ALBERT

Description: High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p

Description: Induction of EBV lytic-phase gene expression, combined with exposure to an antiherpes viral drug, represents a promising targeted therapeutic approach to EBV-associated lymphomas. Short-chain fatty acids or certain chemotherapeutics have been used to induce EBV lytic-phase gene expression in cultured cells and mouse models, but these studies generally have not translated into clinical application. The recent success of a clinical trial with the pan-histone deacetylase (pan-HDAC) inhibitor arginine butyrate and the antiherpes viral drug ganciclovir in the treatment of EBV lymphomas prompted us to investigate the potential of several HDAC inhibitors, including some new, highly potent compounds, to sensitize EBV+ human lymphoma cells to antiviral agents in vitro. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids (oxamflatin, Scriptaid, suberoyl anilide hydroxamic acid, panobinostat [LBH589], and belinostat [PXD101]); the benzamide MS275; the cyclic tetrapeptide apicidin; and the recently discovered HDAC inhibitor largazole. With the exception of suberoyl anilide hydroxamic acid and PXD101, all of the other HDAC inhibitors effectively sensitized EBV+ lymphoma cells to ganciclovir. LBH589, MS275, and largazole were effective at nanomolar concentrations and were 104 to 105 times more potent than butyrate. The effectiveness and potency of these HDAC inhibitors make them potentially applicable as sensitizers to antivirals for the treatment of EBV-associated lymphomas.

Description: Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction. Disclosures Faller: Phoenicia BioSciences: Employment, Membership on an entity's Board of Directors or advisory committees. Perrine:Phoenicia BioSciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1831 Strong epidemiological association of Epstein-Barr Virus (EBV) with various human lymphoid malignancies and in vitro studies demonstrating tumorigenic activity of many EBV latent gene products suggest a causal relationship between EBV and these diseases. However, as EBV maintains a latent state of infection in these lymphomas, typical anti-herpesviral drugs, such as the nucleoside analogs ganciclovir (GCV) or acyclovir, are ineffective as these pro-drugs require expression of a lytic phase EBV protein, thymidine kinase (TK), for their activity. Therefore, selective induction of EBV lytic-phase gene expression in lymphoma cells that harbor latent EBV, coupled with simultaneous exposure to anti-herpesviral drugs, has been advanced as promising targeted therapy, because of resulting targeting of cytotoxicity to the EBV-infected tumor cells. A variety of agents including short-chain fatty acids and chemotherapeutic drugs, have been used to induce EBV lytic-phase infection in cultured cells, but these in vitro studies have generally not translated into clinical application. We have successfully used arginine butyrate and GCV to treat EBV-positive lymphoid malignancies in a recent Phase I/II clinical trial. In this study of 15 patients with relapsed or refractory EBV-positive lymphoid tumors, 4 patients achieved complete tumor remissions and 6 patients partial tumor remissions. However, the rapid metabolism of butyrate requires continuous IV administration of high doses. Butyrate has pan-HDAC inhibitory activity, and we have established that this activity is responsible for the induction of the EBV-TK protein. In recent years, several potent HDAC inhibitors (HDACi) have been tested in the clinic as anti-cancer agents. In the current study, we have investigated a number of HDACi, including some new, highly-potent compounds, for their potential to induce EBV lytic phase gene expression and to kill EBV-infected cells in combination with anti-herpesviral drugs. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids [Oxamflatin, Scriptaid, Suberoyl anilide hydroxamic acid (SAHA), Panobinostat (LBH589) and Belinostat (PXD101)]; the benzamide MS275; cyclic tetrapeptide Apicidin, and newly-identified HDAC inhibitor Largazole, which was originally isolated from a marine cyanobacterium. We assayed the induction of lytic phase in EBV-positive lymphoma cell lines exposed to different HDACi for 24–48 hrs, then quantitated the expression of EBV TK and other EBV transcripts by RT-PCR analysis. To determine tumor cytotoxic activity of the combination of HDACi and GCV, EBV+ lymphoma cells were exposed to a range of concentrations of HDACi and GCV for 3 days and then to GCV alone for another 3 days. Efficacy of a particular HDACi in the combination treatment approach was then determined by enumerating living cells. With the exception of SAHA and PXD101, the other HDACi had synergistic activity with anti-viral agents in killing EBV+ lymphoma cells. The hydroxamic acid LBH589, the benzamide MS275, and synthetic largazole derivatives 234a and 234b were 104 to 105-times more potent in killing EBV+ lymphoma cells in presence of GCV, compared to sodium butyrate. The effective concentration of LBH589 was in the range of 50–100 nM, MS275 at 200–500 nM and Largazole 234a and 234b at 100–200 nM. Of note, at these concentrations, the drugs as single agents produced no growth inhibitory activity in the tumor cells. LBH589, MS275 and Largazole 234a and 234b also strongly induced EBV-TK expression in the tumor cells. The effectiveness of these HDACi compounds at such low concentrations makes them potentially applicable as sensitizers to anti-viral therapeutics for the treatment of EBV-associated lymphomas. Our finding therefore provides an intriguing possibility that these novel HDACi may be used as an alternative therapeutic option, in combination with nucleoside antivirals, for the treatment of EBV-associated tumors. Disclosures: Faller: HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Perrine:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Williams:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Berenson:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: Abstract 4277 Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (〉1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3188 The clinical severity of the beta thalassemia syndromes and hemoglobinopathies can be dramatically modified by up-regulation of basal fetal globin expression through co-inheritance of 3 influential genetic modifiers, or by fetal globin-inducing therapeutics. One approved therapeutic, Hydroxyurea (HU), benefits some patients, but does not reduce anemia enough to achieve transfusion-independence in many thalassemia patients, and additional HbF-enhancing therapies are needed which can be applied in combination regimens without added toxicity. A challenge to successful application of HbF-inducing therapies is the broad genetic heterogeneity, which produces variable baseline HbF levels, and, to date, unpredictable responsiveness to therapeutics among individual patients. Therapies tailored to genetic modifiers (QTL) which modulate baseline HbF levels would allow rational treatment selection for specific subsets of patients. GWAS screening of 1000 patients in diverse populations, and analyses of patients enrolled on two recent clinical trials in sickle cell disease and beta thalassemia, demonstrated that modifiers in the 3 most influential QTLs which increase HbF or F-cells occur commonly, in 30–50% of patients, with BCL11A polymorphisms occurring in 20–30%. Using molecular modeling and high-throughput screening of chemical libraries, we recently discovered 8 therapeutics (6 clinical-stage or approved) which enhance HbF expression through different mechanisms, including displacement of a repressor complex containing HDAC-3 (RB7) or suppression of BCL11A mRNA (MS-275). Two pan-HDAC inhibitors (Butyrate and MS-275) profoundly decrease the 3 BCL11A isoforms by 80–100% in normal erythroid progenitors, while other HDAC inhibitors (LBH389, SAHA) have no effect or even increase BCL11A expression. Decreases in BCL11A provide a favorable effect, similar to the BCL11A QTL, increasing both baseline HbF levels and responsiveness to HbF-inducing drugs. Other inducers (DLT, Benserazide, NSC95397, RB16, ST20) induce the gamma-globin promoter without epigenetic effects. MS-275, DLT, and RB7 increase the promoter activity by 5–6 fold in normal progenitors, while prior generation short chain fatty acid agents induced by 2-fold. This small but diverse therapeutic pipeline, encompassing different mechanisms of action, now allows: 1) epigenetic and/or non-epigenetic gamma-globin induction; 2) significantly higher induction than prior generation inducers; and 3) potential drug combinations which may benefit those patients who do not have favorable QTL polymorphisms. Individual therapeutics can now be investigated in genotyped subjects to provide a basis for tailoring specific inducer combinations to QTL profiles, offering potential for higher therapeutic benefit. Disclosures: No relevant conflicts of interest to declare.