Increased Histone Acetylation Research Articles

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Increased histone acetylation levels or a serotonin precursor reinstate the context memory impaired by the serotonin receptor blocker methiothepin

Increased histone acetylation levels or a serotonin precursor reinstate the context memory impaired by the serotonin receptor blocker methiothepin

Pantothenate and L-Carnitine Supplementation Improves Pathological Alterations in Cellular Models of KAT6A Syndrome.

Mutations in several genes involved in the epigenetic regulation of gene expression have been considered risk alterations to different intellectual disability (ID) syndromes associated with features of autism spectrum disorder (ASD). Among them are the pathogenic variants of the lysine-acetyltransferase 6A (KAT6A) gene, which causes KAT6A syndrome. The KAT6A enzyme participates in a wide range of critical cellular functions, such as chromatin remodeling, gene expression, protein synthesis, cell metabolism, and replication. In this manuscript, we examined the pathophysiological alterations in fibroblasts derived from three patients harboring KAT6A mutations. We addressed survival in a stress medium, histone acetylation, protein expression patterns, and transcriptome analysis, as well as cell bioenergetics. In addition, we evaluated the therapeutic effectiveness of epigenetic modulators and mitochondrial boosting agents, such as pantothenate and L-carnitine, in correcting the mutant phenotype. Pantothenate and L-carnitine treatment increased histone acetylation and partially corrected protein and transcriptomic expression patterns in mutant KAT6A cells. Furthermore, the cell bioenergetics of mutant cells was significantly improved. Our results suggest that pantothenate and L-carnitine can significantly improve the mutant phenotype in cellular models of KAT6A syndrome.

Elevated propionate/propionyl-CoA signalling drives Pde9a overexpression and contractile dysfunction through increased histone acetylation and propionylation

Elevated propionate/propionyl-CoA signalling drives Pde9a overexpression and contractile dysfunction through increased histone acetylation and propionylation

Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5'-3' imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5'-3' imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

EXTH-25. EXPLOITING EPIGENETIC DYSREGULATION IN ISOCITRATE DEHYDROGENASE MUTANT GLIOMA VIA THE USE OF HISTONE DEACETYLASE INHIBITORS

Abstract Isocitrate dehydrogenase 1/2 mutations (IDHmut) define a unique subtype of glioma that exhibits profound epigenetic dysregulation through DNA and histone hypermethylation. Though IDHmutgliomas are less aggressive than their IDH wildtype (IDHwt) counterparts, these tumors are still incurable and are invariably fatal. New treatments are therefore needed. Histone deacetylase (HDAC) enzymes compose a class of epigenetic drug targets already used in the clinic to treat other malignancies, but their potential against gliomas, specifically IDHmutgliomas, have not been extensively studied. We previously showed that IDHwt gliomas are more sensitive than IDHwt gliomas to the cytotoxic and antiproliferative effects mediated by the FDA-approved HDAC inhibitor panobinostat, and that this sensitivity was associated with a greater increase in histone acetylation in IDHmut glioma cultures. Follow-up RNA-Seq showed that panobinostat more greatly affected gene expression in IDHmut glioma cells, with 1999 significant differentially expressed genes showing a 10% or greater change in gene expression in IDHmut cells compared to 388 genes for IDHwt counterparts. This included the known HDAC target and cell-cycle inhibitor CDKN1A, with panobinostat causing a 1.9-fold greater upregulation of CDKN1A in IDHmut cells than IDHwt cells (p= 0.045). In immunocompromised mice intracranially engrafted with IDHmut or IDHwt glioma, 15 mg/kg intraperitoneally-administered panobinostat was detectable after 6 hours within the intracerebral tumors via mass spectrometry at a concentration of 52.4 ng/g (150.1 nM), and extended the survival of IDHmut glioma-engrafted mice (Median: 4 days; Mean: 9.4 days), but not IDHwt glioma-engrafted mice (IDHmut: logrank p= 0.044; IDHwt: logrank p= 0.18). These results suggest that HDAC inhibition via panobinostat may be an effective therapy to treat IDHmutglioma patients. Ongoing studies, including ChIP-Seq, will determine specific sites of histone acetylation and HDACs that are responsible for the greater sensitivity of IDHmut gliomas to HDAC inhibition.

