Microtubule Dynamics Inhibitor Research Articles

Axonal growth on surfaces with periodic geometrical patterns.

The formation of neuron networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system, and for creating novel bioinspired materials for tissue engineering and neuronal repair. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body towards target neurons. Axonal growth is guided by environmental stimuli that include intercellular interactions, biochemical cues, and the mechanical and geometrical features of the growth substrate. The dynamics of the growing axon and its biomechanical interactions with the growing substrate remains poorly understood. In this paper, we develop a model of axonal motility which incorporates mechanical interactions between the axon and the growth substrate. We combine experimental data with theoretical analysis to measure the parameters that describe axonal growth on surfaces with micropatterned periodic geometrical features: diffusion (cell motility) coefficients, speed and angular distributions, and axon bending rigidities. Experiments performed on neurons treated Taxol (inhibitor of microtubule dynamics) and Blebbistatin (disruptor of actin filaments) show that the dynamics of the cytoskeleton plays a critical role in the axon steering mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrates that promote neuronal growth and nerve repair.

The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human cancer xenograft models.

Eribulin, an inhibitor of microtubule dynamics, is known to show antitumor effects through its remodeling activity in the tumor vasculature. However, the extent to which the improvement of tumor hypoxia by eribulin affects radio-sensitivity remains unclear. We utilized 1-(2,2-dihydroxymethyl-3-18F-fluoropropyl)-2-nitroimidazole (18F-DiFA), a new PET probe for hypoxia, to investigate the effects of eribulin on tumor hypoxia and evaluate the radio-sensitivity during eribulin treatment. Mice bearing human breast cancer MDA-MB-231 cells or human lung cancer NCI-H1975 cells were administered a single dose of eribulin. After administration, mice were injected with18F-DiFA and pimonidazole, and tumor hypoxia regions were analyzed. For the group that received combined treatment with radiation, 18F-DiFA PET/CT imaging was performed before tumors were locally X-irradiated. Tumor size was measured every other day after irradiation. Eribulin significantly reduced18F-DiFA accumulation levels in a dose-dependent manner. Furthermore, the reduction in 18F-DiFA accumulation levels by eribulin was most significant 7days after treatment. These results were also supported by reduction of the pimonidazole-positive hypoxic region. The combined treatment showed significant retardation of tumor growth in comparison with the control, radiation-alone, and drug-alone groups. Importantly, tumor growth after irradiation was inversely correlated with 18F-DiFA accumulation. These results demonstrated that 18F-DiFA PET/CT clearly detected eribulin-induced tumor oxygenation and that eribulin efficiently enhanced the antitumor activity of radiation by improving tumor oxygenation.

980P Phase Ib study of a liposomal formulation of eribulin (E7389-LF) + nivolumab (Nivo) in patients (pts) with advanced solid tumors

Eribulin is a halichondrin-class microtubule dynamics inhibitor with cytotoxic and vascular remodeling effects leading to tumor immune modulation. E7389-LF is a liposomal formulation of eribulin designed to enhance antitumor activity and improve pharmacokinetic (PK) profiles. The combination of E7389-LF + Nivo is expected to show antitumor activity by cytotoxic and antitumor immune effects.

304P Incidence and resolution of eribulin-induced peripheral neuropathy (IRENE) in patients with locally advanced or metastatic breast cancer

Eribulin (ERI), a halichondrin-class microtubule dynamics inhibitor, is a typical chemotherapeutic regimen for patients (pts) with previously treated locally advanced or metastatic breast cancer (MBC). Results of pivotal trials showed that about one-third of pts with locally advanced or MBC treated with ERI develop peripheral neuropathy (PN), a common risk with BC chemotherapies. This postauthorization safety study assessed the incidence and severity of ERI-induced PN (EIPN) in pts with locally advanced or MBC.

Disassembly of microtubules by intense terahertz pulses.

