Microtubule Destabilization Research Articles

Dual PET imaging of microtubules and synaptic density in a mouse model of Alzheimer’s Disease

AbstractBackgroundWhile microtubule (MT)‐destabilization and synaptic loss hallmark AD pathological events, no study has systematically quantified their in vivo levels in the same brain. Any therapeutic intervention relying on MT destabilization and synaptic vesicle protein (SV2A) levels will benefit from a dual PET imaging strategy, providing target engagement, dosing, and occupancy studies for new drugs. Here, we report MT destabilization and synaptic density imaging results using [11C]MPC‐6827 and [18F]UCB‐H respectively in the same transgenic (TG) and wild‐type (WT) APP/PS1 mice.MethodMale APP/PS1 TG and WT mice (12‐14 mo, 20± 2g, n = 8/group) underwent 30‐min dynamic brain microPET/CT imaging with [11C]MPC‐6827 (0.2±0.02 mCi, iv tail). Four hours later, the same animals underwent another 30‐min brain PET/CT with [18F]UCB‐H (0.2±0.01 mCi, iv tail). A sub‐set of TG and WT mice (n = 4/group) were used to measure ex vivo destabilized MTs and SV2A levels using western blot to confirm MT destabilization and synaptic loss. Whole‐brain standard uptake values (SUVs) were calculated from co‐registered PET‐CT using PMOD. Ex vivo post‐PET biodistribution studies were performed, with percent injected dose‐pe‐ gram (%ID/g) calculated. 48h post‐biodistribution, in vitro autoradiography studies were performed on frozen brain samples from the same animals with [11C]MPC‐6827 and [18F]UCBH; regional uptake was quantified using phosphor‐stimulated luminescence.Result[11C]MPC‐6827 and [18F]UCBH were produced in high (>97±2%) radiochemical purities (n = 20 productions) and specific activities (∼3980±100 mCi/µmol), decay‐corrected to end‐of‐synthesis. Western blot analysis confirmed high destabilized MTs, and low SV2A levels in TG vs. WT. SUV analysis (Fig. 1) showed 56% higher [11C]MPC‐6827 and 66% lower [18F]UCB‐H in vivo brain uptake in TG compared to WT (**p = 0.0032). %ID/g analysis showed 58% higher [11C]MPC‐6827 and 61% lower [18F]UCB‐H ex vivo brain radioactive uptake in TG vs WT (*p = 0.04). Importantly, autoradiography demonstrated 44% higher [11C]MPC‐6827 and 34% lower [18F]UCB‐H in vitro uptake in cortical and hippocampal regions of TG vs. WT (*p = 0.05).ConclusionTG APP/PS1 mice demonstrated higher [11C]MPC‐6827 and lower [18F]UCB‐H brain uptake compared to WT littermates. In vivo PET, ex vivo biodistribution, and in vitro autoradiography data corroborated each other. Preliminary [11C]MPC‐6827 and [18F]UCB‐H PET evaluations showed negative correlations (*r = ‐0.778), suggesting MT destabilization and synaptic loss happen concurrently.

Dual PET imaging of microtubules and synaptic density in a mouse model of Alzheimer’s Disease

AbstractBackgroundWhile microtubule (MT)‐destabilization and synaptic loss hallmark AD pathological events, no study has systematically quantified their in vivo levels in the same brain. Any therapeutic intervention relying on MT destabilization and synaptic vesicle protein (SV2A) levels will benefit from a dual PET imaging strategy, providing target engagement, dosing, and occupancy studies for new drugs. Here, we report MT destabilization and synaptic density imaging results using [11C]MPC‐6827 and [18F]UCB‐H respectively in the same transgenic (TG) and wild‐type (WT) APP/PS1 mice.MethodMale APP/PS1 TG and WT mice (12‐14 mo, 20± 2g, n = 8/group) underwent 30‐min dynamic brain microPET/CT imaging with [11C]MPC‐6827 (0.2±0.02 mCi, iv tail). Four hours later, the same animals underwent another 30‐min brain PET/CT with [18F]UCB‐H (0.2±0.01 mCi, iv tail). A sub‐set of TG and WT mice (n = 4/group) were used to measure ex vivo destabilized MTs and SV2A levels using western blot to confirm MT destabilization and synaptic loss. Whole‐brain standard uptake values (SUVs) were calculated from co‐registered PET‐CT using PMOD. Ex vivo post‐PET biodistribution studies were performed, with percent injected dose‐pe‐ gram (%ID/g) calculated. 48h post‐biodistribution, in vitro autoradiography studies were performed on frozen brain samples from the same animals with [11C]MPC‐6827 and [18F]UCBH; regional uptake was quantified using phosphor‐stimulated luminescence.Result[11C]MPC‐6827 and [18F]UCBH were produced in high (>97±2%) radiochemical purities (n = 20 productions) and specific activities (∼3980±100 mCi/µmol), decay‐corrected to end‐of‐synthesis. Western blot analysis confirmed high destabilized MTs, and low SV2A levels in TG vs. WT. SUV analysis (Fig. 1) showed 56% higher [11C]MPC‐6827 and 66% lower [18F]UCB‐H in vivo brain uptake in TG compared to WT (**p = 0.0032). %ID/g analysis showed 58% higher [11C]MPC‐6827 and 61% lower [18F]UCB‐H ex vivo brain radioactive uptake in TG vs WT (*p = 0.04). Importantly, autoradiography demonstrated 44% higher [11C]MPC‐6827 and 34% lower [18F]UCB‐H in vitro uptake in cortical and hippocampal regions of TG vs. WT (*p = 0.05).ConclusionTG APP/PS1 mice demonstrated higher [11C]MPC‐6827 and lower [18F]UCB‐H brain uptake compared to WT littermates. In vivo PET, ex vivo biodistribution, and in vitro autoradiography data corroborated each other. Preliminary [11C]MPC‐6827 and [18F]UCB‐H PET evaluations showed negative correlations (*r = ‐0.778), suggesting MT destabilization and synaptic loss happen concurrently.

Oligomeric tau enters brain vascular endothelial cells and induces cellular senescence and brain microvascular dysfunction in tauopathy

AbstractBackgroundVascular mechanisms of Alzheimer’s disease (AD) may constitute the most therapeutically addressable biological pathway underlying dementia. Soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, particularly in microvascular endothelial cells.MethodsPrimary human brain microvascular endothelial cells (HBEC) were exposed to tau oligomers. Microtubule stability, eNOS activation, and markers of senescence were measured. Mice expressing P301S‐mutant human tau (P301S(PS19) mice), a model of tauopathy, were given immunotherapy to selectively remove tau oligomers, or non‐specific IgGs. After measurements of vascular reactivity, eNOS activity and markers of senescence were measured in microvasculature purified from brain.ResultOligomeric tau significantly decreased microtubule stability and eNOS activity, and induced cellular senescence in primary HBECs. Microtubule destabilization, reduced eNOS activity, and increased markers of cellular senescence in brain microvasculature of P301S(PS19) mice were associated with impaired vascular reactivity, and the latter was negated by immunotherapy that specifically removed oligomeric tau from brain.ConclusionBrain microvascular deficits in P301S(PS19) mice modeling tauopathy, mediated by tau oligomers, may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of tau oligomers. Pathogenic tau oligomers and senescent brain microvascular endothelial cells may thus be targets for intervention in AD and other tauopathies.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p<0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p<0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

Glaucoma as a Tauopathy—Is It the Missing Piece in the Glaucoma Puzzle?

Glaucoma is a chronic neurodegenerative disorder affecting the visual system which can result in vision loss and blindness. The pathogenetic mechanisms underlying glaucomatous optic neuropathy are ultimately enigmatic, prompting ongoing investigations into its potential shared pathogenesis with other neurodegenerative neurological disorders. Tauopathies represent a subclass of neurodegenerative diseases characterized by the abnormal deposition of tau protein within the brain and consequent microtubule destabilization. The extended spectrum of tauopathies includes conditions such as frontotemporal dementias, progressive supranuclear palsy, chronic traumatic encephalopathy, and Alzheimer’s disease. Notably, recent decades have witnessed emerging documentation of tau inclusion among glaucoma patients, providing substantiation that this ocular disease may similarly manifest features of tauopathies. These studies found that: (i) aggregated tau inclusions are present in the somatodendritic compartment of RGCs in glaucoma patients; (ii) the etiology of the disease may affect tau splicing, phosphorylation, oligomerization, and subcellular localization; and (iii) short interfering RNA against tau, administered intraocularly, significantly decreased retinal tau accumulation and enhanced RGC somas and axon survival, demonstrating a crucial role for tau modifications in ocular hypertension-induced neuronal injury. Here, we examine the most recent evidence surrounding the interplay between tau protein dysregulation and glaucomatous neurodegeneration. We explore the novel perspective of glaucoma as a tau-associated disorder and open avenues for cross-disciplinary collaboration and new treatment strategies.

P-072 - Dual PET imaging of microtubule destabilization and synaptic integrity in a mouse model of AD

P-072 - Dual PET imaging of microtubule destabilization and synaptic integrity in a mouse model of AD

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

Kinesin family member 2A gates nociception

Nociceptive axons undergo remodeling as they innervate their targets during development and in response to environmental insults and pathological conditions. How is nociceptive morphogenesis regulated? Here, we show that the microtubule destabilizer kinesin family member 2A (Kif2a) is a key regulator of nociceptive terminal structures and pain sensitivity. Ablation of Kif2a in sensory neurons causes hyperinnervation and hypersensitivity to noxious stimuli in young adult mice, whereas touch sensitivity and proprioception remain unaffected. Computational modeling predicts that structural remodeling is sufficient to explain the phenotypes. Furthermore, Kif2a deficiency triggers a transcriptional response comprising sustained upregulation of injury-related genes and homeostatic downregulation of highly specific channels and receptors at the late stage. The latter effect can be predicted to relieve the hyperexcitability of nociceptive neurons, despitepersisting morphological aberrations, and indeed correlates with the resolution of pain hypersensitivity. Overall, we reveal a critical control node defining nociceptive terminal structure, which is regulating nociception.

Application of Pluronics for Enhancing Aqueous Solubility of Lipophilic Microtubule Destabilizing Compounds on the Sea Urchin Embryo Model.

In screening, the dilution of DMSO stock solution of a lipophilic molecule with an assay medium often causes compound precipitation. To overcome the issue, the application of Pluronics as cosolvents was examined using a phenotypic sea urchin embryo assay that allows for the quick and facile evaluation of the antiproliferative effect together with systemic toxicity. Maximum tolerated concentration values for Pluronics L121, P123, and F127 were 1.4 μM, 8.6 μM, and 39.7 μM, respectively, and correlated directly with their hydrophilicity. Pluronics L121 and P123 suppressed cleavage and blastomeres retained the round shape, unlike hydrophilic Pluronic F127, which induced fertilization envelope creasing and embryo deformation that could be associated with the interaction of hydrophilic PEO units with mucopolysaccharides at the surface of sea urchin embryos. The toxicity of P123, but not of L121 and F127, was temperature-dependent and markedly increased at lower temperatures. CMC values obtained at different temperatures confirmed that the toxic effect of P123 was associated with both unimers and micelles, whereas F127 toxicity was related mainly to micelles. Evaluation using phenotypic sea urchin embryo assay revealed that potent microtubule destabilizers, namely albendazole, diarylisoxazole, and two chalcones, retained antimitotic activity after the dilution of their DMSO or 2-pyrrolidone stock solutions with 1.25% w/v Pluronic P123 or 5% w/v Pluronic F127. It was suggested that Pluronic P123 and Pluronic F127 could be used as cosolvents to improve the solubility of lipophilic molecules in aqueous medium.

Preliminary PET Imaging of Microtubule-Based PET Radioligand [11C]MPC-6827 in a Nonhuman Primate Model of Alzheimer's Disease.

The microtubule (MT) instability observed in Alzheimer's disease (AD) is commonly attributed to hyperphosphorylation of the MT-associated protein, tau. In vivo PET imaging offers an opportunity to gain critical information about MT changes with the onset and development of AD and related dementia. We developed the first brain-penetrant MT PET ligand, [11C]MPC-6827, and evaluated its in vivo imaging utility in vervet monkeys. Consistent with our previous in vitro cell uptake and in vivo rodent imaging experiments, [11C]MPC-6827 uptake increased with MT destabilization. Radioactive uptake was inversely related to (cerebrospinal fluid) CSF Aβ42 levels and directly related to age in a nonhuman primate (NHP) model of AD. Additionally, in vitro autoradiography studies also corroborated PET imaging results. Here, we report the preliminary results of PET imaging with [11C]MPC-6827 in four female vervet monkeys with high or low CSF Aβ42 levels, which have been shown to correlate with the Aβ plaque burden, similar to humans.

Why kiss-and-hop explains that tau does not stabilize microtubules and does not interfere with axonal transport (at physiological conditions).

Tau is a microtubule-associated protein that is enriched in the axonal process of neurons. Post-translational modifications of tau have been implicated in the development of tauopathies characterized by defects in axonal transport, neuronal atrophy, and microtubule disassembly. Although tau is almost quantitatively bound to microtubules under physiological conditions, it does not significantly affect axonal transport. Furthermore, acute or chronic tau deficiency does not result in significant destabilization of neuronal microtubules, challenging the classical view that disease-related tau modifications directly cause axonal microtubule collapse. Here, we discuss how the rapid interaction kinetics of the tau-microtubule interaction, which we previously termed the kiss-and-hop interaction, explains why tau does not affect microtubule-dependent axonal transport but still allows tau to modulate microtubule polymerization. In contrast, tau modifications that slow down the kinetics of the tau-microtubule interaction and increase the residence time of tau at a microtubule interaction site can disrupt axonal transport and cause dendritic atrophy. We discuss the consequences of such a gain-of-toxicity mechanism in terms of the development of disease-modulating drugs that target the tau protein.

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.

N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aβ42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aβ42 (GMR > Aβ42) in developing Drosophila eye retinal neurons results in Aβ42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aβ42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aβ42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. We found that gain-of-function of Mnat9 rescues GMR > Aβ42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. Here, we propose a new neuroprotective function of Mnat9 in downregulating the JNK signaling pathway to ameliorate Aβ42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aβ42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9 or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aβ42 and may provide new avenues for targeting neurodegeneration.

Prospective mechanism of action of the tubulysin synthetic derivative (TAM 1344) in HCT116 colon cancer cell line

Tubulin is still a highly valued target in cancer chemotherapy. Agents that target tubulin and microtubule dynamic are considered to be of high therapeutic potential. We conducted a study to assess the effects of TAM1344, a synthetic cytolysin that is derived from the natural tubulysins on the proliferation of cancer cell lines. Tubulysins are a group of naturally occurring cytotoxic compounds that are produced by Myxobacteria. Our results show that TAM1344 exhibit strong antiproliferative activity against different cancer cell lines at low nanomolar concentration. The measured IC50 values in HCT116, A549 and MCF7 cancer cell lines were 0.14 nM, 0.24 nM & 0.09 nM, respectively. In a direct comparison, the three cell lines were more sensitive to the drugs than the myxobacterial natural products tubulysin-A and –B. Additionally, in HCT116 cells, TAM1344 induces destabilization and depletion of the interphase microtubules as indicated by Immunofluorescence staining. Furthermore, the spindle pools of dividing cells show unusual, condensed phenotyping, a characteristic phenotype of many anti-tubulin agents. The nuclei of treated cells look fragmented in comparison to control cells, as detected with DAPI or PI staining. Furthermore, at low concentrations, TAM1344 induces an accumulation of the cells in the G2/M phase of the cell cycle, and therefore apoptotic induction, as indicated with flow cytometry analysis. In addition, it provoked an apoptotic process, marked by elevated caspase-3 activity. To conclude, the results indicate that TAM1344 is a novel, highly effective microtubule-targeting agent.

Lisavanbulin (BAL101553), a novel, oral microtubule destabilizer plus radiation in patients with newly diagnosed, MGMT promoter unmethylated glioblastoma: A phase 1 Adult Brain Tumor Consortium study (ABTC1601).

2058 Background: Lisavanbulin (BAL101553, prodrug of BAL27862) is a novel, oral microtubule destabilizer that leads to cell death mediated by modulating the spindle assembly checkpoint. It has promising antitumoral activity in orthotopic glioblastoma (GBM) models in combination with radiation (RT) ± temozolomide (TMZ), including in MGMT promoter unmethylated (uMGMT) tumors. Lisavanbulin is a lipophilic and small molecule (MW 387), and data in rodents showed its ability to cross the blood-brain barrier (brain-plasma ratio 1:1). Methods: This multicenter phase 1 study was to determine the MTD of oral Lisavanbulin in combination with standard RT (60 Gy in 30 fractions) in patients with newly diagnosed uMGMT GBM. Dose escalation followed a modified 3+3 design. The safety evaluation period ended 4 weeks after completion of radiation, which was also the end of study. Patients were allowed to continue treatment with standard adjuvant TMZ as per treating physician’s recommendation. Results: Twenty-six patients with uMGMT GBM (median age 64y, 42.3% male, 61.5% with gross total resection, median KPS 80) were enrolled. Two GBM harbored an IDH1 mutation. Patients were treated at 5 different dose levels of Lisavanbulin administered once daily for 6 weeks concomitantly with RT: Lisavanbulin 4 mg (5 pts), 6 mg (5 pts), 8 mg (7 pts), 12 mg (5 pts), and 15 mg (4 pts). The initial starting dose was 8 mg. Due to Grade 4 aseptic meningoencephalitis in the first patient, the study was held and the dose decreased to 4 mg. Dose escalation resumed and continued to 15 mg with 1 DLT of Grade 2 confusion and memory impairment observed at 12 mg. A planned dose expansion was not conducted due to discontinuation of funding. Grade 3 AEs were hypertension (2), seizure (2), cognitive disturbance (1), cerebral edema (1), hyponatremia (1), and lymphopenia (1); no additional grade 4 AE was observed other than the 1 case of aseptic meningoencephalitis. Median OS was 12.8 months (95%CI: 9.1-18.3 months). At data cut-off, 8 subjects (30.8%) were still alive. 38.5% subjects had no information on disease progression and were censored during the estimation of PFS. The median PFS was 7.7 months (95%CI: 3.0-9.5 months). Conclusions: The maximum studied safe dose for Lisavanbulin in combination with RT in newly diagnosed uMGMT GBM was determined at 15 mg daily during radiation. Overall, the safety of this combination was acceptable. Next steps in developing Lisavanbulin in newly diagnosed GBM include safety studies in combination with TMZ and of TMZ+RT in MGMT promoter methylated GBM prior to formally studying efficacy in a prospective randomized trial. Clinical trial information: NCT03250299 .

Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy

Vascular mechanisms of Alzheimer’s disease (AD) may constitute a therapeutically addressable biological pathway underlying dementia. We previously demonstrated that soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, including prominently in microvascular endothelial cells. Here we show that soluble pathogenic tau accumulates in brain microvascular endothelial cells of P301S(PS19) mice modeling tauopathy and drives AD-like brain microvascular deficits. Microvascular impairments in P301S(PS19) mice were partially negated by selective removal of pathogenic soluble tau aggregates from brain. We found that similar to trans-neuronal transmission of pathogenic forms of tau, soluble tau aggregates are internalized by brain microvascular endothelial cells in a heparin-sensitive manner and induce microtubule destabilization, block endothelial nitric oxide synthase (eNOS) activation, and potently induce endothelial cell senescence that was recapitulated in vivo in microvasculature of P301S(PS19) mice. Our studies suggest that soluble pathogenic tau aggregates mediate AD-like brain microvascular deficits in a mouse model of tauopathy, which may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of soluble tau aggregates.

Abstract 6397: Eribulin acts as an immune adjuvant to enhance the antitumor efficacy of STING agonists in triple-negative breast cancer models

Abstract Triple-negative breast cancer (TNBC) is a heterogenous subtype of breast cancer lacking effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment. Strategies to improve the response of TNBC patients to immunotherapy include the upregulation of the cGAS-STING innate immune sensing pathway and STING agonists are in clinical development. We previously showed that eribulin, a microtubule destabilizer used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. In separate studies, we demonstrated that eribulin also significantly enhances type I interferon expression induced by STING agonists through a second TBK1-dependent mechanism downstream of STING activation. Both mechanisms of eribulin-mediated activation of interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizer paclitaxel. Our current studies demonstrate the in vivo immunological effects of the microtubule destabilizer eribulin with the STING agonist ADU-S100 that lead to a significant decrease in tumor growth in the spontaneous MMTV-PyVT mammary tumor model upon combination treatment. The immunostimulatory effects of eribulin with or without concurrent administration of a STING agonist were determined in the spleen, tumor draining lymph nodes, and tumors with distinct effects observed in the activation of innate and adaptive responses. We further demonstrate the immunological and antitumor efficacy of combinations of eribulin and ADU-S100 in the syngeneic 4T1 model that is resistant to the antimitotic effects of eribulin as a single agent. Even in the absence of direct antimitotic effects on the tumor, eribulin was sufficient to promote activation of CD4+CD25− effector T-cells in the spleen and lymph nodes specifically in tumored animals but not untumored controls. Additionally, eribulin was able to promote a similar increase in the activation of CD4+ effector T-cells even in untumored animals when administered in combination with a STING agonist. Together, these data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC that could inform on their more rational use in the clinic as single agents or as chemotherapeutic backbones in combination with targeted therapy, including immunotherapeutics. More specifically, our data demonstrate that eribulin can act as an immune adjuvant to promote STING signaling and T-cell activation in multiple in vivo TNBC models in addition to its well established anti-mitotic effects to enhance antitumor efficacy. Citation Format: Leila Takahashi-Ruiz, Charles S. Fermaintt, Nancy Wilkinson, Peter Y. Chan, Susan L. Mooberry, April L. Risinger. Eribulin acts as an immune adjuvant to enhance the antitumor efficacy of STING agonists in triple-negative breast cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6397.

429 Eribulin Synergizes with STING Agonists by Enhancing Type 1 Interferon Expression and Improves Antitumor Efficacy as Combination Treatment

OBJECTIVES/GOALS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks effective targeted treatment options. TNBC's greater degree of immunogenicity than other breast tumors makes immunotherapy a viable strategy. Strategies to improve the immunotherapy response includes targeting the cGAS-STING innate immune pathway with STING agonists. METHODS/STUDY POPULATION: We have previously shown in vitro that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Separately, eribulin also significantly enhances type I interferon expression induced by STING agonists measured by qRT-PCR through a second TBK1-dependent mechanism downstream of STING activation through detecting higher amounts of phosphorylated IRF-3 by western blot protein analysis. Mechanisms of eribulin-mediated interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizing agent paclitaxel. RESULTS/ANTICIPATED RESULTS: We determine that the enhancement of type I interferon expression by eribulin is pharmacologically synergistic with multiple STING agonists. The significant enhancement by eribulin led us to evaluate the antitumor efficacy of eribulin in combination the STING agonist ADU-S100 in a challenging spontaneous mammary tumor model MMTV-PyVT. We show that the combination treatment significantly decreased tumor growth which allowed for longer survival compared to other groups. This is particularly interesting because of our previous studies showing that eribulin alone, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleen and draining lymph nodes of BALB/c mice with 4T1 tumors through flow cytometric analysis. DISCUSSION/SIGNIFICANCE: These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin can act as an immune adjuvant in addition to its anti-mitotic effect.

Binding Parameters of [11C]MPC-6827, a Microtubule-Imaging PET Radiopharmaceutical in Rodents.

Impairment and/or destabilization of neuronal microtubules (MTs) resulting from hyper-phosphorylation of the tau proteins is implicated in many pathologies, including Alzheimer's disease (AD), Parkinson's disease and other neurological disorders. Increasing scientific evidence indicates that MT-stabilizing agents protect against the deleterious effects of neurodegeneration in treating AD. To quantify these protective benefits, we developed the first brain-penetrant PET radiopharmaceutical, [11C]MPC-6827, for in vivo quantification of MTs in rodent and nonhuman primate models of AD. Mechanistic insights revealed from recently reported studies confirm the radiopharmaceutical's high selectivity for destabilized MTs. To further translate it to clinical settings, its metabolic stability and pharmacokinetic parameters must be determined. Here, we report in vivo plasma and brain metabolism studies establishing the radiopharmaceutical-binding constants of [11C]MPC-6827. Binding constants were extrapolated from autoradiography experiments; pretreatment with a nonradioactive MPC-6827 decreased the brain uptake >70%. It exhibited ideal binding characteristics (typical of a CNS radiopharmaceutical) including LogP (2.9), Kd (15.59 nM), and Bmax (11.86 fmol/mg). Most important, [11C]MPC-6827 showed high serum and metabolic stability (>95%) in rat plasma and brain samples.

Discovery of podofilox as a potent cGAMP-STING signaling enhancer with antitumor activity.

Cyclic GMP-AMP (cGAMP) is a second messenger that activates the stimulator of interferon genes (STING) innate immune pathway to induce the expression of type I interferons (IFNs) and other cytokines. Pharmacological activation of STING is considered a potent therapeutic strategy in cancer. In this study, we employed a cell-based phenotypic screen and identified podophyllotoxin (podofilox), a microtubule destabilizer, as a robust enhancer of the cGAMP-STING signaling pathway. We found that podofilox enhanced the cGAMP-mediated immune response by increasing STING-containing membrane puncta and the extent of STING oligomerization. Furthermore, podofilox changed the trafficking pattern of STING and delayed trafficking-mediated STING degradation. Importantly, the combination of cGAMP and podofilox had profound antitumor effects on mice by activating the immune response through host STING signaling. Together, these data provide insights into the regulation of cGAMP-STING pathway activation and demonstrate what we believe to be a novel approach for modulating this pathway and thereby promoting antitumor immunity.