pan-HDAC Inhibitor Research Articles

Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

Procainamide-SAHA Fused Inhibitors of hHDAC6 Tackle Multidrug-Resistant Malaria Parasites.

Epigenetic post-translational modifications are essential for human malaria parasite survival and progression through its life cycle. Here, we present new functionalized suberoylanilide hydroxamic acid (SAHA) derivatives that chemically combine the pan-histone deacetylase inhibitor SAHA with the DNA methyltransferase inhibitor procainamide. A three- or four-step chemical synthesis was designed starting from cheap raw materials. Compared to the single drugs, the combined molecules showed a superior activity in Plasmodium and a potent inhibition against human HDAC6, exerting no cytotoxicity in human cell lines. These new compounds are fully active in multidrug-resistant Plasmodium falciparum Cambodian isolates. They target transmission of the parasite by inducing irreversible morphological changes in gametocytes and inhibiting exflagellation. The compounds are slow-acting and have an additive antimalarial effect in combination with fast-acting epidrugs and dihydroartemisinin. The lead compound decreases parasitemia in mice in a severe malaria model. Taken together, this novel fused molecule offers an affordable alternative to current failing antimalarial therapy.

Everolimus Shows Synergistic Anti-Myeloma Effects with Bortezomib Via the AKT/mTOR Pathway

Introduction: Multiple myeloma (MM) is characterized by the proliferation of malignant plasma cells and a subsequent overabundance of monoclonal paraprotein (M protein). Everolimus ,as an oral mammalian target of rapamycin (mTOR) inhibitor, plays an important role in multiple myeloma. In the phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients ,the median time to progression was 90 days (range, 13 to 278 days),and no dose-limiting toxicity was observed at daily dose of 10 mg. At the same time, the observed responses are promising in some testing combination strategies. The pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with everolimus caused potent G0/G1 arrest and induced pronounced apoptosis, the combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. Everolimus enhances the cytotoxicity of bendamustine in multiple myeloma cells through a network of pro-apoptotic and cell-cycle-progression regulatory proteins.Down regulation of vascular endothelial cell growth factor and everolimus synergistic antitumor activity in multiple myeloma cells. Furthermore, In a phase I study of everolimus in combination with lenalidomide in relapsed or refractory multiple myeloma patients, the overall response rate was 65% and median progression-free survival was 5.5 months. Bortezomib was the first therapeutic proteasome inhibitor to be tested in humans with MM. However, the combination of bortezomib and everolimus for the treatment of MM is still less reported.Methods: To evaluate the in vitro and in vivo anti-tumor activity of the combination of everolimus with bortezomib.CCK-8 was used to explore the proliferation of MM cells. Flow cytometry was used to determine the effect on apoptosis of cells.Western blot and Immunohistochemical staining was used to determine the epression of proteins.Results: In our study, the experimental results demonstrated that everolimus combined with bortezomib suppressed cell proliferation. Treatment with 20 nM everolimus in combination with 10 nM bortezomib for 72h achieved the lowest cell survival rate in both U266 and LP-1 MM cell lines. The combination of everolimus and bortezomib was synergistic in both cell lines indicated by combination index (CI) values of <0.8.Flow cytometry was used to determine the effect on apoptosis of bortezomib in combination with everolimus. everolimus alone and bortezomib alone induced approximately 22.5% and 17.2% apoptosis, combination treatment induced28.8% apoptosis in U266 cells, and the results were 15.6%,10.0% and 16.7% in LP-1 cells. Western blot was used to explore the mechanisms. The expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated while the expression of Caspase-3was significantly up regulated in combination group than other alone group. Furthermore, Synergistic antitumor effect of everolimus and bortezomib was observed in the animal model carrying the myeloma xenograft on the 25th day after tumor implantation . The results of immunohistochemical staining further demonstrated that the expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated and the expression of Caspase-3 was significantly up regulated in combination group.Conclusion:Our results shows synergistic antitumor effects of the combination therapy of bortezomib and everolimus induced myeloma cell apoptosis in vitro and in vivo xenograft mouse model ,which mainly through the inhibition of the AKT/mTOR pathway. Our results provided provide a rationale application for the combinatorial treatment of MM with proteasome inhibitor agents and mTOR inhibitors in future clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Immunomodulatory Effects of HDACi in Combination with Checkpoint Blockade and Lenalidomide in the Immunosuppressive Multiple Myeloma Bone Marrow Microenvironment

