Class IIa Histone Deacetylases Research Articles

Abstract 5147: Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy

Abstract Salt-inducible kinase 2 (SIK2) is an AMPK family kinase involved in multiple signaling pathways including the LBK1-SIK-HDAC, LKB1-SIK-CRTC/CREB, PI3K-PKA-mTOR and Hippo pathways. The expression of SIK2 is significantly upregulated in fraction of ovarian and breast cancers, and associated with poor prognosis. Inhibition of SIK2 kinase activity suppresses growth of ovarian and breast cancers as well as progression of acute myeloid leukemia. SIK2 inhibitors synergistically enhance sensitivity of ovarian and breast cancer cells to paclitaxel, carboplatin and PARP inhibitors. Utilization of SIK2 inhibitors to treat cancers has been investigated in preclinical studies and in a Phase I clinical trial (NCT04711161). To enable pharmacodynamic (PD) analysis in clinical studies, we have developed biomarkers that measure the effect of GRN-300, a SIK2/3 inhibitor, on SIK2 signaling in ovarian cancer cells. Class IIa histone deacetylases (HDAC4/5/7) are SIK2 substrates in cancer cells; and are also expressed in peripheral blood mononuclear cells (PBMCs). We found that GRN-300 decreased phosphorylated HDAC4/5/7 levels in ovarian and breast cancer cells (Lu et al. J Clin Invest, 2022) and now report that this SIK2 inhibitor also decreases pHDAC4/5/7 in PBMCs ex vivo in a dose-dependent manner using Western blot analysis. We also identified three mRNA markers in PBMCs by RNAseq analysis - SEPP1, RNASE1 and MS4A6A - which are up-regulated by GRN-300 in PBMCs isolated from healthy donors. However, these mRNA markers were either expressed at very low levels or not dramatically changed in cancer cells. We identified an additional mRNA marker panel using RNAseq technology by screening seven ovarian and six breast cancer cell lines: A2780, Caov3, IGROV1, OC316, OVCAR3, OVCAR8, SKOv3ip, BT549, Cal51, HCC1937, MDA-MB-231, MDA-MB-436 and MDA-MB-468. mRNA expression of three genes - DDIT4, NUPR1 and IL6 - is upregulated by GRN-300 in more than 9 of 13 cell lines tested. Five genes - ID1, ID2, ID3, ID4 and TGFBI - are down-regulated in response to GRN-300 treatment in more than 10 of 13 cell lines. The decrease in HDAC4/5/7 phosphorylation, and alterations in mRNA markers we discovered could serve as PD biomarkers for clinical trials to evaluate the effects of SIK2 inhibitors and provide insights in the mechanisms of drug action. Citation Format: Hailing Yang, Weiqun Mao, Philip Rask, Markus Peter, Gayathri Swaminath, Stephan W. Morris, Zhen Lu, Robert C. Bast. Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy [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 5147.

Folding of Class IIa HDAC Derived Peptides into α-helices Upon Binding to Myocyte Enhancer Factor-2 in Complex with DNA

Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein–protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Metabolic remodeling in cardiac hypertrophy and heart failure with reduced ejection fraction occurs independent of transcription factor EB in mice.

A metabolic shift from fatty acid (FAO) to glucose oxidation (GO) occurs during cardiac hypertrophy (LVH) and heart failure with reduced ejection fraction (HFrEF), which is mediated by PGC-1α and PPARα. While the transcription factor EB (TFEB) regulates the expression of both PPARGC1A/PGC-1α and PPARA/PPARα, its contribution to metabolic remodeling is uncertain. Luciferase assays were performed to verify that TFEB regulates PPARGC1A expression. Cardiomyocyte-specific Tfeb knockout (cKO) and wildtype (WT) male mice were subjected to 27G transverse aortic constriction or sham surgery for 21 and 56 days, respectively, to induce LVH and HFrEF. Echocardiographic, morphological, and histological analyses were performed. Changes in markers of cardiac stress and remodeling, metabolic shift and oxidative phosphorylation were investigated by Western blot analyses, mass spectrometry, qRT-PCR, and citrate synthase and complex II activity measurements. Luciferase assays revealed that TFEB increases PPARGC1A/PGC-1α expression, which was inhibited by class IIa histone deacetylases and derepressed by protein kinase D. At baseline, cKO mice exhibited a reduced cardiac function, elevated stress markers and a decrease in FAO and GO gene expression compared to WT mice. LVH resulted in increased cardiac remodeling and a decreased expression of FAO and GO genes, but a comparable decline in cardiac function in cKO compared to WT mice. In HFrEF, cKO mice showed an improved cardiac function, lower heart weights, smaller myocytes and a reduction in cardiac remodeling compared to WT mice. Proteomic analysis revealed a comparable decrease in FAO- and increase in GO-related proteins in both genotypes. A significant reduction in mitochondrial quality control genes and a decreased citrate synthase and complex II activities was observed in hearts of WT but not cKO HFrEF mice. TFEB affects the baseline expression of metabolic and mitochondrial quality control genes in the heart, but has only minor effects on the metabolic shift in LVH and HFrEF in mice. Deletion of TFEB plays a protective role in HFrEF but does not affect the course of LVH. Further studies are needed to elucidate if TFEB affects the metabolic flux in stressed cardiomyocytes.

Abstract P2039: Alteration In Intercalated Disc Composition Impacts Cardiac Disease Progression

Intercalated discs (IDs) connect cardiomyocytes electro-mechanically and ID dysfunction has been associated with the onset of cardiomyopathies and arrhythmias. The ID- and myofibril-associated protein nebulin-related anchoring protein (NRAP) was identified as a putative novel interaction partner of protein kinase D1 (PKD). PKD1 phosphorylates class IIa histone deacetylases (HDAC), leading to their cytosolic export. This causes a de-repression of HDAC-mediated inhibition of myocyte enhancer factor 2 (MEF2)-dependent fetal and pro-hypertrophic gene expression. We investigated the regulation of NRAP by PKD1 in the developing and diseased heart. PKD activity was modulated by using the pharmacological PKD inhibitor BPKDi and/or the pro-hypertrophic stimulus phenylephrine (PE) in neonatal rat ventricular myocytes (NRVMs) and -derived engineered heart tissues (EHTs). NRVMs were analyzed by immunofluorescence, western blotting and stable isotope labelling with amino acids in cell culture/mass spectrometry. The outcome was studied by exposure to an autophagy (bafilomycin A1) or a proteasomal (MG132) inhibitor or performing proteome analysis from hearts of MuRF-1/3 double-knock-out (DKO) mice. Exposure of NRVMs to PE enhanced myofibril maturation and NRAP translocation towards IDs while BPKDi led to perinuclear accumulation of NRAP and improved force in EHTs. Combined BPKDi and PE disturbed sarcomeric structure and prevented NRAP localization at the ID. Proteomic analysis revealed that BPKDi reduced NRAP abundance and enhanced the abundance of proteins involved in proteasomal, lysosomal and spliceosomal pathways. Inhibition of proteasomal protein degradation by MG132 increased NRAP levels, whereas NRAP was 42-fold more abundant in hearts of MuRF-1/3-DKO mice that show a protein surplus cardiomyopathy. In conclusion, PKD1 impacts NRAP expression and translocation, and negatively affects EHT force development while proteasomal inhibition increased NRAP abundance suggesting a regulation via the PKD1-HDAC-MEF2 axis. Further investigations aim to study pathways involving PKD1 to regulate myofibril assembly and maintenance in the development and progression of cardiac disease.

