Vehicle-treated Controls Research Articles

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics

BackgroundDespite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge...

Abstract 4207: Zoledronic acid reduces quiescent bone-disseminated human ER+ breast cancer tumor cell burden and estrogen-responsiveness in a pre-clinical model

Abstract Unique to early-stage ER+ breast cancer, ER+ tumor cells already bone disseminated at time of diagnosis can remain dormant at this site until an initiating event (e.g., activating ESR1 mutation acquisition) allows for osteolytic bone metastases (BMET) progression even decades later. Adjuvant use of anti-resorptive bisphosphonates, but not denosumab, reduces BMET risk in post-menopausal women with early stage breast cancer, suggesting protective effects may be unrelated to changes in bone turnover. Pre-clinical modeling of dormant, bone-disseminated ER+ breast cancer cells may enhance our understanding and thus our ability to better target these inherently treatment-resistant cells. In studies reported here, a model of bone-disseminated ER+ dormancy was characterized and used to query effects of zoledronic acid (ZA) on dormant cells. When female nude mice were inoculated (intracardiac) with human ER+ MCF-7 cells labelled with DiD, a membrane dye that dissipates with each cell division, DiD-positive tumor cell number in harvested tibial bone marrow remained unchanged from 24 hours to 28 days post-tumor cell inoculation. When 17ß-estradiol (E2) treatment (0.72 mg 60-day pellets) was initiated on D28 following tumor cell inoculation, detectable DiD+ tumor cell number in harvested tibial bone marrow increased 1.8-fold within one week (D35 vs. D28, p < 0.0001), consistent with initiation of E2-stimulated proliferation, and osteolytic, histology-confirmed BMET appeared subsequently with 60% incidence by 6-weeks post-start of E2 treatment. When mice were instead zoledronic acid-treated during the 28d dormancy period (70 ug/kg subcutaneously on D7 and on D21 post-inoculation), DiD-positive cell number in marrow harvested on D28 was reduced by more than half vs. vehicle-treated controls (p< 0.05). Moreover, DiD-positive cell numbers on D35, one week after start of E2-treatment, remained unchanged in tibial marrow of ZA-treated mice and were thus substantially (3.8-fold) lower than controls (p< 0.001). When followed for 6-weeks after initiation of E2 treatment, no osteolytic BMET were detected in ZA-treated mice. In toto, these results recapitulate clinical findings, thus validating the methodologic approach, and suggest that in addition to reducing dormant ER+ tumor cell burden in bone, bisphosphonates may also alter their estrogen responsiveness. Acknowledgements: Special thanks to undergraduates Alyssa Magee and Geethika Ameneni for assistance with data acquisition, and the METAvivor Foundation and NIH-NCI (NCI R03 CA181893, R01 CA174926, P30 CA023074, T32 CA009213) for financial support. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Janet L. Funk. Zoledronic acid reduces quiescent bone-disseminated human ER+ breast cancer tumor cell burden and estrogen-responsiveness in a pre-clinical model [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 4207.

Abstract 2084: Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3

Abstract Vitamin D3 supplementation is becoming widely accepted as a therapeutic in many common estrogen responsive cancers. Previous studies in breast cancer showed that 24R,25-dihydroxyvitamin D3 (24,25) regulates tumorigenesis via a caveolae membrane-associated signaling pathway involving phospholipase D [PLD] and palmitoylation of an unknown receptor. The goal of this study was to examine the effects of 24,25 in estrogen responsive laryngeal cancer tumorigenesis, investigate the mechanisms involved, and determine the ability of the tumor cells to produce 24,25 locally. Expression of CYP24A1 and CYP27B1 was measured in ERα66+ UM-SCC-12 (UM12) and ERα66- UM-SCC-11A (UM11A) laryngeal cancer cells. Cells were treated with 25(OH)D3, and production of the vitamin D3 metabolites 1,25(OH)2D3, 24,25(OH)2D3, and 1,24,25(OH)3D3 was measured using HPLC. The therapeutic potential of 24,25 was then assessed by treating the cells with 24,25 and measuring proliferation and apoptosis. In vivo, UM12 and UM11A cells were implanted into the cheek of NSG mice; the mice were treated with 24,25, and evaluated for survival and tumor volume. The signaling pathways involving PLD, palmitoylation, and caveolin-1 were investigated as driving mechanisms. CYP24A1 and CYP27B1 were present in ERα66+ UM12 and ERα66- UM11A cells, both of which are responsive to estrogen. When treated with 25(OH)D3, UM11A cells produced more 24,25 than UM12s, comparable to the physiologically relevant levels in blood. Both cells produced similar levels of 1,25. 24,25 increased proliferation and inhibited apoptosis in ERα66+ UM12 cells in vitro, but had the opposite effect in ERα66- UM11A cells. 24,25 increased PLD activity in UM12 cells but decreased PLD activity in UM11A cells. Inhibition of PLD activity or palmitoylation, as well as silencing expression of caveolin-1, reduced the effects of 24,25 on P53 in both cell types, indicating they played a role. Mice with UM11A tumors given 24,25 exhibited reduced tumor burden and increased survival compared to vehicle-treated controls. In contrast, mice with UM12 tumors given 24,25 had an increased tumor burden and reduced survival compared to controls. The presence of CYP24A1 and CYP27B1, local production of 24,25, and demonstration that laryngeal cancer cells respond to 24,25, indicate that 24,25 acts in an autocrine/paracrine manner. 24,25 was pro-tumorigenic in ERα66+ laryngeal cancer cells while having the opposite effect in ERα66- cells, both in vitro and in vivo, demonstrating a dependence on ERα66 expression. 24,25 acts via a mechanism involving PLD, caveolae, and palmitoylation. These data show that 24,25 regulates laryngeal cancer tumorigenesis, which is mediated by membrane associated signaling pathways that may depend on ERα66 level, and suggest a major role of 24R,25-dihydroxyvitamin D3 in laryngeal cancer. Citation Format: Cydney D. Dennis, D. Joshua Cohen, Jonathan T. Dillon, Barbara D. Boyan, Zvi Schwartz. Autocrine regulation of laryngeal cancer tumorigenesis by 24R,25(OH)2D3 [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 2084.

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study

BackgroundOxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. MethodsMLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. ResultsThe experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. ConclusionsNrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Eugenol ameliorates uveitis in mice with experimental autoimmune encephalomyelitis through the suppression of key inflammatory genes

ABSTRACT Visual impairment associated with uveitis is among the potential complications in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Bioinformatics analyses have shown that some hub genes are closely associated with the molecular mechanisms underlying uveitis in EAE. This study evaluated whether 4-allyl-2-methoxyphenol (eugenol) can mitigate the pathogenesis of uveitis in EAE through the interruption of key uveitogenic gene expression. Myelin oligodendrocyte glycoprotein35–55 (MOG) peptide-immunized C57BL/6 mice were injected intraperitoneally with eugenol. The eyeballs and spinal cords of EAE mice with or without eugenol treatment were collected simultaneously and immunohistochemical and molecular biological analyses were conducted. Eugenol treatment significantly ameliorated hindlimb paralysis. Ionized calcium-binding adapter molecule 1 (Iba-1) immunohistochemistry showed that the inflammatory response was significantly reduced in the uvea of eugenol-treated EAE mice compared with vehicle-treated controls. Eugenol also significantly reduced the expression of key uveitogenic genes including C1qb and Tyrobp. The suppressive effect of eugenol on inflammation was also observed in the spinal cord, as determined by the suppression of Iba-1-positive microglial cells. Together, these results suggest that the ameliorative effect of eugenol against EAE uveitis is associated with the suppression of key proinflammatory genes, which may represent targets for the treatment of uveitis.

Disruptive effects of plasticizers bisphenol A, F, and S on steroidogenesis of adrenocortical cells.

Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

Enhancing human islet xenotransplant survival and function in diabetic immunocompetent mice through LRH-1/NR5A2 pharmacological activation.

The intricate etiology of type 1 diabetes mellitus (T1D), characterized by harmful interactions between the immune system and insulin-producing beta cells, has hindered the development of effective therapies including human islet transplantation, which requires strong immunosuppressants that impair beta cell survival and function. As such alternative immunomodulating therapies are required for successful transplantation. The discovery that pharmacological activation of the nuclear receptor LRH-1/NR5A2 can reverse hyperglycemia in mouse models of T1D by altering, and not suppressing the autoimmune attack, prompted us to investigate whether LRH-1/NR5A2 activation could improve human islet function/survival after xenotransplantation in immunocompetent mice. Human islets were transplanted under the kidney capsule of streptozotocin (STZ)-induced diabetic mice, and treatment with BL001 (LRH-1/NR5A2 agonist) or vehicle was administered one week post-transplant. Our study, encompassing 3 independent experiments with 3 different islet donors, revealed that mice treated for 8 weeks with BL001 exhibited lower blood glucose levels correlating with improved mouse survival rates as compared to vehicle-treated controls. Human C-peptide was detectable in BL001-treated mice at both 4 and 8 weeks indicating functional islet beta cells. Accordingly, in mice treated with BL001 for 8 weeks, the beta cell mass was preserved, while a significant decrease in alpha cells was observed compared to mice treated with BL001 for only 4 weeks. In contrast, vehicle-treated mice exhibited a reduction in insulin-expressing cells at 8 weeks compared to those at 4 weeks. These results suggest that BL001 significantly enhances the survival, engraftment, and functionality of human islets in a STZ-induced diabetic mouse model.

Autophagy in the corpus luteum correlates with tissue growth in pregnant rats.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

Brain antioxidant status and gene expressions of nicotinic and dopamine receptors are improved by black seed oil administration in cigarette smoke or nicotine vapour-exposed rats.

Smoking is associated with dysregulation of the antioxidant system and addiction. This study sought to ascertain the effect of Nigella Sativa (NS) oil on the antioxidant system, nicotine/tobacco addiction as well as the expressions of α4β2 nicotinic (nAChR) and dopamine type-2 (DRD2) receptors in selected brain regions of the rat. Thirty male Sprague-Dawley rats were divided into 6 groups comprising of vehicle-treated control, NS oil only, Smoke only, Smoke + NS oil, Nicotine only and Nicotine + NS oil. Animals were passively exposed to cigarette smoke or nicotine vapour for 12 weeks, however, NS oil treatment commenced from 9th-12th week of the experimental duration. Nicotine vapour and cigarette smoke-induced increase in cotinine level were significantly ameliorated by NS treatment. Cigarette smoke or nicotine vapour exposure significantly (p<0.05) decreased the level of antioxidant enzymes while increasing malondialdehyde level in the brain homogenates of the rats. Administration of NS oil significantly (p<0.05) reversed the reduced antioxidant level. Cigarette-smoke also significantly increased α4-nAChR expression in the frontal cortex and olfactory bulb compared to control. Nicotine vapour significantly increased DRD2 expression only in the olfactory cortex. NS oil administration reduced both the cigarette-smoke-induced increase in α4-nAChR and nicotine vapour-induced increase in DRD2 gene expression only in the olfactory cortex. Findings from this study suggest that NS oil improves brain antioxidant status while ameliorating nicotine vapour and cigarette smoke addiction through down-regulation of α4-nAChR and DRD2 gene expressions in discrete brain regions in Sprague-Dawley rats.

Effects of Mycobacterium vaccae NCTC 11659 and Lipopolysaccharide Challenge on Polarization of Murine BV-2 Microglial Cells.

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an "adjuvant-like" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced "adjuvant-like" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.

Normotensive placental extracellular vesicles provide long-term protection against hypertension and cardiovascular disease

BackgroundWomen with normotensive pregnancy are at a reduced risk of developing cardiovascular disease postpartum compared to those who experience hypertensive conditions during pregnancy. However, the underlying mechanisms remain poorly understood. During normotensive pregnancy, vast numbers of placental extracellular vesicles are released into the maternal circulation, which protect endothelial cells from activation and alter maternal vascular tone. We hypothesized that placental extracellular vesicles play a mechanistic role in lowering risk of cardiovascular disease following normotensive pregnancy. ObjectiveThis study aimed to investigate the long-term effects of placental extracellular vesicles derived from normotensive term placentae on the cardiovascular system and explore the mechanisms underlying their biological effects. Study DesignSpontaneously hypertensive rats were injected with placental EVs from normotensive term pregnancies (2 mg /kg each time, n=8) or vehicle control (n=9) at 3 months of age. Blood pressure and cardiac function were regularly monitored from 3 months to 15 months of age. The response of mesenteric resistance arteries to vasoactive substances was investigated to evaluate vascular function. Cardiac remodelling, small artery remodelling, and renal function were investigated to comprehensively assess the impact of placental extracellular vesicles on cardiovascular and renal health. ResultsCompared to vehicle-treated control animals, rats treated with normotensive placental extracellular vesicles exhibited a significantly lower increase in blood pressure and improved cardiac function. Furthermore, the vasodilator response to the endothelium-dependent agonist acetylcholine was significantly enhanced in the normotensive placental extracellular vesicle-treated spontaneously hypertensive rats compared to the control. Moreover, treatment with placental extracellular vesicles reduced wall thickening of small renal vessels and attenuated renal fibrosis. ConclusionPlacental extracellular vesicles from normotensive term pregnancies have long-lasting protective effects reducing hypertension and mitigating cardiovascular damage in vivo.

The Efficacy of a Human-Ready miniMECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome.

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Vanillylacetone attenuates cadmium chloride-induced hippocampal damage and memory loss through up-regulation of nuclear factor erythroid 2-related factor 2 gene and protein expression.

JOURNAL/nrgr/04.03/01300535-202412000-00030/figure1/v/2024-04-08T165401Z/r/image-tiff Memory loss and dementia are major public health concerns with a substantial economic burden. Oxidative stress has been shown to play a crucial role in the pathophysiology of hippocampal damage-induced memory impairment. To investigate whether the antioxidant and anti-inflammatory compound vanillylacetone (zingerone) can protect against hippocampal damage and memory loss induced by cadmium chloride (CdCl2) administration in rats, we explored the potential involvement of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is known to modulate oxidative stress and inflammation. Sixty healthy male Wistar rats were divided into five groups: vehicle-treated (control), vanillylacetone, CdCl2, vanillylacetone + CdCl2, vanillylacetone + CdCl2 + brusatol (a selective pharmacological Nrf2 inhibitor) groups. Vanillylacetone effectively attenuated CdCl2-induced damage in the dental gyrus of the hippocampus and improved the memory function assessed by the Morris Water Maze test. Additionally, vanillylacetone markedly decreased the hippocampal tissue levels of inflammatory biomarkers (interleukin-6, tumor necrosis factor-α, intracellular cell adhesive molecules) and apoptosis biomarkers (Bax and cleaved caspase-3). The control and CdCl2-treated groups treated with vanillylacetone showed reduced generation of reactive oxygen species, decreased malondialdehyde levels, and increased superoxide dismutase and glutathione activities, along with significant elevation of nuclear Nrf2 mRNA and protein expression in hippocampal tissue. All the protective effects of vanillylacetone were substantially blocked by the co-administration of brusatol (a selective Nrf2 inhibitor). Vanillylacetone mitigated hippocampal damage and memory loss induced by CdCl2, at least in part, by activating the nuclear transcription factor Nrf2. Additionally, vanillylacetone exerted its potent antioxidant and anti-inflammatory actions.

Abstract B028: Antiproliferative effect of a cathepsin L inhibitor on hepatocellular carcinoma

Abstract Liver cancer has emerged as one of the fastest-growing malignancies in the United States, despite an overall decline in cancer incidence and mortality rates. Hepatocellular carcinoma (HCC) exhibits significant gender and race/ethnicity differences in incidence and mortality. The need to address this escalating problem necessitates the development of novel and effective chemotherapeutic agents for HCC. The antiproliferative actions of two cathepsin L inhibitors (compounds 1 and 2) using in vitro and mice HCC xenograft models were investigated in this study. In addition, the ability of the compounds to inhibit CatB and generate ROS in HCC cell lines was investigated. The inhibitors have antiproliferative effects on Hep G2 and Hep 3B cell lines with low micromolar IC 50 values. Compound 1 preferentially inhibits both recombinant and endogenous CatL in a time-dependent manner compared to CatB. The levels of ROS produced when cells were treated with compound 1 were non-significant compared with vehicle-treated controls . Given the superior efficacy of compound 1 over its analog in antiproliferative studies, it was advanced to in vitro ADME studies. The inhibitor has bidirectional cellular permeability and is not a substrate for the P-glycoprotein efflux pump (P-gp). In mice, it produced a significant reduction in the sizes of subcutaneous tumors with no gross toxicity. Transcriptomics analysis using RNA-Seq revealed that the cell migration pathway was enriched in a time and dose-dependent manner. In addition, genes responsible for apoptosis and cell structural organization were overexpressed. Overall, the CatL inhibitors under investigation appear as promising starting points for investigation as potential chemotherapy against HCC. Citation Format: Olamide O. Crown, Ifedayo V. Ogungbe, Felicite K. Noubissi. Antiproliferative effect of a cathepsin L inhibitor on hepatocellular carcinoma [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B028.

Abstract B006: High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC

Abstract KRASG12C inhibitors such as sotorasib and adagrasib have shown efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. Therefore, there is a clear need for predictive biomarkers to guide treatment. The proximity ligation assay (PLA) is an immunofluorescent-based approach utilized to analyze endogenous protein-protein interactions. The method allows for the assessment of signaling-associated protein complexes, which can serve as potential targets for specific inhibitors. In particular, KRASG12C inhibitors can disrupt these complexes by interfering with the interaction between RAS and its effector RAF. By providing insights into the presence of these protein complexes, PLA offers a predictive readout for inhibitor sensitivity. We thus conducted a preclinical study to evaluate if a RAS-CRAF PLA could inform on the presence of RAS-RAF protein complexes and predict the response to KRASG12C inhibitors. To assess the specificity of the panRAS-CRAF PLA, we employed RNA interference in a KRASG12C-mutant H358 NSCLC cell line, wherein knockdown of either panRAS or CRAF resulted in a reduction in the panRAS-CRAF PLA signal. Pharmacological inhibition of RAS-RAF interaction with sotorasib also reduced the panRAS-CRAF PLA signal. We next performed panRAS-CRAF PLA in a panel of 11 KRASG12C-mutant NSCLC cell lines, revealing a higher number of panRAS-CRAF PLA spots in H358, LU65, and HCC1171. To further investigate the RAS-RAF interaction, we assessed the abundance of CRAF-bound RAS-GTP by quantifying the binding of RAS-GTP to the RAS-binding domain of CRAF. H358, LU65, and HCC1171 exhibited the highest levels of RAS-GTP among the 11 cell lines. We observed a strong correlation between the number of panRAS-CRAF PLA foci and the levels of CRAF-bound RAS-GTP (r = 0.87, P = 0.0002). These results indicated that the enhanced level of RAS-GTP for CRAF proteins facilitates the formation of RAS-CRAF complexes, thereby augmenting the panRAS-CRAF PLA signals. We then evaluated the in vitro antitumor activity of sotorasib across the G12C-mutated cell lines and found that the 50% inhibitory concentration values of sotorasib for H358, LU65, and HCC1171 were below 30 nmol/L. The panRAS-CRAF PLA was strongly associated with the sensitivity to sotorasib (r = 0.81, P = 0.002). We subsequently assessed the association of panRAS-CRAF PLA with efficacy of adagrasib in a patient-derived xenograft (PDX) cohort. PanRAS-CRAF PLA was assessed on sections of formalin-fixed paraffin-embedded tumors derived from vehicle-treated control groups at the end of efficacy studies in 18 NSCLC PDX models. Our findings revealed a significant association between high panRAS-CRAF PLA signal and increased sensitivity to adagrasib (r = 0.61, P = 0.008). Our results suggest that panRAS-CRAF PLA may serve as a predictive marker to identify patients who could derive optimal benefit from KRASG12C inhibitors, and may direct combination therapy approaches to those patients less likely to benefit from single-agent therapy. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Harika Gundlapalli, Ida Aronchik, Joseph Johnson, Eric B. Haura. High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B006.

Aurantio-obtusin alleviates allergic responses in ovalbumin-induced rhinitis

Allergic rhinitis (AR) is a growing global health concern. Despite being non-fatal, it has a detrimental effect on people's quality of life and poses a significant economic burden. In this study, we aimed to evaluate the anti-allergic effects of aurantio-obtusin against ovalbumin-induced murine model of allergic rhinitis. Aurantio-obtusin was administered to rats orally after sensitization to ovalbumin. Results of the study suggested that aurantio-obtusin causes a significant reduction in nose rubs and sneeze as compared to the vehicle-treated disease control group. The aurantio-obtusin treated group showed a dose-dependent significant reduction in serum TNF-α, IL-4, and IL-6 levels as well as histamine levels in the nasal mucosa. Additionally, it decreased neutrophil and eosinophil numbers in blood of AR rats'. In the nasal tissues of the vehicle-treated rats that were subjected to an OVA challenge, the histology of the nasal mucosa of AR rats revealed aberrant histological changes. A variety of histological abnormalities, including vascular congestion in the nasal mucosa, substantial inflammatory cell infiltration, and obvious alveolar wall oedema were present. In the rats treated with aurantio-obtusin, these characteristics were reversed. Collectively, the findings suggests that aurantio-obtusin could be used to enhance current AR treatment approaches.

Synergistic Activity of Adefovir Dipivoxil and Venetoclax to Target the Bioenergetic Metabolism of Acute Myeloid Leukemia

Adefovir dipivoxil (ADV) is an oral prodrug of the nucleotide analogue adefovir that inhibits viral DNA polymerase and is an FDA-approved drug for the treatment of hepatitis B virus. Preliminary evidence of the antileukemic activity of ADV was recently reported in a patient with acute promyelocytic leukemia (APL; Gong, X., et al. Exp Hematol Oncol, 2022). Herein we report on the antileukemic activity of ADV, alone and in combination with venetoclax (VEN), an oral FDA-approved BCL-2 inhibitor for the treatment of acute myeloid leukemia (AML). Firstly, a significant dose-dependent reduction of cell growth and increase in apoptosis were observed in FLT3-ITD+ MV4-11 (IC50: 97nΜ) and Molm-13 (IC50: 0.816μM) cells, and in primary CD34+ blasts from refractory/relapsed (R/R) AML patients (n=5; IC50: 1.285-3.703μM), exposed to serial concentrations of ADV for 48 hours (hrs). In contrast, no significant changes were observed in ADV-treated CD34+ cells from healthy donors (n=5). Combination of ADV and VEN were synergistic (combination index &amp;lt; 1.0) in reducing cell viability of AML cells (i.e., MV-4-11, U937, KG1 and THP-1) and CD34+ blasts from R/R AML patients (n=3). To gain insights into the molecular mechanisms underlying the antileukemic activity of ADV alone and in combination with VEN, we performed RNA-Seq on AML CD34+ blasts from R/R AML patients (n=6) treated with vehicle, ADV (5 μΜ), VEN (100 nΜ), or ADV+VEN for 24 hrs. Gene set enrichment analysis (GSEA) highlighted upregulation of p53 pathway and apoptosis signaling, and downregulation of oxidative phosphorylation (OXPHOS), glycolysis, and MYC target hallmark gene sets in ADV- and ADV+VEN-treated cells compared with vehicle-treated controls. These results were confirmed by western blot analysis showing a dose-dependent reduction in c-MYC, anti-apoptotic proteins (i.e., BCL-2 and MCL-1), and carnitine palmitoyltransferase-1a [CPT1A, a key enzyme for fatty acid oxidation (FAO), a process that feeds into OXPHOS], and an increase in pro-apoptotic proteins (i.e., BAX and BAK) in ADV- and ADV+VEN-treated MV4-11 and primary AML CD34+ cells, compared with vehicle-treated control cells. Seahorse analysis confirmed a significant reduction of OXPHOS [measured by oxygen consumption rate (OCR)], associated with decreased mitochondrial numbers and sizes evaluated by electron microscopy imaging of AML CD34+ blasts treated with ADV or ADV+VEN compared with vehicle-treated controls. To test in vivo the antileukemic activity of ADV+VEN, we treated a cohort of CD45.1 recipient mice transplanted with BM cells from congenic CD45.2 Mll PTD/WT/Flt3 ITD/ITD AML mice with vehicle, ADV (100mg/kg, oral gavage), VEN (100mg/kg, oral gavage), or ADV+VEN (n=10 per group) for 4 weeks. ADV+VEN-treated mice lived longer (median survival: 60 days after treatment completion) than ADV (20 days, p=0.0001), VEN (20 days, p=0.0001), or vehicle (14 days, p=0.0002) -treated controls (Figure 1). We then performed secondary transplants by engrafting 10 6 BM cells from treated donors into CD45.1 recipient mice (n=10 per group). Recipients of BM from ADV+VEN-treated donors survived longer (median survival: 83 days) than recipients of BM from ADV (60.5 days, p&amp;lt;0.01), VEN (45.5 days, p&amp;lt;0.0001), and vehicle (42.5 days, p&amp;lt;0.0001) -treated donors (Figure 2), confirming a decrease in leukemic stem cell (LSC) burden by ADV+VEN. Next, we examined the antileukemic activity of ADV+VEN in human AML models. We first treated a cohort of NSG mice (n=10 per group) transplanted with luciferase-expressing Molm-13 cells. ADV+VEN-treated mice survived longer (median survival: 59.5 days) than ADV (52 days, p=0.04), VEN (40 days, p&amp;lt;0.0001), and vehicle (40.5 days, p&amp;lt;0.0001) -treated mice. We then treated a cohort of NSG mice (n=8 per group) transplanted with FLT3-ITD+ blasts from a patient with R/R AML. ADV+VEN-treated mice survived longer (median: 38.5 days) than ADV (34.5 days, p=0.035), VEN (25 days, p&amp;lt;0.0001), and vehicle (18.5 days, p&amp;lt;0.0001) -treated mice. In summary, ADV has in vivo anti-leukemic activity in AML likely by interfering with the bioenergetic metabolism of leukemic cells and decreasing their apoptotic threshold. The combination of ADV and VEN is seemingly synergistic and prolonged survival of murine and human AML models, providing a rationale of translating this “all oral” regimen to the clinic for treatment of AML patients, especially those requiring low intensity therapy.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=&amp;lt;0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Plasma Transforming Growth Factor-β1 (TGF-β1) Levels Increase with Age and Aortic Stenosis Progression and Low-Dose Galunisertib Attenuates Disease Progression in Mice

Objective: Aortic stenosis (AS) is an age-related degenerative valvular disease that ultimately leads to heart failure and sudden death. Platelet-derived TGF-β1 is a major source of plasma TGF-β1 and contributes to AS progression in LDLR -/-ApoB 100/100(LA100), a mouse model that spontaneously develops AS that simulates human pathology, including fibrosis and high shear gradient. Although risk factors, such as hypercholesteremia and hyperglycemia, are like those for coronary artery disease, lowering cholesterol and glucose has not been successful in stopping progression, and there is no biomarker for early detection of AS. In this study, we measured plasma TGF-β1 levels as a function of age in LA100 mice and human AS patients and tested a new approach of partial inhibition of TGF-β signaling to inhibit AS progression in LA100 mice, while limiting cardiotoxicity. Methods: We measured plasma TGF-β1 levels in controls, severe AS patients, and LA100 mice before they developed AS (2-months of age), when they developed moderate AS (1-year), and when they developed severe AS (2-years). LA100 mice at different stages of AS were treated with low-dose galunisertib, an inhibitor of the TGF-β receptor, ALK5. Results: Plasma levels of total TGF-β1 were significantly higher in AS patients (n=34) compared to healthy controls (n=22) and patient controls (n=47) (p&amp;lt;0.001 for AS patients vs. healthy controls and p=0.049 for AS patients vs. patient controls). Age was positively correlated with total TGF-β1 levels in AS patients (r=0.475; p=0.005), but not patient or healthy controls (r=0.12, p=0.43). LA100mice developed AS with aging and plasma TGF-β1 levels in LA100mice (n=21-52; ages 2 months-2 years) were significantly higher than those in age-matched WT mice (n=23); (p&amp;lt;0.0001). Plasma TGF-β1 levels correlated with age in LA100 mice (R=0.6, p=&amp;lt;0.0001). Low-dose oral galunisertib was administered at a concentration of 1 µM in drinking water, giving an average daily dose of ~0.06 mg/kg, compared to a human cancer treatment dose of ~4.2 mg/kg daily. Galunisertib treatment: a) halted AS progression at all stages in LA100 mice compared to vehicle-treated control mice (p&amp;lt;0.05, n=9-13), b) did not change plasma TGF-β1 levels, c) inhibited TGF-β1 signaling as shown by decreased Smad2 phosphorylation in valve cells compared to vehicle-treated animals. An age-dependent increase in neutrophil to lymphocyte ratio (NY/LY), a marker of inflammation, was observed in LA100 mice compared to control mice (n=12-31; p&amp;lt;0.01), and the NY/LY ratio correlated with total TGF-β1 plasma levels (R=0.16; p=0.020). Galunisertib treatment reduced NY/LY ratios in LA100 mice (p=0.03). Conclusions: 1. Increases in plasma TGF-β1 levels may be a valuable surrogate marker of AS progression. 2. Low-dose galunisertib attenuates AS progression in the LA100 mouse model without producing the cardiotoxicity associated with higher doses and thus may warrant further study in human AS patients.

Discovery of JNJ-88549968, a Novel, First-in-Class CALRmutxCD3 T-Cell Redirecting Antibody for the Treatment of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal malignant disorders of hematopoiesis arising in the hematopoietic stem cell (HSC) compartment that are characterized by excessive production of mature blood cells of the myeloid lineage. Transformation to secondary acute myeloid leukemia (sAML) represents a significant cause of death among MPN patients and this transformation occurs mainly from the clone carrying the disease phenotype driver mutation. Current treatment options for MPN patients are not curative and are limited to symptomatic treatment. Therefore, identification of novel therapeutic approaches with a clear disease-modifying effect for the treatment of MPNs and intercepting their progression to sAML is an unmet medical need. Mutations in JAK2, thrombopoietin receptor (MPL), and calreticulin (CALR) are phenotypic drivers in the pathogenesis of MPN. CALR mutations (CALRmut) are the second most frequent in MPN. CALRmut are insertions or deletions resulting in a frameshift in the last exon of the gene, causing a loss of the KDEL ER-retention motif and generation of a 36 amino acid positively charged C-terminal neoantigen. Due to loss of the KDEL motif, CALRmut are not confined to the ER and through interaction with MPL are trafficked to the cell surface where they induce persistent MPL activation and oncogenicity. Immunotherapies engaging T cells, such as bispecific cluster of differentiation 3 (CD3) redirection antibodies, show promising response rates in the clinic. Here, we describe JNJ-88549968, a novel first in class T-cell redirecting bispecific antibody that selectively targets CALRmut with the potential to achieve cures by eliminating MPN clones. The mechanism of action of JNJ-88549968 is to act as a bridge between CALRmut MPN cancer cells and T cells, inducing T-cell activation with subsequent T-cell-mediated cytotoxicity to CALRmut cancer cells in vitro and in vivo. JNJ-88549968 recognizes CALRmut epitopes common to all known CALRmut types. Cell surface localization of CALRmut was confirmed in CD34 + cells from CALRmut MPN patients. In-depth characterization of T cells from CALRmut MPN patients, using CyTOF and functional assays, confirmed their fitness and functionality. These data validate T-cell redirection as a rational therapeutic strategy for MPN patients carrying CALRmut. JNJ-88549968 demonstrated selective binding to CALRmut cell lines and no measurable binding to CALR wild type cells. JNJ-88549968 led to CALRmut-selective T-cell activation and cytotoxicity to CALRmut-engineered cell lines in vitro. The activity of JNJ-88549968 was also explored in an autologous setting using CD34 + cells isolated from CALRmut MPN patients as target cells together with T cells isolated from the same patients. JNJ-88549968 elicited concentration-dependent cytotoxicity of patient-derived CALRmut CD34 + cells. JNJ-88549968-mediated cytotoxicity was observed against all tested CALRmut CD34 + cancer cells and was independent of the type of CALR mutation. Moreover, JNJ-88549968 mediated robust in vivo efficacy in two independent CALRmut-positive xenograft murine leukemia models. In an established disseminated model, treatment with JNJ-88549968 significantly increased lifespan (ILS) compared to vehicle-treated control mice. Secreted CALRmut protein can be found in CALRmut MPN patient plasma. Evaluation of the impact of soluble CALRmut on JNJ-88549968 activity, including CALRmut-patient derived whole blood studies, indicated no effect on the activity of JNJ-88549968 in vitro. Taken together, JNJ-88549968 is a novel first-in-class bispecific T-cell redirection antibody investigated for the treatment of CALRmut MPN. JNJ-88549968 is currently being advanced for clinical investigation in patients with MPN.