Inhibition Of Microtubule Polymerization Research Articles

A methodology for specific disruption of microtubule polymerization into dendritic spines.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [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 PO3-25-10.

A pharmacovigilance study on antibody-drug conjugate (ADC)-related neurotoxicity based on the FDA adverse event reporting system (FAERS).

Background: Antibody-drug conjugates (ADCs) are a relatively new class of anticancer agents that use monoclonal antibodies to specifically recognize tumour cell surface antigens. However, off-target effects may lead to severe adverse events. This study evaluated the neurotoxicity of ADCs using the FDA Adverse Event Reporting System (FAERS) database. Research design and methods: Data were extracted from the FAERS database for 2004 Q1 to 2022 Q4. We analysed the clinical characteristics of ADC-related neurological adverse events (AEs). We used the reporting odds ratio (ROR) and proportional reporting ratio (PRR) for the disproportionality analysis to evaluate the potential association between AEs and ADCs. Results: A total of 562 cases of neurological AEs were attributed to ADCs. The median age was 65years old [(Min; Max) = 3; 92]. Neurotoxic signals were detected in patients receiving brentuximab vedotin, enfortumab vedotin, polatuzumab vedotin, trastuzumab emtansine, gemtuzumab ozogamicin, inotuzumab ozogamicin, and trastuzumab deruxtecan. The payloads of brentuximab vedotin, enfortumab vedotin, polatuzumab vedotin, and trastuzumab emtansine were microtubule polymerization inhibitors, which are more likely to develop neurotoxicity. We also found that brentuximab vedotin- and gemtuzumab ozogamicin-related neurological AEs were more likely to result in serious outcomes. The eight most common ADC-related nervous system AE signals were peripheral neuropathy [ROR (95% CI) = 16.98 (14.94-19.30), PRR (95% CI) = 16.0 (14.21-18.09)], cerebral haemorrhage [ROR (95% CI) = 9.45 (7.01-12.73), PRR (95% CI) = 9.32 (6.95-12.50)], peripheral sensory neuropathy [ROR (95% CI) = 47.87 (33.13-69.19), PRR (95% CI) = 47.43 (32.93-68.30)], polyneuropathy [ROR (95% CI) = 26.01 (18.61-36.33), PRR (95% CI) = 25.75 (18.50-35.86)], encephalopathy [ROR (95% CI) = 5.16 (3.32-8.01), PRR (95% CI) = 5.14 (3.32-7.96)], progressive multifocal leukoencephalopathy [ROR (95% CI) = 22.67 (14.05-36.58), PRR (95% CI) = 22.52 (14.01-36.21)], taste disorder [ROR (95% CI) = 26.09 (15.92-42.76), PRR (95% CI) = 25.78 (15.83-42.00)], and guillain barrier syndrome [ROR (95% CI) = 17.844 (10.11-31.51), PRR (95% CI) = 17.79 (10.09-31.35)]. The mortality rate appeared to be relatively high concomitantly with AEs in the central nervous system. Conclusion: ADCs may increase the risk of neurotoxicity in cancer patients, leading to serious mortality. With the widespread application of newly launched ADC drugs, combining the FAERS data with other data sources is crucial for monitoring the neurotoxicity of ADCs. Further studies on the potential mechanisms and preventive measures for ADC-related neurotoxicity are necessary.

SEPTIN10-mediated crosstalk between cytoskeletal networks controls mechanotransduction and oncogenic YAP/TAZ signaling

The transcriptional co-activators of the Hippo pathway, YAP and TAZ, are regulated by mechanotransduction, which depends on dynamic actin cytoskeleton remodeling. Here, we identified SEPTIN10 as a novel cytoskeletal protein, which is transcriptionally regulated by YAP/TAZ and whose overexpression correlates with poor survival and vascular invasion in hepatocellular carcinoma (HCC) patients. Functional characterization demonstrated that SEPTIN10 promotes YAP/TAZ-dependent cell viability, migration and invasion of liver cancer cells. Mechanistically, SEPTIN10 interacts with actin and microtubule filaments supporting actin stress fiber formation and intracellular tension through binding to CAPZA2 while concurrently inhibiting microtubule polymerization through the blockage of MAP4 function. This functional antagonism is important for cytoskeleton-dependent feedback activation of YAP/TAZ, as microtubule depolymerization induces actin stress fiber formation and subsequently YAP/TAZ activity. Importantly, the crosstalk between microfilaments and microtubules is mediated by SEPTIN10 as its loss abrogates actin stress fiber formation after microtubule disruption. Together, the YAP/TAZ target gene SEPTIN10 controls the dynamic interplay between actin and microtubule filaments, which feeds back on Hippo pathway activity in HCC cells and thus acts as molecular switch with impact on oncogenic signaling and cancer cell biology.

Novel dual action PARP and microtubule polymerization inhibitor AMXI-5001 powerfully inhibits growth of esophageal carcinoma both alone and in combination with radiotherapy

Novel dual action PARP and microtubule polymerization inhibitor AMXI-5001 powerfully inhibits growth of esophageal carcinoma both alone and in combination with radiotherapy

The discovery of water-soluble indazole derivatives as potent microtubule polymerization inhibitors

Taking a previously discovered indazole derivative 1 as a lead, systematic structural modifications were performed with an indazole core at the 1- and 6-positions to improve its aqueous solubility. Among the designed indazole derivatives, 6-methylpyridin-3-yl indazole derivative 8l and 1H-indol-4-yl indazole derivative 8m exhibited high potency in the low nanomolar range against A549, Huh-7, and T24 cancer cells, including Taxol-resistant variant cells (A549/Tax). As a hydrochloride salt, 8l exhibited much improved aqueous solubility, and its log P value fell into a favorable range. In mechanistic studies, 8l impeded tubulin polymerization through interacting with the colchicine site, resulting in cell cycle arrest and cellular apoptosis. In addition, compared to lead compound 1, 8l reduced cell migration and led to more potent inhibition of tumor growth in vivo without apparent toxicity. In summary, indazole derivative 8l could work as a potential anticancer agent and deserves further investigation for cancer therapy.

Design, Synthesis, and Biological Evaluation of SSE1806, a Microtubule Destabilizer That Overcomes Multidrug Resistance.

Microtubules are dynamic structures that form spindle fibers during cell division; pharmacological inhibition of microtubule dynamics arrests cells in mitosis, leading to apoptosis, and they have been extensively used to treat various cancers. However, the efficacy of such drugs is often limited by multidrug resistance. This study synthesized and evaluated 30 novel derivatives of podophyllotoxin, a natural antimitotic compound, for their antiproliferative activities. Compound SSE1806 exhibited the most potent antiproliferative activity with GI50 values ranging from 1.29 ± 0.01 to 21.15 ± 2.1 μM in cancer cell lines of different origins; it directly inhibited microtubule polymerization, causing aberrant mitosis and G2/M arrest. Prolonged treatment with SSE1806 increased p53 expression, induced cell death in monolayer cultures, and reduced the growth of mouse- and patient-derived human colon cancer organoids. Importantly, SSE1806 overcame multidrug resistance in a cell line overexpressing MDR-1. Thus, SSE1806 represents a potential anticancer agent that can overcome multidrug resistance.

Anti-tumor and cardiotoxic effects of microtubule polymerization inhibitors: The mechanisms and management strategies.

Microtubule polymerization inhibitors (MPIs) have long been used as anticancer agents because they inhibit mitosis. Microtubules are thought to play an important role in the migration of tumor cells and the formation of tumor blood vessels, and new MPIs are being developed. Many clinical trials of novel MPIs have been conducted in humans, while some clinical studies in dogs have also been reported. More attempts to apply MPIs not only in humans but also in the veterinary field are expected to be made in the future. Meanwhile, MPIs have a risk of cardiotoxicity. In this paper, we review findings on the pharmacological effects and cardiotoxicity of MPIs, as well as the mechanisms of their cardiotoxicity. Cardiotoxicity of MPIs involves not only the direct effects of MPIs on cardiomyocytes but also their effects on vascular function. For example, hypertension induced by impaired vascular function also contributes to the exacerbation of myocardial damage, and blood pressure control may be useful in reducing cardiotoxicity. By combined administration of MPIs and other anticancer agents, MPI efficacy may be enhanced, thereby potentially allowing to keep MPI dosage low. Measurement of myocardial injury markers in blood and echocardiography may be useful for monitoring cardiotoxicity. In particular, two-dimensional speckle tracking may have high sensitivity for the early detection of MPI-induced cardiac dysfunction. The exploration of the potential of new MPIs while understanding their toxicity and how to deal with them will lead to the further development of cancer chemotherapy.

DIPG-54. UPDATED FINDINGS FROM THE PHASE IB STUDY OF UNESBULIN (PTC596) IN CHILDREN WITH NEWLY-DIAGNOSED DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE NETWORK FOR NEURO-ONCOLOGY CLINICAL TRIALS (CONNECT)

Abstract BMI-1 represents a potential therapeutic target in pediatric HGG given its role in self-renewal and DNA-damage signaling, high expression levels in these tumors, and resultant proliferation blockade, mitotic abnormalities, and radiosensitization when modulated by unesebulin, a microtubule polymerization inhibitor. This phase Ib study sought to determine the recommended phase 2 dose (RP2D) and pharmacokinetic (PK) profiles of unesbulin administered concurrently with radiotherapy and as maintenance therapy in children with newly-diagnosed DIPG or HGG, as well as assess intratumoral PKs and target inhibition within a surgical cohort. As previously reported, the established RP2D from the dose-finding cohort (parts A [capsule] and C [tablet]), consisting of 27 patients (median age: 9 years; 18 with DIPG, 9 with HGG) is 200mg/m2 twice weekly. Eleven additional patients (5 with DIPG, 6 with HGG) have been enrolled in an expansion cohort (part D [tablet]), with no dose-modifying toxicities to date. Among 38 total patients, most common drug-related grade 3/4 adverse effects observed are neutropenia (49%), leukopenia (31%), and elevated ALT (18%). Pharmacokinetic profiles are comparable between capsule and tablet formulations, and corroborate adult data. Genomics correlatives are underway on 13 biopsy and 5 autopsy specimens (2 paired samples). Five patients (median age: 5 years; 4 with DIPG, 1 with HGG) have enrolled in the surgical cohort (part B [tablet]) at the RP2D. PK analyses performed on the first three samples confirm intratumoral unesbulin exposure. In summary, the RP2D of unesbulin at 200mg/m2 twice weekly concurrent with and post-radiotherapy is feasible and well-tolerated, with evidence of blood-brain barrier and tumor penetrance. Survival outcomes as well as ongoing analyses of intratumoral BMI-1 pathway inhibition in surgical cohort samples, plus comprehensive molecular profiling (focusing on BMI-1 and cell cycle signaling) of biopsy and autopsy specimens from the entire cohort, will be shared at time of presentation.

Lactate: A critical regulator of cell proliferation via anaphase promoting complex remodeling

Lactate: A critical regulator of cell proliferation via anaphase promoting complex remodeling

Phase 2 study of telisotuzumab vedotin (Teliso-V) monotherapy in patients with previously untreated MET-amplified locally advanced/metastatic non-squamous non-small cell lung cancer (NSQ NSCLC).

TPS9149 Background: Teliso-V is a first-in-class anti–c-Met antibody-drug conjugate composed of the monoclonal antibody telisotuzumab (ABT-700), a cleavable valine-citrulline (dipeptide) linker, and cytotoxic monomethylauristatin E (MMAE; potent microtubule polymerization inhibitor). Direct MMAE payload delivery to c-Met protein overexpressing (OE) cells enables Teliso-V to work independently of c-Met signaling. In NSCLC, while c-Met OE is more common than MET gene amplification (Amp), about 90% of MET Amp tumors will also be c-Met OE. In an ongoing phase 2 trial, Teliso-V showed encouraging efficacy and acceptable safety in previously treated c-Met OE NSQ NSCLC population (Camidge et al. JCO.2022.40.16_suppl.9016). From this study, a retrospective analysis of 10 patients with MET Amp (≥1.8 MET gene to chromosome 7 copy number ratio by fluorescence in situ hybridization [FISH]) demonstrated an objective response rate (ORR) of 80%. Described here is an ongoing phase 2 study evaluating efficacy and safety of Teliso-V monotherapy in patients with previously untreated MET Amp locally advanced/metastatic NSQ NSCLC. Methods: This is a phase 2, single-arm, open-label, global study (NCT05513703). Eligible patients (≥18 years) have histologically documented advanced/metastatic NSQ NSCLC, measurable disease by RECIST v1.1, confirmed MET Amp determined centrally by FISH or locally by FISH, tissue next-generation sequencing (NGS), or plasma NGS using sponsor-approved assays. Additional eligibility criteria include Eastern Cooperative Oncology Group performance status 0 or 1, no alterations in EGFR, ALK, ROS1, or BRAF that predict sensitivity to targeted therapy, no prior systemic therapy for locally advanced or metastatic NSCLC, no prior c-Met–targeted antibody therapy. Teliso-V is administered intravenously at 1.9 mg/kg every 2 weeks until disease progression, intolerable toxicity, or other study drug discontinuation criteria are met. Primary endpoint is ORR assessed by independent central review (ICR). Secondary endpoints include duration of response and disease control rate, progression-free survival per ICR, overall survival, patient-reported outcomes (PROs). Tumor assessments (per RECIST v1.1) are performed at baseline and approx. every 6 (year 1), 8 (year 2), and 12 weeks (year 3 and beyond). PROs are assessed through validated questionnaires (EORTC QLQ-LC13, EORTC QLQ-C30, EQ-5D-5L). Safety and tolerability are evaluated by adverse events, physical examinations, laboratory data, and vital signs. Pharmacokinetic and biomarker evaluations may be used for exploratory analyses. The trial intends to enroll approx. 70 efficacy-evaluable patients, with interim analyses planned after 20 and 50 patients are able to be followed for ≥6 months. The study was open to enrollment as of Nov 2022. Clinical trial information: NCT05513703 .

Abstract CT214: Preliminary efficacy of telisotuzumab vedotin (Teliso-V) treatment in the 2L/3L setting in MET gene amplified (MET Amp), c-Met protein overexpressing (c-Met OE), non-squamous, non-small cell lung cancer (NSQ NSCLC): Retrospective analysis of LUMINOSITY

Abstract Background: Teliso-V is a first-in-class c-Met-directed antibody-drug conjugate comprising the monoclonal antibody telisotuzumab, a cleavable valine-citrulline linker, and the potent microtubule polymerization inhibitor monomethyl auristatin E. In a Phase 2 study (LUMINOSITY, NCT03539536), Teliso-V has shown an acceptable safety profile and encouraging efficacy in c-Met OE NSQ NSCLC. In NSCLC, while c-Met OE is more common than MET Amp, occurring in 25% of patients (pts) versus 1-5%, respectively, ~90% of MET Amp tumors are c-Met OE. We performed a retrospective analysis of LUMINOSITY to characterize the efficacy of Teliso-V in previously treated pts with MET Amp, c-Met OE NSQ NSCLC. Methods: Pts had locally advanced/metastatic NSQ NSCLC, ≤2 prior lines of systemic therapy, ≤1 line of chemotherapy, MET Amp by fluorescence in situ hybridization (FISH), and tumors that were c-Met OE by central immunohistochemistry (IHC). MET Amp was defined as a ratio of ≥1.8 MET gene copy number (GCN) to chromosome 7 (CEP7) CN by FISH with levels of amplification defined as high, ≥5; intermediate (int), >2.2 - <5; or low, ≥1.8 - ≤2.2. MET GCN by FISH was classified as high, ≥10; int, 5 - <10; or low, <5. c-Met OE was defined as ≥25% of tumor cells at 3+ intensity by IHC; patients were classified as MET IHC 3/25 (25 - 49% tumor cells at 3+) or MET IHC 3/50 (≥50% tumor cells at 3+). Teliso-V was dosed at 1.9 mg/kg IV Q2W. Results: As of 27 May 2021, 10 pts with MET Amp were treated with Teliso-V. The median age was 69 (48-75), 9 pts were male, 5 were White, 9 had a history of smoking (current or former), and 9 had an ECOG score of 0-1. The median MET/CEP7 ratio was 3.3 (1.84-12.17) with 4, 1, and 5 pts having high, int, and low levels of amplification, respectively. The median MET GCN was 10.98 (6.95-65.00) with 6, 3, and 1 pts having high, int, and low GCN, respectively. 7 pts were MET IHC 3/50 and 3 were MET IHC 3/25. Pts with MET Amp treated with Teliso-V monotherapy had an ORR of 80% (95% CI [44.4, 97.5]; n=8; all partial response) by independent central review, and a disease control rate of 90%. The median duration of response was 6.9 months. Among the 8 responders, 62.5% (n=5/8) are still event-free and being followed up for disease assessment. Teliso-V monotherapy resulted in median progression-free survival (PFS) of 8.0 months (95% CI [1.3, -]; n=10), compared with 5.7 months (95% CI [2.6, 9.8]) on the last prior line of systemic cancer therapy (n=10). Conclusions: Teliso-V demonstrated a promising ORR in previously treated pts with MET Amp NSQ NSCLC with c-Met OE and improved PFS when compared to last prior systemic cancer therapy. These preliminary data support the ongoing Phase 2 trial of Teliso-V monotherapy in pts with previously untreated MET Amp NSCLC (TeliMET NSCLC-02; NCT05513703), which is currently enrolling. Citation Format: D. Ross Camidge, Jonathan Goldman, Athan Vasilopoulos, Peter Ansell, Summer Xia, Ellen Bolotin, Jim Looman, Christine Ratajczak, Elysa Noon, Shun Lu. Preliminary efficacy of telisotuzumab vedotin (Teliso-V) treatment in the 2L/3L setting in MET gene amplified (MET Amp), c-Met protein overexpressing (c-Met OE), non-squamous, non-small cell lung cancer (NSQ NSCLC): Retrospective analysis of LUMINOSITY [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT214.

Abstract 5729: Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation

Abstract Background: The survival rate for patients with Pancreatic Ductal Adenocarcinoma (PDAC) is dramatically poor with a five-year survival rate less than 10%. The research of new treatments, which could complement the current therapeutic arsenal constituted by Gemcitabine, FOLFIRINOX (fluorouracile, leucovorin, irinotecan, oxaliplatin) and nab-paclitaxel, is a major challenge. Imiqualines are new original small heterocyclic chemical molecules based on the quinoxalinic moiety. Among these first in class compounds, the lead EAPB02303 (1) displays outstanding nanomolar activities comparable to those of the current best anticancer agents on a panel of human cancer cell lines, notably on poorly sensitive cancer like PDAC and melanoma. We tested here if EAPB02303 could be an attractive first in class molecule in PDAC and we conducted in-deep molecular characterization and bioinformatics studies to decipher its mechanism of action. Methods: We characterized EAPB02303 effect on tumor growth in-vitro by conducting sulforhodamine B assay on a panel of PDAC cells including cells derived from PDX (Patient Derived Xenograft) and 3D models with Cancer Associated Fibroblasts. We assessed in-vivo activity on subcutaneous PDAC xenografts mouse models. We then studied EAPB02303 effect on cell cycle, apoptosis and microtubule polymerisation by flow cytometry and immunofluorescence. We analyzed mRNAseq and Reverse Phase Protein Assay (RPPA) data of PDAC cell lines treated with EAPB02303 at multiple time points and concentrations to identify signaling pathways and key proteins implicated in EAPB02303 effect. We performed differential gene expression and gene set enrichment analysis by using EdgeR, Deseq2 and fgsea packages. We also used PharmacoGx package to seek for similar transcriptomic profiles among the CMAP perturbational database. Results: We showed that EAPB02303 exerts activity at low nanomolar concentrations in-vitro in PDAC cell lines and 3D models, and is able to reduce tumor growth in our xenografts in-vivo mouse models. We also found a potent synergy with Paclitaxel at lower concentrations of both compounds. Furthermore, we found that EAPB02303 induces mitosis arrest and impairment of spindle assembly after 24h treatment. Cells also underwent apoptosis after 48h treatment. mRNAseq and RPPA data showed activation of several signaling pathways including MAPK kinases. CMAP database mining revealed a high connectivity score of transcriptomic signatures between EAPB02303 and inhibitors of microtubule polymerization. All these data suggest that EAPB02303 is a new microtubule-disrupting agent with in-vivo activity in PDAC and in-vitro synergy with Paclitaxel, showing potential for future clinical investigations. (1) Imidazo[1,2a]quinoxalines and derivatives thereof for treating cancers. WO 2009 043934A1. Deleuze-Masqeufa C. et al. Citation Format: Kevin Bigot, Véronique Garambois, Nadia Vie, Marine Bruciamacchie, Pierre-Emmanuel Colombo, Diego Tosi, Cindy Patinote, Yann Maggipinto, Pierre-Antoine Bonnet, Céline Gongora, Carine Deleuze-Masquefa, Christel Larbouret. Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation. [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 5729.

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