Academic literature on the topic 'PCI-34051'

Abstract Inhibitors of histone deacetylases (HDACs) which are currently in clinical testing for treating various cancers typically inhibit multiple isoforms of the 11-member HDAC family. We have developed an isoform-selective HDAC inhibitor, PCI-34051, that inhibits HDAC8 with a Ki of 10 nM and greater than 200-fold selectivity over other HDAC isoforms (Balasubramanian et al. (2008) Leukemia,22:1026–34). We have shown that PCI-34051 selectively induced apoptosis in cell lines derived from T-cell lymphomas and leukemias, but not in other tumor or normal cell types. Here we show that it potently inhibits the secretion of the pro-inflammatory cytokine interleukin-1 beta (IL-1b) in lipopolysaccharide (LPS)-stimulated peripheral mononuclear blood cells (PBMC) and isolated monocytes. PCI-34051 inhibited IL-1b secretion (by 80% compared to control) from LPS-stimulated human PBMC with an IC50 of 0.6 uM, which is much lower than the growth inhibitory concentrations of 2.4–4 uM required in T-cell lymphomas. We found that PCI-34051 also inhibited the secretion of interleukin-18 (IL-18) to a similar extent as IL-1b, but secretion of other pro-inflammatory cytokines, including MIP-1b, MCP-1, TNFa, and IL-6, was inhibited to a smaller extent. Interestingly, IL-18, like IL-1b, is synthesized without a signal peptide, and also utilizes the same non-classical endosomal secretory pathway as IL-1b including cleavage of the pro-form by caspase-1. Thus, we theorized that the modulatory effect of PCI-34051 is likely to involve modulation of the post-translational secretory process. In accordance, we found that the IL-1b mRNA levels were reduced by only 20% compared to control, but the intracellular protein levels of the pro-form was increased by >50% in primary monocytes after treatment with PCI-34051, indicating that the mechanism was due to inhibition of the processing from the pro- to the mature form of the cytokine. We showed that this was not due to a direct inhibition of caspase-1 or TACE (TNF-alpha converting enzyme), but is likely due to an as-yet unidentified substrate of HDAC8. In vivo, PCI-34051 inhibited ear swelling induced by oxazolone in a model of contact hypersensitivity in mice, and we showed that this was accompanied by a reduction in IL-1b at both the protein and mRNA levels. Based on this result, we examined the effect of PCI-34051 on IL-1b secretion in human keratinocytes, as well as in PBMC from psoriasis patients, and found that it could reduce IL-1b secretion in both. We found that PCI-34051 decreased IL-1b by 60% in LPS-stimulated PBMC from rheumatoid arthritis (RA) patients, but the pan-HDAC inhibitors which were only weakly inhibitory to HDAC8 did not have this effect, indicating a specific role for HDAC8 in the secretory process. Finally, we found that in unstimulated PBMC from RA patients that had basal production of IL-1b that this could be decreased by 90% by treatment with PCI-34051. Taken together, these findings indicate that PCI-34051 is an active drug that could be useful for the treatment of T-cell lymphoma as well as for autoinflammatory diseases such as RA and psoriasis.

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.

Abstract The tumor microenvironment plays an instrumental role in cancer development and treatment response/resistance. Accumulating evidence is underscoring the fundamental importance of epigenetic regulation in tumor immune evasion. Epigenetic modification agents represent a ‘double-edged sword’, as they can exert divergent effects on cancer cells and the milieu of immune and stromal cells. Our integrative epigenomics analysis has elucidated previously unexplored functions of histone deacetylase 8 (HDAC8) in promoting β-catenin-dependent hepatocarcinogenesis through interacting with another critical chromatin regulator enhancer of zeste homology 2 (EZH2). Given the strong oncogenicity of HDAC8, we further investigated the therapeutic potential of a potent and highly-selective HDAC8-specific inhibitor PCI-34051. Using a hepatocellular carcinoma (HCC) orthotopic model in immunocompetent mice, we demonstrated that HDAC8 inhibition exerted a strong anti-tumorigenic effect (Ctrl vs. PCI: 6.32 vs. 0.33*107 ROI intensity; p<0.01), which was comparable with programmed death-ligand 1 (PD-L1) blockade. Intriguingly, PCI-34051 significantly increased tumor-infiltrating multi-functional CD8+ T cells (~3-fold) and specifically reduced regulatory T cells (~2-fold). However, the level of PD-1+CD8+ T cells also increased, implicating potential T cell exhaustion. Our data suggest that selective chromatin modifications by HDAC8 and EZH2 alter the tumor immune surveillance program. By understanding the impact of epigenetic control on the liver tumor microenvironment, rational combinatorial epigenetic and immune checkpoint targeting has the potential to fully unleash T cell responses against HCC.

