Microtubule Destabilization Research Articles

Elraglusib induces cytotoxicity via direct microtubule destabilization independently of GSK3 inhibition.

Elraglusib (9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK3) with pre-clinical studies demonstrating broad activity against many tumour types. Promising early-phase clinical trial data led to FDA orphan drug status, and a randomized phase 2 study in combination with cytotoxic chemotherapy in pancreatic cancer has recently completed its recruitment. Similarly, single-agent responses in adult T-cell leukaemia/lymphoma and melanoma, and combination treatment data in several other tumour types have been encouraging. The elraglusib mechanism of action is unknown, but it is unlikely to act through GSK3 inhibition because cytotoxicity is observed below the IC50 for GSK3 and other small molecule GSK3 inhibitors do not produce cytotoxic effects, at least in lymphoma cells. We show here that elraglusib perturbs chromosomal alignment to cause a mitotic arrest in multiple tumour lines. This arrest is caused by direct microtubule depolymerization, which prevents the attachment of kinetochores to microtubules. At clinically relevant doses, these mitotically arrested cells eventually undergo mitotic slippage, leading to gross chromosome missegregation, DNA damage and apoptosis. These effects explain the cytotoxicity of elraglusib because temporarily pausing cell cycle progression with the CDK4/6 inhibitor palbociclib abolishes any drug-induced genotoxicity and apoptosis. In summary, elraglusib acts as a direct microtubule destabilizer both in vitro and across multiple cancer types, resulting in mitotic arrest, DNA damage and apoptosis. These effects likely account for its broad pan-cancer activity, which does not rely upon GSK3 inhibition as they are not replicated by other GSK3 inhibitors.

Localization and regulation of yeast aldehyde dehydrogenase Ald4p structures

Previously, we identified yeast strains, namely SWORD, showing more robust Ald4p-GFP filament formation than the typical ALD4::GFP strains. Here, we report that Ald4p-GFP in SWORD strains favorably polymerize into gigantic structures in the cytoplasm, despite the enzyme being established as a mitochondrial resident. In addition, we have found that nocodazole, a microtubule destabilizer, has no effect on Ald4p high-order assembly, suggesting no direct association between microtubule dynamics and Ald4p structure formation. Ald4p assembly cannot be induced by sodium azide treatment, indicating that ATP is not a primary effector of Ald4p polymerization. Interestingly, addition of exogenous acetaldehyde, a substrate of the enzyme, can significantly enhance the structure formation of Ald4p, implying that structure formation may be related to enzymatic activity.

5H-benzo[c]fluorene derivative exhibits antiproliferative activity via microtubule destabilization

Present study aimed at a single component cyclization of 2-benzylidene-1-tetralones for the preparation of 5H-benzo[c]fluorenes and their antiproliferative activity. This ring closure reaction underwent via reductive cyclization in the presence of a sodium borohydride-aluminium chloride system. Ten diverse 5H-benzo[c]fluorene derivatives were prepared and evaluated for antiproliferative activity against three human cancer cell lines by SRB assay. Four of these benzofluorenes exhibited significant antiproliferative effect with an IC50 > 10.75 µM. The best representative compound 21, exhibited IC50 against K562 leukemic cells at 3.27 µM in SRB assay and 7.68 µM in Soft agar colony assay. It exhibited a microtubule destabilization effect in tubulin kinetics and inhibited 82.9 % microtubule polymer mass at 10 µM concentration in Protein Sedimentation assay (Microtubule). Compound 21 exerted G0/G1 phase arrest in cell division cycle analysis in K562 cells. It also induced apoptosis in K562 cells via activation of Caspase cascade pathway. Furthermore, compound 21 also possessed anti-inflammatory activity by inhibiting TNF-α and IL-6 moderately. It exhibited significant in vivo efficacy and reduced K562 tumour in xenograft mice by 47 % at an 80 mg/kg oral dose. Further, it was found to be safe and well tolerable up to 1000 mg/kg in Swiss albino mice. Compound 21 needs to be optimized for better in vivo efficacy in rodent models for further development.

Vinblastine Resistance Is Associated with Nephronophthisis 3-Mediated Primary Cilia via Intraflagellar Transport Protein 88 and Apoptosis-Antagonizing Transcription Factor.

Primary cilia (PC) are microtubule-based organelles that function as cellular antennae to sense and transduce extracellular signals. Nephronophthisis 3 (NPHP3) is localized in the inversin compartment of PC. Mutations in NPHP3 are associated with renal-hepatic-pancreatic dysplasia. In this study, we investigated whether vinblastine (VBL), a microtubule destabilizer, induces anticancer drug resistance through NPHP3-associated PC formation in HeLa human cervical cancer cells. A considerable increase in PC frequency was observed in HeLa cells under serum-deprived (SD) conditions, which led to the inhibition of VBL-induced cell death. VBL-resistant cells were established by repetitive treatments with VBL and showed an increase in PC frequency. NPHP3 expression was also increased by VBL treatment under serum starvation as well as in VBL-resistant cells. NPHP3 expression and PC-associated resistance were positively correlated with apoptosis-antagonizing transcription factor (AATF) and negatively correlated with inhibition of NPHP3. In addition, AATF-mediated NPHP3 expression is associated with PC formation via the regulation of intraflagellar transport protein 88 (IFT88). VBL resistance ability was reduced by treating with ciliobrevin A, a well-known ciliogenesis inhibitor. Collectively, cancer cell survival following VBL treatment is regulated by PC formation via AATF-mediated expression of IFT88 and NPHP3. Our data suggest that the activation of AATF and IFT88 could be a novel regulator to induce anticancer drug resistance through NPHP3-associated PC formation.

Lisavanbulin (BAL101553), a novel microtubule inhibitor, plus radiation in patients with newly diagnosed, MGMT promoter unmethylated glioblastoma.

Lisavanbulin (BAL101553) is a small, lipophilic, oral microtubule destabilizer with promising antitumoral activity observed in preclinical glioblastoma (GBM) models. This multicenter phase 1 study sought to determine the MTD of oral Lisavanbulin in combination with standard RT (60 Gy/30 fractions) but without temozolomide in patients with newly diagnosed MGMT promoter unmethylated GBM (uGBM). Dose escalation followed a modified 3 + 3 design. Secondary objectives included estimation of OS and PFS and pharmacokinetic analysis. Twenty-six patients with uGBM (median age, 63 years, 42.3% male, 61.5% with gross total resection, median Karnofsky performance status 80) were enrolled; 2 tumors had an IDH1 mutation. Predefined dose levels of Lisavanbulin, administered daily concomitantly with RT, were: 4mg (5 pts), 6mg (5 pts), 8mg (7 pts), 12mg (5 pts), and 15mg (4 pts). The initial starting dose was 8mg. Due to grade 4 aseptic meningoencephalitis in the first patient, the dose was decreased to 4mg. Dose escalation resumed and continued to 15mg with dose-limiting toxicities of grade 2 confusion and memory impairment observed at 12mg. Avanbulin exposures increased in a relatively dose-proportional manner with increasing oral dose of Lisavanbulin from 4 to 15mg. Lisavanbulin in combination with RT was considered safe up to the highest predefined oral dose level of 15mg daily.

Exploring microtubule dynamics in Alzheimer's disease: Longitudinal assessment using [11C]MPC-6827 PET imaging in rodent models of Alzheimer's-related pathology.

Microtubule (MT) stability is crucial for proper neuronal function. Understanding MT dysregulation is critical for connecting amyloid beta (Aβ) and tau-based degenerative events and early changes in presymptomatic Alzheimer's disease (AD). Herein we present positron emission tomography (PET) imaging properties of our MT-PET radiotracer, [11C]MPC-6827, in multiple established AD mouse models. Longitudinal PET, biodistribution, autoradiography, immunohistochemistry, and behavioral studies were conducted at multiple time points in APPswe/PSEN1dE9 (APP/PS1), P301S-PS19 (P301S), 5xFAD, and age-matched control mice. Longitudinal [11C]MPC-6827 brain imaging showed significant increases in APP/PS1, P301S, and 5xFAD mice compared to controls. Longitudinal MT-PET correlated positively with biodistribution, autoradiography, and immunohistochemistry results and negatively with behavior data. Our study demonstrated significant longitudinal [11C]MPC-6827 PET increases in multiple AD mouse models for the first time. Strong correlations between PET and biomarker data underscored the interplay of MT destabilization, amyloid, and tau pathology in AD. These results suggest [11C]MPC-6827 PET as a promising tool for monitoring MT dysregulation early in AD progression. Longitudinal positron emission tomography (PET) imaging studies using [11C]MPC-6827 in multiple established Alzheimer's disease (AD) mouse models revealed an early onset of microtubule dysregulation, with significant changes in brain radiotracer uptake evident from 2 to 4 months of age. Intra-group analysis showed a progressive increase in microtubule dysregulation with increasing AD burden, supported by significant correlations between PET imaging data and biodistribution, autoradiography, and molecular pathological markers. [11C]MPC-6827 PET imaging demonstrated its efficacy in detecting early microtubule alterations preceding observable behavioral changes in AD mouse models, suggesting its potential for early AD imaging. The inclusion of the 5xFAD mouse model further elucidated the impact of amyloid beta (Aβ) toxicity on inducing tau hyperphosphorylation-mediated microtubule dysregulation, highlighting the versatility of [11C]MPC-6827 in delineating various aspects of AD pathology. Our study provides immediate clarity on high uptake of the microtubule-based radiotracer in AD brains in a longitudinal setting, which directly informs clinical utility in Aβ/tau-based studies.

Microtubule-induced restriction of nuclear deformability in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq foundation Background Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the left ventricular (LV) wall in conjunction with diastolic dysfunction with a prevalence of ~1:200. HCM pathophysiology is incompletely understood. We hypothesized that misregulated mechanotransduction plays an important role in the disease process. Extensive cytoskeletal remodeling has been observed in HCM patients, with a robust increase in tubulin and desmin levels, with a concomitant increase in stabilizing post-translational modifications, which we previously showed contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex. Little is known about the interaction of the non-sarcomeric cytoskeleton with the nucleus and the role this may have in the development of HCM. Purpose Relate cytoskeleton changes to nuclear and chromatin morphology in HCM patient myocardium and assess nuclear deformability in contracting cardiomyocytes with and without microtubule destabilizing drugs. Methods Nuclear morphology and chromatin organization was assessed using electron microscopy in cardiac septal tissue from 19 patients with obstructive HCM and compared to samples from 4 non-failing donors (NF). Further nuclear changes were studied using wild type (WT) and homozygous Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, KI), which have a HCM phenotype. Nuclear morphology was assessed in slices of 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. To study nuclear deformability during contractions, adult cardiomyocytes were isolated, stained with live cell dyes and imaged live while electrically paced. Lastly, DNA damaged was assessed in 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. Results Cardiomyocyte nuclei of HCM patients had a significantly higher number of invaginations (0.32±0.09 invaginations/µm in HCM versus 0.18±0.03 invaginations/µm in NF hearts), were more irregular of shape and had altered chromatin organization compared to those from NF hearts. Nuclei of Mybpc3c.2373InsG mice were considerably larger (322±75 µm³ versus 221±24 µm³). While sarcomere shortening is similar (7.0±1.4% in Mybpc3c.2373InsG versus 6.5±1.7% in WT), nuclei from Mybpc3c.2373InsG mice are less deformable than WT nuclei (figure). Addition of microtubule destabilizer nocodazole or detyrosination inhibitor EPO-Y restored nuclear deformability to WT levels, indicating a role of detyrosinated microtubules in nuclear deformability. In addition, nuclei from Mybpc3c.2373InsG have more extensive DNA damage (19.7±3.4 foci in Mybpc3c.2373InsG versus 9.1±3.2 foci in WT). Conclusion Extensive nuclear changes are observed in HCM patients and a HCM mouse model. Cytoskeletal remodeling results in less deformable nuclei that might contribute to chromatin alterations and increased DNA damage. Nuclear deformability can be restored by microtubule modifying drugs, specifically those that decrease microtubule detyrosination.

Antiproliferative and Pro-Apoptotic Activity and Tubulin Dynamics Modulation of 1H-Benzimidazol-2-yl Hydrazones in Human Breast Cancer Cell Line MDA-MB-231.

(1) Background: The aim of the work is the evaluation of in vitro antiproliferative and pro-apoptotic activity of four benzimidazole derivatives containing colchicine-like and catechol-like moieties with methyl group substitution in the benzimidazole ring against highly invasive breast cancer cell line MDA-MB-231 and their related impairment of tubulin dynamics. (2) Methods: The antiproliferative activity was assessed with the MTT assay. Alterations in tubulin polymerization were evaluated with an in vitro tubulin polymerization assay and a docking analysis. (3) Results: All derivatives showed time-dependent cytotoxicity with IC50 varying from 40 to 60 μM after 48 h and between 13 and 20 μM after 72 h. Immunofluorescent and DAPI staining revealed the pro-apoptotic potential of benzimidazole derivatives and their effect on tubulin dynamics in living cells. Compound 5d prevented tubulin aggregation and blocked mitosis, highlighting the importance of the methyl group and the colchicine-like fragment. (4) Conclusions: The benzimidazole derivatives demonstrated moderate cytotoxicity towards MDA-MB-231 by retarding the initial phase of tubulin polymerization. The derivative 5d containing a colchicine-like moiety and methyl group substitution in the benzimidazole ring showed potential as an antiproliferative agent and microtubule destabilizer by facilitating faster microtubule aggregation and disrupting cellular and nuclear integrity.

Characterizing immune-mediated neuronal microtubule destabilization and axonal transport defects in a mouse model of optic neuritis

Abstract Multiple sclerosis (MS) is a central nervous system autoimmune disease that often presents with inflammation of the optic nerve, optic neuritis (ON). The mechanisms of neuronal damage in MS and ON are unknown. In vitro studies modeling MS neurodegeneration demonstrated that cytolytic lymphocytes drive microtubule destabilization in human induced pluripotent stem cell-derived spinal motor neurons. Microtubules are essential for axonal transport, a key function for neuron health. We hypothesize that immune-mediated neuronal microtubule destabilization drives development of ON through disruption of axonal transport. MOG35-55 T cell receptor transgenic mice were used as a model of ON. The non-invasive ophthalmologic technique optical coherence tomography (OCT) allows for confirmation of symptomatic ON. Using OCT, increasing severity of progressive ON was observed over 5 months. To examine transport efficiency, cholera toxin subunit B (CTB), an axonal transport tracer, was injected into the eyes of mice with ON. Optic nerves were collected and stained for β3-tubulin, a marker of microtubules, and imaged by immunofluorescence. Preliminary data found decreased β3-tubulin staining and markedly reduced CTB transport in the presence of immune cell infiltration in mice with ON. These data confirm that OCT can be used to monitor ON progression and provide evidence that immune-mediated neuronal microtubule destabilization causes axonal transport deficits that underly ON development.

Abstract 7141: The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents

Abstract Our previous work showed that the prenylated hydroxy-stilbene AUS_001 exerts a strong safety profile and potent anti-proliferative responses, as assessed in 30 types of cancer cell lines, stemming from its microtubule destabilization activity. The aim of the current study was to delineate the molecular mechanism of microtubule destabilization by AUS_001 and to provide insight into its high potency and low toxicity. Surface plasmon resonance (SPR) experiments revealed the direct interaction of AUS_001 with tubulin with an apparent equilibrium dissociation constant, Kd, of 2.13 × 10−5M. Size exclusion competition assays and an X-ray crystal structure of the tubulin-AUS_001 complex at 2.1 Å resolution establish ligand binding within the colchicine site on tubulin. Since colchicine-site ligands are well known to inhibit the curved-to-straight conformational transition of tubulin, which is an essential process for microtubule formation, these results readily explain the microtubule-destabilizing activity of the compound. Notably, while both combretastatin A4 (CA-4) and AUS_001 are both colchicine-site stilbene ligands, CA-4 binds in the cis configuration, while AUS_001 maintains a trans configuration leading to a substantially different pocket engagement. Comparison of the tubulin-AUS_001 complex structure with the ones of tubulin-colchicine and tubulin-plinabulin revealed that AUS_001 elicits different rearrangements in the T7 loop of β-tubulin versus colchicine and occupies a different zone of the pocket relative to plinabulin, further elaborating its unique binding properties. Interestingly, AUS_001 exhibits a higher relative affinity than colchicine itself, as assessed by i) an ultrafiltration-based tubulin-ligand release assay and ii) a radioligand colchicine competition assay. However, fitting steady state SPR data for AUS_001 binding to tubulin revealed fast association and dissociation rates and was indicative of reversible binding. The latter finding is corroborated by AUS_001-triggered reverse biological effects including morphological alterations, cell cycle G2/M arrest, and by a decrease of cell viability upon discontinuation of the drug treatment in glioma and pancreatic cancer cells. Conversely, colchicine, CA-4 or paclitaxel administered at the same doses as those applied for AUS_001 failed to reverse the drug-induced phenotypes upon removal resulting in sustained cytotoxicity. In conclusion, we fully characterized the unique binding mode of AUS_001 to the colchicine site of tubulin and elucidated the reversible nature of the target engagement. AUS_001 features a distinctive and reversible molecular interaction with tubulin which provides a plausible explanation for its favorable safety profile compared to other microtubule targeting agents. Citation Format: Herman Lelie, Yao-Chieh Chou, Alastair J. King, Zlata Boiarska, Andrea E. Prota, Michel O. Steinmetz, Marina Koutsioumpa. The novel microtubule-destabilizing compound AUS_001 maintains unique binding to the colchicine site of tubulin and elicits reversible cellular effects relative to other anti-tubulin agents [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 7141.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [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 7144.

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [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 4701.

Abstract 5259: Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics

Abstract Microtubule-targeted agents (MTAs) are some of the most widely used and effective chemotherapeutics and they continue to find utility as the payloads of antibody-drug conjugates and in combination with targeted agents to improve response, including with immune checkpoint inhibitors. While all MTAs have shared antiproliferative effects, over the last decade it has become increasingly clear that individual drugs of this class have distinct effects on microtubule-dependent signaling and trafficking that are critical for their anticancer efficacy. We and others have demonstrated that eribulin is unique from other MTAs in its ability to promote vascular remodeling, reverse epithelial-to-mesenchymal transition (EMT), and increase immune infiltration into tumors both in animal models and in the clinic. However, the mechanism linking eribulin-mediated microtubule destabilization to these phenotypes, which are associated with improved overall survival of eribulin-treated patients, is unclear. We found that acute eribulin treatment at clinically relevant concentrations promotes the production of antitumor cytokines in both immune and cancer cells and synergizes with direct STING agonists to promote enhanced immune infiltration and improved antitumor efficacy in vivo in both breast and bladder cancer models. This effect was shown to involve activation of natural killer cells and is associated with TBK1-mediated IRF3 phosphorylation, which is regulated downstream of the microtubule associated GTPase GEF-H1. Intriguingly, GEF-H1 release from destabilized microtubules has also been shown to enhance the formation of focal adhesions and promote the differentiation of mesenchymal cells to a more epithelial phenotype. We show that eribulin is indeed sufficient to promote the formation and activation of focal adhesion complexes in mesenchymal triple-negative breast cancer cells, which was not observed when cells were subjected to treatment with microtubule stabilizing chemotherapeutics. Both genetic and pharmacological inhibitors of GEF-H1 were utilized to interrogate the relationship between eribulin-mediated microtubule destabilization and TBK1/IRF3-dependent immunological activation as well as the formation of RhoA-dependent focal adhesions and reversal of EMT-associated phenotypes. Together, our data provide a mechanistic rationale for unique effects of eribulin as compared to other classes of MTAs that could inform on the use of these widely used, but mechanistically underappreciated drugs in a more personalized manner and inform on their rational combination with targeted agents, including immunotherapy. Citation Format: Zaineb Hassouneh, Nancy Wilkinson, Leila Takahashi-Ruiz, Neelam Mukherjee, April L. Risinger. Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics [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 5259.

Pathogenic soluble tau peptide disrupts endothelial calcium signaling and vasodilation in the brain microvasculature

The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary calcium (Ca2+) signals to direct blood flow. The initiation and amplification of endothelial cell Ca2+ signals require an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.

Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity.

The activation of stimulator of interferon genes (STING) signaling induces the production of type I interferons (IFNs), which play critical roles in protective innate immunity for the host to defend against viral infections. Therefore, achieving sustained or enhanced STING activation could become an antiviral immune strategy with potential broad-spectrum activities. Here, we discovered that various clinically used microtubule-destabilizing agents (MDAs) for the treatment of cancer showed a synergistic effect with the activation of STING signaling in innate immune response. The combination of a STING agonist cGAMP and a microtubule depolymerizer MMAE boosted the activation of STING innate immune response and showed broad-spectrum antiviral activity against multiple families of viruses. Mechanistically, MMAE not only disrupted the microtubule network, but also switched the cGAMP-mediated STING trafficking pattern and changed the distribution of Golgi apparatus and STING puncta. The combination of cGAMP and MMAE promoted the oligomerization of STING and downstream signaling cascades. Importantly, the cGAMP plus MMAE treatment increased STING-mediated production of IFNs and other antiviral cytokines to inhibit viral propagation in vitro and in vivo. This study revealed a novel role of the microtubule destabilizer in antiviral immune responses and provides a previously unexploited strategy based on STING-induced innate antiviral immunity.

Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma.

Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (Cmax of 7807 ng/mL (20 μM), Tmax at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC50s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC50 values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.

Investigating LIM (Lin-11, Isl-1, and Mec-3) Kinases and Their Correlation with Pathological Events and Microtubule Dynamics in an Experimental Model of Spinal Cord Injury in Rats.

The spinal cord injury (SCI) and the neurodegenerative processes accompanying it follow an intricate pathway with very limited options for treatment strategies until now. Microtubules, essential for the growth and maintenance of neurons, are mostly disorganized and destabilized due to neurodegeneration. Regeneration or plasticity is restricted to the adult central nervous system (CNS) due to several intrinsic and extrinsic mechanisms. Some fundamental or injury-induced expressions of specific molecules can be inhibited or antagonized pharmacologically to protect neurons to a certain extent after neurodegeneration. Accordingly, these molecules offer an excellent target as a therapeutic approach to promote neuroprotection. LIM kinases (LIMKs) are one of these molecules that phosphorylates members of the actin-depolymerizing factor (ADF)/cofilin family of actin-binding and filament-severing proteins. The individual role of LIMKs has not yet been studied in the pathology of SCI. In this study, we targeted LIMK and checked its role in microtubule destabilization in vitro. LIMK1 was found to be upregulated after microtubule depolymerization and inhibition of LIMK with specific inhibitor-protected neurons. Then, we checked the expressions of individual LIMKs throughout different time points across SCI in a rat contusion model, correlating with established pathophysiological markers. The phosphorylated form of LIMK1 was found to be elevated at chronic time points after injury, where scar formation and diminution of neurons prevail. Finally, we targeted the LIMK pathway with its specific inhibitor BMS-5, which showed neuroprotection after SCI. Overall, our results provided a concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat SCI-associated neurodegeneration.

The microtubule cytoskeleton: A validated target for the development of 2-Aryl-1H-benzo[d]imidazole derivatives as potential anticancer agents

In this study, a series of 2-Aryl-1H-benzo[d]imidazole derivatives were developed to target intra- and extracellular microtubule networks. Compounds O-7 and O-10 showed impressive anti-proliferative activity across various tested cell lines, demonstrating selectivity indexes of 151.7 and 61.9, respectively. O-7 achieved an IC50 value of 0.236 ± 0.096 μM, while O-10 showed an IC50 value of 0.622 ± 0.13 μM against A549 cell lines. The induction of early-stage apoptosis in a dose-dependent manner further underscored the potential of O-7 and O-10 as effective anti-proliferative agents. O-7 and O-10 exhibited substantial inhibition of wound closure, with wound closure percentages decreasing from 23% at 0 μM to 0.43% and 2.62% at 20 μM, respectively. Colony formation reduction rates were impressive, with O-7 at 74.2% and O-10 at 81.2%. These results indicate that the O-7 and O-10 can impede cancer cell migration and have a high potential to curtail colony formation. The mode of action investigations for O-7 and O-10 revealed that O-7 could inhibit in vitro tubulin polymerization and disrupt the intracellular microtubule cytoskeleton. This disruption led to cell cycle arrest in the G2/M phase, indicating that O-7 exerts its anticancer activity through microtubule destabilization. However, O-10 shows a different mode of action than O-7 and requires further investigation. Overall, our study showcases the potential of the synthesized benzimidazole derivatives as novel and selective anticancer agents, motivating further exploration of their pharmacological properties and therapeutic applications.

Design, synthesis, and bioevaluation of diarylpyrimidine derivatives as novel microtubule destabilizers.

In this work, a series of new diarylpyrimidine derivatives as microtubule destabilizers were designed, synthesized, and evaluated for anticancer activities. Based on restriction configuration strategy, we introduced the pyrimidine moiety containing the hydrogen-bond acceptors as cis-olefin bond of CA-4 analogs to improve structural stability. Compounds 11a-t exerted antiproliferative activities against three human cancer cell lines (SGC-7901, HeLa, and MCF-7), due to tubulin polymerization inhibition, showing high selectivity toward cancer cells in comparison with non-tumoral HSF cells, as evidenced by MTT assays. In mechanistic investigations, compound 11s remarkably inhibited tubulin polymerization and disorganized microtubule in SGC-7901 cells by binding to tubulin. Moreover, 11s caused G2/M phase cell cycle arrest in SGC-7901 cells in a concentration-dependent manner. Furthermore, molecular modeling analysis revealed that 11s interacts with tubulin through binding to the colchicine site. In addition, the prediction of physicochemical properties disclosed that 11s conformed well to the Lipinski's rule of five. This work offered a fresh viewpoint for the discovery of new tubulin-targeting anticancer drugs.

How do neurons age? A focused review on the aging of the microtubular cytoskeleton.

Aging is the leading risk factor for Alzheimer's disease and other neurodegenerative diseases. We now understand that a breakdown in the neuronal cytoskeleton, mainly underpinned by protein modifications leading to the destabilization of microtubules, is central to the pathogenesis of Alzheimer's disease. This is accompanied by morphological defects across the somatodendritic compartment, axon, and synapse. However, knowledge of what occurs to the microtubule cytoskeleton and morphology of the neuron during physiological aging is comparatively poor. Several recent studies have suggested that there is an age-related increase in the phosphorylation of the key microtubule stabilizing protein tau, a modification, which is known to destabilize the cytoskeleton in Alzheimer's disease. This indicates that the cytoskeleton and potentially other neuronal structures reliant on the cytoskeleton become functionally compromised during normal physiological aging. The current literature shows age-related reductions in synaptic spine density and shifts in synaptic spine conformation which might explain age-related synaptic functional deficits. However, knowledge of what occurs to the microtubular and actin cytoskeleton, with increasing age is extremely limited. When considering the somatodendritic compartment, a regression in dendrites and loss of dendritic length and volume is reported whilst a reduction in soma volume/size is often seen. However, research into cytoskeletal change is limited to a handful of studies demonstrating reductions in and mislocalizations of microtubule-associated proteins with just one study directly exploring the integrity of the microtubules. In the axon, an increase in axonal diameter and age-related appearance of swellings is reported but like the dendrites, just one study investigates the microtubules directly with others reporting loss or mislocalization of microtubule-associated proteins. Though these are the general trends reported, there are clear disparities between model organisms and brain regions that are worthy of further investigation. Additionally, longitudinal studies of neuronal/cytoskeletal aging should also investigate whether these age-related changes contribute not just to vulnerability to disease but also to the decline in nervous system function and behavioral output that all organisms experience. This will highlight the utility, if any, of cytoskeletal fortification for the promotion of healthy neuronal aging and potential protection against age-related neurodegenerative disease. This review seeks to summarize what is currently known about the physiological aging of the neuron and microtubular cytoskeleton in the hope of uncovering mechanisms underpinning age-related risk to disease.