Vehicle-treated Controls Research Articles

Abstract C020: Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors

Abstract While adoptive cellular therapies (ACT) such as CAR-T and NK therapy have shown clinical efficacy against hematologic malignancies, their use against solid tumors (such as ovarian or pancreatic cancer) has had limited success to date. In particular, CAR-T therapy for solid cancers has led to severe toxicities, including on-target, off-tumor killing of healthy cells and graft vs. host disease (GVHD) with allogeneic transplant. NK cell therapies, in contrast, have fewer associated toxicities due to specific and innate recognition/killing of cancer cells. This greater safety profile makes NK therapies an attractive treatment option for solid cancers. NK therapies are currently limited by insufficient in vivo tumor clearance, caused largely by their lower cytotoxicity and in vivo expansion compared to CAR-T cells. To address this, we have screened 300,000 small molecule compounds for their ability to enhance NK cytotoxicity against ovarian cancer (OVCAR) cells. Our primary screen identified 11 lead compounds which increase NK killing activity against OVCAR cells >= 10% over baseline (vehicle-treated control). Secondary in vitro assays have confirmed that at least 3 out of 11 lead compounds enhance NK cytotoxicity by at least 10% over baseline. As such, we hypothesize that our lead compounds will increase in vivo NK cytotoxicity and tumor clearance, through mechanisms which might include I) upregulation of NK activating receptors, II) downregulation of NK inhibitory receptors, and/or III) modulation of NK metabolism in the immunosuppressive tumor microenvironment. Immediate future studies include in vivo testing of our lead compounds’ ability to enhance NK cytotoxicity, using immunodeficient mouse models of subcutaneous OVCAR tumors. Moreover, we will utilize 2D and 3D in vitro culture techniques alongside flow cytometry and RNA sequencing to identify how lead compounds impact markers of NK activation and metabolism. This will include expression of activating/inhibitory receptors, exhaustion markers, and key metabolic factors. Finally, we will assess the effect of lead compounds on NK function within an immunosuppressive tumor environment. Our findings will contribute to effective future NK cell therapies for solid tumors. Citation Format: Indrani Das, Olubukola Abiona, Riufu Liu, Grace K. Lee, David N. Wald. Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C020.

Exposure to the antiretroviral drug dolutegravir impairs structure and neurogenesis in a forebrain organoid model of human embryonic cortical development

IntroductionFor many therapeutic drugs, including antiretroviral drugs used to treat people living with HIV-1 (PLWH), we have little data on the potential effects on the developing human brain due to limited access to tissue and historical constraints on the inclusion of pregnant populations in clinical trials. Human induced pluripotent stem cells (iPSCs) offer a new avenue to gain insight on how drugs may impact human cell types representative of the developing central nervous system. To prevent vertical transmission of HIV and promote the health of pregnant PLWH, antiretroviral therapy must be initiated and/or maintained throughout pregnancy. However, many antiretroviral drugs are approved for widespread use following clinical testing only in non-pregnant populations and there may be limited information on potential teratogenicity until pregnancy outcomes are evaluated. The integrase strand transfer inhibitor dolutegravir (DTG) is a frontline antiretroviral drug that is effective in viral suppression of HIV but was previously reported to be associated with a slight increase in the risk for neural tube defects in one study, although this has not been replicated in other cohorts.MethodsTo directly investigate the potential impact of DTG on human cortical neurogenesis, we measured the effects of daily drug exposure on the early stages of corticogenesis in a human iPSC-based forebrain organoid model. We quantified organoid size and structure and analyzed gene and protein expression to evaluate the impact of several doses of DTG on organoid development.ResultsWe observed deficits in organoid structure and impaired neurogenesis in DTG-treated organoids compared to vehicle-treated control organoids after 20 or 40 days in culture. Our highest dose of DTG (10 μM) resulted in significantly smaller organoids with a reduced density of neural rosette structures compared to vehicle-treated controls. Mechanistically, RNA-sequencing and immunohistological analysis suggests dysregulated amino acid transport and activation of the integrated stress response in the DTG-treated organoids, and functionally, a small molecule integrated stress response inhibitor (ISRIB) could partially rescue increased expression of proteins related to cell cycle regulation.DiscussionTogether, these results illustrate the potential for human iPSC-based strategies to reveal biological processes during neurogenesis that may be affected by therapeutic drugs and provide complementary data in relevant human cell types to augment preclinical investigations of drug safety during pregnancy.