Pan-histone deacetylase (HDAC) inhibition with valproic acid (VPA) has beneficial effects after spinal cord injury (SCI), although with side effects. We focused on specific HDAC8 inhibition, because it is known to reduce anti-inflammatory mediators produced by macrophages (Mφ). We hypothesized that HDAC8 inhibition improves functional recovery after SCI by reducing pro-inflammatory classically activated Mφ. Specific HDAC8 inhibition with PCI-34051 reduced the numbers of perilesional Mφ as measured by histological analyses, but did not improve functional recovery (Basso Mouse Scale). We could not reproduce the published improvement of functional recovery described in contusion SCI models using VPA in our T-cut hemisection SCI model. The presence of spared fibers might be the underlying reason for the conflicting data in different SCI models.

Abstract JAK2-V617F mutation in hematopoietic stem cells (HSC) is a common finding in myeloproliferative neoplasms (MPNs). Although alterations in the hematopoietic microenvironment have been described in these entities, information on the functional and genetic characteristics of bone marrow (BM) derived mesenchymal stromal cells (BM-MSC) from JAK2+ MPNs patients is scarce. The aim of the current study was to characterize and compare BM-MSC from 24 MPNs patients with JAK2V617F mutation (14 BM-MSC from essential thrombocythemia-ET and 10 BM-MSC from polycythemia vera-PV) with those from 14 healthy donors-HD. For this purpose BM-MSC expansion, multilineage differentiation, apoptosis, inmunophenotyping, gene expression profiling, RT-PCR and Western Blot analysis were performed. Compared with HD, BM-MSC from MPNs patients showed similar morphology and differentiation capacity, but an increased proliferation rate with less apoptosis cells. BM-MSC from MPNs expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for hematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPNs patients (p <.05) when compared with BM-MSC from HD. Gene expression profile of BM-MSCs from 8 JAK2V617F (4 PV/4 TE) patients, and from 10 HD showed a total of 169 genes that were differentially expressed in BM-MSC from MPNs patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and CXCL12 genes were up-regulated. To analyze whether these changes in MPNs-MSC conferred an alteration in their functional capacity, co-cultures with CD34+ cells from MPNs and BM-MSC were performed. A significant increase in the CFU-GM clonogenic supporting capacity of MPNs-MSC when compared with HD-MSC was observed. To evaluate whether a Histone deacetylase (HDAC) inhibitor could modify the behavior of MPNs-MSC an HDAC8 specific inhibitor, PCI-34051 was used. A decrease in HDAC8 gene (RT-PCR) and protein (WB analysis) expression was observed in BM-MSC from MPNs treated with PCI-34051 at a concentration of 25µM for 48 hours. HDAC8-selective inhibition also induced a cell cycle arrest in the MPNs BM-MSC with an increase of the proportion of apoptotic cells. To assess the impact of this inhibition on the capacity of MPNs-MSC to support hematopoiesis, BM mononuclear cells (BM-MNC) were co-cultured in transwell for 48 hours with PCI-34051-treated and non-treated BM-MSC. After co-culture, cell viability, clonogenic (CFU-GM) assays and TP53 expression were analyzed. A decrease in cell viability (p=0.028) and CFU-GM (p=0.018) was demonstrated when BM-MNC from MPNs had been in culture with MPNs BM-MSC treated with the HDAC8 inhibitor, as well as an increase in TP53 expression. These results suggest that MPNs-MSC display different proliferative rate, MSC markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support hematopoietic cells from MPNs patients, suggesting that HDAC8 may be a potential therapeutic target in this setting. Disclosures Sánchez-Guijo: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Abstract Abstract 772 Chromosomal inversion inv(16)(p13.1q22) is found in approximately 12% of acute myeloid leukemia (AML) patients, and leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, and encodes a fusion protein CBFβ-SMMHC. Previous studies revealed that CBFβ-SMMHC is a dominant inhibitor of core-binding factor (CBF) function, and impairs hematopoietic differentiation. Expression of CBFβ-SMMHC predisposes for leukemia transformation, however, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC remains elusive. The tumor suppressor p53 is considered the master genomic guardian that is frequently mutated in a wide variety of tumors but is rarely mutated in inv(16) AML. Thus, we examined whether CBFβ-SMMHC fusion protein might impair p53 function. We found that p53 acetylation (Ac-p53) level was reduced in the presence of CBFβ-SMMHC fusion protein in the myeloid progenitor 32D cell line as well as in primary pre-leukemic bone marrow progenitor cells isolated from our conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre) mice (Kuo et al, Cancer Cell 2006, 9:1,57-68). We assessed the effect of CBFβ-SMMHC on p53 transcriptional activity by quantitative RT-PCR analysis of p53 target genes including TP53 and p21 Cdkn1a, Mdm2, Bid, Bax, Stag1, LincRNA-p21, Gadd45b in 32D cells. The result showed that expression of these p53 target genes are reduced in the presence of CBFβ-SMMHC fusion protein, consistent with the impaired Ac-p53 by CBFβ-SMMHC. To understand how CBFβ-SMMHC impairs p53 function, we tested whether CBFβ-SMMHC fusion protein might interact with the p53 protein by co-immunoprecipitation (co-IP) assays. We found that CBFβ-SMMHC fusion protein interacts with p53 both in 32D cells and primary bone marrow cells. Although CBFβ-SMMHC fusion protein is detected both in the nucleus and the cytoplasm, the complex with p53 is present exclusively in the nucleus. It has been reported that CBFβ-SMMHC interacts with histone deacetylase 8 (HDAC8) through the C-terminal SMMHC region. Therefore, we assessed the interaction between CBFβ-SMMHC, p53 and HDAC8 in 32D cell line by co-IP and sequential co-IP. We were able to detect a multimeric protein complex containing CBFβ-SMMHC, p53, and Hdac8. To access whether HDAC8 contributes to the deacetylation of p53, we used two independent small-hairpin (sh)-RNA to knock-down Hdac8 in 32D-CBFβ-SMMHC cells. Hdac8 knock-down led to robust increase in Ac-p53 levels while total p53 levels were modestly stabilized. To test whether this effect is dependent on the deacetylase function of HDAC8, we used HDAC8 selective pharmacological inhibitors (HDAC8i including PCI-34051 and PCI-48012) directed against its catalytic sites (Balasubramanian et al Leukemia 2008, 22:5,1026-34). Treatment with HDAC8i remarkably increased Ac-p53 in both control and CBFβ-SMMHC cells. Since p53 protein levels were also increased upon HDAC8i treatment, we included Mdm2 inhibitor Nutlin-3 to stabilize p53. HDAC8i treatment alone or in combination with Nutlin-3 was able to enhance Ac-p53 compared to Nutlin-3 treatment, confirming its effect in restoring p53 acetylation. Collectively, our study shows that the CBFβ-SMMHC fusion protein forms an aberrant complex with p53 and HDAC8, leading to the aberrant deacetylation and impaired activity of p53. In addition, this deacetylation of p53 conferred by CBFβ-SMMHC is mediated by HDAC8. Our study reveals a novel leukemogenic mechanism in which CBFβ-SMMHC disrupts p53 activation through aberrant protein-protein interaction and recruitment of HDAC8. Disclosures: No relevant conflicts of interest to declare.

Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

The use of pan-HDACi for disease treatment has gained an interest in recent years, albeit exhibiting low specificity, variable efficacy and side effects. Each HDAC with its own activity could be considered as an independent pharmacological target to develop an effective therapy that circumvents the adverse effects of pan-HDACi treatment. HDAC8 belongs to class I HDAC and is known to modulate cohesin complex activity through deacetylation of SMC3. It possesses a unique structure among HDACs, which allowed the development of highly specific inhibitors, such as the PCI-34051. However, HDAC8 function and its involvement in pathological conditions is still largely unknown. To examine in depth HDAC8 physiological and pathological roles and assess whether it can represent a valuable pharmacological target, we analysed its function and the effect of its inhibition by using both in vitro (cell lines) and in vivo (zebrafish) models. In particular, we assessed HDAC8 function in three different tissues and related disorders: i) haematopoietic stem and progenitor cells (HSPC) and acute myeloid leukemia (AML); ii) neural stem cells (NSC) and Cornelia de Lange syndrome (CdLS); iii) skeletal muscle and Duchenne muscular dystrophy (DMD). We found that HDAC8 overexpression increased the proliferation of HSPCs in zebrafish and that its inhibition with PCI treatment restored normal phenotype, favouring cell cycle arrest, and induced apoptosis of AML cells. By contrast, HDAC8 knockdown lead to impairment of both central nervous system development and skeletal muscle differentiation. Furthermore, we found that HDAC8 overexpression is also associated with DMD phenotype and demonstrated that treatment with PCI inhibitor almost restored normal condition. Such positive effects were underlined by multiple mechanisms, which included cell cycle arrest, apoptosis induction and modulation of canonical Wnt pathway. Moreover, by acetylome profiling we identified α-tubulin as HDAC8 target thus revealing HDAC8 involvement in regulation of microtubule structure. Additionally, to confirm the involvement of HDAC8 in the aforementioned pathologies, we investigated also the role of its partner NIPBL. By RNA-seq analysis we assessed the effect of NIPBL knockdown on gene expression revealing a number of differentially expressed genes linked to pathways altered in CdLS or AML.