Inhibition Of Microtubule Polymerization Research Articles

Abstract 556: Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression

Abstract An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC50 values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC50 values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p<0.0001) or 40H3-MMAE (p<0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p<0.0001) and 26% with 40H3-MMAE (p<0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs. Citation Format: Sonia Jain, Eric Chun Ho, Kendra A. Porath, Antonella Antignani, David J. FitzGerald, Jann N. Sarkaria. Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression [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 556.

Abstract P2-20-17: Eribulin is an immune potentiator in breast cancer by up-regulation of human leukocyte antigen class I

Abstract Background: Eribulin, an anticancer drug that increases the overall survival (OS) of patients with metastatic recurrent breast cancer, inhibits microtubule polymerization, although the mechanism is different from that of other microtubule inhibitors (taxanes). A subgroup analysis revealed a low neutrophil-to-lymphocyte ratio (NLR) (< 3) to be a prognostic factor of eribulin treatment. NLR has also been reported to be a predictive marker for cancer immunotherapy. Checkpoint inhibitor-based cancer immunotherapy is currently available for breast cancer. We hypothesized that eribulin improves breast cancer cell immune response. Immunological effector cytotoxic T lymphocytes (CTLs) recognize antigenic peptides presented by human leukocyte antigen (HLA) class I; HLA class I downregulation is frequently observed in breast cancer. Here, we evaluated the CTL response in eribulin-treated breast cancer cells. Materials and Methods: HLA class I expression before and after eribulin treatment was evaluated using immunohistochemistry in tumors from patients, and by immunofluorescence, flow cytometry, western blotting, and quantitative RT-PCR (qRT-PCR) in breast cancer cells (MDA-MB231 and MCF7). Factors that upregulate HLA class I were screened using RNA-seq. To evaluate T cell recognition, we generated cancer testis antigen (NY-ESO-1)-specific T cell receptor transduced-T cells (TCR-T cells). NY-ESO-1 cDNA was stably transduced into MCF7 and MDA-MB-231 cells. TCR-T cell reactivity with MCF7/NY-ESO-1 cells was analyzed using an ELISPOT assay. A combinatorial therapy model was established using eribulin and TCR-T. Results: HLA class I was upregulated after eribulin treatment in clinical samples. To confirm this, we treated breast cancer cells with eribulin and evaluated HLA class I expression. Eribulin increased HLA class I expression, as evidenced by immunofluorescence, flow cytometry, western blotting, and qRT-PCR. RNA-seq results on eribulin-treated cells showed that eribulin upregulated NLRC5, a master regulator of HLA class I. IFNγ ELISPOT assay revealed that eribulin increased IFNγ secretion by TCR-T cells (p< 0.01), indicating that eribulin enhanced the immune response. These results suggest that eribulin and immunotherapy had a synergistic effect. Therefore, we established an eribulin and NY-ESO-1 combinatorial therapy model and showed that the combination group had a significantly lower number of viable cancer cells than the eribulin- and TCR-T-only groups (p< 0.01). Conclusions: Eribulin increased the expression of HLA class I, probably by upregulating NLRC5 in breast cancer cells. It also enhanced TCR-T recognition in breast cancer cells. The combinatorial therapy model revealed the synergistic effect of eribulin and TCR-T. These results indicate that eribulin might be an immune potentiator and that combination therapy with immunotherapy can be effective for the treatment of breast cancer. Citation Format: Asaka Wada, Goro Kutomi, Yoshihiko Hirohashi, Daisuke Kyuno, Hiroaki Shima, Yoko Kuga, Toshihiko Torigoe, Ichiro Takemasa. Eribulin is an immune potentiator in breast cancer by up-regulation of human leukocyte antigen class I [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-17.

Design, synthesis, and biological activity evaluation of novel tubulin polymerization inhibitors based on pyrimidine ring skeletons

A library of new pyrimidine analogs was designed and synthesized of these, compound K10 bearing a 1,4‑benzodioxane moiety and 3,4,5‑trimethoxyphenyl group, exhibiting the most potent activity, with IC50 values of 0.07–0.80 μM against four cancer cell lines. Cellular-based mechanism studies elucidated that K10 inhibited microtubule polymerization, blocked the cell cycle at the G2/M phase, and eventually induced apoptosis of HepG2 cells. Additionally, K10 inhibited the migration and invasion of HepG2 cells in a dose-dependent manner. Overall, our work indicates that the tubulin polymerization inhibitor incorporating pyrimidine and the 3,4,5‑trimethoxyphenyl ring may deserve consideration for cancer therapy.

Tau-RNA complexes inhibit microtubule polymerization and drive disease-relevant conformation change.

Alzheimer's disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent structural biology studies of tau proteinopathy, aggregated tau forms a distinct set of conformational variants specific to the different types of tauopathy disorders. However, the constituents driving the formation of distinct pathological tau conformations on pathway to tau-mediated neurodegeneration remain unknown. Previous work demonstrated RNA can serve as a driver of tau aggregation, and RNA associates with tau containing lesions, but tools for evaluating tau/RNA interactions remain limited. Here, we employed molecular interaction studies to measure the impact of tau/RNA binding on tau microtubule binding and aggregation. To investigate the importance of tau/RNA complexes (TRCs) in neurodegenerative disease, we raised a monoclonal antibody (TRC35) against aggregated tau/RNA complexes. We showed that native tau binds RNA with high affinity but low specificity, and tau binding to RNA competes with tau-mediated microtubule assembly functions. Tau/RNA interaction in vitro promotes the formation of higher molecular weight tau/RNA complexes, which represent an oligomeric tau species. Coexpression of tau and poly(A)45 RNA transgenes in Caenorhabditis elegans exacerbates tau-related phenotypes including neuronal dysfunction and pathological tau accumulation. TRC35 exhibits specificity for Alzheimer's disease-derived detergent-insoluble tau relative to soluble recombinant tau. Immunostaining with TRC35 labels a wide variety of pathological tau lesions in animal models of tauopathy, which are reduced in mice lacking the RNA binding protein MSUT2. TRC-positive lesions are evident in many human tauopathies including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease. We also identified ocular pharyngeal muscular dystrophy as a novel tauopathy disorder, where loss of function in the poly(A) RNA binding protein (PABPN1) causes accumulation of pathological tau in tissue from post-mortem human brain. Tau/RNA binding drives tau conformational change and aggregation inhibiting tau-mediated microtubule assembly. Our findings implicate cellular tau/RNA interactions as modulators of both normal tau function and pathological tau toxicity in tauopathy disorders and suggest feasibility for novel therapeutic approaches targeting TRCs.

Co-administration of JQ1, a bromodomain-containing protein 4 inhibitor, enhances the antitumor effect of combretastatin A4, a microtubule inhibitor, while attenuating its cardiotoxicity

Combretastatin A4 (CA4) inhibits microtubule polymerization, and clinical trials of the prodrug, CA4 disodium phosphate (CA4DP), as an anti-cancer agent have been conducted. However, CA4DP has not been marketed to date because the margin between the effective dose and the cardiotoxic dose is insufficient. Meanwhile, bromodomain-containing protein 4 (BRD4) has been reported to be required for recovery from mitotic arrests induced by anti-microtubule drugs. BRD4 has also been reported to be involved in the progression of heart failure. Therefore, we hypothesized that the combined use of CA4DP with BRD4 inhibitors can enhance the antitumor effect and attenuate CA4DP-induced cardiotoxicity. In this study, the antitumor effect and cardiotoxicity caused by the co-administration of CA4DP with JQ1, a BRD4 inhibitor, were evaluated.CA4 or JQ1 alone reduced the viability of cultured canine mammary tumor cells (CHMp-13a). Viability was further reduced by co-administration, through the suppression of c-Myc. BRD4 positivity in CHMp-13a cytoplasm showed a significant increase when treated with CA4 alone, while the increase was not significant following co-administration. In CHMp-13a xenograft-transplanted mice, co-administration of CA4DP and JQ1 suppressed tumor growth significantly.In CA4DP-induced cardiac injury model rats, echocardiography showed a CA4DP-induced decrease in cardiac function and histopathology showed cardiomyocyte necrosis. Meanwhile, these cardiac changes tended to be milder following the co-administration of CA4DP and JQ1.These results suggest that CA4DP-JQ1 co-administration enhances the antitumor effect of CA4DP while attenuating its cardiotoxicity and therefore potentially open the doors to the development of a novel cancer chemotherapy with reduced cardiotoxicity risks.

Molecular Docking of Cryptoconcatones to α-Tubulin and Related Pironetin Analogues.

Cryptoconcatones A-L represent a series of 12 dihydropyrone derivatives isolated from the evergreen tree Cryptocarya concinna Hance, which is well distributed in southeast Asia. The lead compound in the series, cryptoconcatone L, has revealed antiproliferative activity against cultured cancer cells but its mechanism of action remains unknown. Based on a structural analogy with the anticancer natural product pironetin, which is well known for binding covalently to α-tubulin and for functioning as a microtubule polymerization inhibitor, we investigated the interaction of cryptoconcatones with tubulin dimers using molecular docking. The α-tubulin binding capacity of each compound was quantified (through calculation of the empirical energy of interaction ΔE) and structure-binding relationships were delineated. Two compounds were found to interact with α-tubulin much more potently than pironetin: cryptoconcatones F and L. In both cases, the facile formation of a covalent bond with Cys316 was evidenced, as observed with the parent compound pironetin. A few other pironetin analogues were investigated, including spicigerolide, which is an analogue of another known α-tubulin binder. Altogether, this study points to the identification of a series of 5,6-dihydro-α-pyrones as α-tubulin-binding agents. The study contributes to a better understanding of the mechanism of action of cryptoconcatones and should help the design of analogues targeting the pironetin site of α-tubulin.

Abstract 1291: Synthesis of Hemithioindigo Compounds as Microtubule Polymerization Inhibitors

Abstract 1291: Synthesis of Hemithioindigo Compounds as Microtubule Polymerization Inhibitors

Design and Synthesis of Novel Phenylahistin Derivatives Based on Co-Crystal Structures as Potent Microtubule Inhibitors for Anti-Cancer Therapy.

Phenylahistin is a naturally occurring marine product with a diketopiperazine structure that can bind to the colchicine site of microtubulin as a possible anticancer agent. To develop more potent microtubule inhibitors, novel phenylahistin derivatives were designed and synthesized based on the co-crystal complexes of phenylahistin derivatives and microtubulin. We established a focused library of imidazole-type molecules for the introduction of different groups to the C-ring and A-ring of phenylahistin. Structure-activity relationship studies indicated that appropriate hydrocarbon substituents and unsaturated alkenyl substituents at the 1-position of the imidazole group are important for improving the activity of such compounds. In addition, this study found that propylamine groups could maintain the activity of these compounds, as exemplified by compound 16d (IC50 = 5.38 nM, NCI-H460). Compound 15p (IC50 = 1.03 nM, NCI-H460) with an allyl group exhibited potent cytotoxic activity at the nanomolar level against human lung cancer cell lines. Immunofluorescence assay indicated that compound 15p could efficiently inhibited microtubule polymerization and induced a high expression of caspase-3. 15p also displayed good pharmacokinetic characteristics in vitro. Additionally, the growth of H22 transplanted tumors was significantly inhibited in BALB/c mice when 15p alone was administered at 4 mg/kg, and the tumor inhibition rate was as much as 65%. Importantly, the continuous administration of 15p resulted in a lower toxicity than that of docetaxel (10 mg/kg) and cyclophosphamide (20 mg/kg). Overall, the novel allyl-imidazole-diketopiperazine-type derivatives could be considered safe and effective potential agents for cancer treatment.

Evolution of Potential Antimitotic Stapled Peptides from Multiple Helical Peptide Stretches of the Tubulin Heterodimer Interface: Helix-Mimicking Stapled Peptide Tubulin Inhibitors

Protein-protein interactions play a crucial role in microtubule dynamics. Microtubules are considered as a key target for the design and development of anticancer therapeutics, where inhibition of tubulin-tubulin interactions plays a crucial role. Here, we focused on a few key helical stretches at the interface of α,β-tubulin heterodimers and developed a structural mimic of these helical peptides, which can serve as potent inhibitors of microtubule polymerization. To induce helicity, we have made stapled analogues of these sequences. Thereafter, we modified the lead sequences of the antimitotic stapled peptides with halo derivatives. It is observed that halo-substituted stapled peptides follow an interesting trend for the electronegativity of halogen atoms in interaction patterns with tubulin and a correlation in the toxicity profile. Remarkably, we found that para-fluorophenylalanine-modified stapled peptide is the most potent inhibitors, which perturbs microtubule dynamics, induces apoptotic death, and inhibits the growth of melanoma.

PH-sensitive nanomedicine of novel tubulin polymerization inhibitor for lung metastatic melanoma.

Microtubule binding agents such as paclitaxel and vincristine have activity in metastatic melanoma. However, even responsive tumors develop resistance, highlighting the need to investigate new drug molecules. Here, we showed that a new compound, CH-2-102, developed by our group, has high anti-tumor efficacy in human and murine melanoma cells. We confirmed that CH-2-102 robustly suppresses the microtubule polymerization process by directly interacting with the colchicine binding site. Our results unveil that CH-2-102 suppresses microtubule polymerization and subsequently induces G2 phase cell arrest as one of the possible mechanisms. Notably, CH-2-102 maintains its efficacy even in the paclitaxel resistance melanoma cells due to different binding sites and a non-Pgp substrate. We developed a pH-responsive drug-polymer Schiff bases linker for high drug loading into nanoparticles (NPs). Our CH-2-102 conjugated NPs induced tumor regression more effectively than Abraxane® (Nab-paclitaxel, N-PTX), free drug, and non-sensitive NPs in B16-F10 cell-derived lung metastasis mouse model. Furthermore, our results suggest that the formulation has a high impact on the in vivo efficacy of the drug and warrants further investigation in other cancers, particularly taxane resistant. In conclusion, the microtubule polymerization inhibitor CH-2-102 conjugated pH-responsive NPs induce tumor regression in lung metastasis melanoma mice, suggesting it may be an effective strategy for treating metastatic melanoma.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ β-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

Discovery of a novel piperlongumine analogue as a microtubule polymerization inhibitor with potent anti-angiogenic and anti-metastatic efficacy

In an effort to discover anticancer agents with simultaneous effects on tubulin and angiogenesis, we designed and synthesized two series of piperlongumie (PL) derivatives by replacing of phenyl group with a variety of benzoheterocycle (series II) or cyclizing the C7–C8 olefin into an aromatic heterocycle (series I). Most of the new compounds showed better antiproliferative activities against six cancer cell lines than the parent drug PL. Compound II-14b had the best cytotoxic profile of these two series in cancer cells, whilst being relatively low cytotoxicity against normal human cells and high potency against drug-resistant cells. It disrupted cellular microtubule networks and inhibited tubulin assembly with an IC50 value of 5.8 μM. Further studies elucidated that II-14b showed antitumor activities through multiple mechanisms, including the pruduction of abundant ROS, the dissipation of mitochondrial membrane potential, the accumulation of DNA double-strand breaks, and the induction of cell cycle in G2/M phase. More importantly, we have observed that it possesses potential anti-angiogenesis capabilities, including suppression of HUVECs cell migration, invasion, and endothelial tube formation in vitro and in vivo. In vivo assessment indicated that II-14b inhibits the growth and metastasis of MGC-803 xenograft tumour in zebrafish. These findings show that II-14b is a high-efficacy and non-toxic antitumor agent.

1528TiP A phase II/III study evaluating the efficacy and safety of unesbulin in advanced leiomyosarcoma (SUNRISELMS)

Leiomyosarcoma (LMS) is one of the most common and aggressive subtypes of soft tissue sarcoma in adults, with the highest risk of recurrence and decreased disease-specific survival (George S, et al. J Clin Oncol. 2018; 36:144–150). A high unmet need remains for treatment of patients with unresectable or metastatic, relapsed or refractory LMS. In a Phase 1b study (NCT03761095), unesbulin, a microtubule polymerization inhibitor, plus dacarbazine (DTIC), was evaluated in patients with unresectable or metastatic, relapsed or refractory LMS, with acceptable benefit/risk ratio to support the start of a Phase 2/3 study.

Discovery of a Novel Vascular Disrupting Agent Inhibiting Tubulin Polymerization and HDACs with Potent Antitumor Effects.

Most vascular disrupting agents (VDAs) fail to prevent the regrowth of blood vessels at the edge of tumors, causing tumor rebound and relapse. Herein, a series of novel multifunctional vascular disrupting agents (VDAs) capable of inhibiting microtubule polymerization and histone deacetylases (HDACs) were designed and synthesized using the tubulin polymerization inhibitor TH-0 as the lead compound. Among them, compound TH-6 exhibited the most potent antiproliferative activity (IC50 = 18-30 nM) against a panel of cancer cell lines. As expected, TH-6 inhibited tubulin assembly and increased the acetylation level of HDAC substrate proteins in HepG2 cells. Further in vivo antitumor assay displayed that TH-6 effectively inhibited tumor growth with no apparent toxicity. More importantly, TH-6 disrupted both the internal and peripheral tumor vasculatures, which contributed to the persistent tumor inhibitory effects after drug withdrawal. Altogether, TH-6 deserves to be further investigated for the new approach to clinical cancer therapy.

Host nuclear repositioning and actin polarization towards the site of penetration precedes fungal ingress during compatible pea-powdery mildew interactions.

Actin polarization and actin-driven host nuclear movement towards the fungal penetration site facilitates successful host colonization during compatible pea-Erysiphe pisi interactions. Proper nuclear positioning in plant cells is crucial for developmental processes and response to (a)biotic stimuli. During plant-fungal interactions, the host nucleus moves toward the infection site, a process regulated by the plant cytoskeleton. Notably, rearrangement of the plant cytoskeleton is one of the earliest cellular responses to pathogen invasion and is known to impact penetration efficiency. Yet, the connection between host nuclear movement and fungal ingress is still elusive, particularly in legumes. Here, we investigated the host nuclear dynamics during compatible interactions between Pisum sativum (pea) and the adapted powdery mildew (PM) fungus Erysiphe pisi to gain insights into the functional relevance of PM-induced nuclear movement in legumes. We show that the host nucleus moves towards the fungal appressorium before penetration and becomes associated with the primary haustorium. However, the nucleus migrates away from the primary infection site as the infection progresses toward colony expansion and sporulation. Treatment of pea leaves with the actin-polymerization inhibitor, cytochalasin D, abolished host nuclear movement towards the fungal penetration site and restricted PM growth. In contrast, treatment with oryzalin, a microtubule-polymerization inhibitor, had no effect. In addition to nuclear movement, strong polarization of host actin filaments towards the site of appressorial contact was evident at early infection stages. Our results suggest that actin focusing mediates host nuclear movement to the fungal penetration site and facilitates successful colonization during compatible pea-PM interactions.

Abstract 5326: Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers

Abstract Extracellular vesicles (EV), facilitated with cancer-targeted antibody or peptide, have been demonstrated as an effective delivery vehicle of chemotherapy and gene therapy. This study aimed to develop and evaluate a targeted EV to deliver a newly identified natural cytotoxic marine compound verrucarin A (Ver-A) or combination with highly potent microtubule polymerization inhibitor mertansine (DM1). The tumor models, glioblastoma (GBM), triple-negative breast cancer (TNBC) and others, were used to validate the developed targeted therapy. First, the stirred-tank bioreactor-based EV biomanufacturing was optimized by developing a two-step purification procedure using size exclusion (300 kDa) and affinity (CD63) columns in liquid chromatography system. Second, both single and dual tumor-targeting monoclonal antibodies (mAbs), such as anti-SSTR2, CD276, EGFR and/or CD47 mAb either developed in house or used in clinics, were tagged on the surface of PEGylated EV to construct mAb-EV-drugs. The flow cytometry analysis, live-cell confocal images and IC50 assay revealed the effective surface binding, intracellular drug delivery and high anti-cancer cytotoxicity of mAb-EV-drugs in cancer cell lines, and the biodistribution study confirmed the tumor targeting in xenograft mouse models. Furthermore, the maximum tolerated dose study showed minimal systemic toxicity, as confirmed by hematoxylin and eosin staining of multiple important organs. Finally, the anti-tumor efficacy study demonstrated significant inhibition of tumor growth by mAb-EV-Ver-A or Ver-A/DM1 in cell line-derived xenograft models, patient-derived xenograft models, and/or immunocompetent xenograft models. This study suggested that the targeted EV is an efficient platform to deliver combined potent chemotherapies to treat cancer or tumor. Citation Format: Kai Chen, Yingnan Si, Seulhee Kim, Zhuoxin Zhou, Lufang Zhou, X. Margaret Liu. Targeted extracellular vesicle to deliver combined chemotherapies to treat cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5326.

Recent Advances in Combretastatin A-4 Inspired Inhibitors of Tubulin Polymerization: An Update.

Cancer is one of the leading causes of fatality and mortality worldwide. Investigations on developing therapeutic strategies for cancer are supported throughout the world. The massive achievements in molecular sciences involving biochemistry, molecular chemistry, medicine, and pharmacy, and high throughput techniques such as genomics and proteomics have helped create new potential drug targets for cancer treatment. Microtubules are very attractive targets for cancer therapy because of the crucial roles they play in cell division. In recent years, lots of efforts have been put into the identification of new microtubule-targeting agents (MTAs) in anticancer therapy. Combretastatin A-4 (CA-4) is a natural compound that binds to microtubules' colchicine binding site and inhibits microtubule polymerization. Due to CA-4's structural simplicity, many analogs have been synthesized. This article summarises the new molecule development efforts to reach CA-4 analogues by pharmacophore group modifications, which have been reported since 2015.

A phase 1b study of unesbulin (PTC596) plus dacarbazine for the treatment of patients with locally recurrent, unresectable, or metastatic relapsed/refractory leiomyosarcoma.

11507 Background: Leiomyosarcoma (LMS) is one of the most common subtypes of soft tissue sarcoma and is associated with high risk of relapse and a poor prognosis for advanced disease. In preclinical LMS models, unesbulin, a microtubule polymerization inhibitor, potentiated the activity of dacarbazine (DTIC) (Jernigan F, et al. Mol Cancer Ther. 2021;20:1846–1857). Here, we report preliminary safety and efficacy results from a Phase 1b dose escalation study evaluating the combination of unesbulin with DTIC in patients with advanced LMS (NCT03761095). Methods: In this single-arm, open-label, Phase 1b clinical trial, patients with advanced LMS received unesbulin orally at 200, 300, or 400 mg twice weekly (BIW) in combination with intravenous DTIC at 1,000 mg/m2 once every 21 days. The primary objectives were to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) of unesbulin in combination with DTIC and to characterize the safety profile of the combination. The time-to-event continual reassessment model (TITE-CRM) was used for dose finding. An expansion cohort with up to 12 additional patients is currently being enrolled. Results: As of the data cutoff on January 6, 2022, 29 LMS patients have been treated. Median prior lines of therapy were 3 (range 1–6). Of 27 evaluable patients, 12 had non-uterine and 15 had uterine LMS. The MTD/RP2D of unesbulin was determined to be 300 mg BIW with DTIC 1,000 mg/m2 every 21 days using the TITE-CRM. At the RP2D, the most common treatment-related adverse events included fatigue, diarrhea, neutropenia, and thrombocytopenia. In the intent-to-treat population, the overall response rate (ORR) was 17.2% (5/29) and the disease control rate (DCR) (DCR = complete response + partial response + stable disease at 12 weeks) was 58.6%. At the 300 mg dose level, the ORR was 19% (4/21) and the DCR was 57.1%. Patients received a median of four cycles (range 1–12). The study is ongoing, with patients continuing to receive treatment. Conclusions: Unesbulin 300 mg BIW in combination with DTIC 1,000 mg/m2 every 21 days was well tolerated and demonstrated promising efficacy in a heavily pre-treated patient population with advanced LMS; these results support further investigation. Updated clinical results will be presented as the data mature. A randomized, placebo-controlled, Phase 2/3 trial is planned. Clinical trial information: NCT03761095.

Phase 3 VERACITY clinical study of sabizabulin in men with metastatic castration-resistant prostate cancer who have progressed on an androgen receptor targeting agent.

TPS5102 Background: Sabizabulin is a first-in-class, oral agent that inhibits microtubule polymerization disrupting the cytoskeleton and arresting cellular proliferation. A Phase 1b/2 clinical study was conducted to establish the MTD and evaluate the preliminary efficacy in men with metastatic castrate resistant prostate cancer (mCRPC) who progressed on at least one androgen receptor targeting agent (ARTA). The most common AEs reported were mild to moderate diarrhea, fatigue, nausea, and vomiting with no clinically relevant neurotoxicity or neutropenia. In the Phase 1b/2, the median radiographic progression free survival for the 63mg dose (n = 55) was 11.4 months (range 6-36+ months) in the Phase 1b portion including responses > 2.75 years. The Phase 3 reformulation which had improved bioavailability is 32mg PO qd. Methods: VERACITY is an ongoing Phase 3 multicenter, randomized, active-control study designed to evaluate sabizabulin in the treatment of mCRPC who have progressed on at least one ARTA. Patients on the study will be chemotherapy naive. Subjects (n = 245) are being randomized in a 2:1 ratio to receive sabizabulin (32 mg/d oral) or active control (alternative ARTA) and will remain on study until radiographic progression-free survival (rPFS). Randomization will be stratified by: measurable disease vs bone-only disease and by prior exposure to ARTA (progressed on one vs more than one prior ARTA). The primary efficacy endpoint of the study is median rPFS. Secondary endpoints include: objective response rate (ORR), duration of objective response, overall survival and time to intravenous (IV) chemotherapy. The Phase 3, VERACITY registration clinical trial is current ongoing in approximately 45 clinical sites with enrollment anticipated to be completed in 2022 and with unblinded results presented in 2023 Based upon the Phase 1b/2 clinical trial, sabizabulin daily chronic oral dosing has a favorable safety profile, is feasible to administer chronically, and has significant and durable antitumor activity. mCRPC that has progressed following ARTAs and prior to taxane chemotherapy, remains an urgent unmet medical need for patients with advanced disease. Sabizabulin is an exciting first-in-class agent that may add to the armamentarium for the treatment of mCRPC following progression on ARTA’s. Clinical trial information: NCT04844749.

A computational analysis of colchicine structural analogs as potential microtubule destabilizing agents

Microtubules are important for the formation of the cytoskeleton and mitotic spindles, and they aid in cell movement and intracellular transport. Therefore, they are important for cell survival and cell function. Microtubules are composed of the alpha‐beta tubulin heterodimer protein. During polymerization tubulin proteins experience phases of dynamic instability, which is characterized by microtubule growth and shrinkage at varying rates. Microtubule formation can be inhibited by compounds such as colchicine, a known microtubule destabilizing agent, that binds to tubulin to block polymerization. In this study we investigated one hundred eleven compounds that had a similar structure to colchicine. We used AutoDock Vina to dock each compound including colchicine to tubulin in the same binding site as colchicine. The binding energy of colchicine was ‐10.9 kcal/mol, and the binding energies of the top 10% binding affinity compounds were between ‐9 kcal/mol and ‐7.9 kcal/mol. The pharmacokinetic properties of these drugs, computational binding affinity and similarity to colchicine show promise as novel inhibitors of microtubule polymerization, which will be further explored through laboratory experiments.