Mediterranean G6PD variant mitigates expression of DNA methyltransferases and right heart pressure in experimental model of pulmonary hypertension

DNA methylation potentially contributes to the pathogenesis of pulmonary hypertension (PH). However, the role of DNA methyltransferases (DNMTs: 1, 3a, and 3b), the epigenetic writers, in modulating DNA methylation observed in PH remains elusive. Our objective was to determine DNMT activity and expression in the lungs of experimental rat models of PH. Because the activity of DNMTs is metabolically driven, another objective was to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in regulating DNMT expression and activity in the lungs of novel loss-of-function Mediterranean G6PD variant (G6PDS188F) rats. As outlined for modeling PH, rats injected with sugen5416 (SU) were placed in a hypoxia (Hx) chamber set at 10% oxygen for 3weeks and then returned to normoxia (Nx) for 5weeks (SU/Hx/Nx). Rats kept in atmospheric oxygen and treated with SU were used as controls. We assessed the activity and expression of DNMTs in the lungs of rats exposed to SU/Hx/Nx. WT rats exposed to SU/Hx/Nx developed hypertension and exhibited increased DNMT activity and Dnmt1 and Dnmt3b expression. In G6PDS188F rats, which developed less of a SU/Hx/Nx-induced increase in right ventricle pressure and hypertrophy than WT rats, we observed a diminished increase in expression and activity of DNMTs, DNA hypomethylation, increased histone acetylation and methylation, and increased expression of genes encoding NOS3 and SOD2-vascular-protective proteins. Collectively, increased DNMTs contribute to reduced expression of protective genes and to the pathogenesis of SU/Hx/Nx-induced experimental PH. Notably, G6PD regulates the expression of DNMTs and protective proteins in the lungs of hypertensive rats.

Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

The tumor microenvironment (TME) represents a milieu enabling cancer cells to develop malignant properties, while concerted interactions between cancer and stromal cells frequently shape an “activated/reprogramed” niche to accelerate pathological progression. Here we report that a soluble factor epiregulin (EREG) is produced by senescent stromal cells, which non-cell-autonomously develop the senescence-associated secretory phenotype (SASP) upon DNA damage. Genotoxicity triggers EREG expression by engaging NF-κB and C/EBP, a process supported by elevated chromatin accessibility and increased histone acetylation. Stromal EREG reprograms the expression profile of recipient neoplastic cells in a paracrine manner, causing upregulation of MARCHF4, a membrane-bound E3 ubiquitin ligase involved in malignant progression, specifically drug resistance. A combinational strategy that empowers EREG-specific targeting in treatment-damaged TME significantly promotes cancer therapeutic efficacy in preclinical trials, achieving response indices superior to those of solely targeting cancer cells. In clinical oncology, EREG is expressed in tumor stroma and handily measurable in circulating blood of cancer patients post-chemotherapy. This study establishes EREG as both a targetable SASP factor and a new noninvasive biomarker of treatment-damaged TME, thus disclosing its substantial value in translational medicine.

Nickel nanoparticles induce epithelial-mesenchymal transition in human bronchial epithelial cells via the HIF-1α/HDAC3 pathway

We and others have previously demonstrated that exposure to nickel nanoparticles (Nano-Ni) caused fibrogenic and carcinogenic effects; however, the underlying mechanisms are still not fully understood. This study aimed to investigate the effects of Nano-Ni on epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells (BEAS-2B) and its underlying mechanisms since EMT is involved in both cancer pathogenesis and tissue fibrosis. Our results showed that exposure to Nano-Ni, compared to the control Nano-TiO2, caused a remarkable decrease in the expression of E-cadherin and an increase in the expression of vimentin and α-SMA, indicating an inducible role of Nano-Ni in EMT development in human bronchial epithelial cells. HIF-1α nuclear accumulation, HDAC3 upregulation, and decreased histone acetylation were also observed in the cells exposed to Nano-Ni, but not in those exposed to Nano-TiO2. Pretreatment of the cells with a specific HIF-1α inhibitor, CAY10585, or HIF-1α-specific siRNA transfection prior to Nano-Ni exposure resulted in the restoration of E-cadherin and abolished Nano-Ni-induced upregulation of vimentin and α-SMA, suggesting a crucial role of HIF-1α in Nano-Ni-induced EMT development. CAY10585 pretreatment also attenuated the HDAC3 upregulation and increased histone acetylation. Inhibition of HDAC3 with specific siRNA significantly restrained Nano-Ni-induced reduction in histone acetylation and restored EMT-related protein expression to near control levels. In summary, our findings suggest that exposure to Nano-Ni promotes the development of EMT in human bronchial epithelial cells by decreasing histone acetylation through HIF-1α-mediated HDAC3 upregulation. Our findings may provide information for further understanding of the molecular mechanisms of Nano-Ni-induced fibrosis and carcinogenesis.

A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Abstract OEC103: ATP-dependent Citrate Lyase Drives Metabolic Remodeling In The Heart During Cancer

Cardiovascular diseases and cancer are the leading causes of death worldwide. Recurrent mutations of metabolic enzymes in cancer cells provide benefits for tumor growth, while creating a systemic metabolic phenotype and increasing the risk for adverse cardiac events. We recently showed that tumors producing the oncometabolite D-2-hydroxyglutarate increase ATP citrate lyase activity and histone acetylation in the heart. How cancer cells drive metabolic remodeling in the heart, and whether ATP citrate lyase is critical for cardiac adaptation during cancer, remains poorly understood. We co-cultured wildtype adult mouse ventricular cardiomyocytes with colorectal cancer cells overexpressing ATP citrate lyase. Co-culture of cardiomyocytes with colorectal cancer cells was associated with reduced expression of ATP citrate lyase, pyruvate dehydrogenase, and mitochondrial complex I, suggesting that the cancer cell environment impaired mitochondrial function in vitro. In vitro modulation of ATP citrate lyase activity abrogated the metabolic alterations in cultured cardiomyocytes indicating that ATP citrate lyase drives remodeling during cancer. Accordingly, we created mice lacking ATP citrate lyase in the heart using CRISPR-Cas 9 genomic knockout. Loss of ATP citrate lyase leads to left ventricular systolic dysfunction and causes global gene and protein expression changes in the heart. Targeted mass spectrometry-based metabolomics and lipidomics showed decreased acetyl-CoA and succinyl-CoA levels in isolated cardiomyocytes, while the abundance of cholesterol esters increased. In vivo [ 18 F]-FDG-positron emission tomography revealed that animals lacking ATP citrate lyase increased plasma glucose uptake compared to animals with cardiac ATP citrate lyase expression. These findings are consistent with maladaptive metabolic remodeling and impaired oxidative metabolism during heart failure. Manipulating the activity of ATP citrate lyase using BMS303141 (0.5 μM) in ex vivo perfused wildtype rat hearts caused a rapid decline in cardiac power and increased utilization of glucose confirming our in vivo mouse studies. Conversely, inhibition of ATP citrate lyase during oncometabolic stress via D-2-hydroxyglutarate improved cardiac contractile function via redirection of Krebs cycle flux and provision of ATP levels. Together these data demonstrate that ATP citrate lyase regulates oxidative metabolism and structural remodeling in the heart and creates a targetable metabolic liability during cancer.

Abstract P2076: β-hydroxybutyrate Administered At Reperfusion Alleviates Myocardial Ischemia-reperfusion Injury By Enhancing Autophagy And Mitochondrial Homeostasis

Introduction: ST-elevation myocardial infarction (STEMI) is the leading cause of heart failure. Ischemia/reperfusion (I/R) injury after revascularization contributes ~50% of infarct size, which we don’t have a clinical therapy. β-hydroxybutyrate (β-OHB) is a metabolite that increases during stress. In addition to providing energy, it serves as a signaling molecule (such as histone deacetylase inhibitor) to regulate many protective processes. Increasing the β-OHB level before I/R is cardiac protective. However, it has not been tested when administered at reperfusion. Hypothesis: The physiological concentration of β-OHB administered at the time of reperfusion reduces I/R injury by enhancing autophagy and mitochondrial homeostasis. Methods: C57BL6 wild-type mice were randomized into 2 groups and subjected to I/R surgery (I: 45 mins; R: 24 hrs). At reperfusion, normal saline or 10 mmol/kg of β-OHB (4-6 mM blood level) was given intraperitoneally. Echo was used to evaluate cardiac functions. We measured Infarct size, autophagic flux, and mitochondrial DNA levels in the heart. CAG-RFP-GFP-LC3 (RFLC3) mice were used to detect autophagic flux. Neonatal rat ventricular myocytes (NRVMs) were subjected to simulated I/R to test the effect of β-OHB in vitro . Small interfering RNA of ATG7 gene was used to assess the role of autophagy. Seahorse assay was performed to evaluate mitochondrial function after I/R. Results: β-OHB gave at reperfusion reduced infarct size by 50% (N=15 in control and N=11 in β-OHB group), and preserved systolic function in mice. In the border zone, the LC3II and the mtDNA level increased, and β-OHB increased autophagic flux in RFLC3 mice (N=5). In NRVMs subjected to I/R, β-OHB increased histone acetylation, cell viability, and maintained mitochondrial homeostasis (mitochondrial mass, membrane potential), and reduced ROS generation (N=4-6). Seahorse assay showed β-OHB enhanced ATP production after I/R. ATG7 knockdown blocked the protective effect of β-OHB in NRVMs. Conclusions: β-OHB administered at reperfusion reduces infarct size by increasing autophagic flux and maintaining mitochondrial homeostasis. Since β-OHB has been used in heart failure patients safely, it is a viable therapeutics to reduce infarct size in STEMI patients.

Extracellular matrix derived from Wharton’s Jelly-derived mesenchymal stem cells promotes angiogenesis via integrin αVβ3/c-Myc/P300/VEGF

BackgroundAngiogenesis is required in many physiological conditions, including bone regeneration, wound healing, and tissue regeneration. Mesenchymal stem cells-derived extracellular matrix (MSCs-ECM) could guide intricate cellular and tissue processes such as homeostasis, healing and regeneration.MethodsThe purpose of this study is to explore the effect and mechanism of ECM derived from decellularized Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) on endothelial cell viability and angiogenesis. The human umbilical vein endothelial cells (HUVECs) were pretreated with WJ-MSCs ECM for 2d/7d/14d, respectively. After pretreatment, the angiogenesis ability of HUVECs was detected.ResultsIn this study, we found for the first time that WJ-MSCs ECM could improve the angiogenesis ability of HUVECs with a time-dependent manner in vitro. Mechanically, WJ-MSCs ECM activated the focal adhesion kinase (FAK)/P38 signaling pathway via integrin αVβ3, which further promoted the expression of the cellular (c)-Myc. Further, c-Myc increased histone acetylation levels of the vascular endothelial growth factor (VEGF) promoter by recruiting P300, which ultimately promoting VEGF expression.ConclusionsECM derived from Wharton’s Jelly-derived mesenchymal stem cells promotes angiogenesis via integrin αVβ3/c-Myc/P300/VEGF. This study is expected to provide a new approach to promote angiogenesis in bone and tissue regeneration.

The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer.

Objective:The novel compound GCJ-490A has been discovered as a pan-histone deacetylase (HDAC) inhibitor that exerts potent inhibitory activity against HDAC1, HDAC3, and HDAC6. Because of the important roles of HDACs in lung cancer development and the high distribution of GCJ-490A in lung tissue, we explored the anti-tumor potency of GCJ-490A against non-small cell lung cancer (NSCLC) in vitro and in vivo in this study.Methods:The in vitro effects of GCJ-490A alone or combined with the EGFR inhibitor gefitinib against NSCLC were measured with proliferation, apoptosis, and colony formation assays. NSCLC xenograft models were used to investigate the efficacy of GCJ-490A combined with gefitinib for the treatment of NSCLC in vivo. Western blot assays, luciferase reporter assays, chromatin immunoprecipitation assays, quantitative real time-PCR, immunohistochemistry, and transcription factor activity assays were used to elucidate possible mechanisms.Results:GCJ-490A effectively inhibited NSCLC cell proliferation and induced apoptosis in vitro and in vivo. Interestingly, inhibition of HDAC1 and HDAC6 by GCJ-490A increased histone acetylation at the IKKα promoter and enhanced IKKα transcription, thus decreasing c-Met. Moreover, this c-Met downregulation was found to be essential for the synergistic anti-tumor activity of GCJ-490A and gefitinib.Conclusions:These findings highlight the promising potential of HDAC inhibitors in NSCLC treatment and provide a rational basis for the application of HDAC inhibitors in combination with EGFR inhibitors in clinical trials.

Abstract 2673: Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model

Abstract Background: Treating colorectal cancer (CRC) using targeted therapies based on each patient’s individual mutational profile continues to gain interest. Oncogenic mutations of the PIK3CA gene leads to tumor initiation and progression. Copanlisib inhibits the PI3K pathway and has proven to be an effective treatment strategy for various cancers characterized by overactivation of the PI3K signaling cascade. However, resistance mechanisms to targeted therapies such as copanlisib are frequently encountered, and can be overcome through combination with another molecule such as romidepsin, a histone deacetylase (HDAC) inhibitor. Here we aim to identify a novel therapeutic regimen for improved treatment of the molecular subtypes of CRC that can be targeted with PI3K inhibition. Methods: Organoids were derived from colorectal cancer tumor tissue from two different biopsy sites from one patient with PIK3CA mutant rectal cancer (RC46A and RC46B), as well as from a patient with APC and TP53 mutant CRC (MTB74) under approved IRB protocols. All organoids were plated and allowed to mature for 24 to 48 hours before treatment and baseline images were obtained. The organoids were imaged again 48 hours later, and response was determined by measuring the change in organoid diameter using ImageJ. Additionally, organoids were collected for immunoblotting to examine alterations in the PI3K pathway as well as histone acetylation, and proteins involved in apoptosis. Results: Control organoids demonstrated increased median relative changes in diameter of +53.38%, +43.04% and +46.2% for RC46A, RC46B and MTB74, respectively. In comparison, the combination treatment group exhibited median relative changes for RC46A, RC46B and MTB74 as -29.19%, -46.46% and -100% (p<0.001). These results were supported with immunoblot analysis that revealed a synergistic increase in histone acetylation with the combination treatment. Single agent copanlisib treatment elicited alterations in PI3K signaling including downregulation of pS6, pAKT, p4EBP1 as expected. Lastly, an increase in cleaved PARP was observed in combination treated organoids which is indicative of increased apoptosis. Conclusions: As molecular diagnostic technology continues to advance, treating each individual cancer with a targeted therapy based on molecular profiling is becoming more feasible. Dual inhibition of the PI3K pathway and HDAC is effective in reducing cell growth and proliferation of CRC organoids characterized by PIK3CA mutations, as well as other molecular subtypes such as cancers with APC and TP53 mutations. This combination strategy shows promise for improving treatment response and reducing resistance in patients across these molecular subtypes. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Susan N. Payne, Autumn M. Olson, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2673.

Abstract 6060: Can the gut microbiota both radiosensitize tumors and spare normal tissue toxicity

Abstract Background: New non-toxic radiosensitizers are needed in the treatment of muscle-invasive bladder cancer because elderly patients are vulnerable to chemotherapy-related toxicity of currently available radiosensitizers. Our previous study showed that high fiber diets sensitized RT112 xenografts to ionizing radiation (IR) by modifying the gut microbiome and this phenotype was positively correlated with B.acidifaciens (BA) abundance. It is noted that gut microbiota can enhance anti-tumoral responses, so we hypothesise that gut microbiota including BA may radiosensitize tumors via secretion of metabolites and immunomodulation. Methods: UPPL1591 mouse bladder cancer cell line flank xenografts were implanted into C57BL/6 mice (an immune-competent setting) at the same time as starting low (0.2% cellulose) and high fiber (5% psyllium, 5% psyllium plus 10% resistant starch/10% inulin) diets for their lifetime (N=30). 16S rRNA sequencing, IC-MS, IHC and the Nanostring platform were used for gut microbiota, untargeted metabolomics, and local tumor immunity analyses. For normal tissue toxicity studies, mice received 10-14 Gy IR (N=84). We used an intestinal crypt assay and FACS analysis of spleen to assess acute normal effects in small intestine and systemic immunity. Cell viability, clonogenic assay and Western blotting were used to validate the cytotoxic and radiosensitizing effects of bacterial supernatants on RT112 human bladder cancer cells (N=3). Results: Psyllium plus either resistant starch (RS, p=0.007) or inulin (p<0.001) significantly decreased tumors size. Psyllium plus inulin raised inosine-producing bacteria (p<0.001) and faecal inosine levels (p<0.001) which has been shown to enhance immunotherapy efficacy. Increased systemic (p=0.189) and local (p=0.024) anti-tumoral T cell responses were observed in the psyllium plus inulin group. Furthermore, the two diets mitigated the radiation injury from 14 Gy in intestinal crypt assays (p=0.011) and increased systemic immunity, with higher populations of B (p=0.024), cytotoxic T (p=0.019) and helper T (p<0.001) cells. Psyllium plus RS or inulin significantly increased caecal size (p<0.001), implying higher fermentation levels. We previously found inulin can enhance the relative abundance of BA. The supernatants of co-cultures of BA and F.praunitzii (FP, butyrate-producer) conferred greater cytotoxity than BA alone (p<0.001) or co-cultures of Bifidobacterium and FP (p<0.001), and also increased histone acetylation levels. Bacterial supernatants of BA increased DNA damage with 5Gy IR (p<0.05) and radiosensitivity of bladder tumour cells (p=0.008 at 8Gy). Conclusions: B.acidifaciens is a potential radiosensitiser with enhanced efficacy in combination with butyrate-producing bacteria. Our in vivo experiments suggest a role for inosine and cytotoxic T cells generated by the gut microbiota in radiosensitisation of bladder tumors. Citation Format: Chee Kin Then, Salome Paillas, Xuedan Wang, Mariya Misheva, James McCullagh, Kevin R. Foster, Anne E. Kiltie. Can the gut microbiota both radiosensitize tumors and spare normal tissue toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6060.