported that microtubule dynamics control the translation of HIF-1α mRNA. We showed that treatment with microtubule-targeting drugs (MTDs) shifts HIF-1α mRNA from actively translating polysomes to non-translating ribosomes resulting in inhibition of HIF-1α transcriptional activity. Using cell lines with acquired mutations at different MTD binding sites we conclusively showed that microtubule disruption was required for the inhibition of HIF translation. However, additional mRNAs susceptible to MTD treatment or the exact signaling pathway that senses microtubule disruption and leads to inhibition of translation are not known. Polysome RNA-Seq analyses of untreated and drug treated cells has identified additional mRNAs whose polysome association is shifted towards inactive translation similar to HIF-1α mRNA. We are using bioinformatics analyses to identify a common microtubule-susceptibility motif in addition to validation experiments at the protein and pathway level. The polysome analysis showed that protein synthesis is inhibited at the stage of translation initiation. In an effort to better understand the underlying mechanism of translation inhibition of HIF-1α mRNA and additional microtubule-dependent mRNAs, we assessed 4E-BP1 protein as its phosphorylation is a key regulator of cap dependent mRNA translation. We observed that taxanes or Eribulin treatment hyper-phosphorylates 4E-BP1 without affecting ribosomal protein S6 (rpS6) phosphorylation. Using the taxane-resistant breast cancer cell line MB-231/K20T which harbors an acquired mutation at the taxane binding site of β-tubulin we showed that Taxol treatment has no effect on 4E-BP1 hyper- phosphorylation, consistent with its lack of microtubule activity. In contrast, Eribulin, which has a distinct binding site on tubulin, is able to overcome taxol-resistance by fully engaging the microtubule-4E-BP1 axis. Interestingly, in renal cell carcinoma (RCC) cells, for which treatment with microtubule inhibitors has no clinical benefit, we didn't observe 4E-BP1 hyper-phosphorylation after taxane or Eribulin treatment, even though microtubules were affected. These results suggest that the microtubule-4E-BP1 axis is compromised in RCC and that there is a link between 4E-BP1 phosphorylation and MTD efficacy. Hence, we propose that identifying the link of 4E-BP1 dependent signaling with microtubules will have important therapeutic implications in MTD chemotherapy. Citation Format: Prashant Khade, Paraskevi Giannakakou. Microtubule-regulated mRNA translation signaling and therapeutic implications in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2121. doi:10.1158/1538-7445.AM2015-2121

Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

Abstract The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600