Histone deacetylase inhibitors (HDACi) may engage host immunity as one basis for their antitumor effects. Herein, we demonstrate an application of this concept using the HDACi panobinostat to augment ...the antitumor efficacy of trastuzumab (anti-HER2) therapy, through both tumor cell autonomous and nonautonomous mechanisms. In HER2
tumors that are inherently sensitive to the cytostatic effects of trastuzumab, cotreatment with panobinostat abrogated AKT signaling and triggered tumor regression in mice that lacked innate and/or adaptive immune effector cells. However, the cooperative ability of panobinostat and trastuzumab to harness host anticancer immune defenses was essential for their curative activity in trastuzumab-refractory HER2
tumors. In trastuzumab-resistant HER2
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xenografts and BT474 tumors expressing constitutively active AKT, panobinostat enhanced the antibody-dependent cell-mediated cytotoxicity function of trastuzumab. IFNγ-mediated, CXCR3-dependent increases in tumor-associated NK cells underpinned the combined curative activity of panobinostat and trastuzumab in these tumors. These data highlight the immune-enhancing effects of panobinostat and provide compelling evidence that this HDACi can license trastuzumab to evoke NK-cell-mediated responses capable of eradicating trastuzumab-refractory HER2
tumors.
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Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 ...domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, ...we investigate the therapeutic, antimelanoma potential of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma. Utilizing murine and human melanoma cell lines, we analyzed the effects of LBH589 on proliferation and survival. In addition, we analyzed the expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589-treated melanoma to activate antigen-specific T cells. Finally, we assessed the in-vivo effects of LBH589 in a mouse melanoma model. Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of major histocompatibility complex and costimulatory molecules on melanoma cells leading to an increased ability to activate antigen-specific T cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost. Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.
Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been ...found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.
Disequilibrium in the human debilitated immune system favors proliferation of invasive Candida species, a major therapeutic challenge due to development of resistance to several conventional ...antifungal agents (CAA) worldwide. Multiple mutations observed at specific loci that are targets for CAA are recognized as sources of drug resistance. This has prompted a shift from CAA, to diverse combination therapies, photodynamic and short peptide therapies capable of triggering specific apoptotic reactions within candidal cells. In this review, new designs and combination of short peptide (SP) with CAA as well as current application of photodynamic inactivation (PDI) against Candida species geared at generating reactive species of oxygen (ROS) and nitrogen (RNS) are discussed. It is observed that oxidative and nitrosative stresses provides a superior broad candidacidal effects for eradication of drug-resistant Candida species. The mechanism and limitations in these strategic approaches over CAA is also discussed.
We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome–negative (Ph−) acute lymphoblastic leukemia ...(ALL). Two model human Ph− ALL cell lines (T-cell MOLT-4 and pre–B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC50: 5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph− ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph− ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph− ALL.
Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of ...integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Insulin-like growth factor-1 (IGF-1) is an important growth and survival factor in multiple myeloma (MM). Here, we demonstrate that IGF-1 induces significant down-regulation of the proapoptotic ...BH3-only protein Bim in MM cells. Reduced Bim levels by RNA interference (RNAi) protected cells from drug-induced cell death. The IGF-1–mediated down-regulation of Bim was the result of (1) reduced transcription by activation of the Akt pathway and inactivation of the transcription factor FoxO3a, (2) increased proteasome-mediated degradation of the Bim extra-long protein by activation of the mitogen-activated protein kinase pathway, and (3) epigenetic regulation of both the Bim and the FoxO3a promoter. Treatment of cells with the histone deacetylase inhibitor LBH589 resulted in a clear up-regulation in the expression of Bim. Furthermore, the methylation inhibitor 5-aza-2′deoxycytidine (decitabine) significantly increased the effects of LBH589. On IGF-1 treatment, the Bim promoter region was found to be unmethylated, whereas chromatin immunoprecipitation analysis of the IGF-1–treated cells showed both a reduced histone H3 tail Lys9 (H3K9) acetylation and an increased H3K9 dimethylation, which contributed actively to its silencing. These data identify a new mechanism in the IGF-1–dependent survival of MM cells and emphasize the need for IGF-1–targeted drug therapy.
Purpose: The molecular chaperone heat shock protein (hsp)-90 maintains estrogen receptor (ER)-α in an active conformation, allowing
it to bind 17β-estradiol (E 2 ) and transactivate genes, including ...progesterone receptor (PR)-β and the class IIB histone deacetylase HDAC6. By inhibiting
HDAC6, the hydroxamic acid analogue pan-HDAC inhibitors (HA-HDI; e.g., LAQ824, LBH589, and vorinostat) induce hyperacetylation
of the HDAC6 substrates α-tubulin and hsp90. Hyperacetylation of hsp90 inhibits its chaperone function, thereby depleting
hsp90 client proteins. Here, we determined the effect of HA-HDIs on the levels and activity of ERα, as well as on the survival
of ERα-expressing, estrogen-responsive human breast cancer MCF-7 and BT-474 cells.
Experimental Design: Following exposure to HA-HDIs, hsp90 binding, polyubiquitylation levels, and transcriptional activity of ERα, as well as
apoptosis and loss of survival, were determined in MCF-7 and BT-474 cells.
Results: Treatment with HA-HDI induced hsp90 hyperacetylation, decreased its binding to ERα, and increased polyubiquitylation and
depletion of ERα levels. HA-HDI treatment abrogated E 2 -induced estrogen response element-luciferase expression and attenuated PRβ and HDAC6 levels. Exposure to HA-HDI also depleted
p-Akt, Akt, c-Raf, and phospho-extracellular signal–regulated kinase-1/2 levels, inhibited growth, and sensitized ERα-positive
breast cancer cells to tamoxifen.
Conclusions: These findings show that treatment with HA-HDI abrogates ERα levels and activity and could sensitize ERα-positive breast
cancers to E 2 depletion or ERα antagonists.
A deregulated epigenome contributes to the transformed phenotype of mantle cell lymphoma (MCL). This involves activity of the polycomb repressive complex (PRC) 2, containing three core proteins, ...EZH2, SUZ12, and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. We determined the effects of 3-deazaneplanocin A (DZNep), an S-adenosylhomocysteine hydrolase inhibitor, and/or pan-histone deacetylase inhibitor panobinostat (PS) on cultured and primary MCL cells.
Following treatment with DZNep and/or PS, apoptosis and the levels and activity of EZH2 and PRC2 proteins in cultured and primary MCL cells were determined.
Treatment with DZNep depleted EZH2, SUZ12, and 3MeK27H3 in the cultured human MCL cells. DZNep also increased expression of p21, p27, and FBXO32, whereas it depleted Cyclin D1 and Cyclin E1 levels in MCL cells. In addition, DZNep treatment induced cell-cycle arrest and apoptosis in cultured and primary MCL cells. Furthermore, as compared with treatment with each agent alone, cotreatment with DZNep and PS caused greater depletion of EZH2, SUZ12, 3MeK27H3, and Cyclin D1 levels, whereas it induced greater expression of FBXO32, p16, p21, and p27. Combined treatment with DZNep and PS synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34 + cells. Cotreatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice.
These preclinical in vitro and in vivo findings show that cotreatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL.
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