Background: The interaction of myeloma (MM) cells with bone marrow (BM) accessory cells and/or the extracellular matrix induces genomic, epigenomic, and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most effective anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. We have recently shown that WT161, a novel small molecule inhibitor of HDAC6, alone or in combination with proteasome inhibitors induces MM cytotoxicity even in the bortezomib resistant MM cells. To target the MM BM immune suppressive microenvironment, we here have assessed the immunomodulatory effects of a novel triple-combination strategy using HDACi with checkpoint blockade and lenalidomide (Len) in MM BM ex vivo.Methods: Differential effects of Class I and Class II HDACi including pan-HDAC inhibitor Trichostatin A (TsA, 5-100nM), HDAC1 selective inhibitor Romidepsin (Rom, 1.5-15nM), HDAC3 inhibitor BG45 (10-40uM), HDAC6 inhibitor Tubastatin A HCl (TubA) and WT161 (0.5-10uM) alone or in combination with anti-PD1 (20ug/ml) and Len (0.5-1uM) were evaluated in MM-BM. HDACi direct effect on MM (MM1.S, H929, and RPMI8226) cell growth was measured by MTT. In the autologous coculture settings, BMMC from patients with newly diagnosed (ND)-MM and relapsed (R)-MM were cultured with HDACi alone or in combination with anti-PD1 and Len, with or without drug-wash-out for 1-5 days. Effector cell activation, proliferation, and cytotoxic activity was measured by flow cytometry analysis. Triple-combination effects on MDSC-mediated immune suppression was determined in cultures of autologous T cells with CD11b+CD14+HLA-DR- mMDSC, CD11b+CD14+HLA-DR-CD33+CD15+nMDSC, and CD11b+CD14+HLA-DR+APCs.Results: Healthy PBMC and MM cell line cultures demonstrated a dose-dependent toxicity and inhibition of proliferation in both CD4 and CD8 T cells induced by Rom; in contrast, TubA, WT161, and BG45 significantly induced T cell proliferation and cytotoxic activity even after wash out of HDACi. Additionally, HDAC6 inhibitors TubA and WT161 markedly increased expression of T cell exhaustion antigens TIM3 and LAG3, and these effects were partially reversed by drug washout. Moreover, triple-combination of HDACi with PD-1 blockade and Len significantly induced CD25+CD69+ activated T and NK cell mediated cytotoxicity of CD138+ MM cells. Interestingly, in the autologous co-cultures of MM-BMMC, TubA, BG45 and Rom induced CD25+CD69+ activated T cells, and the PD-1 blockade and Len further enhanced T cell activation. However, HDAC inhibitors, with the exception of HDAC3 inhibitor BG45, expanded immunosuppressive mMDSC populations within MM-BM myeloid compartment. Specifically, WT161 induced differentiation of myeloid cells by shifting CD14-HLA-DR-CD33+CD15+ nMDSCs to CD14+HLA-DR- mMDSCs and CD14+HLA-DR+APCs. Moreover, Rom significantly increased frequency of immunosuppressive IFNα producing plasmacytoid dendritic cells (pDCs), while decreasing Tregs, in MM-BM. Importantly, WT161 and TubA in combination with PD-1 blockade and Len reversed MDSC-mediated immunosuppressive activity, as measured by T cell proliferation. Finally, inhibition of HDAC6 induced MM cell cytotoxicity, and addition of PD-1 blockade and Len further enhanced TubA- and WT161 induced anti-MM immune responses.Conclusion: These data suggest that distinct classes of HDACi may induce differential immunomodulatory effects in the MM BM. Inhibition of HDAC6 induces changes in immune suppressor cells leading to enhanced anti-MM immune response, and addition of PD-1 blockade with Len further enhances HDAC6 inhibition-induced effector cell mediated anti-MM immune response. Ongoing analysis of triple combination of HDAC6 inhibition with checkpoint blockade and lenalidomide in the MM BM microenvironment will further define the role of HDAC6 in disease pathogenesis, and suggest novel immune-based epigenetic and targeted combination therapies for MM. DisclosuresHofmeister:Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Bradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees. Gorgun:Bristol Mayer Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding.

Selective Inhibition of HDAC1 and HDAC3 Inhibits Survival of Diffuse Large Cell Lymphoma Via Epigenetic Modulation of Death Associated Protein (DAPK)

Histone deacetylases (HDACs) are key players of epigenetic regulation of gene expression and are therefore attractive targets for cancer therapy. Many epigenetic drugs including HDAC inhibitors (HDACi) have shown anti-tumor activity in several types of cancers. Recently SAHA and belinostat Pan-HDACi have been FDA-approved for the treatment of T cell lymphoma. However, the capacity of Pan-HDACi for distinguishing different classes and isoforms of HDACs is limited, and may produce profound side effects. Therefore, it is important to identify cancer-relevant HDACs to promote selective HDAC inhibition for therapy in other malignancies such as diffuse large B cell lymphoma (DLBCL), the most aggressive non-Hodgkin lymphoma. Recent studies have shown that HDAC1 (HDACs class I) are commonly overexpressed in multiple cancer types and contribute to poor overall survival. However, the role of HDAC3 is not well known. The goal of this study was to determine the mechanistic role of HDAC3 and HDAC1 in DLBCL.Immunohistochemistry performed on DLBCL tumors (n=29) demonstrate 42% positivity for HDAC3 and 100% positivity for HDAC1. Ectopically expressed HDAC3 led to increased thymidine uptake, whereas HDAC3 knockdown using siRNA led to a significant decrease in thymidine uptake (p<0.001) suggesting that HDAC3 could be necessary for DLBCL cell growth. Moreover, inhibition of HDAC3 using a selective pharmacological inhibitor RGFP966 suppressed cell proliferation in a dose dependent manner in several DLBCL cell lines (SUDHL6, OCILY1, OCILy10 and OCILy3). However, the effect of RGFP966 on cell survival as determined by annexin V/ propidium iodide was underwhelming. We sought to target both HDAC1 and HDAC3 using the pharmacological inhibitor RG283 and assessed biological effect of dual inhibition. Interestingly, dual inhibition of HDAC1 and HDAC3 with RG2833 significantly suppressed both cell proliferation (<0.0001) and survival (p<0.001) in most of the DLBCL cell lines tested. As expected, treatment with RG2833 increased acetylation of H3K9, H3K14, H3K27 and H4K5 histone marks and led to P21 upregulation in DLBCL cells. Interestingly, exogenously overexpressed HDAC3 but not HDAC1 increased sensitivity of DLBCL cells to Pan-HDAC inhibitors vorinostat and belinostat, which suggest that endogenous HDAC3 in DLBCL cells may exist in a protein complex that may evade targeting by Pan-HDACi. In order to identify a potential mechanism of action for dual HDAC3 and HDAC1 inhibition, we have focused on tumor suppressor gene DAPK (Death associated protein), which has been previously shown to be silenced in DLBCL patients via DNA methylation. DAPK is a serine/threonine kinase and possesses pro-apoptotic and tumor-suppressive functions. We have confirmed by RT-PCR that DAPK was suppressed/silenced in DLBCL cell lines (n=8) as compared to CD19+ (n=3) normal B cells. HDAC inhibition alone or in combination with DNA methyltransferase inhibitors (DNMTi) can induce apoptosis by modulating expression of tumor suppressor genes. We next asked whether HDAC3 and HDAC1 dual inhibition through RG2833 alone or its combination with DNMTi, azacytidine (AZA) will be able to restore expression of DAPK in DLBCL cells. Quantitative RT-PCR analysis in OCILy3 and OCILY10 cells demonstrated that treatment with RG2833 alone modestly increased DAPK expression; however, co-treatment with RG283 and AZA led to marked increase in DAPK expression. As expected, the combination of RG2833 and Aza had significantly greater pro-apoptotic effect in DLBCL cells than either drug alone suggesting that DAPK expression may be an important determinant of response to HDAC1/HDAC3 dual inhibitor in DLBCL cells.In summary, our study provides first evidence of antitumor activity of dual HDAC3 and HDAC1 targeted agent therapy in DLBCL patients. Moreover, we have identified epigenetic modulation of DAPK as potential modulator of cellular response to HDAC1/HDAC3 dual inhibition. This study suggests for further clinical trials and/or development of selective inhibitors of HDAC3 or HDAC1/HDAC3 in DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Blocking HDAC3 in Bone Marrow Stromal Cells Has Direct Anti-Multiple Myeloma Effect and Modulates T Cell Function

IntroductionHistone deacetylases (HDAC) are therapeutic targets in Multiple Myeloma (MM) and the pan-HDAC inhibitor Panobinostat is FDA approved. However, clinical trials showed that non-selective HDAC inhibitors have a narrow therapeutic index, prompting the development of isoform-specific HDAC inhibitors such as the HDAC6 inhibitor Ricolinostat. Preclinical studies have shown that HDAC3 downregulation/inhibition exerts an anti-myeloma effect, suggesting that HDAC3 may be a novel molecular target in MM. However, the effect of targeting HDAC3 in the bone marrow (BM) microenvironment has not been investigated. The BM microenvironment, particularly stromal and immune cells, have been shown to be key in supporting MM growth, immune escape, and resistance to therapy. We sought to study whether targeting HDAC3 in BM stroma cells (BMSC) has direct cytotoxic effects against stromal cells and/or impacts MM cell and T cell function through secreted factors and/or cell-to-cell contact mechanisms.MethodsWe targeted HDAC3 in human BMSC line HS-5 and primary-derived BMSC from MM patients (MM-BMSC) via siRNA or via the HDAC3-selective inhibitor BG45. MTT assay and flow cytometry with annexin V/PI staining were used to assess viability and apoptosis post-HDAC3 KD/pharmacologic inhibition. Downregulation of HDAC3 was confirmed by immunoblotting. To assess the effect of HDAC3 silencing in MM-BMSC on MM cells, we performed siRNA-mediated HDAC3 KD in HS-5 and MM-BMSC, removed transfection media after 48 hours, and performed co-culture experiments with MM1S.Luc and H929.Luc MM cell lines. After 96 hours MM cell proliferation was measured using the Luciferase Assay. Scrambled siRNA transfected HS-5 and MM-BMSC were used as control. To assess the effect of HDAC3 silencing in BMSC on T cells, we isolated peripheral blood mononuclear cells from healthy donors by Ficoll density gradient separation. CD3+ T cells were isolated using EasySep™ Human T Cell Isolation Kit and activated using Dynabeads Human T-Activator CD3/CD28. Cytokine array profiling was performed on the cell-culture supernatant using the Raybiotech C-series Human Cytokine Array according to manufacturer's protocol.ResultsFirst, we showed that pharmacological inhibition of HDAC3 via BG45 does not have direct anti-proliferative or cytotoxic effect on the HS-5 stromal cell line at doses up to 5-fold higher than the EC50 of MM cell lines. Consistent with this data, siRNA-mediated HDAC3 KD does not have direct anti-proliferative and/or cytotoxic effects on HS-5 or MM-BMSC. However, when MM cell lines were co-cultured with HDAC3-silenced HS-5 or MM-BMSC, there was a statistically significant decrease in BMSC-induced MM proliferation compared to control (HS-5: 38% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.1-27.5% decrease in MM proliferation in HDAC3 KD, p < 0.05). To assess whether cell-to-cell contact is necessary to mediate this growth arrest, we cultured MM cell lines in conditioned media from HDAC3 or scrambled siRNA transfected BMSC and discovered that the former was sufficient to induce growth/proliferation arrest (HS-5: 31.5% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.3% decrease in MM proliferation in HDAC3 KD, p < 0.05), suggesting a paracrine effect.We next showed that conditioned supernatant from HDAC3-KD HS-5, cultured alone or in combination with MM cell lines, significantly inhibited the proliferation of activated T-cells (HS-5 supernatant: 47.8% decrease in MM proliferation in HDAC3 KD; HS-5 + MM co-culture supernatant: 32.6% decrease in MM proliferation in HDAC3 KD). To identify the soluble factors responsible for this paracrine effect, we subjected HDAC3-KD and scrambled-transfected HS-5 supernatant to cytokine profiling. We identified a number of cytokines modulated by HDAC3 KD, which we are currently analyzing. Among these, we identified a 1.37-fold increase in soluble PD-L1 when HDAC3 was silenced in HS-5 cells.ConclusionsOur results show that inhibiting HDAC3 in BMSC mediates anti-MM and anti-T cell effect via soluble factors. Gene expression profiling and proteomic studies are currently underway to define the molecular mechanisms supporting these effects. Our preliminary data suggest that targeting HDAC3 in BMSC may alter the checkpoint repertoire in the BM milieu, suggesting potential utility of therapeutic strategies combining HDAC3 and checkpoint inhibitors. DisclosuresHideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Mazitschek:Acetylon: Equity Ownership. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

The Crucial Role of ROS-HO-1 Dependent Apoptosis Induced By Pan-HDACi in T-Cell Acute Lymphoblastic Leukemia

Histone hypoacetylation occurs in many hematological malignances and histone deacetylase inhibitors (HDACi) is a promising approach to treat hematological malignances. The purpose of our study was to further investigate the mechanisms and efficiency of pan-HDAC inhibition (AR-42) in primary human TALL cells and T-ALL cell line. Our previous study demonstrated significantly increased mRNA and protein expression of HDAC1 HDAC2 HDAC3 HDAC7 and HO-1 in T-ALL patients(p <0.01); the expression of HDAC1and HDAC2 and HO-1 were associated with the risk stratification. Furthermore HO-1 overexpression was mediated by constitutively activated NF-κB, which were confirmed by EMSA, immunofluorescence and immunoblotting. In present study, primary cell of 25 T-ALL patients and Jurkat were treated with AR-42, and cell viability, apoptosis, cell cycle, mitochondrial membrane potential(MMP) and effects on signaling pathways were examined. Our data showed AR-42 was cytotoxic to T-ALL cells with IC50 values of 0.325±0.082 and 0.296±0.064μM for T-ALL primary cell and Jurkat; AR-42 arrested Jurkat cells at G0/G1 phase with the increasing expression of p21; AR-42 induced apoptosis with cleavage of caspases3,8 and 9, and cell apoptosis was largely prevented by caspase inhibition (Z-VAD-FMK). AR-42 induced apoptosis through an increase in the ROS production and the disruption of mitochondrial membrane potential, and cell apoptosis induced by AR-42 was significantly diminished by ROS scavengers NAC (N-acetylcysteine). AR-42 downregulated the expression of HO-1 and inhibited activation of NF-κB. Overexpression of HO-1 by a lentivirus construct partly prevent apoptosis induced by AR-42. Together, these data demonstrate that AR-42 can induce caspase-dependent apoptosis mainly through ROS-(NF-κB)- (HO-1) pathway in T-cell acute lymphoblastic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Panobinostat penetrates the blood-brain barrier and achieves effective brain concentrations in a murine model.

Panobinostat, an orally bioavailable pan-HDAC inhibitor, has demonstrated potent activity in multiple malignancies, including pediatric brain tumors such as DIPG, with increased activity against H3K27M mutant cell lines. Given limited evidence regarding the CNS penetration of panobinostat, we sought to characterize its BBB penetration in a murine model. Panobinostat 15mg/kg was administered IV to 12 CD-1 female mice. At specified time points, mice were euthanized, blood samples were collected, and brains were removed. LC-MS was performed to quantify panobinostat concentrations. Cmax and AUC were estimated and correlated with previously published pharmacokinetic analyses and reports of IC-50 values in DIPG cell lines. Mean panobinostat plasma concentrations (ng/mL) were 27.3 ± 2.5 at 1h, 7.56 ± 1.8 at 2h, 1.48 ± 0.56 at 4h, and 2.33 ± 1.18 at 7h. Mean panobinostat brain concentrations (ng/g) were 60.5 ± 6.1 at 1h, 42.9 ± 5.4 at 2h, 33.2 ± 6.1 at 4h, and 28.1 ± 4.3 at 7h. Brain-to-plasma ratio at 1h was 2.22 and the brain to plasma AUC ratio was 2.63. Based on the published human pharmacokinetic data, the anticipated Cmax in humans is expected to be significantly higher than the IC-50 identified in DIPG models. It is expected that panobinostat would be effective in CNS tumors where the IC-50 is in the low nanomolar range. Thus, our data demonstrate panobinostat crosses the BBB and achieves concentrations above the IC-50 for DIPG and other brain tumors and should be explored further for clinical efficacy.

Treatment with Histone Deacetylase Inhibitor Attenuates Peripheral Inflammation-Induced Cognitive Dysfunction and Microglial Activation: The Effect of SAHA as a Peripheral HDAC Inhibitor.

It has been demonstrated that peripheral inflammation induces cognitive dysfunction. Several histone deacetylase (HDAC) inhibitors ameliorate cognitive dysfunction in animal models of not only peripheral inflammation but also Alzheimer's disease. However, it is not clear which HDAC expressed in the central nervous system or peripheral tissues is involved in the therapeutic effect of HDAC inhibition on cognitive dysfunction. Hence, the present study investigated the effect of peripheral HDAC inhibition on peripheral inflammation-induced cognitive dysfunction. Suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor that is mainly distributed in peripheral tissues after intraperitoneal administration, was found to prevent peripheral inflammation-induced cognitive dysfunction. Moreover, pretreatment with SAHA dramatically increased mRNA expression of interleukin-10, an anti-inflammatory cytokine, in peripheral and central tissues and attenuated peripheral inflammation-induced microglial activation in the CA3 region of the hippocampus. Minocycline, a macrophage/microglia inhibitor, also ameliorated cognitive dysfunction. Furthermore, as a result of treatment with liposomal clodronate, depletion of peripheral macrophages partially ameliorated the peripheral inflammation-evoked cognitive dysfunction. Taken together, these findings demonstrate that inhibition of peripheral HDAC plays a critical role in preventing cognitive dysfunction induced by peripheral inflammation via the regulation of anti-inflammatory cytokine production and the inhibition of microglial functions in the hippocampus. Thus, these findings could provide support for inhibition of peripheral HDAC as a novel therapeutic strategy for inflammation-induced cognitive dysfunction.

EMBR-28. CNS DISTRIBUTIONAL KINETICS OF PANOBINOSTAT IN THE MOUSE

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and has the potential for leptomeningeal spread, often responsible for the preponderance of morbidity and mortality in MB patients. Adverse sequelae following high dose craniospinal radiation and aggressive chemotherapies, including neurocognitive deficits and endocrine disorders, impairs patient quality of life and thus call for new therapeutic strategies. Panobinostat is a pan-histone deacetylase (HDAC) inhibitor that has been shown to be effective against MBs in vitro. The objective of the current study is to evaluate the key pharmacokinetic (PK) parameters and the mechanisms influencing the brain penetration of panobinostat in the mouse model, often used in MB preclinical efficacy trials. Pharmacokinetic studies were conducted using FVB wild-type (WT) mice and triple-knockout (TKO, Mdr1a/b–/–Bcrp1–/–) transporter-deficient mice. Panobinostat was administered intravenously (10 mg/kg) and concentrations at selected time points in plasma, brain and spinal cord were determined using LC-MS/MS. PK parameters, including systemic clearances, volumes of distribution, half-lives, and areas-under-the-concentration time curve (AUC) were calculated by using non-compartmental analysis. In WT mice, the CNS penetration was initially limited; however, it increased with time and the brain-to-plasma (B/P) and spinal cord-to-plasma (SC/P) distributional partition coefficients (AUC ratios) were 3.7 and 1.4, respectively. The B/P and SC/P ratios of panobinostat in BBB transporter-deficient TKO mice were 1.7- and 2.3-fold higher than those in WT mice, respectively. Our data suggest that P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp) efflux transporters, localized in BBB, may influence the delivery of panobinostat to tumors in the brain following systemic intravenous administration. These systemic and CNS distributional parameters will inform future preclinical efficacy studies of panobinostat for MB. As such, studies investigating other administration routes that can bypass the BBB, such as the intrathecal injection of novel formulations, are ongoing.

HGG-30. BRAIN PENETRANT HDAC INHIBITOR RG2833 SYNERGIZES WITH LOMUSTINE AND RADIATION TO INDUCE DIPG CELL DEATH

Diffuse intrinsic pontine glioma (DIPG) is driven by epigenetic dysregulation. The pan-HDAC inhibitor panobinostat showed pre-clinical efficacy against DIPG, but was limited by toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. RG2833 reverses temozolomide-resistance in glioblastoma through downregulation of the NFĸB pathway. As this pathway is upregulated in DIPG and may contribute to tumorigenesis, we hypothesized that RG2833 would kill DIPG cells. We found that RG2833 treatment inhibits cell growth in the 4 to 9μM range in both autopsy and biopsy-derived DIPG cell lines (MTS assay for HSJD007 p=0.0004, JHH-DIPG1 p=0.001, SF-7761 p=0.04, SU-DIPG13 p=0.01 by t-test). Western blot confirmed on target activity by increased histone 3 acetylation at IC50 doses. RG2833 induces apoptosis (cPARP Western blot, cleaved caspase 3 immunofluorescence HSJD007 p<0.003, JHH-DIPG1 p=0.0026 by t-test) and slows cell proliferation (phospho-Rb Western blot, BrdU immunofluorescence HSJD007 p=0.008, JHH-DIPG1 p=0.0002 by t-test) in multiple DIPG cell lines. RG2833 disrupts the NFĸB pathway through acetylation of p65, resulting in decreased expression of NFĸB regulated pro-survival genes (Western blot for BCL2, BCL-xL, and XIAP). In a DIPG flank tumor mouse model, treatment with RG2833 alone for 1 week suppresses flank tumor growth (p< 0.005 by t-test) and induces apoptosis (Western blot for cPARP). We next assessed whether RG2833 combines synergistically with conventional therapies. We found that RG2833 has strong synergism with both lomustine and radiation to slow DIPG cell growth in vitro (ZIP synergy scores by SynergyFinder for RG2833+lomustine in JHH-DIPG1 17.8 and HSJD007 17.7, RG2833+radiation in JHH-DIPG1 9.7 and JHH-DIPG16A 10.9). Evaluation of combination treatment for apoptotic effects in vitro and in vivo are ongoing. This data identifies selective HDAC inhibitor RG2833 as a promising therapy for DIPG that combines synergistically with conventional therapies.

Repurposing Vorinostat for the Treatment of Disorders Affecting Brain.

Based on the findings in recent years, we summarize the therapeutic potential of vorinostat (VOR), the first approved histone deacetylase (HDAC) inhibitor, in disorders of brain, and strategies to improve drug efficacy and reduce side effects. Scientific evidences provide a strong case for the therapeutic utility of VOR in various disorders affecting brain, including stroke, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, X-linked adrenoleukodystrophy, epilepsy, Niemann-Pick type C disease, and neuropsychiatric disorders. Further elucidation of the neuroprotective and neurorestorative properties of VOR using proper clinical study designs could provide momentum towards its clinical application. To improve the therapeutic prospect, concerns on systemic toxicity and off-target actions need to be addressed along with the improvement in formulation and delivery aspects, especially with respect to solubility, permeability, and pharmacokinetic properties. Newer approaches in this regard include poly(ethylene glycol)-b-poly(DL-lactic acid) micelles, VOR-pluronic F127 micelles, encapsulation of iron complexes of VOR into PEGylated liposomes, human serum albumin bound VOR nanomedicine, magnetically guided layer-by-layer assembled nanocarriers, as well as convection-enhanced delivery. Even though targeting specific class or isoform of HDAC is projected as advantageous over pan-HDAC inhibitor like VOR, in terms of adverse effects and efficacy, till clinical validation, the idea is debated. As the VOR treatment-related adverse changes are mostly found reversible, further optimization of the therapeutic strategies with respect to dose, dosage regimen, and formulations of VOR could propel its clinical prospects.

Down‐regulation of Ca 2+ ‐Activated K + Channel K Ca 3.1 in mouse preosteoblast cells treated with vitamin D receptor agonists

Background Bone tissue is a dynamic and living organ, constantly renewed through the process of bone remodeling. The intricate balance between bone-forming and bone-resorbing activity is vital for maintenance of bone homeostasis. Vitamin D (VD) plays pivotal roles in calcium homeostasis, mineral metabolism, and bone development indirectly via control calcium absorption in the intestine and reabsorption in the kidney. However, several in vitro studies showed that VD can directly suppress the cell proliferation of mouse osteoblasts. The direct effect of VD receptor (VDR) in osteoblasts is poorly understood. The intermediate-conductance Ca2+-activated K+ channel KCa3.1 regulates intracellular Ca2+ signaling and is associated with cell proliferation in various non-excitable cells including preosteoblasts. In this study, we examined the effects of treatments with VDR agonists on the expression and activity of KCa3.1 in mouse preosteoblast MC3T3-E1 cells. Methods Intracellular Ca2+ changes were measured in MC3T3-E1 cells by Ca2+ indicator, fura-2/AM. Cell viability was measured by a WST-1 assay. MC3T3-E1 cells were transiently transfected with HDAC2, Fra-1, and VDR Silencer Select pre-designed siRNAs using a Lipofectamine RNAiMAX reagent. The Silencer Select negative control No.1 siRNA was used as a control. Results Treatments with calcitriol, a VDR agonist, for 48 h markedly decreased the expression levels of KCa3.1 transcripts and proteins in MC3T3-E1 cells, resulting in the significant inhibition of Ca2+ rises induced by DCEBIO, a specific KCa3.1 activator. Treatments with calcitriol also significantly decreased the expression of several transcriptional regulators of KCa3.1 such as histone deacetylase 2 (HDAC2) and Fra-1 composed of activation protein 1. In addition, the pharmacological blockade of HDAC with vorinostat, a pan-HDAC inhibitor, significantly decreased the expression of KCa3.1. The knockdown of Fra-1 using siRNA decreased the expression of KCa3.1 mRNA without the suppression of HDAC2 mRNA. The knockdown of VDR also recovered the expression of KCa3.1, HDAC2 and Fra-1 in calcitriol-treated MC3T3-E1 cells. Conclusion Our results suggest that KCa3.1 is a new downstream target of VDR signaling and the down-regulation of KCa3.1 through the transcriptional repression of KCa3.1 contribute, at least partly, to the antiproliferative effects of VDR agonists in mouse pre-osteoblasts.

A novel dual HDAC and HSP90 inhibitor, MPT0G449, downregulates oncogenic pathways in human acute leukemia in vitro and in vivo

Acute leukemia is a highly heterogeneous disease; therefore, combination therapy is commonly used for patient treatment. Drug–drug interaction is a major concern of combined therapy; hence, dual/multi-target inhibitors have become a dominant approach for cancer drug development. HDACs and HSP90 are involved in the activation of various oncogenic signaling pathways, including PI3K/AKT/mTOR, JAK/STAT, and RAF/MEK/ERK, which are also highly enriched in acute leukemia gene expression profiles. Therefore, we suggest that dual HDAC and HSP90 inhibitors could represent a novel therapeutic approach for acute leukemia. MPT0G449 is a dual effect inhibitor, and it showed cytotoxic effectiveness in acute leukemia cells. Molecular docking analysis indicated that MPT0G449 possessed dual HDAC and HSP90 inhibitory abilities. Furthermore, MPT0G449 induced G2 arrest and caspase-mediated cell apoptosis in acute leukemia cells. The oncogenic signaling molecules AKT, mTOR, STAT3, STAT5, MEK, and ERK were significantly downregulated after MPT0G449 treatment in HL-60 and MOLT-4 cells. In vivo xenograft models confirmed the antitumor activity and showed the upregulation of acetyl-histone H3 and HSP70, biomarkers of pan-HDAC and HSP90 inhibition, with MPT0G449 treatment. These findings suggest that the dual inhibition of HDAC and HSP90 can suppress the expression of oncogenic pathways in acute leukemia, and MPT0G449 represents a novel therapeutic for anticancer treatment.

468 Is topical remetinostat gel an effective and safe treatment for basal cell carcinoma? Results of a phase 2, open label, single arm trial

Surgical excision remains the gold standard treatment for basal cell carcinoma (BCC). However, it is associated with potential morbidity, particularly for patients with multiple or recurrent tumors. To identify novel treatment option for BCC, we previously used molecular data from human BCCs to conduct a drug repositioning screen. Our results identified histone deacetylase (HDAC) inhibitors as the leading predicted therapeutic, strongly opposing the BCC gene-expression signature. We conducted a phase 2, open label, single arm and institution trial of the topical pan-HDAC inhibitor, remetinostat, as the first proof-of-principle study of this medication class for BCC. Participants with at least one BCC referred for excision at Stanford were recruited and instructed to apply 1% remetinostat gel three times daily for six weeks. Measurements of tumor diameter and photography were conducted at baseline and week 8, and remaining tumor was then excised for histological examination, with a subset also sent for immunohistochemistry (IHC). 25 participants with 33 BCCs were included in the per-protocol analysis. The objective response rate (the proportion of tumors achieving at least a 30% decrease in the longest diameter from baseline to week 8) was 69.7% (90% confidence interval 54-82.5%), and 54.8% of the tumors demonstrated complete resolution on histological examination. IHC staining supported the pharmacodynamic effect of the remetinostat, demonstrating increased acetylated histone H3 in the excised biopsies compared to baseline. No systemic adverse events were reported, and the medication was well tolerated. Remetinostat, a topical pan-HDAC inhibitor, is a safe and effective topical treatment for BCC, reducing disease burden in a clinically significant manner. Our results provide the first in-human validation of HDAC inhibitors as a potential therapeutic.

A Pan-Histone Deacetylase Inhibitor Enhances the Antitumor Activity of B7-H3-Specific CAR T Cells in Solid Tumors.

The limited efficacy of chimeric antigen receptor (CAR) T-cell therapies with solid malignancies prompted us to test whether epigenetic therapy could enhance the antitumor activity of B7-H3.CAR T cells with several solid cancer types. We evaluated B7-H3 expression in many human solid cancer and normal tissue samples. The efficacy of the combinatorial therapy with B7-H3.CAR T cells and the deacetylase inhibitor SAHA with several solid cancer types and the potential underlying mechanisms were characterized with in vitro and ex vivo experiments. B7-H3 is expressed in most of the human solid tumor samples tested, but exhibits a restricted expression in normal tissues. B7-H3.CAR T cells selectively killed B7-H3 expressing human cancer cell lines in vitro. A low dose of SAHA upregulated B7-H3 expression in several types of solid cancer cells at the transcriptional level and B7-H3.CAR expression on human transgenic T-cell membrane. In contrast, the expression of immunosuppressive molecules, such as CTLA-4 and TET2, by T cells was downregulated upon SAHA treatment. A low dose of SAHA significantly enhanced the antitumor activity of B7-H3.CAR T cells with solid cancers in vitro and ex vivo, including orthotopic patient-derived xenograft and metastatic models treated with autologous CAR T-cell infusions. Our results show that our novel strategy which combines SAHA and B7-H3.CAR T cells enhances their therapeutic efficacy with solid cancers and justify its translation to a clinical setting.

Development and Validation of High-Content Analysis for Screening HDAC6-Selective Inhibitors.

Throughout recent decades, histone deacetylase (HDAC) inhibitors have shown encouraging potential in cancer treatment, and several pan-HDAC inhibitors have been approved for treating malignant cancers. Numerous adverse effects of pan-HDAC inhibitors have been reported, however, during preclinical and clinical evaluations. To avoid undesirable responses, an increasing number of investigations are focusing on the development of isotype-selective HDAC inhibitors. In this study, we present an effective and quantitative cellular assay using high-content analysis (HCA) to determine compounds' inhibition of the activity of HDAC6 and Class I HDAC isoforms, by detecting the acetylation of their corresponding substrates (i.e., α-tubulin and histone H3). Several conditions that are critical for HCA assays, such as cell seeding number, fixation and permeabilization reagent, and antibody dilution, have been fully validated in this study. We used selective HDAC6 inhibitors and inhibitors targeting different HDAC isoforms to optimize and validate the capability of the HCA assay. The results indicated that the HCA assay is a robust assay for quantifying compounds' selectivity of HDAC6 and Class I HDAC isoforms in cells. Moreover, we screened a panel of compounds for HDAC6 selectivity using this HCA assay, which provided valuable information for the structure-activity relationship (SAR). In summary, our results suggest that the HCA assay is a powerful tool for screening selective HDAC6 inhibitors.

Downregulation of Mcl-1 by Panobinostat Potentiates Proton Beam Therapy in Hepatocellular Carcinoma Cells.

Epigenetic modulation by histone deacetylase (HDAC) inhibitors is an attractive anti-cancer strategy for diverse hematological and solid cancers. Herein, we explored the relative effectiveness of the pan-HDAC inhibitor panobinostat in combination with proton over X-ray irradiation in HCC cells. Clonogenic survival assays revealed that radiosensitization of Huh7 and Hep3B cells by panobinostat was more evident when combined with protons than X-rays. Panobinostat increased G2/M arrest and production of intracellular reactive oxygen species, which was further enhanced by proton irradiation. Immunofluorescence staining of γH2AX showed that panobinostat enhanced proton-induced DNA damage. Panobinostat dose-dependently decreased expression of an anti-apoptotic protein, Mcl-1, concomitant with increasing acetylation of histone H4. The combination of panobinostat with proton irradiation enhanced apoptotic cell death to a greater extent than that with X-ray irradiation. Depletion of Mcl-1 by RNA interference enhanced proton-induced apoptosis and proton radiosensitization, suggesting a potential role of Mcl-1 in determining proton sensitivity. Together, our findings suggest that panobinostat may be a promising combination agent for proton beam therapy in HCC treatment.

The Effect of Histone Deacetylase Inhibitors Panobinostat or Entinostat on Motor Recovery in Mice After Ischemic Stroke.

Using rigorous and clinically relevant experimental design and analysis standards, in this study, we investigated the potential of histone deacetylase (HDAC) inhibitors panobinostat and entinostat to enhance recovery of motor function after photothrombotic stroke in male mice. Panobinostat, a pan-HDAC inhibitor, is a FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor in late stage of clinical investigation. The drugs were administered every other day (panobinostat-3 or 10mg/kg; entinostat-1.7 or 5mg/kg) starting from day 5 to 15 after stroke. To imitate the current standard of care in stroke survivors, i.e., physical rehabilitation, the animals run on wheels (2h daily) from post-stroke day 9 to 41. The predetermined primary end point was motor recovery measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. In addition, we evaluated the running distance and speed throughout the study, and the number of parvalbumin-positive neurons in medial agranular cortex (AGm) and infarct volumes at the end of the study. Both sensorimotor tests revealed that combination of physical exercise with either drug did not substantially affect motor recovery in mice after stroke. This was accompanied by negligible changes of parvalbumin-positive neurons recorded in AGm and comparable infarct volumes among experimental groups, while dose-dependent increase in acetylated histone 3 was observed in peri-infarct cortex of drug-treated animals. Our observations suggest that add-on panobinostat or entinostat therapy coupled with limited physical rehabilitation is unlikely to offer therapeutic modality for stroke survivors who have motor dysfunction.

Abrogation of histone deacetylases (HDACs) decreases survival of chronic myeloid leukemia cells: New insight into attenuating effects of the PI3K/c-Myc axis on panobinostat cytotoxicity.

Although the identification of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of many cancer types including chronic myeloid leukemia (CML), still adjustment of neoplastic cells to cytotoxic effects of anticancer drugs is a serious challenge. In the area of drug resistance, epigenetic alterations are at the center of attention and the present study aimed to evaluate whether blockage of epigenetics mechanisms using a pan-histone deacetylase (HDAC) inhibitor induces cell death in CML-derived K562 cells. We found that the abrogation of HDACs using panobinostat resulted in a reduction in survival of the K562 cell line through p27-mediated cell cycle arrest. Noteworthy, the results of the synergistic experiments revealed that HDAC suppression could be recruited as a way to potentiate cytotoxicity of Imatinib and to enhance the therapeutic efficacy of CML. Here, we proposed for the first time that the inhibitory effect of panobinostat was overshadowed, at least partially, through the aberrant activation of the phosphoinositide 3-kinase (PI3K)/c-Myc axis. Meanwhile, we found that upon blockage of autophagy and the proteasome pathway, as the main axis involved in the activation of autophagy, the anti-leukemic property of the HDAC inhibitor was potentiated. Taken together, our study suggests the beneficial application of HDAC inhibition in the treatment strategies of CML; however, further in vivo studies are needed to determine the efficacy of this inhibitor, either as a single agent or in combination with small molecule inhibitors of PI3K and/or c-Myc in this malignancy.