The biological effects of terahertz (THz) radiation have been observed across multiple levels of biological organization, however the sub-cellular mechanisms underlying the phenotypic changes remain to be elucidated. Filamentous protein complexes such as microtubules are essential cytoskeletal structures that regulate diverse biological functions, and these may be an important target for THz interactions underlying THz-induced effects observed at the cellular or tissue level. Here, we show disassembly of microtubules within minutes of exposure to extended trains of intense, picosecond-duration THz pulses. Further, the rate of disassembly depends on THz intensity and spectral content. As inhibition of microtubule dynamics is a mechanism of clinically-utilized anti-cancer agents, disruption of microtubule networks may indicate a potential therapeutic mechanism of intense THz pulses.

BMS will acquire Eisai ADC for cancer

Bristol Myers Squibb will pay Eisai $650 million to jointly develop the Tokyo-based firm’s first antibody-drug conjugate (ADC) for cancer. Eisai also could earn up to $2.45 billion in milestone payments. The antibody targets cancers expressing folate receptor α. It then delivers a small-molecule payload called eribulin, Eisai’s simplified and synthetic version of halichondrin B, a toxic microtubule dynamics inhibitor derived from sea sponges. The therapy is in Phase 1 studies as a possible treatment for endometrial, ovarian, lung, and breast cancers.

Microtubule-targeting agents impair kinesin-2-dependent nuclear transport of β-catenin: Evidence of inhibition of Wnt/β-catenin signaling as an important antitumor mechanism of microtubule-targeting agents.

An aberrant accumulation of nuclear β-catenin is closely associated with the augmentation of cancer malignancy. In this work, we report that several microtubule-targeting agents (MTAs) such as vinblastine, taxol, and C12 (combretastatin-2-aminoimidazole analog) inhibit Wnt/β-catenin signaling in oral squamous cell carcinoma (OSCC). We showed that the inhibition of microtubule dynamics by MTAs decreased the level of β-catenin by increasing Axin and adenomatous polyposis coli levels and reducing the level of dishevelled. Furthermore, MTAs strongly reduced the localization of β-catenin in the nucleus. The reduction in the level of nuclear β-catenin was neither due to the degradation of β-catenin in the nucleus nor due to an increase in the export of nuclear β-catenin from the nucleus. A motor protein kinesin-2 was found to assist the nuclear transportation of β-catenin. Interestingly, Wnt/β-catenin signaling antagonist treatment synergized with MTAs and the activators of Wnt/β-catenin signaling antagonized with the MTAs. C12 potently suppressed the growth of 4-Nitroquinoline 1-oxide-induced OSCC in the tongue of C57 black 6 mice and also abrogated Wnt/β-catenin signaling pathway in the tumor. Our results provide evidence that the decrease in Wnt/β-catenin signaling is an important antitumor effect of MTAs and the combined use of MTAs with Wnt/β-catenin signaling antagonists could be a promising strategy for cancer chemotherapy.

Eribulin in combination with bevacizumab as second-line treatment for HER2-negative metastatic breast cancer progressing after first-line therapy with paclitaxel and bevacizumab: a multicenter, phase II, single arm trial (GIM11-BERGI)

BackgroundWe evaluated the efficacy and safety of the nontaxane microtubule dynamics inhibitor eribulin plus the humanized anti-VEGF monoclonal antibody bevacizumab in a novel second-line chemotherapy scheme in HER2-negative metastatic breast cancer (MBC) patients progressing after first-line paclitaxel and bevacizumab.Patients and methodsThis is a multicenter, single-arm, Simon's two-stage, phase II study. The primary endpoint was the overall response rate, considered as the sum of partial and complete response based on the best overall response rate (BORR). The secondary endpoints were progression-free survival (PFS), overall survival (OS), and clinical benefit rate.ResultsA total of 58 of the 61 patients enrolled in the study were evaluable for efficacy. The BORR was 24.6% (95% CI 14.5-37.3). The clinical benefit rate was 32.8% (95% CI 21.3-46.0). The median PFS was 6.2 months (95% CI 4.0-7.8), and median OS was 14.8 months (95% CI 12.6-22.8). Overall, adverse events (AEs) were clinically manageable and the most common AEs were fatigue, paresthesia, and neutropenia. Quality of life was well preserved in most patients.ConclusionsThe results of this study suggest that second-line therapy with bevacizumab in combination with eribulin has a meaningful clinical activity and may represent a potential therapeutic option for patients with HER2-negative MBC.

Abstract PS4-20: Low levels of interleukin-6 at baseline were significantly associated with improved overall survival of patients treated with eribulin for locally advanced or metastatic breast cancer

Abstract (Background)Eribulin, a non-taxane inhibitor of microtubule dynamics, is a unique chemotherapy agent for locally advanced or metastatic breast cancer (MBC). The treatment has been demonstrated to extend overall survival (OS) without extending progression-free survival (PFS). This effect seems to result from the suppression of epithelial-mesenchymal transition (EMT) and improvement of the hypoxic microenvironment by vascular normalization induced by eribulin, directly or indirectly. In a previous study, we identified that high absolute lymphocyte count (ALC) at the baseline was significantly associated with longer OS in the eribulin group, but not in the treatment of physician’s choice (TPC) group, in the phase III EMBRACE trial (Miyoshi et al., Breast Cancer, 2020). These results strongly suggest an association between eribulin efficacy and immune response. Therefore, we focused on serum levels of immune and inflammatory cytokines,which are expected to have greater utility as a predictive factor for eribulin than ALC. Interleukin (IL) 6, an inflammatory cytokine associated with cancerprogression, and soluble interleukin-2 receptor (sIL-2R), a receptor of IL-2 released from various immune cells including T cells, B cells, and natural killer cells, were selected for the investigation. (Patients and methods)A total of 44 patients treated with eribulin for MBC were recruited for the study. We examined the predictive values of IL-6 and sIL-2R in addition to the neutrophil-to-lymphocyte ratio (NLR) and ALC at baseline. The cutoff values of NLR and ALC were set at 3.0 and 1500/μL, respectively. We used the normal ranges of IL-6 (4.0 pg/mL) and sIL-2R (474 U/mL) as cutoff values. (Results)The OS of patients with low NLR (n=28) and high ALC (n=17) were significantly longer than that of patients with high NLR (n=16, p=0.0287) and low ALC (n=27, p=0.0234). There were no significant associations between PFS and NLR or ALC (p=0.0852 and p=0.2231, respectively). Patients with normal IL-6 levels (n=17) had significantly longer PFS (p=0.0023) and OS (p=0.0013) than those with elevated IL-6 levels (n=27). Regarding sIL-2R, patients with normal sIL-2R levels (n=18) had longer OS (p=0.0483) than those with elevated sIL-2R levels (n=26), but their PFS was similar (p=0.2435). Multivariate analysis showed that IL-6 levels were only significantly associated with OS (hazard ratio, 0.093; 95% confidence interval, 0.012-0.720; p=0.0106). There was no significant association between IL-6 levels and NLR or ALC. (Conclusion and discussion)Patients treated with eribulin demonstrated a significantly longer OS if their baseline IL-6 levels were within the normal range. This predictive efficacy for eribulin was more accurate than that of NLR or ALC. As there was no significant association between IL-6 levels and NLR or ALC, IL-6 appears to predict whether the tumor microenvironment is favorable or unfavorable for eribulin treatment, mediated through different mechanisms. Therefore, IL-6 levels may be useful for selecting patients who will benefit from the administration of eribulin in terms of improved OS. Citation Format: Ayako Bun, Natsuko Inoue, Yoshimasa Miyagawa, Reiko Fukui, Yukie Fujimoto, Tomoko Higuchi, Atsushi Sata, Hiromi Ozawa, Michiko Imamura, Yasuo Miyoshi. Low levels of interleukin-6 at baseline were significantly associated with improved overall survival of patients treated with eribulin for locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-20.

Abstract 4179: E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Natural products have been a rich source of inspiration for drug discovery as demonstrated by the fact that over one-third of current therapeutic agents are natural products or compounds derived from them. Particularly, when the supply of a natural product from the natural source is limited, organic synthesis can offer a solution. However, with increasing structural complexity, this approach becomes exponentially more challenging, as a synthesis must meet various requirements, including high overall efficiency, scalability, cost-effectiveness, and product purity as well as conforming to good manufacturing practice (GMP) regulations. Despite the outstanding in vivo antitumor activity in mice, the limited supply from the natural sources has prevented drug development based on intact halichondrins. Methods: We successfully synthesized 19.5 g of C52-halichondrin-B alcohol with 99.84% purity via a total synthesis. From 15.0 g of this material, we obtained 11.5 g of C52-halichondrin-B amine (E7130) with 99.81% purity under GMP conditions. With totally synthetic E7130, a novel microtubule dynamics inhibitor, we studied the activities in both of in vitro and in vivo. The inhibitory activity of E7130 towards tubulin polymerization was analyzed in the cell-free system in which tubulin polymerization could be monitored by fluorescence enhancement due to the incorporation of a fluorescence reporter into the microtubules during polymerization. We analyzed in vivo antitumor activities using both a human cancer cell line orthotopic transplantation mouse model and subcutaneous xenograft models. The immunohistochemical analyses were performed with tumor tissues collected from mouse models to analyze histological changes after treatment with E7130. Results: E7130 is a novel microtubule dynamics inhibitor with exceedingly potent in vitro and in vivo anticancer activities. Significantly, E7130 not only is cytotoxic, but can also increase CD31-positive endothelial cells in tumors and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. Notably, this dual activity has been recognized for the first time through this study. According to these unique tumor microenvironment ameliorative effects, E7130 can augment the effect of other antitumor treatments in mouse models. In particular, a dose of 90 µg/kg, one-half of the maximum tolerated dose in mice, showed a prominent combinational effect with cetuximab, which suggests that E7130 has a different mechanism than other microtubule-targeted drugs. Conclusions: E7130 ameliorates the tumor microenvironment to improve cancer treatment when used in combination with other compounds. The data provide compelling evidence that E7130 is a promising molecular-targeting anticancer agent. Citation Format: Satoshi Kawano, Takanori Abe, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator [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 4179.

Abstract 1924: Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE)

Abstract Background: Eribulin mesylate, a synthetic analog of the marine sponge natural product halichondrin B, is a microtubule dynamics inhibitor that acts via both cytotoxic antimitotic mechanisms as well as non-cytotoxic effects on tumor vasculature, tumor phenotype and the tumor microenvironment. Eribulin is approved in the US for treatment of certain patients with advanced breast cancer or liposarcoma. Here we examined in vitro antiproliferative responses to eribulin versus vinorelbine and paclitaxel in 100 human cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE), followed by analysis of CCLE baseline gene expression data with the goal of assessing overlapping and non-overlapping pathways and genes associated with drug response and resistance. Methods: In vitro proliferation assays were conducted on 100 CCLE cell lines. Cancer types represented by ≥5 cell lines include breast, lung, blood, colorectum, ovary, pancreas, skin and kidney. Comparative systems-level analyses of genes, networks and pathways associated with drug response and/or resistance were performed to identify gene and pathway-level determinants of drug response that were either unique to eribulin or shared with vinorelbine and/or paclitaxel. Results: We evaluated genes and molecular pathways driving sensitivity to eribulin versus vinorelbine and paclitaxel in a panel of 100 CCLE cell lines and identified key molecular determinants of response and resistance unique to eribulin. Pathway expression analysis revealed key pathways significantly associated with response to eribulin, including those associated with oxidative stress-induced senescence, histone deacetylation, DNA methylation and Notch signaling. The oxidative stress-induced senescence pathway was significantly enriched for genes whose pretreatment/basal mRNA expression levels were positively associated with response to eribulin but not vinorelbine or paclitaxel. Expression levels of various tubulin isoforms were also identified as positively correlated with eribulin response. Higher and lower pretreatment expression of E-cadherin and N-cadherin, respectively, were also found in cell lines sensitive to eribulin treatment. Conclusion: Pretreatment gene expression analysis demonstrated key gene sets and molecular pathways that may drive response and resistance to eribulin and may differentiate this drug from vinorelbine and paclitaxel. Detailed analysis will be presented at the meeting. Citation Format: Pallavi Sachdev, Roy Ronen, Janusz Dutkowski, Bruce A. Littlefield. Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE) [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 1924.

Abstract 4183: Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [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 4183.

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation.

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions, and oxidative damage has been implicated in several neurodegenerative diseases. End-binding protein 1 (EB1) is a master regulator of microtubule plus-end tracking proteins (+TIPs) and is critically involved in the control of microtubule dynamics and cellular processes. However, the impact of glutathionylation on EB1 functions remains unknown. Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation. In vitro biochemical and cellular assays reveal that EB1 is glutathionylated. Diamide, a mild oxidizing reagent, reduces EB1 comet number and length in cells, indicating the impairment of microtubule dynamics. Three cysteine residues of EB1 are glutathionylated, with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics. In addition, glutaredoxin 1 (Grx1) deglutathionylates EB1, and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration, suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity. Collectively, these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.

Validation of Systemic and Local Tumour Immune Response to Eribulin Chemotherapy in the Treatment of Breast Cancer.

In addition to its cytocidal effects as a microtubule dynamics inhibitor, eribulin mesylate (eribulin) regulates the tumour microenvironment. We examined the clinical significance of tumour infiltrating lymphocytes (TILs) and transforming growth factor-β (TGF-β), which are local markers of host immunity, and of the neutrophil-lymphocyte ratio (NLR) and absolute lymphocyte count (ALC), which are systemic markers. We administered eribulin chemotherapy to 106 patients with locally advanced or metastatic breast cancer. Of these, 21 had their lesions resected. The response to eribulin was significantly associated with ALC (p=0.007). The expression of pSmad2 (an indicator of activation of TGF-β downstream signaling) was significantly decreased before and after eribulin chemotherapy (p<0.001). Moreover, a baseline ALC ≥ 1,500 /μl was observed in a significantly high number of patients with pSmad2 negative conversion (p<0.001). Eribulin improved the tumour immune microenvironment by decreasing TGF-β expression. This demonstrated that local change can be evaluated based on ALC.

A phase I/II study of eribulin mesilate (ERI) plus irinotecan (IRI) in children with refractory or recurrent solid tumors.

10535 Background: ERI is an inhibitor of microtubule dynamics. IRI is used to treat pediatric sarcomas. In the pediatric preclinical testing program, ERI was well tolerated and had promising activity compared with vincristine (a common chemotherapeutic for pediatric cancers) for various solid tumors ( in vivo xenograft panels). Methods: Children with relapsed/refractory solid tumors (excluding active central nervous system tumors) were enrolled. Prior treatment with IRI was allowed. Dose escalation was conducted for 2 schedules with the primary objective (phase 1) of determining the maximum tolerated dose and the recommended phase 2 dose: (A) ERI 1.4 mg/m2 IV infusion (Days 1 + 8 of 21-day cycle) + IRI 20 or 40 mg/m2 IV infusion (Days 1–5 of 21-day cycle); (B) ERI 1.4 mg/m2 IV infusion (Days 1 + 8 of 21-day cycle) + IRI 100 or 125 mg/m2 IV infusion (Days 1 + 8 of 21-day cycle). Safety and pharmacokinetic parameters were assessed. Results: 13 patients (pts) were enrolled (median age: 9 years [range: 3–17]); 4 pts had rhabdomyosarcoma (RMS), 2 had non-RMS soft tissue sarcoma, 2 had Ewing sarcoma, 2 had hepatoblastoma, 2 had nephroblastoma, and 1 had neuroblastoma. Overall, 7 pts previously received IRI. No dose-limiting toxicities (DLTs) were reported for either schedule. At data cut-off (July 14, 2019), 4 pts (with Ewing sarcoma, neuroblastoma, RMS, or hepatoblastoma) continued treatment (A, n = 2; B, n = 2) and 9 pts discontinued treatment (primarily for radiographic progression, n = 7 [A, n = 4; B, n = 3]). All pts experienced ≥1 treatment-emergent adverse event (TEAE); the most common any grade TEAE was neutropenia (n = 10; A, n = 5; B, n = 5). 11 pts had grade ≥3 TEAEs (A, n = 6; B, n = 5); the most frequent grade ≥3 TEAE was neutropenia (n = 9; A, n = 5; B, n = 4). No pt withdrew ERI or IRI due to an AE; 3 pts had dose reductions for ERI (A, n = 1; B, n = 2) and 3 pts had dose interruption of ERI (A, n = 2; B, n = 1) due to AEs. No pt had dose reductions for IRI, and 3 pts had dose interruption of IRI (A, n = 2; B, n = 1) due to AEs. 3 deaths occurred; 2 deaths were the result of tumor progression (A, n = 1; B, n = 1), and the cause of 1 was unknown (B). 1 pt with neuroblastoma treated according to schedule A had a partial response. Systemic exposures of ERI, IRI, and SN-38 (IRI active metabolite) were similar between schedules and doses. Conclusions: No DLTs were observed. Toxicity was manageable. Administration of IRI treatment on Days 1–5 is widely used in pediatric cancers; enrollment for phase 2 is ongoing with treatment Schedule A (ERI 1.4 mg/m2 Days 1 + 8 of 21-day cycle; IRI 40 mg/m2 Days 1–5 of 21-day cycle). Clinical trial information: NCT03245450.

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1-amplification to paclitaxel by targeting APC/C and the pro-survival protein MCL-1.

e18065 Background: Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. Methods: To do this, we used a specific anaphase-promoting complex/ cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the anti-apoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells with CCNE1-amplification that are challenged with clinically relevant dose of paclitaxel. Results: Using time-laps microscopy we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cells and restores death in mitosis (DIM). Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient´s survival. Conclusions: Our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel efficacy in this highly lethal gynecological disease.

A novel benzodiazepine derivative that suppresses microtubule dynamics and impairs mitotic progression.

A novel 2,3-benzodiazepine-4 derivative, named 1g, has recently been shown to function as an anti-proliferative compound. We now show that it perturbs the formation of a functional mitotic spindle, inducing a spindle assembly checkpoint (SAC)-dependent arrest in human cells. Live analysis of individual microtubules indicates that 1g promotes a rapid and reversible reduction in microtubule growth. Unlike most anti-mitotic compounds, we found that 1g does not interfere directly with tubulin or perturb microtubule assembly in vitro The observation that 1g also triggers a SAC-dependent mitotic delay associated with chromosome segregation in Drosophila neural stem cells, suggests that it targets a conserved microtubule regulation module in humans and flies. Altogether, our results indicate that 1g is a novel promising anti-mitotic drug with the unique properties of altering microtubule growth and mitotic spindle organization.

High absolute lymphocyte counts are associated with longer overall survival in patients with metastatic breast cancer treated with eribulin\u2014but not with treatment of physician\u2019s choice\u2014in the EMBRACE study

BackgroundEribulin, a nontaxane synthetic inhibitor of microtubule dynamics, is widely used to manage locally advanced or metastatic breast cancer (MBC). Eribulin has demonstrated immunomodulatory activity on the tumour microenvironment. Baseline neutrophil-to-lymphocyte ratio (NLR), a marker of immune status, may predict progression-free survival in eribulin treatment. This post hoc analysis assessed predictors for overall survival (OS).MethodsThe phase 3 open-label study (EMBRACE) of eribulin versus treatment of physician’s choice (TPC) in patients with MBC provided source data. Baseline absolute lymphocyte counts (ALCs) and NLR were evaluable in 751 and 713 patients, respectively.ResultsEribulin prolonged OS versus TPC in patients with baseline ALC ≥ 1500/µl (hazard ratio [HR] 0.586; 95% confidence interval [CI] 0.437–0.784; P < 0.001). There was no significant difference by treatment for ALC < 1500/µl (HR 1.002; 95% CI 0.800–1.253; P = 0.989). Univariate and multivariate analyses were performed and identified baseline ALC as a potential predictor of OS in eribulin-treated patients. Interaction analysis of OS supported 1500/µl as a potentially differential cutoff value. NLR at a cutoff value of 3 was associated with prolonged OS (eribulin group). However, similar results were also observed in the TPC group, without apparent interaction effect, suggesting that NLR may be a general prognostic marker rather than a specific predictor of OS for eribulin.DiscussionThis hypothesis-generating study speculates that baseline ALC may be an independent predictor for longer OS in eribulin-treated MBC patients and could be clinically impactful because it can be evaluated without the need for additional invasive procedures.Trial Registrationwww.ClinicalTrials.gov code: NCT00388726

Abstract P4-10-08: Absolute lymphocyte count (ALC) is a predictor of eribulin benefit in advanced or metastatic breast cancer (MBC)

Abstract Objective: This post hoc analysis assessed predictors for OS in patients (pts) treated with eribulin using data from 2 phase 3 studies: EMBRACE and Study 301. Background: Eribulin is a nontaxane synthetic inhibitor of microtubule dynamics approved for use in locally advanced or metastatic breast cancer after two previous lines of chemotherapy. In EMBRACE, 508 pts were randomized to eribulin monotherapy and 254 pts to the treatment of physician’s choice (TPC). Median OS of pts in the eribulin arm was significantly better than the TPC arm but no significant difference was seen in progression-free survival (PFS) by independent review. Study 301 randomized 554 pts to eribulin and 548 pts to capecitabine; no significant difference was seen in PFS. Kashiwagi et al, PloS one, 2017, suggested that cells such as tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment may be a predictive marker of eribulin efficacy—pts with a high number of TILs had significantly longer PFS than pts with low TIL counts. Miyagawa et al, Clin Breast Cancer, 2018, found neutrophil-to-lymphocyte ratio to be significantly associated with longer PFS with eribulin. Our analysis focused on ALC, a peripheral immune parameter. Methods: Pts from EMBRACE with baseline ALCs were included in this analysis. The Kaplan-Meier method was used to estimate OS distribution. Interaction analysis of OS was performed between treatment and baseline ALCs to explore differential cutoff values. Hazard ratios (HR) were estimated from the stratified Cox proportional hazard model with the randomization stratification factors: HER2/neu status, prior capecitabine treatment, and geographical region. Multivariate analyses for baseline factors were performed using the Cox proportional hazard model to identify predictors and to confirm baseline ALC effect for OS. Similar methods were used to analyze associations between baseline ALC and OS in Study 301. Results: EMBRACE included 500 pts in the eribulin arm and 251 in the TPC arm with an evaluable baseline ALC. Pts with high baseline ALC consistently experienced greater eribulin benefit vs pts with low baseline ALC across cutoff values from 1300-1750/μL. The greatest difference was observed at the cutoff value of 1500/μL. Median OS was higher in pts with baseline ALC ≥1500/µL vs baseline ALC &amp;lt;1500/µL in the eribulin arm (15.6 vs 11.6 months) but not in the TPC arm (Table). The eribulin group showed prolonged OS compared with the TPC group in pts with ALC ≥1500/µL (HR 0.586; 95% CI: 0.437-0.784; P&amp;lt;0.001). There was no significant difference by treatment for ALC &amp;lt;1500/µL (HR 1.002; 95% CI: 0.800-1.253; P=0.989). Study 301 included 553 pts on eribulin and 547 pts on capecitabine. Similar results were observed in the eribulin arm, median OS was higher in pts with baseline ALC ≥1500/µL vs pts with baseline ALC &amp;lt;1500/µL (19.6 vs 14.6 months). There was a treatment effect favoring eribulin vs capecitabine at ALC ≥1500/µL but not at ALC &amp;lt;1500/µL. Conclusion: ALC at baseline may potentially be an independent predictor for longer OS in pts treated with eribulin. This hypothesis-generating study speculates that baseline ALC may be useful in the selection of pts to be treated with eribulin. Table. Multivariate analysis of OS identified the following predictors for OS in the eribulin group: prior capecitabine, ECOG PS, HR status, number of organs involved, refractory to taxanes, and baseline ALCStudyBaseline ALCArmPatients, nMedian OS (months)HR (95% CI)EMBRACEALC ≥1500/µLEribulin19915.60.586 (0.437-0.784)TPC9211.4ALC &amp;lt;1500/µLEribulin30111.61.002 (0.800-1.253)TPC15910.3Study 301ALC ≥1500/µLEribulin23319.60.811 (0.662-0.993)Capecitabine26916.0ALC &amp;lt;1500/µLEribulin32014.60.907 (0.758-1.084)Capecitabine27812.5ALC, absolute lymphocyte count; CI, confidence interval; HR, hazard ratio; OS, overall survival; TPC, treatment of physician’s choice. Citation Format: Javier Cortes, Jose Manuel Perez-Garcia, Kenichi Nomoto, Karenza Alexis, Kenichi Saito, Yuta Yoshimura, Kenzo Muramoto, Yasuo Miyoshi. Absolute lymphocyte count (ALC) is a predictor of eribulin benefit in advanced or metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-08.