Synthesis and biological evaluation of C-4 substituted phenoxazine-bearing hydroxamic acids with potent class II histone deacetylase inhibitory activities

Class II histone deacetylases (HDACs) are considered as potential targets to treat Alzheimer’s disease (AD). Previously, C-3 substituted phenothiazine-containing compounds with class II HDAC-inhibiting activities was found to promote neurite outgrowth. This study replaced phenothiazine moiety with phenoxazine that contains many C-3 and C-4 substituents. Some resulting compounds bearing the C-4 substituent on a phenoxazine ring displayed potent class II HDAC inhibitory activities. Structure-activity relationship (SAR) of these compounds that inhibited HDAC isoenzymes was disclosed. Molecular modelling analysis demonstrates that the potent activities of C-4 substituted compounds probably arise from π-π stacked interactions between these compounds and class IIa HDAC enzymes. One of these, compound 7d exhibited the most potent class II HDAC inhibition (IC50= 3–870 nM). Notably, it protected neuron cells from H2O2-induced neuron damage at sub-μM concentrations, but with no significant cytotoxicity. These findings show that compound 7d is a lead compound for further development of anti-neurodegenerative agents.

High-Contrast PET Imaging with [18F]NT160, a Class-IIa Histone Deacetylase Probe for In Vivo Imaging of Epigenetic Machinery in the Central Nervous System.

We utilized positron emission tomography (PET) imaging in vivo to map the spatiotemporal biodistribution/expression of class-IIa histone deacetylases (class-IIa HDACs) in the central nervous system (CNS). Herein we report an improved radiosynthesis of [18F]NT160 using 4-hydroxy-TEMPO which led to a significant improvement in radiochemical yield and molar activity. PET imaging with [18F]NT160, a highly potent class-IIa HDAC inhibitor, led to high-quality and high-contrast images of the brain. [18F]NT160 displayed excellent pharmacokinetic and imaging characteristics: brain uptake is high in gray matter regions, tissue kinetics are appropriate for a 18F-tracer, and specific binding for class-IIa HDACs is demonstrated by self-blockade. Higher uptake with [18F]NT160 was observed in the hippocampus, thalamus, and cortex while the uptake in the cerebellum was relatively low. Overall, our current studies with [18F]NT160 will likely facilitate the development and clinical translation of PET tracers for imaging of class-IIa HDACs biodistribution/expression in cancer and the CNS.

Abstract 6256: Inhibition of class IIa HDACs potentiates MYC inhibitor-driven cytotoxicity by inducing MYC depletion and oxidative stress in non-small cell lung cancer

Abstract Lung cancer is the most common cause of cancer mortality worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Dysregulation and/or overexpression of MYC are widely implicated in NSCLC, which suggests that MYC may be a promising therapeutic target for the treatment of NSCLC. Recently, two direct MYC inhibiting agents with improved in vivo efficacy and tolerability, MYCi975 and Omomyc, have been developed. To define new drug strategies that could potentiate the therapeutic effects of MYC inhibitors, transcriptome datasets from MYCi975 and Omomyc studies were reanalyzed. These data revealed the induction of two class IIa histone deacetylases (HDACs), HDAC5 and HDAC9, upon MYC inhibition. Importantly, class IIa HDACs are involved in cancer proliferation, aggressiveness, and prognosis. In addition, novel agents that selectively inhibit class IIa HDACs have demonstrated promising anti-tumor efficacy; thus, suggesting potential therapeutic benefits from dual targeting of MYC and class IIa HDACs. We assessed the treatment efficacy of MYCi plus class IIa HDACi across 18 NSCLC cell lines, 10 of which demonstrated substantial reduction of cell viability upon combination treatment. Querying of driver mutation spectra associated with differential drug response in our cell line panel defined that EGFR mutant cell lines exhibited resistance, while STK11 or Ras mutant cell lines were sensitive to our drug paradigm. Gene set enrichment analysis comparing the transcriptomes of sensitive versus resistant cell lines revealed that MYC Targets, Oxidative Phosphorylation, and Reactive Oxygen Species (ROS) pathways were activated in combination treatment responsive cell lines. RNA sequencing on treated cell lines identified large-scale transcriptional shifts facilitated by combination treatment, including suppression of MYC, cell cycle, and mitochondrial pathways. Furthermore, G1/S arrest and elevated mitochondrial ROS levels in combination drug-treated cells were confirmed using flow cytometry. Concordant with the role of MYC as a key cell cycle regulator, MYC protein levels were decreased in the combination treatment group as compared to the vehicle or mono-treatment groups. Importantly, both MYC overexpression and ROS scavenger treatment partially rescued the reduction of cell viability by combination treatment, suggesting both MYC depletion and ROS elevation as contributors to therapeutic efficacy driven by combination treatment. Expanding these studies to in vivo models, we discern that combination MYC and class IIa HDAC inhibition significantly reduced tumor burden in a patient-derived xenograft model of human NSCLC. In summary, we define here a novel drug paradigm combining MYC and class IIa HDAC inhibitors, which potentiates anti-tumor efficacy in NSCLC via MYC depletion and oxidative stress. Citation Format: Jina Park, Ying-Yu Chen, Jennie Cao, Michelle Vaz, Ray-Whay C. Yen, Stephen B. Baylin, Michael J. Topper. Inhibition of class IIa HDACs potentiates MYC inhibitor-driven cytotoxicity by inducing MYC depletion and oxidative stress in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6256.

