The "Persuadable Middle" on Same-Sex Marriage: Formative Research to Build Support among Heterosexual College Students

Same-sex marriage is a controversial policy issue that affects the welfare of gay and lesbian couples throughout the USA. Considerable research examines opinions about same-sex marriage; however, studies have not investigated the covariates of the “persuadable middle”— those individuals who are neutral or unsure about their views. This group of people is often the target of same-sex marriage campaigns, yet they have received no empirical attention.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89607/1/Woodford et al 2011 Persuadable Middle.pd

HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses

The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds

RNAi-mediated depletion of histone deacetylases highlights the potential for isoform-specific inhibitors in B-cell lymphoma and acute myeloid leukemia

Introduction: Histone deacetylase (HDAC) inhibitors are a novel class of drugs with demonstrated activity in hematological malignancies. Vorinostat and romidepsin are FDA approved for treatment of cutaneous T-cell lymphoma and inhibit HDACs1, 2, 3 and 6 or HDAC1, 2 and 3, respectively. It is unknown which HDACs are most important for the survival of hematological tumors, however a targeted approach using HDAC-selective inhibitors could improve efficacy and reduce toxicity in the clinic. Here we utilised RNAi technology and an HDAC-specific inhibitor to investigate whether depletion/inhibition of individual or multiple HDACs could phenocopy the effects of pan-HDACi in B-cell lymphoma and acute myeloid leukemia (AML). Methods: HDACs were individually knocked down in murine Eμ-Myc (HDACs1, 2, 3, 6) and AML (MLL-AF9+NrasG12D; HDACs 1-11) cells using constitutive (pLMS/pLMN) or Tetracycline-inducible (pTRMPVIN) vectors in vitro and in vivo. Eµ-Myc cells were treated with HDAC3-specific inhibitor RGFP966. Cell proliferation, apoptosis, cell cycle, protein expression and gene knockdown were assessed by FACS, western blot and qRT-PCR. Global gene expression was assessed using RNAseq technology. Results: Loss of HDACs1, 2 or 6 had no long term effects on cell growth, while Eμ-Myc and AML cells depleted of HDAC3 were reproducibly lost from culture in vitro and in vivo. This phenotype was not prevented by Bcl-2 over-expression, caspase inhibition or knockout of p21 in Eμ-Myc but appeared dependent on Trp53 expression, including specific mutants of Trp53. HDAC3 knockdown altered the transcription of <0.05% genes in Eμ-Myc cells. Importantly, HDAC3-specific inhibitor RGFP966 reduced the growth rate of Eμ-Myc cells at low micromolar concentrations (0.5-1µM) while inducing apoptosis above 2µM, also partially dependent on Trp53 status. Conclusions: Our results have revealed exquisite sensitivities of murine B cell lymphoma and AML cells to depletion of HDAC3 in vitro and in vivo. This strongly suggests that HDAC3-specific inhibitors could prove useful for the treatment of various hematological malignancies. Further work investigating the molecular events underpinning the loss of proliferation induced by HDAC3 knockdown/inhibition and the effects of depleting multiple HDACs are currently underway. Ultimately, we aim to use this technology to discover efficacious HDACi with the best toxicity profile for the treatment of hematological malignancies

An intact immune system is required for the anticancer activities of histone deacetylase inhibitors

Cell-intrinsic effects such as induction of apoptosis and/or inhibition of cell proliferation have been proposed as the major antitumor responses to histone deacetylase inhibitors (HDACi). These compounds can also mediate immune-modulatory effects that may contribute to their anticancer effects. However, HDACi can also induce anti-inflammatory, and potentially immunosuppressive, outcomes. We therefore sought to clarify the role of the immune system in mediating the efficacy of HDACi in a physiologic setting, using preclinical, syngeneic murine models of hematologic malignancies and solid tumors. We showed an intact immune system was required for the robust anticancer effects of the HDACi vorinostat and panobinostat against a colon adenocarcinoma and two aggressive models of leukemia/lymphoma. Importantly, although HDACi-treated immunocompromised mice bearing established lymphoma succumbed to disease significantly earlier than tumor bearing, HDACi-treated wild-type (WT) mice, treatment with the conventional chemotherapeutic etoposide equivalently enhanced the survival of both strains. IFN-gamma and tumor cell signaling through IFN-gamma R were particularly important for the anticancer effects of HDACi, and vorinostat and IFN-gamma acted in concert to enhance the immunogenicity of tumor cells. Furthermore, we show that a combination of vorinostat with alpha-galactosylceramide (alpha-GalCer), an IFN-gamma-inducing agent, was significantly more potent against established lymphoma than vorinostat treatment alone. Intriguingly, B cells, but not natural killer cells or CD8(+) T cells, were implicated as effectors of the vorinostat antitumor immune response. Together, our data suggest HDACi are immunostimulatory during cancer treatment and that combinatorial therapeutic regimes with immunotherapies should be considered in the clinic. (C)2013 AACR

Characterization of the apoptotic and therapeutic activities of the histone deacetylase inhibitors LAQ824 and LBH589 using a mouse model of B cell lymphoma

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival including inhibition of tumor cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis and modulation of immune responses and show promising activity against hematological malignancies in clinical trials. Using the Eµ-myc model of B-cell lymphoma and the HDACi LBH589 and LAQ824 we demonstrated a direct correlation between induction of tumor cell death in vivo and therapeutic efficacy. Neither HDACi required p53 activity or a functional death receptor pathway, but mediated lymphoma cell death via the intrinsic apoptotic pathway as demonstrated by decreased apoptosis and therapeutic activity of LBH589 and LAQ825 against Eµ-myc/Bcl-2 and Eµ-myc/Bcl-XL cells. Interestingly, both LBH589 and LAQ824 effectively killed Eµ-myc/caspase-9-/- and Eµ-myc/Apaf-1-/- lymphomas, which lack a functional apoptosome and thus have a defective apoptotic program downstream of the mitochondria. These cells did not display classic morphological or biological features of apoptosis following treatment with LBH589 and LAQ824 however their clonogenic capacity was significantly reduced and interestingly electron microscopy analysis indicated that these HDACi-treated cells underwent autophagy. Importantly, both Eµ-myc/caspase-9-/- and Eµ-myc/Apaf-1-/- responded to LBH589 and LAQ824 in vivo indicating that in the absence of an effective apoptotic program downstream of mitochondrial membrane perturbation, tumor cells can undergo autophagy and this is sufficient to mediate therapeutic efficacy. Our studies provide important information regarding the mechanisms of action of LBH589 and LAQ824 that may have broader implications regarding future stratification of patients receiving therapy with these agents and the use of these compounds in combination with other anti-cancer agents

The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy

LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the E{micro}-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-XL protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. E{micro}-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response

Functional-genetic dissection of HDAC dependencies in mouse lymphoid and myeloid malignancies

Histone deacetylase inhibitors have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat and panobinostat are FDA-approved for hematological malignancies and inhibit class II and/or class I HDACs including HDAC1, 2, 3 and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACi in three genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine AMLs (MLL-AF9;NrasG12D; PML-RARalpha APL) and Emu-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all three tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 over-expression, caspase inhibition or knockout of Cdkn1a in Emu-Myc lymphoma and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Emu-Myc lymphoma and induced differentiation in APL. Genetic co-depletion of Hdac1 with Hdac2 was pro-apoptotic in Emu-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that co-depletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies