Microtubule Dynamics Inhibitor Research Articles

An intracerebral microdialysis study to determine the neuropharmacokinetics of eribulin in patients with metastatic or primary brain tumors.

Eribulin is an inhibitor of microtubule dynamics. It is not as highly protein bound as the taxanes and is less vulnerable to extrusion by P-glycoprotein in the blood-brain barrier (BBB). These features predict that eribulin could play an active role in managing brain tumors. Indeed, the small amount of published clinical data indicates eribulin may have some efficacy against breast cancer brain metastases. To better understand the potential of eribulin for treating brain tumors, we performed an intracerebral microdialysis study to determine the neuropharmacokinetics of eribulin in cancer patients undergoing tumor resection. After tumor removal, two microdialysis catheters were inserted into peritumoral brain tissue. Approximately 24h after surgery, a single dose of eribulin 1.4mg/m2 was administered intravenously. Dialysate samples were collected continuously for 72h, with plasma samples collected in parallel. Eribulin concentrations were analyzed by tandem mass spectrometry. Dialysate samples from 12 intracerebral microdialysis catheters placed in 7 study participants were included in the analysis. A statistically significant difference was observed between eribulin concentrations in brain tissue where BBB was disrupted versus intact, with a difference in mean maximum concentrations on log2 scale of 3.37 (std err = 0.59, p-value = 0.005). Nonetheless, overall brain to plasma ratios of eribulin only ranged from 0.13 to 1.99%. Although we could detect higher concentrations of eribulin in brain tissue where BBB was disrupted, intracerebral eribulin levels were not sufficient to predict eribulin would have consistent clinically meaningful activity against tumors in the brain. NCT02338037 (January 9, 2015).

Abstract A079: SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, with only 12% of patients living longer than five years after diagnosis. Recent studies have shown that inhibiting microtubule dynamics may be a promising therapeutic avenue for treating patients with this cancer. Microtubules are critical in cancer cell growth and division, as well as intracellular cytoskeletal support and internal trafficking. In PDAC, βIII- and βIVb-tubulin are highly upregulated in contrast to normal pancreas cells. Decreased expression of βIII- and βIVb-tubulin, through silencing or inhibition, correlates with reduced PDAC cell growth, tumorigenic potential, metastases in xenograft models, and chemoresistance. We hypothesized that a novel colchicine binding site beta-tubulin inhibitor, SB-216, will decrease cell growth while also decreasing the expression of oncogenic βIII- and βIVb-tubulin. To examine the anti-proliferative effects, PDCL110, a PDAC patient-derived cell line, cells were seeded (6,000 cells/well), and after 24 hours, treated with 0.1% DMSO (control), 2 nM, 5 nM, 10 nM, or 15 nM SB-216. Plates were analyzed on the IncuCyte Live-Cell Analysis System for six days, where phase contrast images of confluency were captured every four hours. To characterize the mRNA expression of βIII- and βIVb-tubulin, Panc-1 and PDCL110 cells were treated with 0.1% DMSO and 5 nM SB-216 for 48 hours. Next, total RNA was extracted using a TRIzol reagent. cDNAs were prepared using the SYBR Green RNA Reverse Transcription kit, and mRNA expression of beta-tubulin isotypes was quantified using the QuantStudio Real-Time PCR system. The expression of tubulin isotypes was normalized with the internal control ACTIN. Cell growth curves illustrated a significant (p<0.006) decrease in proliferation after 48 hours of treatment at all tested concentrations of SB-216. After 72 hours, cell growth inhibition became more significant (p<0.0001) in a dose-dependent manner. SB-216 treatment of a commercially available PDAC cell line (Panc-1) led to decreased mRNA expression of TUBB3 (class βIII, p<0.05) and TUBB4B (class βIVb, p<0.05) compared to DMSO control. Similarly, TUBB4B expression was significantly (p<0.05) decreased in the PDCL110 cells upon SB-216 treatment. These results enhance our confidence in utilizing SB-216 as a therapeutic agent against PDAC. Future studies are directed at elucidating the effects of SB-216 on metastases, chemoresistance, and mitochondrial function. Through an improved understanding of the mechanisms behind microtubule inhibition, we can meet the urgent clinical need of finding targeted treatments for this complex cancer. Citation Format: Lauren C Gattie, Chun Cai, Rui Wang, Wei Li, Evan Glazer. SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A079.

The effects of the combination of temozolomide and Eribulin on T98G human glioblastoma cell line: an ultrastructural study

ABSTRACT Glioblastoma tumors are the most aggressive primary brain tumors that develop resistance to temozolomide (TMZ). Eribulin (ERB) exhibits a unique mechanism of action by inhibiting microtubule dynamics during the G2/M cell cycle phase. We utilized the T98G human glioma cell line to investigate the effects of ERB and TMZ, both individually and in combination. The experimental groups were established as follows: control, E5 (5 nM ERB), T0.75 (0.75 mM TMZ), T1 (1.0 mM TMZ), and combination groups (E5+T0.75 and E5+T1). All groups showed a significant decrease in cell proliferation. Apoptotic markers revealed a time-dependent increase in annexin-V expression, across all treatment groups at the 48-hour time point. Caspase-3, exhibited an increase in the combination treatment groups at the 48-hour mark. Transmission electron microscopy (TEM) revealed normal ultrastructural features in the glioma cells of the control group. However, treatments induced ultrastructural changes within the spheroid glioblastoma model, particularly in the combination groups. These changes included a dose-dependent increase in autophagic vacuoles and apoptotic morphology of the cells. In conclusion, the similarity in the mechanism of action between ERB and TMZ suggests the potential for synergistic effects when combined. Our results highlight that this combination induced severe damage and autophagy in glioma spheroids after 48 hours.

Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer

ObjectiveOvarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. MethodsThe human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with BNZ-111, and cell proliferation, apoptosis, and cell cycle were assessed. ResultsIt demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. ConclusionBNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology.

Efficacy and safety of eribulin-based chemotherapy in patients with advanced breast cancer: A single-centre retrospective study.

e13121 Background: Eribulin, a nontaxane microtubule dynamics inhibitor, which was approved for the third-line treatment of advanced breast cancer (ABC) in China in 2019. This retrospective study aimed to assess efficacy and safety of Eribulin-based chemotherapy in ABC patients, especially for patients exhibiting HER2-0 and HER2-low status (immunohistochemistry [IHC] 1+ or IHC 2+with a negative in situ hybridization assay). Methods: This single-center retrospective study planned to include 126 ABC patients from January 2020 to December 2023. Enrolled participants received Eribulin either as monotherapy or in combination with other regiments such as anti-angiogenic therapy, PD-1 inhibitor, or alternative chemotherapy in clinical practice. The primary outcomes included progression-free survival (PFS) and safety assessments. Results: A total of 84 patients with ABC received Eribulin were enrolled. Patients' baselines were summarized as follows: The median age was 53.3 years (range 30.0–86.0 years). 53 (63.1%) patients were HER2-low while 40 (47.6%) patients were hormone receptor (HR) positive. 63.1% patients have received at least one line chemotherapy before the use of Eribulin. According to the K-M survival analysis, the median PFS was 6.0 (range 3.2-11.3) months for all patients. The median PFS was longer for patients received aromatase inhibitors (AI) for over 6 months than patients with AI naive (median 10.1 vs 6.6 months; p=0.192) in HR positive patients. Patients who received CDK4/6 therapy had longer PFS than patients (median 10.7 vs 6.6 months; p=0.38). There was a trend toward prolonged PFS in the HER2-0 group compared with HER2-low group (median 10.13 vs 6.03 months; p=0.154), although there was no significant difference. A nonsignificant improvement of PFS was observed in HR-/HER2-0 patients, compare with HR-/HER2-low patients (median 16.4 vs 4.7mo; p=0.21). Meanwhile the median PFS of HER2-0 and HER2-Low was similar in HR positive patients. The objective response rate was 35.5% in HER2-0 group and 30.2% in HER2-low group. In multivariate cox regression, Eribulin as first line treatment (HR, 4.028; 95% CI, 1.131-14.337) and HER2-0 (HR, 2.903; 95% CI, 1.257-6.702) showed prognostic values regarding PFS. Common grade 3 or 4 adverse events that were reported in more than 10% were neutropenia (41.6%) and leukopenia (26.2%). No treatment-related deaths were reported. Conclusions: This single-center study demonstrated encouraging efficacy and safety in the ABC patients treated with Eribulin. Significant PFS benefit was associated with Eribulin treatment in patients with HER2-0 compared with HER2-low. This study still needs longer follow-up to evaluate its efficacy more comprehensively.

Abstract PO3-05-10: Efficacy and safety of eribulin plus gemcitabine in second-line or beyond for patients with HER2-negative metastatic breast cancer (MBC): A multi-center, open-label, single-arm, phase II study

Abstract Background: Eribulin is a halichondrin non-taxane inhibitor of microtubule dynamics approved in China for advanced breast cancer patients who have received at least two prior chemotherapy treatments. The combination of eribulin and gemcitabine has demonstrated a similar progression-free survival (PFS) benefit as paclitaxel plus gemcitabine, with less neurotoxicity, for patients with MBC who have not received prior cytotoxic chemotherapy. However, the effect of eribulin plus gemcitabine on PFS in second line or beyond remains unclear. Methods: This open-label, single-arm, phase II study (NCT05263882) was conducted at 13 institutions in China. Eligible patients had histologically confirmed HER2-negative MBC and had received at least one prior taxane-containing chemotherapy regimen for advanced disease, and anthracycline-containing regimens in the adjuvant setting. Patients received intravenous infusions of eribulin (1.4 mg/m2) and gemcitabine (1.0 g/m2) on days 1 and 8 of a 21-day cycle. Efficacy outcomes, including PFS, objective response rate (ORR), and disease control rate (DCR), were assessed using RECIST v1.1. Adverse events (AEs) were graded according to NCI-CTC version 5.0. Results: A total of 65 patients were enrolled from November 2021 to May 2023; 47 (72.3%) had HR+/HER2- and 18 (27.7%) had triple-negative MBC. The median patient age was 50 years (range: 31-68), and the sites of metastasis were the bone (70.8%), liver (58.5%), lymph nodes (50.8%), lung (43.1%) and brain (10.8%). Patients had received a median of 3 prior lines of systemic treatment, 2 lines of chemotherapy, and 1 line of endocrine treatment. Among all patients, the ORR was 52.3%, the DCR was 92.3% and the median PFS was 7.6 months (Table). For the HR-positive subgroup, the median PFS was 8.4 months, while for the triple-negative subgroup, it was 7.6 months. Among patients who had received prior CDK4/6 inhibitor treatment, the median PFS was 7.2 months. In the subgroup of patients who had not received CDK4/6 inhibitor treatment, the median PFS had not been reached. The most common grade 3-4 AEs were hematological, including neutropenia (41.6%), leukopenia (33.9%), anemia (23.1%), and thrombocytopenia (15.4%). No grade ≥3 perceived AEs were reported. Conclusion: Eribulin plus gemcitabine was effective in heavily pretreated patients with HER2- MBC, while maintaining a predictable and manageable safety profile. Table. Summary of efficacy outcomes Citation Format: PeiJian Peng, Xiao-Lu Xu, Jin-Cai Zhong, Jin-Hui Ye, Zhi-Hui Wang, Hong Wang, Hong Lin, Cai-Wen Du, Guorong Zou, Jie Ouyang, Ying-Ying Shi, Fei Xu, Geng-Sheng Yu, Yong-Kui Lu, Yong-Xia Wang, Shi-En Cui, Lu-Zhen Li. Efficacy and safety of eribulin plus gemcitabine in second-line or beyond for patients with HER2-negative metastatic breast cancer (MBC): A multi-center, open-label, single-arm, phase II study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-10.

Abstract PO1-06-14: Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC)

Abstract Background: Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence indicate that PI3K pathway activation maintains the stemness and chemoresistance of breast cancer stem cells (CSCs). PI3K inhibition sensitizes CSCs to chemotherapy. Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed overall survival benefit in metastatic HER2 negative BC. Preclinically, the addition of copanlisib (C), a potent pan-class I PI3K inhibitor that is highly selective against α and δ PI3K isoforms, improved anti-tumor effect in both E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN alteration status. Here we report the preliminary data of the phase I dose escalation trial of E+C in patients with metastatic TNBC. Methods: This trial includes a phase I study to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a randomized phase II study of E+C (at RP2D) versus E (stratified by tumor PIK3CA/PTEN mutation status) with progression-free survival (PFS) as the primary endpoint. Key eligibility included patients with: metastatic TNBC who progressed on ≤5 lines of chemotherapy, prior anthracycline/taxane chemotherapy (unless contraindicated), ECOG 0-1, adequate organ function and known tumor PIK3CA/PTEN/AKT mutation status. Key exclusions include: prior receipt of E or any PI3K/mTOR/AKT inhibitor, grade ≥2 neuropathy, presence of tumor AKT mutation, and uncontrolled diabetes, hypertension or congenital QT prolongation. A 3+3 design was used to determine the DLT and RP2D. Dose level 1 (DL1) started with E at 1.1 mg/m2 mg IV and C at 45 mg IV on days (D) 1/8 of 21-D cycle, with the potential to escalate to 2 additional dose levels – DL2: E 1.4 mg/m2 and C 45 mg on D 1/8; DL3: E 1.4 mg/m2 and C 60 mg on D 1/8. Results: Eight patients were enrolled in the phase I study and received at least 1 dose of E+C. The median age was 48.5 (range, 26-67) years. The majority of patients identified as White (additionally 2 Hispanic, 1 Black) and had an ECOG performance status of 1 (n=6, 75.0%). Tumor PIK3CA and PTEN alterations were each observed in 1 patient. Median number of cycles received were 2 (range, 1-5). All 8 patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all dose levels were: lymphopenia (n=5, 62.5%), neutropenia (n=5, 62.5%), hyperglycemia (n=4, 50.0%), anemia (n=4, 50.0%) and fatigue (n=4, 50.0%); most toxicities were grade (G) 1-2. G3 hyperglycemia and hypertension were observed in 1 patient each, were medically managed and did not lead to treatment interruption. 1 patient was diagnosed with pulmonary embolism, possibly attributed to study therapy and breast cancer. DLTs were reported in 2 of 2 patients treated at DL2 - 1 patient developed G3 rash responsive to oral corticosteroids, and 1 patient developed G3 acute kidney injury in the setting of hypovolemia and discontinued study therapy. No additional patients were enrolled in DL2. No DLTs were observed in all 6 patients treated at DL1. 1 patient treated at DL1 had possible G1 non-infectious pneumonitis that resolved spontaneously on short interval imaging. Stable disease was observed in 2 of 6 response-evaluable patients (33.3%). 1 patient achieved regression of cutaneous disease following 1 cycle of therapy as assessed by the treating investigator. DL1, E at 1.1 mg/m2 plus C at 45 mg IV, was determined to be the RP2D. Tumor tissue biomarker analysis is underway. Conclusion: These data indicate that E+C at DL1 is a well-tolerated regimen warranting further investigation. The study is actively enrolling patients to the phase II study. Clinical trial information: NCT04345913. Funding Source: National Cancer Institute. Citation Format: Nusayba Bagegni, Brittney Haas, Leslie Nehring, Jingqin Luo, Laura Kennedy, Meghna Trivedi, Manali Bhave, Rabih Said, Cynthia Ma. Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-14.

Involvement of ferroptosis in eribulin-induced cytotoxicity in ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a unique clinicopathological subtype of epithelial ovarian cancer that is resistant to standard chemotherapy. Eribulin, a microtubule dynamics inhibitor of halichondrin class, has unique effects in the cancer microenvironment such as induction of epithelization and reduction in metastatic potential in breast cancer cells; however, nothing is known about the effect of eribulin and the detailed mechanisms in OCCC. This study aimed to investigate the involvement of ferroptosis and its mechanism in the antitumor activity of eribulin in OCCC cells and a mouse xenograft model. We found that eribulin-induced cell death was reduced by ferroptosis inhibitors; deferoxamine, an iron chelator and ferrostatin-1, a lipid peroxidation inhibitor. Eribulin increased the levels of intracellular iron, reactive oxygen species (ROS), and lipid peroxides, and increased the mitochondrial membrane potential. Eribulin downregulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), and superoxide dismutase (SOD) activity. The combination of eribulin and ML210, a glutathione peroxidase 4-inhibiting ferroptosis inducer, had a synergistic effect on ferroptosis. Taken together, our findings show firstly that eribulin triggers ferroptosis in OCCC and this effect occurs via the suppression of the Nrf2–HO-1 signaling pathway, SOD activity and the promotion of lipid peroxidation. These findings suggest that eribulin-induced ferroptosis is associated with its anti-tumor effect and also could be a potential therapeutic target in OCCC.

Abstract 7139: Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer

Abstract Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. With improved pharmacokinetic parameters, BNZ-111 effectively disrupts tubulin polymerization through hydrogen bonds and hydrophobic interactions. It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. This study suggests that BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology Citation Format: Jeong-Won Lee, Kwangho Lee, Byumseok Koh, Yoo-Young Lee, Duk-Soo Bae. Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer [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 7139.

Abstract 5260: Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling activity. In the previous meeting (AACR2022, 2023), we reported that ERI and liposomal formulation of ERI (ERI-LF) also have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF showed more potent immune modulation and combination activities with anti-PD-1 antibody (PD-1 Ab) than ERI in syngeneic mice models and humanized mice models. Although immune-checkpoint inhibitors (ICI) are using as a key drug for various types of cancers in clinical setting, several clinical questions regarding suitable therapies after ICI therapy and efficacy of ICI rechallenge are remaining. In this study, we evaluated anti-tumor activities of combination of ERI-LF and PD-1 Ab in two syngeneic transplantation models using mouse non-small lung cancer cells, P-glycoprotein-knockout (Pgp-KO) KLN205 cells and Pgp-KO LL2 cells. As a result, combination of 1mg/kg ERI-LF (Q7D×2) with PD-1 Ab (200 ug/head, twice a week ×2 weeks) showed significant stronger antitumor activity compared with each monotherapy in only Pgp-KO KLN205 model, in which ERI-LF increased intratumoral microvascular density as a vascular remodeling activity, not in Pgp-KO LL2 model. We next conducted continuous treatment of PD-1 Ab in Pgp-KO KLN205 model to investigate tumor regrowth during treatment of PD-1 Ab. In this model, tumor growth was inhibited by PD-1 Ab until about two weeks after initiation of the treatment and then this inhibitory activity disappeared after two weeks even by continuous treatment of PD-1 Ab. We performed flow cytometry analysis (FCM) for tumor-infiltrating immune cells and RNAseq analysis using regrowing tumors compared with non-treatment tumors. Expressions of mRNA of several immune inhibitory receptors were upregulated in tumors with regrowth, although CD4+ cells and CD8+ cells were also increased in FCM using tumor samples. Furthermore, we investigated anti-tumor activity of combination of ERI-LF and PD-1 Ab against tumors which changed from inhibitory phase to regrowth phase during PD-1 Ab treatment in Pgp-KO KLN205 model. The combination of ERI-LF at 1mg/kg (Q7D×3) and PD-1 Ab (200 ug/head, twice a week ×3 weeks) also showed potent anti-tumor activity against tumors with regrowth by prior PD-1 Ab treatment as well as PD-1 Ab-naïve tumor, suggesting that this combination therapy might be effective after prior ICI therapy. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers (NCT04078295) is underway. Patients with or without prior ICI therapy were enrolled in a small cell lung cancer cohort of this clinical study. Our preclinical results might provide a scientific rationale for a uniqueness of ERI-LF as a post ICI therapy. Citation Format: Moe Tamura, Yuki Niwa, Taro Semba. Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice [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 5260.

Biosynthesis of the highly oxygenated tetracyclic core skeleton of Taxol

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9α-hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1β-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5α-ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.

Impaired Microtubule Dynamics Are a Driver of Tau Phosphorylation

AbstractBackgroundThe microtubule cytoskeleton is both stable and dynamic, and both states are necessary for normal neuronal function. Stable microtubules support long‐term structures like dendrites and axons, while dynamic microtubules promote synapse maintenance and are mostly composed of tyrosinated a‐tubulin, a condition that affects microtubule associated protein binding and motor protein recruitment. We have recently shown that human iPSC neurons carrying the familial AD APP London V717I mutation have impaired microtubule dynamics and significant increases in tubulin post‐translational modification (PTMs) associated with microtubule longevity. These changes result from decreased levels of the enzyme tubulin tyrosine ligase (TTL), which refreshes detyrosinated tubulin to its original, tyrosinated state. In human AD brains, TTL levels also decrease, while long‐lived microtubule PTMs increase. Here, we report that by compromising normal microtubule dynamics or by reducing TTL expression alone we can drive tau phosphorylation in rodent hippocampal neurons and explore the mechanisms by which this regulation occurs.MethodHuman cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Inhibition of microtubule dynamics was performed with taxol or through lentiviral knockdown of TTL. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure protein levels.ResultWe found that in addition to increased levels of p262 tau, APP London neurons possess higher levels of active AMPK, a kinase that can phosphorylate serine 262 on tau and an important regulator of tau pathology in vitro and in vivo. We also demonstrate that decreasing microtubule dynamics with taxol or TTL silencing alone is sufficient to promote tau phosphorylation and that inhibition of AMPK or knockdown of its regulatory subunit can prevent it.ConclusionThese findings demonstrate that reduced levels of TTL and loss of dynamic microtubules drive AMPK‐mediated tau phosphorylation and suggest that TTL‐dependent loss of dynamic microtubules may contribute to tau pathology in AD.

Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

Amelioration of Tumor-promoting Microenvironment via Vascular Remodeling and CAF Suppression Using E7130: Biomarker Analysis by Multimodal Imaging Modalities.

E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFβ1 production, a precursor of TGFβ1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFβ1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFβ1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.

Incidence and Resolution of Eribulin-Induced Peripheral Neuropathy (IRENE) in Locally Advanced or Metastatic Breast Cancer: Prospective Cohort Study.

Eribulin, a halichondrin-class microtubule dynamics inhibitor, is a preferred treatment option for patients with advanced breast cancer who have been pretreated with an anthracycline and a taxane. Peripheral neuropathy (PN) is a common side effect of chemotherapies for breast cancer and other tumors. The Incidence and Resolution of Eribulin-Induced Peripheral Neuropathy (IRENE) noninterventional postauthorization safety study assessed the incidence and severity of PN in patients with breast cancer treated with eribulin. IRENE is an ongoing observational, single-arm, prospective, multicenter, cohort study. Adult patients (≥18 years of age) with locally advanced or metastatic breast cancer and disease progression after 1-2 prior chemotherapeutic regimen(s) for advanced disease were treated with eribulin. Patients with eribulin-induced PN (new-onset PN or worsening of preexisting PN) were monitored until death or resolution of PN. Primary endpoints included the incidence, severity, and time to resolution of eribulin-induced PN. Secondary endpoints included time to disease progression and safety. In this interim analysis (data cutoff date: July 1, 2019), 67 (32.4%) patients experienced any grade eribulin-induced PN, and 12 (5.8%) patients experienced grade ≥3 eribulin-induced PN. Median time to resolution of eribulin-induced PN was not reached. Median time to disease progression was 4.6 months (95% CI, 4.0-6.5). Treatment-emergent adverse events (TEAEs) occurred in 195 (93.8%) patients and serious TEAEs occurred in 107 (51.4%) patients. The rates of any grade and grade ≥3 eribulin-induced PN observed in this real-world study were consistent with those observed in phase III randomized clinical trials. No new safety findings were observed.

A randomized trial of eribulin monotherapy versus eribulin plus anlotinib in patients with locally recurrent or metastatic breast cancer

Eribulin mesylate is a novel, nontaxane, microtubule dynamics inhibitor. In this study, we assessed the efficacy and safety of eribulin versus eribulin plus the oral small-molecule tyrosine kinase inhibitor anlotinib in patients with locally recurrent or metastatic breast cancer. In this single-center, open-label, phase II clinical study (NCT05206656) conducted in a Chinese hospital, patients with human epidermal growth factor receptor 2 (HER2)-negative, locally recurrent or metastatic breast cancer previously treated with anthracycline- or taxane-based chemotherapy were randomized (1 : 1) to receive eribulin alone or in combination with anlotinib. The primary efficacy endpoint was investigator-assessed progression-free survival (PFS). From June 2020 to April 2022, a total of 80 patients were randomly assigned to either eribulin monotherapy or eribulin plus anlotinib combination therapy, with 40 patients in each group. The data cut-off was 10 August 2022. The median PFS was 3.5 months [95% confidence interval (CI) 2.8-5.5 months] for eribulin and 5.1 months (95% CI 4.5-6.9 months) for eribulin plus anlotinib (hazard ratio= 0.56, 95% CI 0.32-0.98; P= 0.04). The objective response rates were 32.5% versus 52.5% (P= 0.07), respectively, and disease control rates were 67.5% versus 92.5% (P= 0.01), respectively. Patients <50 years of age, with an Eastern Cooperative Oncology Group performance status score of 0, visceral metastasis, number of treatment lines of four or more, hormone receptor negative (triple-negative), and HER2 low expression appeared to benefit more from combined treatment. The most common adverse events in both groups were leukopenia (n= 28, 70.0%, patients in the eribulin monotherapy group versus n= 35, 87.5%, patients in the combination therapy group), aspartate aminotransferase elevations (n= 28, 70.0%, versus n= 35, 87.5%), neutropenia (n= 25, 62.5%, versus n= 31, 77.5%), and alanine aminotransferase elevations (n= 25, 62.5%, versus n= 30, 75.0%). Eribulin plus anlotinib can be considered an alternative treatment option for HER2-negative locally advanced or metastatic breast cancer.

Phase 2 small cell lung cancer (SCLC) cohort of a phase 1b/2 trial of a liposomal formulation of eribulin in combination with nivolumab.

8593 Background: E7389-LF is a liposomal formulation of the microtubule dynamics inhibitor eribulin. Combining E7389-LF with an immune checkpoint inhibitor (ICI; eg, nivolumab) may improve antitumor response via vascular remodeling. The phase 1b part of the open-label Study 120 evaluated the dosing and safety of E7389-LF in combination with nivolumab in patients (pts) with solid tumors. The phase 2 part assessed efficacy and safety in disease cohorts; herein, we report results from the SCLC cohort. Methods: Pts with unresectable and measurable SCLC who had progression during or after 1st-line (1L) platinum-based chemotherapy with/without an ICI were enrolled to the SCLC cohort of the phase 2 part of Study 120. Pts received E7389-LF 2.1 mg/m2 in combination with nivolumab 360 mg IV Q3W. The primary objective of the phase 2 part was to assess the objective response rate (ORR). Secondary objectives included assessments of safety and progression-free survival (PFS); exploratory objectives included the disease control rate (DCR), overall survival (OS), and biomarker analysis. Tumor response was assessed by investigators per RECIST v1.1 Q6W. All adverse events (AEs) were monitored and recorded. Results: In the SCLC cohort, 34 pts were enrolled; the median age was 66.0 years (range 46–81). At data cutoff (May 31, 2022), 5 pts (14.7%) were undergoing treatment. Discontinuations occurred in 29 pts (85.3%)—26 (76.5%) primarily due to disease progression, 3 (8.8%) due to an AE. Efficacy analyses included 33 pts, as 1 pt had their diagnosis of SCLC changed to a different cancer type after being enrolled. Of the 33 evaluable pts, 27 (81.8%) had an ICI as part of their prior 1L therapy. The ORR of E7389-LF in combination with nivolumab was 24.2% (95% CI 11.1–42.3) in the evaluable population; the DCR was 75.8% (95% CI 57.7–88.9). Median PFS was 3.98 mos (95% CI 2.63–4.40); the 6-mo PFS rate was 27.7% (95% CI 13.0–44.6). At a median follow-up period of 10.6 mos, median OS was not reached (95% CI not estimable); the 6-mo OS rate was 90.9% (95% CI 74.4–97.0). Treatment-related, treatment-emergent (TE)AEs of any grade and of grade ≥3 severity occurred in 97.1% (n = 33) and 82.4% (n = 28) of the 34 enrolled pts, respectively; the most common treatment-related TEAE was neutropenia (any grade: 58.8% [n = 20]; grade ≥3: 52.9% [n = 18]). The neutrophil count nadir occurred around cycle 1 day 8. TEAEs led to dose reduction of E7389-LF in 19 pts (55.9%). Withdrawal of E7389-LF or nivolumab occurred in 5 pts (14.7%): acute kidney injury, cough, myocarditis, pneumonitis, and radiation pneumonitis (n = 1 each). Changes in vascular and immune-related plasma markers were observed. Conclusions: E7389-LF in combination with nivolumab showed notable antitumor activity as 2L therapy in pts with SCLC, as evidenced by the notable ORR of 24.2%. No new safety signals were observed for this combination. Clinical trial information: NCT04078295 .

Abstract 5075: Antitumor activity of liposomal formulation of eribulin combined with anti-human PD-1 antibody using hPBMC-humanized mouse models

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling and reversal of epithelial-mesenchymal transition activities. In the previous meeting (AACR2022), we reported that ERI and liposomal formulation of ERI (ERI-LF) have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF has greater immunomodulatory activity than ERI. Furthermore, we also reported that these immunomodulatory activities contribute combination antitumor activity of ERI and ERI-LF with anti-PD-1 antibody (Ab) using a P-glycoprotein-knockout 4T1 mouse breast cancer syngeneic model; however, evaluation of antitumor activity of immune-checkpoint inhibitor (ICI) using mouse cancer syngeneic model has limitation because numbers and cancer types of mouse cancer cell lines are limited. In this study, we developed humanized mouse model bearing both human cancer cell lines and human peripheral blood mononuclear cells (hPBMC) to evaluate antitumor activity of ICI for various cancer types. Furthermore, we also evaluated combination antitumor activity of ERI-LF with anti-human PD-1 Ab using this hPBMC-humanized mouse models to verify generality of this combination. We first isolated hPBMC from healthy volunteers and the obtained hPBMC was intravenously injected into NOG mice. To confirm transplantation of hPBMC in the mice, we performed flow cytometric analysis of peripheral blood of mice in a time course manner. In the peripheral blood of humanized mice, chimera ratio of hCD45+ cells gradually increased until 5 weeks after hPBMC-injection and then decreased after 6 weeks after hPBMC-injection. Because tumor growth of some cancer cell lines was rejected by hPBMC transplantation, we evaluated some human cancer cell lines that are able to grow in hPBMC-humanized condition and selected MKN45 and NCI-H526 cell lines for human gastric cancer and small cell lung cancer models, respectively. Finally, we verified combination antitumor activity of ERI-LF with anti-human PD-1 Ab in hPBMC-humanized MKN45 and NCI-H526 models. In both models, combination of ERI-LF with anti-human PD-1 Ab showed significantly stronger antitumor activity compared with each monotherapy. In summary, we demonstrated that combination of ERI-LF with anti-PD-1 Ab is effective for gastric and small cell lung cancers, suggesting that this combination therapy is effective for various cancer types. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers including gastric and small cell lung cancers (NCT04078295) is underway. Citation Format: Yuki Niwa, Taro Semba. Antitumor activity of liposomal formulation of eribulin combined with anti-human PD-1 antibody using hPBMC-humanized mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5075.

Identification of Kinase Targets for Enhancing the Antitumor Activity of Eribulin in Triple-Negative Breast Cell Lines.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype of breast cancer, and current treatments are only partially effective in disease control. More effective combination approaches are needed to improve the survival of TNBC patients. Eribulin mesylate, a non-taxane microtubule dynamics inhibitor, is approved by the U.S. Food and Drug Administration to treat metastatic breast cancer after at least two previous chemotherapeutic regimens. However, eribulin as a single agent has limited therapeutic efficacy against TNBC. High-throughput kinome library RNAi screening, Ingenuity Pathway Analysis, and STRING analysis were performed to identify target kinases for combination with eribulin. The identified combinations were validated using in vivo and ex vivo proliferation assays. We identified 135 potential kinase targets whose inhibition enhanced the antiproliferation effect of eribulin in TNBC cells, with the PI3K/Akt/mTOR and the MAPK/JNK pathways emerging as the top candidates. Indeed, copanlisib (pan-class I PI3K inhibitor), everolimus (mTOR inhibitor), trametinib (MEK inhibitor), and JNK-IN-8 (pan-JNK inhibitor) produced strong synergistic antiproliferative effects when combined with eribulin, and the PI3K and mTOR inhibitors had the most potent effects in vitro. Our data suggest a new strategy of combining eribulin with PI3K or mTOR inhibitors to treat TNBC.

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease.