Polycomb Target Genes Are Silenced in Multiple Myeloma

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

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.</p> <p>Methods</p> <p>HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^®Pathway Analysis.</p> <p>Results</p> <p>Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.</p> <p>Conclusion</p> <p>This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.</p

Correction to: Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia (Clinical Epigenetics, (2015), 7, 1, (2), 10.1186/s13148-014-0034-4)

Following publication of the original article [1], the authors identified an error in Fig. 2e. The correct Fig. 2 is given below. (Figure presented.)

Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process

Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia

Background: One of the most frequently found abnormalities in acute myeloid leukemia (AML) is the t(8;21)(q22;q22) translocation, which is seen in around 15% of patients. This translocation results in the production of the AML1/ETO (A/E) fusion protein and commonly involves cooperating activating mutations of RAS. AE9a encodes a C-terminally truncated A/E protein of 575 amino acids that retains the ability to recruit histone deacetylases (HDACs). Expression of AE9a leads to rapid development of leukemia in experimental mouse systems. We have recently shown that treatment of mice bearing A/E9a;Nras(G12D) tumors with the histone deacetylase inhibitor (HDACi) panobinostat leads to degradation of the A/E9a fusion protein, cell cycle arrest, differentiation of AML blasts into mature granulocytes and prolonged survival. Herein, we sought to enhance this therapeutic effect. Findings: Combined treatment of mice bearing A/E9a;Nras(G12D) leukemias with panobinostat and arsenic trioxide (ATO) resulted in a significant survival advantage compared to mice treated with either agent alone. Moreover, some of the mice treated with the panobinostat/ATO combination showed complete tumor responses and remained in remission for over 220 days. Panobinostat caused differentiation of A/E9a;Nras(G12D) cells while ATO induced apoptosis of the leukemic cells, an effect that was enhanced following co-treatment with panobinostat. Conclusions: Our results indicate that leukemic blast differentiation mediated by panobinostat combined with induction of apoptosis by ATO could be therapeutically beneficial and should be considered for patients with t(8;21) AM

Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat

The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimen

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 Eμ-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death

HDAC inhibitor panobinostat engages host innate immune defenses to promote the tumoricidal effects of trastuzumab in HER2+ Tumors

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 AU565 xenografts and BT474 tumors expressing constitutively active AKT, panobinostat enhanced the antibody-dependent cell-mediated cytotoxicity function of trastuzumab. IFNg-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