Cardiomyocyte-Specific Overexpression of Activated Yes-Associated Protein Modified-RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration.

The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system (YAP5SACM-SMRTs or GFPCM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via invitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SACM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SACM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFPCM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFPCM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SACM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SACM-SMRTs appeared to promote cardiomyocyte proliferation. The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Microglia ameliorate delirium-like phenotypes in a murine model of acute ventilator-induced lung injury

BackgroundDelirium affects 50–85% of patients on mechanical ventilation and is associated with increased mortality, prolonged hospitalization, and a three-fold higher risk of dementia. Microglia, the resident immune cells of the brain, exhibit both neuroprotective and neurotoxic functions; however, their effects in mechanical ventilation-induced acute lung injury (VILI) are unknown. We hypothesize that in a model of short-term VILI, microglia play a neuroprotective role to ameliorate delirium-like phenotypes.MethodsMicroglia depletion (n = 18) was accomplished using an orally administered colony stimulating factor 1 receptor inhibitor, while controls received a vehicle diet (n = 18). We then compared extent of neuronal injury in the frontal cortex and hippocampus using cleaved caspase-3 (CC3) and multiple delirium-like behaviors in microglia depleted and non-microglia depleted male mice (C57BL/6 J aged 4–9 months) following VILI. Delirium-like behaviors were evaluated using the Open Field, Elevated Plus Maze, and Y-maze assays. We subsequently evaluated whether repopulation of microglia (n = 14 repopulation, 14 vehicle) restored the phenotypes.ResultsFrontal/hippocampal neuronal CC3 levels were significantly higher in microglia depleted VILI mice compared to vehicle-treated VILI controls (p < 0.01, p < 0.01, respectively). These structural changes were accompanied by worse delirium-like behaviors in microglia depleted VILI mice compared to vehicle controls. Specifically, microglia depleted VILI mice demonstrated: (1) significantly increased time in the periphery of the Open Field (p = 0.01), (2) significantly increased coefficient of variation (p = 0.02), (3) trend towards reduced time in the open arms of the Elevated Plus Maze (p = 0.09), and (4) significantly decreased spontaneous alternations on Y-maze (p < 0.01). There was a significant inverse correlation between frontal CC3 and percent spontaneous alternations (R2 = 0.51, p < 0.01). Microglia repopulation showed a near-complete return to vehicle levels of delirium like-behaviors.ConclusionsThis study demonstrates that microglia depletion exacerbates structural and functional delirium-like phenotypes after VILI, while subsequent repopulation of microglia restores these phenotypes. These findings suggest a neuroprotective role for microglia in ameliorating neuronal and functional delirium-like phenotypes and call for consideration of interventions that leverage endogenous microglia physiology to mitigate delirium.

MELATONIN ENHANCES TEMOZOLOMIDE-INDUCED APOPTOSIS IN GLIOBLASTOMA AND NEUROBLASTOMA CELLS.

The combination of temozolomide (TMZ) and paclitaxel (PTX) is the most commonly used chemotherapy regimen for glioblastoma, but there is no specific treatment for neuroblastoma due to the acquired multidrug resistance. Approximately half of treated glioblastoma patients develop resistance to TMZ and experience serious side effects. Melatonin (MEL), a multifunctional hormone long known for its antitumor effects, has a great advantage in combination cancer therapy thanks to its ability to affect tumors differently than normal cells. This study aims to evaluate the in vitro inhibitory effects of MEL in combination with TMZ on cancer cell viability and to elucidate the underlying mechanisms in the glioblastoma and neuroblastoma cell lines. C6 (Rattus norvegicus) and N1E-115 (Mus musculus) cancer cell lines and C8-D1A (mice) healthy cell lines were used. Cell proliferation was evaluated using the MTT test. IC50 values were determined by probit analysis. Two concentrations of TMZ (IC50 and 1/2 IC50) were used to induce cytotoxicity in the C6 and N1E-115 cell lines, both alone and in combination with PXT and MEL (all at IC50). The viable, dead, and apoptotic cells were determined by image-based cytometry using Annexin V/PI staining. The gene expression related to signaling pathways was assessed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), and key proteins were identified by the Western blot analysis. MTT assay showed that the combination of TMZ and MEL significantly reduces the viability of both glioblastoma and neuroblastoma cells compared to the vehicle-treated controls. Notably, MEL combined with 1/2 IC50 TMZ showed a significant death rate of cancer cells compared to controls and PTX. According to qRT-PCR data, the TMZ + MEL combination resulted in the upregulation of the genes of antioxidative enzymes (Sod1 and Sod2) and DNA repair genes (Mlh1, Exo1, and Rad18) in both cell lines. Moreover, the levels of Nfkb1 and Pik3cg were significantly reduced following the TMZ + MEL treatment. The combination of MEL with TMZ also enhanced the cell cycle arrest and increased the expression of p53 and pro-apoptotic proteins (Bax and caspase-3), while significantly decreasing the expression of anti-apoptotic protein Bcl-2. Our findings indicate that the combination of MEL with a low dose of TMZ may serve as an upstream inducer of apoptosis. This suggests the potential development of a novel selective therapeutic strategy as an alternative to TMZ for the treatment of both glioblastoma and neuroblastoma.

Silmitasertib in Combination With Cabozantinib Impairs Liver Cancer Cell Cycle Progression, Induces Apoptosis, and Delays Tumor Growth in a Preclinical Model.

The rising incidence of hepatocellular carcinoma (HCC) is a global problem. Several approved treatments, including immune therapy and multi-tyrosine kinase inhibitors, are used for treatment, although the results are not optimum. There is an unmet need to develop highly effective chemotherapies for HCC. Targeting multiple pathways to attack cancer cells is beneficial. Cabozantinib is an orally available bioactive multikinase inhibitor and has a modest effect on HCC treatment. Silmitasertib is an orally bioavailable, potent CK2 inhibitor with a direct role in DNA damage repair and is in clinical trials for other cancers. In this study, we planned to repurpose these existing drugs on HCC treatment. We observed a stronger antiproliferative effect of these two combined drugs on HCC cells generated from different etiologies as compared to the single treatment. Global RNA-seq analyses revealed a decrease in the expression of G2/M cell cycle transition genes in HCC cells following combination treatment, suggesting G2 phase cell arrest. We observed G2/M cell cycle phase arrest in HCC cells upon combination treatment compared to the single-treated or vehicle-treated control cells. The downregulation of CCNA2 and CDC25C following combination therapy further supported the observation. Subsequent analyses demonstrated that combination treatment inhibited 70 kDa ribosomal protein S6 kinase (p70S6K) phosphorylation, and increased Bim expression. Apoptosis of HCC cells were accompanied by increased poly (ADP-ribose) polymerase cleavage and caspase-9 activation. Next, we observed that a combination therapy significantly delayed the progression of HCC xenograft growth as compared to vehicle control. Together, our results suggested combining cabozantinib and silmitasertib would be a promising treatment option for HCC.

The glutamate receptor antagonist ifenprodil inhibits hepatitis E virus infection.

Infection with hepatitis E virus (HEV) represents a global problem, with over 20 million people infected annually. No specific antiviral drugs are available for treating HEV infection, necessitating the development of novel targeted therapeutics. Here, we report that the N-methyl-D-aspartate receptor (NMDAR) antagonist ifenprodil, a clinically approved drug used to treat idiopathic pulmonary fibrosis (IPF), is an HEV inhibitor in liver-derived cells. In vitro investigation demonstrates that ifenprodil suppresses viral protein expression in a dose-dependent manner in human hepatoma cells by inhibiting early stages of viral infection. We also found that ifenprodil modulates host cell intrinsic biological processes distinct from virus-induced innate immunity, inhibiting HEV RNA accumulation in primary human hepatocytes. Finally, the inhibitory effect of ifenprodil in vivo was also tested in rabbits challenged with the HEV-3ra CHN-BJ-R14 strain. Fecal virus shedding was below the limit of detection in two animals for both ribavirin-treated and ifenprodil-treated rabbits compared to vehicle-treated control animals. Our data demonstrate that ifenprodil is an effective anti-HEV compound with potential as a therapeutic candidate for the treatment of HEV infection.

Hydroxyurea reduces the levels of the fetal globin gene repressors ZBTB7A/LRF and BCL11A in erythroid cells in vitro

Abstract Objectives Hydroxyurea (HU) is the most widely used therapy for adults and children with sickle cell disease (SCD). It is believed to act largely by inducing the transcription of fetal γ-globin genes to generate fetal hemoglobin (HbF), which inhibits the pathological polymerization of sickle hemoglobin (HbS). The mechanisms by which hydroxyurea elevates HbF are unclear. We explored the hypothesis that hydroxyurea induces HbF expression by inhibiting the expression of 2 γ-globin gene repressors, BCL11A and ZBTB7A (also known as LRF), which normally bind the γ-globin gene promoters to inhibit their expression after birth. Methods We treated immortalized murine erythroleukemia cells and normal human donor CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea and measured the effects on globin, BCL11A and ZBTB7A protein and mRNA expression. Results Treating murine erythroleukemia cells or human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea reduced the protein levels of BCL11A and ZBTB7A compared to the vehicle-treated control. BCL11A mRNA levels were reduced in both cell types upon hydroxyurea treatment. However, ZBTB7A mRNA levels were only reduced in human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts. Conclusions Hydroxyurea can act in erythroid cells to reduce the levels and activity of two direct fetal γ-globin transcriptional repressors with accompanying de-repression of the γ-globin genes and induction of HbF, which may explain the mechanism of action leading to amelioration of symptoms in SCD patients treated with this drug.

Detrimental effect of prenatal progesterone exposure on anxiety and depressive-like responses in adult male and female rat offspring: role of plasma, hippocampal corticosterone and hippocampal progesterone receptors

In clinical practice, the use of exogenous progesterone (Pro) is often required in assisted reproduction programs due to reduced levels of the hormone and the risk of miscarriage. Exposure to the hormone reduces anxiety in rodents, but the long-term effects of prenatal exposure in adult offspring are unknown. Therefore, the present study was designed to investigate the effect of prenatal Pro treatment on anxiety- and depression-like behavior and the effect on plasma, hippocampal corticosterone (CORT) and hippocampal progesterone receptor (PR) in young adult male and female rat offspring. The behavioral responses of offspring of both sexes were tested in the open field, and the elevated plus maze tests, and for depressive-like behavior in the sucrose preference test, the forced swimming test and the splash test. CORT levels and PR expression were measured by ELISA. The results indicate that prenatal Pro exposure at low and high doses (10 and 50 mg/kg, s.c. during G0-G10) induces anxiogenic and depressive-like effects compared to vehicle-treated controls, which are associated with high plasma and hippocampal CORT levels and upregulated hippocampal PR in male and female adult offspring. Our results demonstrate that prenatal Pro exposure has detrimental effects on the emotional status of male and female adult offspring, which may be associated with long-term changes in hormonal homeostasis.

Cadmium-induced fetal erythropoiesis disturbances in mice

Maternal anemia has been identified as a contributing factor to adverse reproductive outcomes associated with cadmium (Cd) exposure, a common heavy metal. Our recent findings suggest that inhibited erythroid differentiation and enucleation also play significant roles in the direct embryonic toxicity resulting from maternal Cd exposure. However, the effects of Cd exposure on lipid metabolism remodeling, which is essential for physiological erythropoiesis, remain poorly understood. In the present study, pregnant mice were administered low doses of CdCl2 via oral exposure from early to late gestation to mitigate Cd-induced maternal anemia. Compared to vehicle-treated controls, embryos from Cd-treated mice exhibited a slight decrease in weight, though without signs of atrophy. Consistent with our previous observations, fetal livers from Cd-exposed embryos demonstrated a dose-dependent inhibition of erythroid differentiation, as confirmed by ex vivo analysis. Notably, an intrinsic decrease in lipid peroxidation during erythroid differentiation was observed in the bone marrow and fetal livers of vehicle-treated mice, attributed to diminished lipid content. In contrast, this decrease in lipid peroxidation was absent in fetal liver erythroblasts from Cd-treated mice, where an increase in lipid peroxidation was instead noted. These findings elucidate a potential mechanism, lipid peroxidation, underlying Cd-induced embryonic toxicity.

Abstract P377: Short-Chain Fatty Acids Improve Cardiac Function in a Hypertensive Rat Model of Heart Failure

Hypertension-induced ventricular remodeling contributes significantly to the progression of heart failure (HF). The global increase in HF prevalence results in heightened cardiovascular morbidity and mortality, yet treatment options remain limited. The gut microbiota ferments indigestible dietary fibers, producing essential metabolites such as short-chain fatty acids (SCFAs), amino acids metabolites. We hypothesize that direct oral administration of SCFAs modulates mucosal immune cells, thereby ameliorating the heart failure with preserved ejection fraction (HFpEF) phenotype in the double-transgenic rat (dTGR) model. Four-week-old male rats, double-transgenic for human renin and angiotensinogen (dTGRs), were utilized. These dTGR rats exhibit classical symptoms of HFpEF, including hypertension, inflammation, and fibrosis in the heart and kidneys. The rats were randomized into five experimental groups: healthy non-transgenic vehicle-treated controls (wild type), vehicle-treated dTGR, and dTGR treated with either acetate, propionate, or butyrate. Following a 3-week treatment period, comprehensive phenotyping was conducted, including echocardiography, Millar-tip catheterization, gene expression analysis, and histological examination. SCFA administration improves diastolic function, as evidenced by alterations in the end-diastolic pressure-volume relationship measured via Millar-tip catheter. Notably, butyrate treatment had the most pronounced effect on alleviating diastolic dysfunction and reducing blood pressure. Additionally, gene expressions such as atrial natriuretic peptide, connective tissue growth factor, and myosin heavy chain alpha and beta showed improvement in SCFA-treated dTGR HFpEF rats compared to vehicle-treated counterparts. Furthermore, SCFA treatment ameliorated cardiac inflammation. Our data suggest that SCFAs, particularly butyric acid, enhance hypertensive diastolic dysfunction and cardiac gene expression in dTGR rats, underscoring their therapeutic potential in HF.

Cannabinoid receptor 2 agonist AM1241 alleviates epileptic seizures and epilepsy-associated depression via inhibiting neuroinflammation in a pilocarpine-induced chronic epilepsy mouse model

Increasing evidence suggests that cannabinoid receptor 2 (CB2R) serves as a promising anti-inflammatory target. While inflammation is known to play crucial roles in the pathogenesis of epilepsy, the involvement of CB2R in epilepsy remains unclear. This study aimed to investigate the effects of a CB2R agonist, AM1241, on epileptic seizures and depressive-like behaviors in a mouse model of chronic epilepsy induced by pilocarpine. A chronic epilepsy mouse model was established by intraperitoneal administration of pilocarpine. The endogenous cannabinoid system (eCBs) in the hippocampus was examined after status epilepticus (SE). Animals were then treated with AM1241 and compared with a vehicle-treated control group. Additionally, the role of the AMPK/NLRP3 signaling pathway was explored using the selective AMPK inhibitor dorsomorphin. Following SE, CB2R expression increased significantly in hippocampal microglia. Administration of AM1241 significantly reduced seizure frequency, immobility time in the tail suspension test, and neuronal loss in the hippocampus. In addition, AM1241 treatment attenuated microglial activation, inhibited pro-inflammatory polarization of microglia, and suppressed NLRP3 inflammasome activation in the hippocampus after SE. Further, the therapeutic effects of AM1241 were abolished by the AMPK inhibitor dorsomorphin. Our findings suggest that CB2R agonist AM1241 may alleviate epileptic seizures and its associated depression by inhibiting neuroinflammation through the AMPK/NLRP3 signaling pathway. These results provide insight into a novel therapeutic approach for epilepsy.

Probiotic treatment with viable α-galactosylceramide-producing Bacteroides fragilis reduces diabetes incidence in female nonobese diabetic mice.

We aimed to investigate whether alpha-galactosylceramide (α-GalCer)-producing Bacteroides fragilis could induce natural killer T (NKT) cells in nonobese diabetic (NOD)mice and reduce their diabetes incidence. Five-week-old female NOD mice were treated orally with B. fragilis, and islet pathology and diabetes onset were monitored. Immune responses were analyzed by flow cytometry and multiplex technology. Effects of ultraviolet (UV)-killed α-GalCer-producing B. fragilis and their culture medium on invariant NKT (iNKT) cells were tested ex vivo on murine splenocytes, and the immunosuppressive capacity of splenocytes from B. fragilis-treated NOD mice were tested by adoptive transfer to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. B. fragilis reduced the diabetes incidence from 69% to 33% and the percent of islets with insulitis from 40% to 7%, which doubled the serum insulin level compared with the vehicle-treated control mice. Furthermore, the early treatment reduced proinflammatory mediators in the serum, whereas the proportion of CD4+ NKT cell population was increased by 33%. B. fragilis growth media stimulated iNKT cells and anti-inflammatory M2 macrophages ex vivo in contrast to UV-killed bacteria, which had no effect, strongly indicating an α-GalCer-mediated effect. Adoptive transfer of splenocytes from B. fragilis-treated NOD mice induced a similar diabetes incidence as splenocytes from untreated NOD mice. B. fragilis induced iNKT cells and M2 macrophages and reduced type 1 diabetes in NOD mice. The protective effect seemed to be more centered on gut-pancreas interactions rather than a systemic immunosuppression. B. fragilis should be considered for probiotic use in individuals at risk of developing type 1 diabetes.

Decline in corepressor CNOT1 in the pregnant myometrium near term impairs progesterone receptor function and increases contractile gene expression

Progesterone (P4), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P4/PR partially exerts these effects by tethering to NF-κB bound to their promoters, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P4/PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a P4-induced corepressor that also interacts within the same chromatin complex as PR-B. In mouse myometrium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of endogenous CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in the enrichment of repressive histone marks and an increase in the enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in the expression of corresponding histone-modifying enzymes. Myometrial tissues from P4-treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. In hTERT-HM cells, P4 treatment enhanced CNOT1 expression and its recruitment to NF-κB-response elements within the CX43 and OXTR promoter regions. Furthermore, knockdown of CNOT1 significantly increased the expression of contractile genes. These novel findings suggest that decreased expression and binding of the transcriptional corepressor CNOT1 at the chromatin level near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition.

Novel 5-HT7 receptor antagonists modulate intestinal immune responses and reduce severity of colitis.

Inflammatory bowel disease (IBD) encompasses several debilitating chronic gastrointestinal (GI) inflammatory disorders, including Crohn's disease and ulcerative colitis. In both conditions, mucosal inflammation is a key clinical presentation associated with altered serotonin (5-hydroxytryptamine or 5-HT) signaling. This altered 5-HT signaling is also found across various animal models of colitis. Of the 14 known receptor subtypes, 5-HT receptor type 7 (5-HT7) is one of the most recently discovered. We previously reported that blocking 5-HT signaling with either a selective 5-HT7 receptor antagonist (SB-269970) or genetic ablation alleviated intestinal inflammation in murine experimental models of colitis. Here, we developed novel antagonists, namely, MC-170073 and MC-230078, which target 5-HT7 receptors with high selectivity. We also investigated the in vivo efficacy of these antagonists in experimental colitis by using dextran sulfate sodium (DSS) and the transfer of CD4+CD45RBhigh T cells to induce intestinal inflammation. Inhibition of 5-HT7 receptor signaling with the antagonists, MC-170073 and MC-230078, ameliorated intestinal inflammation in both acute and chronic colitis models, which was accompanied by lower histopathological damage and diminished levels of proinflammatory cytokines compared with vehicle-treated controls. Together, the data reveal that the pharmacological inhibition of 5-HT7 receptors by these selective antagonists ameliorates the severity of colitis across various experimental models and may, in the future, serve as a potential treatment option for patients with IBD. In addition, these findings support that 5-HT7 is a viable therapeutic target for IBD.NEW & NOTEWORTHY This study demonstrates that the novel highly selective 5-HT7 receptor antagonists, MC-170073 and MC-230078, significantly alleviated the severity of colitis across models of experimental colitis. These findings suggest that inhibition of 5-HT7 receptor signaling by these new antagonists may serve as an alternative mode of treatment to diminish symptomology in those with inflammatory bowel disease.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