The role of class IIa histone deacetylases in regulating endothelial function.

Vascular endothelial cells (ECs) are monolayer cells located in the inner layer of the blood vessel. Endothelial function is crucial in maintaining local and systemic homeostasis and is precisely regulated by sophisticated signaling pathways and epigenetic regulation. Endothelial dysfunctions are the main factors for the pathophysiological process of cardiovascular and cerebrovascular diseases like atherosclerosis, hypertension, and stroke. In these pathologic processes, histone deacetylases (HDACs) involve in epigenetic regulation by removing acetyl groups from lysine residues of histones and regulating downstream gene expression. Among all HDACs, Class IIa HDACs (HDAC4, 5, 7, 9) contain only an N-terminal regulatory domain, exert limited HDAC activity, and present tissue-specific gene regulation. Here, we discuss and summarize the current understanding of this distinct subfamily of HDACs in endothelial cell functions (such as angiogenesis and immune response) with their molecular underpinnings. Furthermore, we also present new thoughts for further investigation of HDAC inhibitors as a potential treatment in several vascular diseases.

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

Synergistic antitumor effect of histone deacetylase class IIa inhibitor with lenvatinib in hepatocellular carcinoma.

Histone deacetylase (HDAC) class I and IIa are highly expressed in hepatocellular carcinoma (HCC) and associated with decreased survival. However, clinically used pan and class I inhibitors have serious adverse events. In this study, we assessed the antitumor effects and tolerability of class IIa HDAC inhibitor (HDACI) with lenvatinib, which is a standard therapy for HCC. Combination therapy with class IIa HDACI and lenvatinib exerted synergistic antitumor effect in human HCC cell lines. In mouse models, this therapy showed significant antitumor effects, and few adverse events occurred. In immunoblotting, the expression of fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) was high in cell lines that showed a high antitumor effect. In addition, class IIa HDACI administration decreased the expression of FGFR4. In the small interfering RNA (siRNA) analysis, knockdown of HDAC9, which is an isoform of HDAC class IIa, reduced the expression of FGFR4 and induced apoptosis. Immunohistochemistry of human clinical specimens showed a positivity rate of 32% for FGFR4 and 84% for HDAC9 in HCC, and all FGFR4-positive patients were HDAC9 positive. Class IIa HDACI and lenvatinib combination therapy induces apoptosis by downregulating FGFR4 and blocking the FGFR signaling in FGFR4-positive HCC cell lines and has demonstrated synergistic antitumor effects and safety. This combination therapy overcomes the problems of conventional therapies and will be beneficial for FGFR4-positive HCC patients.

The multiple roles of salt-inducible kinases in regulating physiology.

Salt-inducible kinases (SIKs), which comprise a family of three homologous serine-threonine kinases, were first described for their role in sodium sensing but have since been shown to regulate multiple aspects of physiology. These kinases are activated or deactivated in response to extracellular signals that are cell surface receptor mediated and go on to phosphorylate multiple targets including the transcription cofactors CRTC1-3 and the class IIa histone deacetylases (HDACs). Thus, the SIK family conveys signals about the cellular environment to reprogram transcriptional and posttranscriptional processes in response. In this manner, SIKs have been shown to regulate metabolic responses to feeding/fasting, cell division and oncogenesis, inflammation, immune responses, and most recently, sleep and circadian rhythms. Sleep and circadian rhythms are master regulators of physiology and are exquisitely sensitive to regulation by environmental light and physiological signals such as the need for sleep. Salt-inducible kinases have been shown to be central to the molecular regulation of both these processes. Here, we summarize the molecular mechanisms by which SIKs control these different domains of physiology and highlight where there is mechanistic overlap with sleep/circadian rhythm control.

Histone deacetylase 5-induced deficiency of signal transducer and activator of transcription-3 acetylation contributes to spinal astrocytes degeneration in painful diabetic neuropathy.

Diabetes patients with painful diabetic neuropathy (PDN) show severe spinal atrophy, suggesting pathological changes of the spinal cord contributes to central sensitization. However, the cellular changes and underlying molecular mechanisms within the diabetic spinal cord are less clear. By using a rat model of type 1 diabetes (T1D), we noted an extensive and irreversible spinal astrocyte degeneration at an early stage of T1D, which is highly associated with the chronification of PDN. Molecularly, acetylation of astrocytic signal transducer and activator of transcription-3 (STAT3) that is essential for maintaining the homeostatic astrocytes population was significantly impaired in the T1D model, resulting in a dramatic loss of spinal astrocytes and consequently promoting pain hypersensitivity. Mechanistically, class IIa histone deacetylase, HDAC5 were aberrantly activated in spinal astrocytes of diabetic rats, which promoted STAT3 deacetylation by direct protein-protein interactions, leading to the PDN phenotypes. Restoration of STAT3 signaling or inhibition of HDAC5 rescued astrocyte deficiency and attenuated PDN in the T1D model. Our work identifies the inhibitory axis of HDAC5-STAT3 induced astrocyte deficiency as a key mechanism underlying the pathogenesis of the diabetic spinal cord that paves the way for potential therapy development for PDN.

Abstract B014: The role of HDAC-MEF2 axis in the epigenetic control of immune tumoral microenvironment

Abstract Epigenetic mechanisms have a main impact on cancer regulation and on microenvironment control. In the context of leiomyosarcoma (LMS), a highly aggressive and immunologically cold cancer, class IIa histone deacetylases (HDACs) and myocyte enhancer factor (MEF2) form a repressive complex that leads to the reorganization of epigenetic landscape. We have recently described the effect of some HDAC inhibitors (HDACi) on chromatin organization and in particular the increase of H3K27Ac genome occupancy near TSS and promoter regions, caused by NKL-54, a HDAC1/2/3 selective inhibitor, and the re-expression of some chemokines can be observed after the treatment with TMP-195, a specific class IIa HDACi. The hypothesis of this project is that the HDAC-MEF2 complex represses the transcription of some immune genes that may have a major role in antitumor response through an epigenetic control of chromatin. To verify this proposal, we knocked-down (KD) MEF2A in SK-UT-1, a high grade LMS cell line, or we treated the same cells with NKL-54 and TMP-195. We compared the impact of MEF2A KD and HDACs inhibition on chemokines and cytokines release by RNA-seq and Luminex profiling, while H3K27ac levels obtained by ChIP-seq were used to quantify enhancers and super-enhancers activation. To evaluate the impact of MEF2A on LMS cells proliferation we performed a clonogenic assay. To test the effectiveness of class IIa HDACs inhibitors in vivo, we aim to generate a syngeneic model of LMS by transforming uterine smooth muscle cells with Large T and Small T antigen, p53175H and by knocking out (KO) ATRX and PTEN. The preliminary data show that KD of MEF2A leads to an increased expression and release of some chemokines and cytokines such as CXCL5, CXCL2 and CXCL8 in SK-UT-1 cells. Class IIa HDACs inhibition further enhances this effect. Furthermore, the absence of MEF2A causes a reduction of colonies formation compared with WT cells, pointing out the fundamental role of MEF2A in LMS cell proliferation. Finally, ChIP-seq analysis of the genomic location of CXCL2 and CXCL5 in HDAC4 and HDAC9 KO shows an increased H3K27Ac signal at the level of the regulative elements of the two genes, indicating the epigenetic reprogramming acting on these loci and achieved by HDACs impairment. High grade LMS are characterized by the formation of HDAC IIa and MEF2 repressive complexes at some specific genomic loci required to sustain LMS malignancy and possibly to weaken LMS immunogenicity. The ablation or the specific inhibition of class IIa HDACs lead to the release of MEF2A from this repressive complex and the subsequent establishment of a transcriptional open chromatin environment that would promote tumor immune infiltration and clearance. These preliminary data, together with previous evidence, highlight the role of HDAC-MEF2 in the context of LMS microenvironment organization, suggesting that the targeting of these partners can boost the immune system activity, and this can be exploited for the development of LMS therapies. Citation Format: Martina Minisini, Emiliano Dalla, Vanessa Tolotto, Claudio Brancolini. The role of HDAC-MEF2 axis in the epigenetic control of immune tumoral microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B014.

Low- and high-cocaine intake affects the spatial and temporal dynamics of class IIa HDAC expression-activity in the nucleus accumbens and hippocampus of male rats as measured by [18F]TFAHA PET/CT neuroimaging

Repeated cocaine alters neuronal function in the nucleus accumbens (NAc), a brain region involved in cocaine taking, and in hippocampus (HC), known for contextual and associative learning. [18F]TFAHA is a histone deacetylase (HDAC) class IIa-specific radiotracer for positron emission tomography (PET)-imaging developed by our group to study epigenetic mechanisms. Here, [18F]TFAHA was used to conduct PET-imaging coupled with computed tomography (CT) of rat brains at baseline and after repeated cocaine intravenous self-administration (cocaine-IVSA) in low-intake versus high-intake cocaine groups. A 3 h-access FR1-schedule of cocaine-IVSA (0.5 mg/kg/infusion) for 12 continuous days was used with male Sprague Dawley rats following jugular vein catheterization. PET/CT neuroimaging with [18F]TFAHA was acquired in a dynamic mode over 40 min post-radiotracer administration at baseline and on day 12 of cocaine-IVSA using a longitudinal, repeated design. This study shows that high-cocaine intake significantly decreases class IIa HDAC expression-activity in NAc, while low-cocaine intake significantly decreases expression-activity in HC in male rats. These findings suggest the individual rats with low-cocaine intake had epigenetic changes in HC, where drug-associative changes occur. Alternatively, individuals with high-cocaine intake had robust epigenetic changes in NAc, where rewared-related behaviors originate. These findings are the first longitudinal data obtained in vivo to implicate class IIa HDACs in the persistent behavioral effects of cocaine. Furthermore, our results are consistent with published research implicating class IIa HDACs in cocaine-induced brain changes and studies suggesting a relationship between an individual's drug-taking behavior and regional pattern of epigenetic changes in the brain.

O.04 Improvement of muscle strength in a mouse model for recessive RYR1-related congenital myopathy treated with HDAC and inhibitors

Congenital myopathies are a genetically diverse group of muscle disorders characterized by severe muscle weakness leading to poor quality of life of affected individuals. In approximately 30% of the cases, patients with congenital myopathies carry either a dominant or recessive mutation in the <i>RYR1</i> gene. The presence of recessive <i>RYR1</i> mutations is accompanied by reduction of RyR1 expression and content in skeletal muscle and is associated with fibre hypotrophy and muscle weakness. In many cases muscles of patients with recessive <i>RYR1</i> mutations also exhibit increased content of class IIa histone de-acetylases (HDACs) and aberrant genomic DNA methylation. We recently developed a mouse model knocked-in for the p.Q1970fsX16+p.A4329D RyR1 mutations, isogenic to those identified in a severely affected patient suffering from a recessive form of <i>RYR1</i>-related multi-minicore disease. The phenotype of p.Q1970fsX16+p.A4329D RyR1 mice recapitulates many aspects of the clinical picture of patients carrying a hypomorphic RYR1 allele + a missense mutation in the other allele. We treated the compound heterozygous Ryr1 mutant mice with a combination of two drugs targeting DNA methyltransferases and class II histone de-acetylases for 15 weeks. Our results show that treatment of the mutant mice with drugs targeting epigenetic enzymes improves in vivo and ex vivo muscle strength, <i>RYR1</i> protein content and muscle ultrastructure. This study provides proof of concept for the pharmacological treatment of patients with congenital myopathies linked to recessive <i>RYR1</i> mutations.

HDAC7 Activates IKK/NF-κB Signaling to Regulate Astrocyte-Mediated Inflammation.

Class IIa histone deacetylases (HDAC) have been shown to drive innate immune cell-mediated inflammation in the peripheral system, but their roles in cerebral inflammatory responses remain largely unknown. Here, we elucidate that HDAC7 is selectively elevated in lipopolysaccharide (LPS)-challenged astrocytes both in vivo and in vitro. We identify that HDAC7 binds to the inhibitory kappa B kinase (IKK) to promote IKKα and IKKβ deacetylation and subsequent activation, leading to the activation of nuclear factor κB (NF-κB). Astrocyte-specific overexpression of HDAC7 results in NF-κB activation, pro-inflammatory gene upregulation and anxiety-like behaviors in mice, while downregulating HDAC7 reserves LPS-induced NF-κB activation and inflammatory responses. Furthermore, pharmacological inhibition of HDAC7 by a class IIa HDAC inhibitor attenuates LPS-induced NF-κB activation, inflammatory responses and anxiety-like behaviors both in vivo and in vitro. Together, our data reveal a novel mechanism of HDAC7 in astrocyte-mediated inflammation and suggest that targeting HDAC7 could be a potential therapeutic strategy for the treatment of anxiety and other inflammation-related diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-022-02965-6.

Class IIa histone deacetylase inhibition ameliorates acute kidney injury by suppressing renal tubular cell apoptosis and enhancing autophagy and proliferation.

Expression and function of histone deacetylases (HDACs) vary with cell types and pathological conditions. Our recent studies showed that pharmacological targeting class IIa HDACs attenuated renal fibrosis, but the effect of class IIa HDAC inhibition on acute kidney injury (AKI) remains unknown. In this study, we found that four class IIa HDACs (4, 5, 7, 9) were highly expressed in the kidney of folic acid (FA) and ischemia/reperfusion (I/R)-induced AKI in mice. Administration of TMP269, a potent and selective class IIa HDAC inhibitor, improved renal function and reduced tubular cell injury and apoptosis, with concomitant suppression of HDAC4 and elevation of acetyl-histone H3. Mechanistical studies showed that TMP269 treatment inhibited FA and I/R-induced caspase-3 cleavage, Bax expression and p53 phosphorylation. Conversely, TMP269 administration preserved expression of E-cadherin, BMP7, Klotho and Bcl-2 in injured kidneys. Moreover, TMP269 was effective in promoting cellular autophagy as indicated by increased expression of Atg7, beclin-1, and LC3II, and promoted renal tubular cell proliferation as shown by increased number of proliferating cell nuclear antigen-positive cells and expression of cyclin E. Finally, blocking class IIa HDACs inhibited FA-and I/R-induced phosphorylation of extracellular signal-regulated kinases 1 and 2, and p38, two signaling pathways associated with the pathogenesis of AKI. Collectively, these results suggest that pharmacological inhibition of class IIa HDACs protects against AKI through ameliorating apoptosis, enhancing autophagy and promoting proliferation of renal tubular cells by targeting multiple signaling pathways.

Abstract 3280: Combination of MYC and class IIa HDAC inhibition potentiates anti-tumor efficacy in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality among both men and women in the United States. Immune checkpoint blockade has emerged as a promising therapy for advanced non-small cell lung cancer (NSCLC); however, only a subset of patients responds to this treatment regimen. Therefore, combinatorial therapeutic strategies to enhance anti-tumor immunity and efficacy of immunotherapy are in urgent need. Our group and others have demonstrated the potential of MYC inhibition in NSCLC to render the tumor microenvironment (TME) more immune permissive by upregulating antigen presentation machinery and chemokines that recruit effector lymphocytes. We performed a dedicated reanalysis of published transcriptome data from two distinct cancer types, prostate and lung, which were treated with novel MYC inhibiting agents, MYCi975 and Omomyc, respectively. These data revealed augmentation of two class IIa histone deacetylases (HDACs), HDAC5 and HDAC9, upon MYC inhibition. In addition to the suggested interaction between MYC and Class IIa HDACs derived from our transcriptional analyses, class IIa HDACs have an established role in tumorigenesis, angiogenesis, and poor prognosis of cancer patients, thus implying that therapeutic benefits could be derived from dual inhibition of MYC and class IIa HDAC. Elucidation of the basal relationship between Class IIa HDACs and the immune microenvironment of NSCLC was derived through correlative analyses of TCGA Lung adenocarcinoma and squamous carcinoma RNA-seq data. These data revealed a conserved positive correlation across non-small cell lung cancer between class IIa HDAC RNA levels, Treg score, and Treg/CD8 ratio by immune deconvolution analysis. Evaluation of combinatorial treatment efficacy of MYCi plus Class IIa HDACi was undertaken using 19 NSCLC cell lines, which recapitulate the mutational and histological landscape of NSCLC. These data identified a genotype-driven drug synergy, wherein EGFR mutant cell lines presented as resistant, and Ras mutant cell lines presented as sensitive to our combination drug paradigm. In summary, we defined a positive association between Class IIa HDAC expression and Treg score in TCGA NSCLC samples, suggesting a possibility of immune-modulation via class IIa HDAC inhibition. Furthermore, we identified transcriptional augmentation of class IIa HDAC induced by MYC repression, which indicates a potential novel therapeutic opportunity. Finally, we determined that inhibition of class IIa HDACs potentiates MYC inhibitor-driven anti-tumor efficacy across a diverse set of NSCLC cell lines. Citation Format: Jina Park, Michelle Vaz, Ray-Whay C. Yen, Stephen B. Baylin, Michael J. Topper. Combination of MYC and class IIa HDAC inhibition potentiates anti-tumor efficacy in non-small cell lung cancer [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 3280.

Evidence that HDAC7 acts as an epigenetic “reader” of AR acetylation through NCoR-HDAC3 dissociation

Histone deacetylase (HDAC) proteins are epigenetic regulators that govern a wide variety of cellular events. With a role in cancer formation, HDAC inhibitors have emerged as anti-cancer therapeutics. Among the eleven metal-dependent class I, II, and IV HDAC proteins targeted by inhibitor drugs, class IIa HDAC4, -5, -7, and -9 harbor low deacetylase activity and are hypothesized to be "reader" proteins, which bind to post-translationally acetylated lysine. However, evidence linking acetyllysine binding to a downstream functional event is lacking. Here, we report for the first time that HDAC4, -5, and -7 dissociated from corepressor NCoR in the presence of an acetyllysine-containing peptide, consistent with reader function. Documenting the biological consequences of this possible reader function, mutation of a critical acetylation site regulated androgen receptor (AR) transcriptional activation function through HDAC7-NCoR-HDAC3 dissociation. The data document the first evidence consistent with epigenetic-reader functions of class IIa HDAC proteins.