Microtubule Formation Research Articles

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2

In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation through enhanced localization to TPX2 condensates, which form the core of the branch site on microtubules. Lastly, we provide a high-resolution cryo-EM structure of HURP on the microtubule, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

Versatile gamma-tubulin complexes contribute to the dynamic organization of MTOCs during Drosophila spermatogenesis

The initiation of microtubule formation is facilitated by γ-tubulin and γ-Tubulin Ring Complex (γ-TuRC) in various microtubule-organizing centers (MTOCs). While the heterogeneity of tissue-specific MTOCs and γ-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. We investigated the testis-specific distribution and biochemical interaction of the canonical γ-TuRC proteins Grip163 and Grip84. We found that while Grip163 is present on the centrosome and basal body, Grip84 localizes to the centrosome and Golgi in spermatocytes and colocalizes with the testis-specific γ-Tubulin complexes (t-γ-TuC) at the basal body, apical nuclear tip, and near the elongated mitochondria after meiosis. We also showed the apical nuclear tip localization of some γ-TuRC interacting partners and proved their binding to t-γ-TuC proteins. These results highlight and prove the importance of the different γ-TuRCs in organizing the diverse MTOCs present during the extensive rearrangement of cell organelles during the spermatogenesis of Drosophila.

Bta-miR-665 improves bovine blastocyst development through its influence on microtubule dynamics and apoptosis.

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture.

Effects of co-culture system and apple seed extract supplementation on apoptosis and microtubule formation in pig IVF embryos with cell cycle arrested

Effects of co-culture system and apple seed extract supplementation on apoptosis and microtubule formation in pig IVF embryos with cell cycle arrested

RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

Forebrain Eml1 depletion reveals early centrosomal dysfunction causing subcortical heterotopia.

Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.

Human cranial bone-derived mesenchymal stem cells cultured under simulated microgravity can improve cerebral infarction in rats

The efficacy of transplanting human cranial bone-derived mesenchymal stem cells (hcMSCs) cultured under simulated microgravity (sMG) conditions has been previously reported; however, their effect on cerebral infarction remains unknown. Here, we examined the efficacy of transplanting hcMSCs cultured in an sMG environment into rat models of cerebral infarction. For evaluating neurological function, hcMSCs cultured in either a normal gravity (1G) or an sMG environment were transplanted in rats 1 day after inducing cerebral infarction. The expression of endogenous neurotrophic, axonal, neuronal, synaptogenic, angiogenic, and apoptosis-related factors in infarcted rat brain tissue was examined using real-time polymerase chain reaction and western blotting 35 days after stroke induction. The RNAs of hcMSCs cultured under 1G or sMG environments were sequenced. The results showed that neurological function was significantly improved after transplantation of hcMSCs from the sMG group compared with that from the 1G group. mRNA expressions of nerve growth factor, fibroblast growth factor 2, and synaptophysin were significantly higher in the sMG group than in the 1G group, whereas sortilin 1 expression was significantly lower. RNA sequencing analysis revealed that genes related to cell proliferation, angiogenesis, neurotrophy, neural and synaptic organization, and inhibition of cell differentiation were significantly upregulated in the sMG group. In contrast, genes promoting microtubule and extracellular matrix formation and cell adhesion, signaling, and differentiation were downregulated. These results demonstrate that hcMSCs cultured in the sMG environment may be a useful source of stem cells for the recovery of neurological function after cerebral infarction.

Herbal Therapies for Cancer Treatment: A Review of Phytotherapeutic Efficacy.

Natural products have proven to be promising anti-cancer agents due to their diverse chemical structures and bioactivity. This review examines their central role in cancer treatment, focusing on their mechanisms of action and therapeutic benefits. Medicinal plants contain bioactive compounds, such as flavonoids, alkaloids, terpenoids and polyphenols, which exhibit various anticancer properties. These compounds induce apoptosis, inhibit cell proliferation and cell cycle progression, interfere with microtubule formation, act on topoisomerase targets, inhibit angiogenesis, modulate key signaling pathways, improve the tumor microenvironment, reverse drug resistance and activate immune cells. Herbal anti-cancer drugs offer therapeutic advantages, particularly selective toxicity against cancer cells, reducing the adverse side effects associated with conventional chemotherapy. Recent studies and clinical trials highlight the benefits of herbal medicines in alleviating side effects, improving tolerance to chemotherapy and the occurrence of synergistic effects with conventional treatments. For example, the herbal medicine SH003 was found to be safe and potentially effective in the treatment of solid cancers, while Fucoidan showed anti-inflammatory properties that are beneficial for patients with advanced cancer. The current research landscape on herbal anticancer agents is extensive. Numerous studies and clinical trials are investigating their efficacy, safety and mechanisms of action in various cancers such as lung, prostate, breast and hepatocellular carcinoma. Promising developments include the polypharmacological approach, combination therapies, immunomodulation and the improvement of quality of life. However, there are still challenges in the development and use of natural products as anti-cancer drugs, such as the need for further research into their mechanisms of action, possible drug interactions and optimal dosage. Standardizing herbal extracts, improving bioavailability and delivery, and overcoming regulatory and acceptance hurdles are critical issues that need to be addressed. Nonetheless, the promising anticancer effects and therapeutic benefits of natural products warrant further investigation and development. Multidisciplinary collaboration is essential to advance herbal cancer therapy and integrate these agents into mainstream cancer treatment.

The Domestication of Wild Boar Could Result in a Relaxed Selection for Maintaining Olfactory Capacity.

Domesticated animals are artificially selected to exhibit desirable traits, however not all traits of domesticated animals are the result of deliberate selection. Loss of olfactory capacity in the domesticated pig (Sus scrofa domesticus) is one example. We used whole transcriptome analysis (RNA-Seq) to compare patterns of gene expression in the olfactory mucosa of the pig and two subspecies of wild boar (Sus scrofa), and investigate candidate genes that could be responsible for the loss of olfactory capacity. We identified hundreds of genes with reductions in transcript abundance in pig relative to wild boar as well as differences between the two subspecies of wild boar. These differences were detected mainly in genes involved in the formation and motility of villi, cilia and microtubules, functions associated with olfaction. In addition, differences were found in the abundances of transcripts of genes related to immune defenses, with the highest levels in continental wild boar subspecies. Overall, the loss of olfactory capacity in pigs appears to have been accompanied by reductions in the expression of candidate genes for olfaction. These changes could have resulted from unintentional selection for reduced olfactory capacity, relaxed selection for maintaining olfactory capacity, pleiotropic effects of genes under selection, or other non-selective processes. Our findings could be a cornerstone for future researches on wild boars, pigs, feral populations, and their evolutionary trajectories, aimed to provide tools to better calibrate species management as well as guidelines for breeders.

Excess microtubule and F-actin formation mediates shortening and loss of primary cilia in response to a hyperosmotic milieu.

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shock-induced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin.

Abstract Mo047: DNA METHYLATION AND TRANSCRIPTIONAL MARKERS OF MYOCARDIAL REVERSE REMODELING IN HF PATIENTS ON LVAD SUPPORT

Heart failure (HF) involves global genome modifications. Adverse structural and function remodeling that occurs in HF was initially considered to be irreversible. However, ~15% of HF patients on left ventricular assist device (LVAD) support show significant structural and functional reverse remodeling, distinguishing them as responders (R), while ~85% are non-responders (NR). DNA methylation and transcriptional changes that exert myocardial recovery is not well understood. To address this, we performed whole-genome bisulphite sequencing and RNA sequencing on paired myocardial biopsies from HF patients (n=84) before (pre-LVAD) and after LVAD unloading (post-LVAD). Post-LVAD samples showed activation of pathways including TGFβ signaling, PTEN and Fatty acid oxidation compared to pre. Integrating DNA methylation and transcriptomics data revealed hypomethylation (upregulation) of genes including Scaper, Frat1 and Magi1 and hypermethylation (downregulation) of Tpp3 and Icam. Overall, suggesting a unique ECM-specific stabilization pathway post-LVAD unloading. Segregating samples based on their response to LVAD unloading, pathways such as inflammation and fibrotic signaling were activated in postNR compared to preNR. Hypomethylation of Magi1 and hypermethylation of Tppp3 was observed in postNR suggesting cross-linking proteins and destabilization of microtubule formation as potential mechanisms of functional regression in NR. Conversly, postR showed activation of deubiquitination, oxidative phosphorylation and NLR signaling compared to preR. Finally, we observed integrin signaling, syndecan interaction, collagen degradation and wound healing pathways were activated in postR compared to postNR. Hypomethylation of actin cytoskeleton ( Myl12a ), microtubule/motor proteins ( Kif5b ) and tropomyosin ( Tpm3 ) were observed in postR, suggesting a plausible reverse remodeling mechanism mediated by ECM degradation and stabilization of myocardium in these patients. Western blot analysis also showed a trend towards an increase in collagen in postNR compared to postR (n=4, p=0.06). Finally, a mouse model of HF and recovery was used and pathways including ECM degradation and cardioprotective signaling were activated in mice that recovered from HF compared to mice that did not. Overall, our data suggests a unique ECM regulation pathway to contribute towards myocardial reverse remodeling in HF patients.

Spindle morphology changes between meiosis and mitosis driven by CK2 regulation of the Ran pathway.

The transition from meiotic divisions in the oocyte to embryonic mitoses is a critical step in animal development. Despite negligible changes to cell size and shape, following fertilization the small, barrel-shaped meiotic spindle is replaced by a large zygotic spindle that nucleates abundant astral microtubules at spindle poles. To probe underlying mechanisms, we applied a drug screening approach using Ciona eggs and found that inhibition of Casein Kinase 2 (CK2) caused a shift from meiotic to mitotic-like spindle morphology with nucleation of robust astral microtubules, an effect reproduced in cytoplasmic extracts prepared from Xenopus eggs. In both species, CK2 activity decreased at fertilization. Phosphoproteomic differences between Xenopus meiotic and mitotic extracts that also accompanied CK2 inhibition pointed to RanGTP-regulated factors as potential targets. Interfering with RanGTP-driven microtubule formation suppressed astral microtubule growth caused by CK2 inhibition. These data support a model in which CK2 activity attenuation at fertilization leads to activation of RanGTP-regulated microtubule effectors that induce mitotic spindle morphology.

Potential Mechanism and Involvement of p120-Catenin in the Malignant Biology of Glioma.

This study analyzed the influence of p120-catenin (catenin [cadherin-associated protein], delta 1 [CTNND1]) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative polymerase chain reaction (QT-PCR) technology was employed to assess the expression of p120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups : siRNA-blank control (BC) group (no RNA sequence was transfected), siRNA-negative control (NC) group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzymelabeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (p<0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r=0.906, p<0.0001) and QT-PCR (F=830.6, p<0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (p<0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (p<0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

The Effects and Mechanism of ATM Kinase Inhibitors in Toxoplasma gondii.

Toxoplasma gondii, an important opportunistic pathogen, underscores the necessity of developing novel therapeutic drugs and identifying new drug targets. Our findings indicate that the half-maximal inhibitory concentrations (IC50) of KU60019 and CP466722 (abbreviated as KU and CP) against T. gondii are 0.522 μM and 0.702 μM, respectively, with selection indices (SI) of 68 and 10. Treatment with KU and CP affects the in vitro growth of T. gondii, inducing aberrant division in the daughter parasites. Transmission electron microscopy reveals that KU and CP prompt the anomalous division of T. gondii, accompanied by cellular enlargement, nuclear shrinkage, and an increased dense granule density, suggesting potential damage to parasite vesicle transport. Subsequent investigations unveil their ability to modulate the expression of certain secreted proteins and FAS II (type II fatty acid synthesis) in T. gondii, as well as including the dot-like aggregation of the autophagy-related protein ATG8 (autophagy-related protein 8), thereby expediting programmed death. Leveraging DARTS (drug affinity responsive target stability) in conjunction with 4D-Label-free quantitative proteomics technology, we identified seven target proteins binding to KU, implicated in pivotal biological processes such as the fatty acid metabolism, mitochondrial ATP transmission, microtubule formation, and Golgi proteins transport in T. gondii. Molecular docking predicts their good binding affinity. Furthermore, KU has a slight protective effect on mice infected with T. gondii. Elucidating the function of those target proteins and their mechanism of action with ATM kinase inhibitors may potentially enhance the treatment paradigm for toxoplasmosis.

Differentially Expressed Proteins in the Serum of Elderly Patients Who Experienced Perioperative Neurocognitive Disorders Following Transurethral Resection of the Prostate.

Perioperative neurocognitive disorders (PND) are a group of prevalent neurological complications that often occur in elderly individuals following major or emergency surgical procedures. The etiologies are not fully understood. This study endeavored to investigate novel targets and prediction methods for the occurrence of PND. A total of 229 elderly patients diagnosed with prostatic hyperplasia who underwent transurethral resection of the prostate (TURP) combined with spinal cord and epidural analgesia were included in this study. The patients were divided into two groups, the PND group and non-PND group, based on the Z-score method. According to the principle of maintaining consistency between preoperative and intraoperative conditions, three patients from each group were randomly chosen for serum sample collection. isobaric tags for relative and absolute quantification (iTRAQ) proteomics technology was employed to analyze and identify the proteins that exhibited differential expression in the serum samples from the two groups. Bioinformatics analysis was performed on the proteins that exhibited differential expression. Among the 1101 serum proteins analyzed in the PND and non-PND groups, eight differentially expressed proteins were identified in PND patients. Of these, six proteins showed up-regulation, while two proteins showed down-regulation. Further bioinformatics analysis of the proteins that exhibited differential expression revealed their predominant involvement in cellular biological processes, cellular component formation, as well as endocytosis and phagocytosis Additionally, these proteins were found to possess the RING domain of E3 ubiquitin ligase. The iTRAQ proteomics technique was employed to analyze the variation in protein expression in serum samples from patients with PND and those without PND. This study successfully identified eight proteins that exhibited differential expression levels between the two groups. Bioinformatics analysis indicates that proteins exhibiting differential expression are primarily implicated in the biological processes associated with microtubules. Investigating the microtubule formation process as it relates to neuroplasticity and synaptic formation may offer valuable insights for enhancing our comprehension and potential prevention of PND. Registered (ChiCTR2000028836). Date (20190306).

Mechanisms of mitotic inhibition in human aorta endothelial cells: Molecular and morphological in vitro spectroscopic studies

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments.Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug’s action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells.The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Abstract 674: Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway

Abstract Aberrant migration and proliferation of cancer cells are the hallmark of metastatic tumors. Protein Regulator of Cytokinesis 1 (PRC1) is a scaffold protein that regulates metabolic pathways of cancer cell proliferation, survival, and metastasis. BKT300 is a small molecule (MW 399.33, C22H25NO6), that selectively inhibits the migration and survival of hematological and solid cancer cells. BKT300 inhibits, at high affinity, both actin and microtubule formation and organization leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis through the caspase 3 pathway. These effects are selective to cancer cells and were not observed on normal cells. BKT300 was found to arrest PRC1 in a phosphorylated form at position T481, down regulates the expression of CDC25C and up regulate p21. Suppression of PRC1 using SiRNA ablation inhibited BKT300 induced apoptosis on several tumor cell lines and inhibited the effect of BKT300 on G2M cell cycle arrest. Furthermore, using Biacore SPR, microscale thermophoresis (MST), and ELISA assays we were able to demonstrate the direct binding of BKT300 to PRC1. Beyond its single agent activity, BKT300 was shown to synergize with standard of care (SoC) treatments, including anti PD-1 immunotherapy, BCL-2 inhibitor (Ventoclax), topoisomerase I inhibitor (Irinotecan) topoisomerase II inhibitor (Doxorubicin), anti-metabolite Fluorouracil (5-FU) and alkylating agent (Temozolomide). BKT300 has a remarkable safety profile when administered at high doses to mice rats, minipigs and non-human primates. Treatment with BKT300 showed almost no effects on hematopoiesis and biochemistry in doses far higher than the affective ones. The in-vivo activity of BKT300 was demonstrated in multiple Xenograft, Syngeneic and PDX models in mice. In-vivo treatment with BKT300 resulted in preventive (small tumors) as well as therapeutic (large tumors) activity in triple negative breast cancer (TNBC) xenograft, MDA-MB-453 and MDA-MB-468 models leading to over then 70%-90% reduction in tumor size following one to two treatments cycles. Similar results were obtained using PDX models derived from patients with Colon, Ovarian and Pancreatic cancer. BKT300 is a novel, first in class, targeted anti-cancer treatment that inhibits the activity of the scaffold protein PRC1. BKT300 is expected to provide a new treatment option to, the high unmet medical need of, advanced difficult to treat cancers, specifically ones that overexpress PRC1 and CDC25C. Citation Format: Amnon Peled, Michal Abraham, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon. Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway [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 674.

Abstract 513: Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). We previously showed that loss of Neurofibromatosis 1 (NF1), a gene frequently mutated across cancers, enhances the activity of DM1, the maytansinoid payload of T-DM1, through a novel function in regulating microtubule (MT) dynamics. Maytansinoids are puzzlingly more effective in cells (in the nanomolar range) vs in vitro (in the micromolar range). Since maytansinoids bind at the interface between tubulin dimers, they are thought to only bind soluble tubulin dimers or MT ends, which would suggest very few binding sites available for pharmacological interaction in vivo, at odds with data. Here we investigated the interaction of DM1 with NF1 and MTs, using cellular and reductionist in vitro systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3-GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In turbidity-based tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization, and completely rescued DM1-induced inhibition. Visual inspection of fluorescent MTs showed that NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a significant increase in MT dynamics (elongation speed, rescue and catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intra-tubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair.In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intra-tubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Zdeněk Lánský, Marcus Braun, Luigi Scietti, Luca Mazzarella. Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload [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 513.