Abstract 3057: Vascular Smooth Muscle Cell Cd47 Contributes To Atherosclerosis Development

Aims: Thrombospondin-1 (TSP1), a secretory protein, has been associated with various cardiovascular diseases. TSP1 mainly functions via its cognate receptors CD47 and CD36. Recently, we observed elevated TSP1 expression in human and murine atherosclerotic arteries. However, the role of vascular smooth muscle cell (VSMC) TSP1-CD47 signaling in regulating VSMC phenotype and atherogenesis is unclear. Methods and Results: Human arterial SMCs were treated with human TSP1 and analyzed for SMC markers and proliferation. The data revealed decreased expression of SMC markers, ACTA, CNN1, and SM22α in TSP1-exposed VSMCs compared with vehicle-treated control cells. Further, TSP1 treatment increased VSMC proliferation in vitro , suggesting TSP1 induces VSMC hyperproliferative phenotype. Additional experiments showed CD47 as the major TSP1’s receptor in VSMCs. To investigate the in vivo role of VSMC Cd47 in atherosclerosis, we generated tamoxifen-inducible SMC-specific Cd47 knockout mice ( Cd47 f/f Myh11 Cre +/– , Cd47 ΔVSMC ) by crossing female Cd47 f/f mice with male Myh11 Cre +/- mice. Male tamoxifen-injected Cd47 ΔVSMC and corn oil-administered Cd47 WT mice were given a single injection of AAV8- hPCSK9 (1х10 11 viral genomes/mouse, IP) and fed a Western diet for 12 weeks. En face oil red O (ORO) staining of whole aortas exhibited reduced atherosclerosis in Cd47 ΔVSMC mice compared with control Cd47 WT mice. Further histochemical staining performed on aortic root sections revealed attenuated neointimal lesion area, lipid accumulation and necrotic area in Cd47 ΔVSMC mice. However, there were no significant differences in weight gain, body composition (fat and lean mass), plasma total cholesterol, and fasting blood glucose between Cd47 ΔVSMC and Cd47 WT mice. Conclusions: Taken together, these findings suggest that TSP1 promotes VSMC phenotype switch and VSMC-specific Cd47 deletion in hypercholesterolemic mice suppresses atherosclerosis progression.

A Role for GABAA Receptor β3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6β3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the β3 subunit for tremor suppression. We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact β3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed β3 cre- mice, but had no effect on tremor in floxed β3 cre+ littermates that have β3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed β3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. As α6β3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express β3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6β3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that β3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6β3δ extra-synaptic GABAA receptors.

Toxicologic Pathology Forum: Apoptosis/Single Cell Necrosis as a Possible Procedural Effect in Primate Brain Following Ice-Cold Saline Perfusion.

Nonclinical studies of test articles (TAs) in nonhuman primates are often designed to assess both biodistribution and toxicity. For this purpose, studies commonly use intravenous perfusion of ice-cold (2°C-8°C) saline to facilitate measurements of TA-associated nucleic acids and proteins, after which tissues undergo later fixation by immersion for histological processing and microscopic evaluation. Intriguingly, minimal apoptosis/single cell necrosis (A/SCN) of randomly distributed neural cells is evident in the cerebral cortex and less often the hippocampus in animals from all groups, including vehicle-treated controls. Affected cells exhibit end-stage features such as cytoplasmic hypereosinophilia, nuclear condensation or fragmentation, and shape distortions, so their lineage(s) generally cannot be defined; classical apoptotic bodies are exceedingly rare. In addition, A/SCN is not accompanied by glial reactions, leukocyte infiltration/inflammation, or other parenchymal changes. The severity is minimal in controls but may be slightly exacerbated (to mild) by TA that accumulate in neural cells. One plausible hypothesis explaining this A/SCN exacerbation is that cold shock (perhaps complicated by concurrent tissue acidity and hypoxia) drives still-viable but TA-stressed cells to launch a self-directed death program. Taken together, these observations indicate that A/SCN in brain processed by cold saline perfusion with delayed immersion fixation represents a procedural artifact and not a TA-related lesion.

CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice.

A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a αCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis.