Histone Deacetylase Inhibitors Resistance Research Articles

Sensitization of hepatocellular carcinoma cells to HDACi is regulated through hsa-miR-342-5p/CFL1

BackgroundIncreased prevalence of hepatocellular carcinoma (HCC) remains a global health challenge. HCC chemoresistance is a clinical obstacle for its management. Aberrant miRNA expression is a hallmark for both cancer progression and drug resistance. However, it is unclear which miRNAs are involved in HCC chemoresistance.MethodsMicroRNA microarray analysis revealed a differential expression profile of microRNAs between the hepatocellular carcinoma HA22T cell line and the HDACi-R cell line, which was validated by quantitative real-time PCR (qRT-PCR). To determine the biological function of miR-342-5p and the mechanism of the microRNA-342-5p/CFL1 axis in hepatocellular carcinoma HDACi resistance, loss- and gain-of-function studies were conducted in vitro.ResultsHere we demonstrated the molecular mechanism of histone deacetylase inhibitor (HDACi) resistance in HCC. Differential miRNA expression analysis showed significant down regulation of miR-342-5p in HDACi-R cells than in parental HA22T cells. Mimics of miR-342-5p enhanced apoptosis through upregulation of Bax, cyto-C, cleaved-caspase-3 expressions with concomitant decline in anti-apoptotic protein (Bcl-2) in HDACi-R cells. Although HDACi did not increase cell viability of HDACi-R, overexpression of miR-342-5p decreased cofilin-1 expression, upregulated reactive oxygen species (ROS) mediated apoptosis, and sensitized HDACi-R to HDACi in a dose-dependent manner.ConclusionOur findings demonstrated the critical role of miR-342-5p in HDACi resistance of HCC and that this mechanism might be attributed to miR-342-5p/cofilin-1 regulation.

Comprehensive analysis of microRNAs modulated by histone deacetylase inhibitors identifies microRNA-7-5p with anti-myeloma effect.

Basic research to expand treatment options for multiple myeloma is greatly needed due to the refractory nature of the disease. Histone deacetylase (HDAC) inhibitors, which are epigenetic regulators, are attractive but have limited applications. MicroRNAs (miRNAs), which are also epigenetic regulators, are important molecules that may lead to future therapeutic breakthroughs. In this study, we comprehensively searched for miRNAs that are altered by HDAC inhibitors in myeloma cells. We identified miR-7-5p (miR-7) as a miRNA downregulated by HDAC inhibitors. Transfection of myeloma cell lines with miR-7 suppressed cell proliferation, induced apoptosis, and enhanced the effects of the HDAC inhibitor panobinostat. Expression of miR-7 was downregulated by c-Myc inhibition, but upregulated by bortezomib. Comprehensive examination of miR-7 targets revealed four candidates: SLC6A9, LRRC59, EXOSC2, and PSME3. Among these, we focused on PSME3, an oncogene involved in proteasome capacity in myeloma cells. PSME3 knockdown increases myeloma cell death and panobinostat sensitivity. In conclusion, miR-7, which is downregulated by HDAC inhibitors, is a tumor suppressor that targets PSME3. This miR-7 downregulation may be involved in HDAC inhibitor resistance. In addition, combinations of anti-myeloma drugs that complement changes in miRNA expression should be considered.

Integrative analyses identified gap junction beta-2 as a prognostic biomarker and therapeutic target for breast cancer.

Increasing evidence has shown that connexins are involved in the regulation of tumor development, immune escape, and drug resistance. This study investigated the gene expression patterns, prognostic values, and potential mechanisms of connexins in breast cancer. We conducted a comprehensive analysis of connexins using public gene and protein expression databases and clinical samples from our institution. Connexin mRNA expressions in breast cancer and matched normal tissues were compared, and multiomics studies were performed. Gap junction beta-2 mRNA was overexpressed in breast cancers of different pathological types and molecular subtypes, and its high expression was associated with poor prognosis. The tumor membrane of the gap junction beta-2 mutated group was positive, and the corresponding protein was expressed. Somatic mutation and copy number variation of gap junction beta-2 are rare in breast cancer. The gap junction beta-2 transcription level in the p110α subunit of the phosphoinositide 3-kinase mutant subgroup was higher than that in the wild-type subgroup. Gap junction beta-2 was associated with the phosphoinositide 3-kinase-Akt signaling pathway, extracellular matrix-receptor interaction, focal adhesion, and proteoglycans in cancer. Furthermore, gap junction beta-2 overexpression may be associated with phosphoinositide 3-kinase and histone deacetylase inhibitor resistance, and its expression level correlated with infiltrating CD8+ T cells, macrophages, neutrophils, and dendritic cells. Gap junction beta-2 may be a promising therapeutic target for targeted therapy and immunotherapy and may be used to predict breast cancer prognosis.

STAT3 Is a Potential Therapeutic Target for Histone Deacetylase Inhibitor-Resistant Cutaneous T-Cell Lymphoma

Background: Histone deacetylase inhibitors (HDACis), such as vorinostat and romidepsin, are effective treatment agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. Despite the promising anti-lymphoma activity of HDACis, drug resistance remains a significant clinical problem. Therefore, novel therapeutic strategies are required to overcome HDACi resistance. In this study, we generated HDACi-resistant CTCL cell lines and investigated the mechanisms of HDACi resistance. Aims: This study aimed to identify the mechanism of resistance to HDACis in CTCLs and to explore new therapeutic targets. Methods: The CTCL cell lines MyLa, MJ, and Hut78 were used in this study. Vorinostat-resistant cell lines, namely MyLa resistant, HUT78 resistant, and MJ resistant, were prepared by repeatedly exposing cells to increasing concentrations of vorinostat. To maintain drug resistance, the cells were continuously cultured in a medium containing 2.5 μM vorinostat. Following this, we conducted a comprehensive gene expression analysis using a microarray platform to investigate the deregulated genes in the resistant cell lines. Results: CTCL cell lines resistant to vorinostat were also resistant to romidepsin, suggesting that vorinostat-resistant cell lines showed cross-resistance to other HDACis. Next, we selected genes whose expression was upregulated more than 1.5 times in the three resistant cell lines and performed pathway analysis. Pathway analysis revealed that the upregulated genes were significantly enriched in the cytokine-mediated signaling pathways. Among the upregulated genes, we focused on interleukin-6 signal transducer (IL6ST)/glycoprotein (gp) 130, which forms a heterogeneous complex with IL6 and IL6 receptors, and activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Furthermore, flow cytometric analysis revealed that surface gp130 expression was significantly upregulated in the resistant cell lines. Moreover, the MyLa-resistant and MJ-resistant cell lines showed increased sensitivity to a gp130 inhibitor (SC144). Gp130 activation could activate the JAK/STAT pathway; therefore, we examined the expression level of the activated forms of STAT3 and p-STAT3 and found that p-STAT3 was significantly upregulated in the resistant cell lines. To investigate whether p-STAT3 upregulation is involved in the development of vorinostat resistance, we performed STAT3 knockdown using siRNA. We confirmed that STAT3 knockdown significantly decreased the survival of the resistant cell lines. Moreover, resistant cell lines showed increased sensitivity to the STAT3-selective inhibitor C188-9. However, compared to the controls, the resistant cell lines showed no difference in their sensitivity towards the JAK1/2 inhibitor ruxolitinib. This suggests that the STAT3 activation occurs not only through the JAK/STAT pathway but also through a pathway that is not mediated by JAK. We also examined the expression level of p-STAT5; however, it was downregulated in the resistant cell lines, which was in contrast to that of p-STAT3. These findings suggest that STAT3 activation, which is mediated by HDAC inhibition, could contribute to the resistance of CTCL to HDACis. Finally, we examined the gp130 and p-STAT3 protein expression in CTCL skin samples, which were obtained from the same patients before and after vorinostat treatment. We found that the gp130 and p-STAT3 expression was significantly increased in the specimens during relapse after vorinostat treatment. Conclusion: Activation of STAT3 in HDACi-resistant CTCL cells was enhanced by gp130, which contributed to their resistance. These results suggest that STAT3 could be the new therapeutic target in HDACi-resistant CTCL and that gp130/STAT3 inhibition could be an important therapeutic strategy. To further elucidate the detailed mechanism of HDACi resistance, analysis of the pathways activating STAT3 without JAK and the target proteins of STAT3 is warranted.

CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells.

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.

Pharmaco-transcriptomic correlation analysis reveals novel responsive signatures to HDAC inhibitors and identifies Dasatinib as a synergistic interactor in small-cell lung cancer.

BackgroundHistone acetylation/deacetylase process is one of the most studied epigenetic modifications. Histone deacetylase inhibitors (HDACis) have shown clinical benefits in haematological malignancies but failed in solid tumours due to the lack of biomarker-driven stratification.MethodsWe perform integrative pharmaco-transcriptomic analysis by correlating drug response profiles of five pan-HDACis with transcriptomes of solid cancer cell lines (n=659) to systematically identify generalizable gene signatures associated with HDACis sensitivity and resistance. The established signatures are then applied to identify cancer subtypes that are potentially sensitive or resistant to HDACis, and drugs that enhance the efficacy of HDACis. Finally, the reproductivity of the established HDACis signatures is evaluated by multiple independent drug response datasets and experimental assays.FindingsWe successfully delineate generalizable gene signatures predicting sensitivity (containing 46 genes) and resistance (containing 53 genes) to all five HDACis, with their reproductivity confirmed by multiple external sources and independent internal assays. Using the gene signatures, we identify low-grade glioma harbouring isocitrate dehydrogenase 1/2 (IDH1/2) mutation and non-YAP1-driven subsets of small-cell lung cancer (SCLC) that particularly benefit from HDACis monotherapy. Further, based on the resistance gene signature, we identify clinically-approved Dasatinib as a synthetic lethal drug with HDACi, synergizing in inducing apoptosis and reactive oxygen species on a panel of SCLC. Finally, Dasatinib significantly enhances the therapeutic efficacy of Vorinostat in SCLC xenografts.InterpretationOur work establishes robust gene signatures predicting HDACis sensitivity/resistance in solid cancer and uncovers combined Dasatinib/HDACi as a synthetic lethal combination therapy for SCLC.

Epigenetics in the Pathogenesis and Treatment of Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphomas (CTCLs) comprise a group of heterogeneous diseases involving malignant T cells. The pathogenesis and etiology of CTCL are still unclear, although a large number of genetic and epidemiological studies on CTCL have been conducted. Most CTCLs have an indolent course, making early diagnosis difficult. Once large-cell transformation occurs, CTCL progresses to more aggressive types, resulting in an overall survival of less than five years. Epigenetic drugs, which have shown certain curative effects, have been selected as third-line drugs in patients with relapsing and refractory CTCL. Many studies have also identified epigenetic biomarkers from tissues and peripheral blood of patients with CTCL and suggested that epigenetic changes play a role in malignant transformation and histone deacetylase inhibitor (HDACi) resistance in CTCL. Single-cell sequencing has been applied in CTCL studies, revealing heterogeneity in CTCL malignant T cells. The mechanisms of HDACi resistance have also been described, further facilitating the discovery of novel HDACi targets. Despite the heterogeneity of CTCL disease and its obscure pathogenesis, more epigenetic abnormalities have been gradually discovered recently, which not only enables us to understand CTCL disease further but also improves our understanding of the specific role of epigenetics in the pathogenesis and treatment. In this review, we discuss the recent discoveries concerning the pathological roles of epigenetics and epigenetic therapy in CTCL.

Platycodin D reverses histone deacetylase inhibitor resistance in hepatocellular carcinoma cells by repressing ERK1/2-mediated cofilin-1 phosphorylation

BackgroundChemoresistance remains the main obstacle in hepatocellular carcinoma (HCC) therapy. Despite significant advances in HCC therapy, HCC still has a poor prognosis. Thus, there is an urgent need to identify a treatment target to reverse HCC chemotherapy resistance. Platycodon grandiflorus (PG) is a perennial herb that has been used as food and traditional Chinese medicine for thousands of years in Northeast Asia. Platycodin D (PD), a main active triterpenoid saponin found in the root of PG, has been reported to possess anticancer properties in several cancer cell lines, including HCC; however, the reversal effect of this molecule on HCC chemoresistance remains largely unknown. PurposeThis study aimed to investigate the role and the mechanism of PD-mediated reversal of the histone deacetylase inhibitor (HDACi) resistance in HCC cells. MethodsHuman HCC cells (HA22T) and HDACi-resistant (HDACi-R) cells were used. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Combination index was used to calculate the synergism potential. Expression of ERK1/2 (total/phospho), cofilin-1 (total/phospho) and apoptosis-related protein was determined using western blotting. Mitochondrial membrane potential was assessed using the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide) probe. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Mitochondrial reactive oxygen species generation was measured using the MitoSOX Red fluorescent probe. ResultsWe found that PD treatment inhibited cell viability both in HA22T HCC and HDACi-R cells. Inhibition of ERK1/2 by PD98059 could reverse drug resistance in HDACi-R cells treated with PD98059 and PD. Nevertheless, pre-treatment with U46619, an ERK1/2 activator, rescued PD-induced apoptosis by decreasing levels of apoptosis-related proteins in HCC cells. The combined treatment of PD with apicidin a powerful HDACi, dramatically enhanced the apoptotic effect in HDACi-R cells. ConclusionFor the first time, we showed that PD reversed HDACi resistance in HCC by repressing ERK1/2-mediated cofilin-1 phosphorylation. Thus, PD can potentially be a treatment target to reverse HCC chemotherapy resistance in future therapeutic trials.

Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy

AbstractDespite the promising antilymphoma activity of histone deacetylase (HDAC) inhibitors as a drug class, resistance is a significant clinical issue. Elucidating the molecular mechanisms driving HDAC inhibitor resistance and/or the specific targets that are altered in drug-resistant cells may facilitate the development of strategies that overcome drug resistance and are more effective for refractory patients. We generated novel T-cell lymphoma (TCL) cell line models of acquired resistance to the HDAC inhibitor belinostat to identify potential effective therapies. Belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including romidepsin, panobinostat, and vorinostat. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones. Drug-resistant cells featured significantly decreased expression of the key antiviral mediators IRF1 and STAT1. On the basis of these findings, we investigated the efficacy of the clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that HDAC inhibitor–resistant cells displayed enhanced vulnerability to reovirus replication and cell death in both in vitro and in vivo models compared with their parental counterparts. Importantly, Reolysin also significantly increased the antilymphoma activity of belinostat in HDAC inhibitor–resistant cells. Our data demonstrate that Reolysin alone or in combination with belinostat is a novel therapeutic strategy to treat TCL patients who develop resistance to HDAC inhibitors.

Abstract 4316: Targeting LIFR overcomes HDAC inhibitor resistance in ovarian cancer

Abstract Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States and a critical need exists for the development of novel therapies for the treatment of OCa. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent clinical studies showed that cancer cells elucidate feedback activation of LIFR that limits response to histone deacetylase (HDAC) inhibitors. Recently, we developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIF-LIFR interactions. Here, we examined whether LIFR inhibitor EC359 abrogate the side effects of histone deacetylase inhibitor SAHA (Vorinostat) for the treatment of OCa. Methods: The effect of EC359 and SAHA as a combination therapy on OCa cell viability was examined by MTT assays. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and caspase3/7 assays respectively. The efficacy of combination therapy on OCa stem cells was determined using extreme limiting dilution assays. Mechanistic studies were performed using western blotting, qRT-PCR and Mass Spectrometry analyses. The effect of combination therapy on STAT3 signaling was examined using reporter gene assays. The in vivo efficacy of combination therapy on tumors was examined using ex vivopatient derived explants and mouse xenograft models. Results: EC359 significantly enhanced the efficacy of SAHA in reducing cell viability, colony formation ability, and apoptosis compared to monotherapy of SAHA in multiple established and primary OCa cells. Further, EC359 enhanced SAHA ability to reduce self-renewal of OCa stem cells. As expected in STAT3 reporter assays, SAHA treatment activated STAT3 reporter and EC359 addition abrogated SAHA mediated STAT3 activation. Mechanistic studies using multiple OCa models and western blot analysis confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359 treatment. Treatment of human primary OCa tumor explants with EC359 enhanced ability of SAHA to decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. DIA based Mass Spectrometry analyses identified unique pathways modulated by combination therapy. Treatment of OCa xenografts with EC359 enhanced the ability of SAHA to reduce in vivotumor growth compared to monotherapy treated tumors. Conclusions: Our results suggest that EC359 has therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDAC inhibitors in treating OCa. Citation Format: Mengxing Li, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Bindu Santhamma, Hui Yan, Zhenming Xu, Edward Kost, Rajeshwar Rao Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Targeting LIFR overcomes HDAC inhibitor resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4316.

IDH1R132H Causes Resistance to HDAC Inhibitors by Increasing NANOG in Glioblastoma Cells

The R132H mutation in isocitrate dehydrogenase 1 (IDH1R132H) is commonly observed and associated with better survival in glioblastoma multiforme (GBM), a malignant brain tumor. However, the functional role of IDH1R132H as a molecular target for GBM treatment is not completely understood. In this study, we found that the overexpression of IDH1R132H suppresses cell growth, cell cycle progression and motility in U87MG glioblastoma cells. Based on cell viability and apoptosis assays, we found that IDH1R132H-overexpressing U87MG and U373MG cells are resistant to the anti-cancer effect of histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA), vorinostat (SAHA), and valproic acid. Octyl-(R)-2-hydroxyglutarate (Octyl-2HG), which is a membrane-permeable precursor form of the oncometabolite (R)-2-hydroxyglutarate (R-2HG) produced in IDH1-mutant tumor cells, significantly increased HDACi resistance in glioblastoma cells. Mechanistically, IDH1R132H and Octyl-2HG enhanced the promoter activation of NANOG via increased H3K4-3Me, consequently increasing NANOG mRNA and protein expression. Indeed, HDACi resistance was attenuated in IDH1R132H-expressing glioblastoma cells by the suppression of NANOG using small interfering RNAs. Furthermore, we found that AGI-5198, a selective inhibitor of IDH1R132H, significantly attenuates HDACi resistance and NANOG expression IDH1R132H-expressing glioblastoma cells. These results suggested that IDH1R132H is a potential molecular target for HDACi-based therapy for GBM.

Discovery of Novel Janus Kinase (JAK) and Histone Deacetylase (HDAC) Dual Inhibitors for the Treatment of Hematological Malignancies

Concurrent inhibition of Janus kinase (JAK) and histone deacetylase (HDAC) could potentially improve the efficacy of the HDAC inhibitors in the treatment of cancers and resolve the problem of HDAC inhibitor resistance in some tumors. Here, a novel series of pyrimidin-2-amino-pyrazol hydroxamate derivatives as JAK and HDAC dual inhibitors was designed, synthesized, and evaluated, among which 8m possessed potent and balanced activities against both JAK2 and HDAC6 with half-maximal inhibitory concentration at the nanomolar level. 8m exhibited improved antiproliferative and proapoptotic activities over SAHA and ruxolitinib in several hematological cell lines. Remarkably, 8m exhibited more potent antiproliferation effect than the combination of SAHA and ruxolitinib in HEL cells bearing JAK2V617F mutation. Pharmacokinetic studies in mice showed that 8m possessed good bioavailability after intraperitoneal administration. Finally, 8m showed antitumor efficacy with no significant toxicity in a HEL xenograft model. Collectively, the results confirm the therapeutic potential of JAK and HDAC dual inhibitors in hematological malignancies and provide valuable leads for further structural optimization and antitumor mechanism study.

Single-cell heterogeneity in Sézary syndrome

AbstractSézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with a median life expectancy of less than 4 years. Although initial treatment responses are often good, the vast majority of patients with SS fail to respond to ongoing therapy. We hypothesize that malignant T cells are highly heterogeneous and harbor subpopulations of SS cells that are both sensitive and resistant to treatment. Here, we investigate the presence of single-cell heterogeneity and resistance to histone deacetylase inhibitors (HDACi) within primary malignant T cells from patients with SS. Using single-cell RNA sequencing and flow cytometry, we find that malignant T cells from all investigated patients with SS display a high degree of single-cell heterogeneity at both the mRNA and protein levels. We show that this heterogeneity divides the malignant cells into distinct subpopulations that can be isolated by their expression of different surface antigens. Finally, we show that treatment with HDACi (suberanilohydroxamic acid and romidepsin) selectively eliminates some subpopulations while leaving other subpopulations largely unaffected. In conclusion, we show that patients with SS display a high degree of single-cell heterogeneity within the malignant T-cell population, and that distinct subpopulations of malignant T cells carry HDACi resistance. Our data point to the importance of understanding the heterogeneous nature of malignant SS cells in each individual patient to design combinational and new therapies to counter drug resistance and treatment failure.

HSP72 functionally inhibits the anti-neoplastic effects of HDAC inhibitors

BackgroundThe anticancer effects of histone deacetylase inhibitors (HDACi) vary between patients, and their molecular mechanisms remain poorly understood. Previously, we have identified heat shock 70 kDa protein 1A (HSPA1A, also known as HSP72) as the most overexpressed protein in valproic acid (VPA)-resistant cell lines. KNK437, an inhibitor of heat shock proteins, enhanced the cytotoxic effects of not only VPA but also vorinostat, another HDACi. However, the mechanisms underlying the role of HSP72 in resistance against HDACi remain largely unknown. ObjectiveThe purpose of this study was to identify the mechanisms underlying the role of HSP72 in HDACi resistance. MethodsWe established an HSP72-overexpressing Jurkat cell line and used it to assess the functional role of HSP72 following treatment with the HDACi vorinostat and VPA. ResultsHDACi-induced apoptosis, assessed using annexin V assays, sub-G1 fraction analysis, and PARP cleavage, was significantly lower in HSP72-overexpressing cells than in control cells. The HDACi-induced upregulation in caspase-3, −8, and −9 activity, as well as the HDACi-induced reduction in mitochondrial membrane potential, were also suppressed following HSP72 overexpression. The basal expression levels of Bcl-2, phosphorylated Bad, and XIAP increased in HSP72-overexpressing cells, whereas HDACi-induced Bid truncation and the suppression of Bad expression. Furthermore, vorinostat-induced histone hyperacetylation was also diminished in HSP72-overexpressing cells. ConclusionThese findings clearly demonstrate that HSP72 inhibits HDACi-induced apoptosis.

Essential Role of DNA Methyltransferase 1-mediated Transcription of Insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors.

Purpose: Histone deacetylase inhibitors (HDI) are promising anticancer therapies; however, drug resistance limits their efficacy. Here, we investigated the molecular mechanisms underlying HDI resistance, focusing on the mechanism of HDI-mediated induction of insulin-like growth factor 2 (IGF2) based on our previous study.Experimental Design: The methylation status of CCCTC-binding factor (CTCF)-binding sites in the IGF2/H19 imprinting control region (ICR) were determined by methylation-specific PCR and bisulfite sequencing. The effectiveness of single or combinatorial blockade of DNA methyltransferase 1 (DNMT1) and histone deacetylase (HDAC) was evaluated using cell viability assay and patient-derived tumor xenograft (PDX) model.Results: HDAC inhibition by vorinostat increased acetylated STAT3 (K685), resulting in transcriptional upregulation of DNMT1 DNMT1-mediated hypermethylation of CTCF-binding sites in the IGF2/H19 ICR decreased CTCF insulator activity, leading to a transcriptional upregulation of IGF2 and activation of the insulin-like growth factor 1 receptor (IGF-1R) pathway in cells with acquired or de novo vorinostat resistance. Strategies targeting DNMT1 diminished the IGF2 expression and potentiated vorinostat sensitivity in preclinical models of lung cancer with hypermethylation in the H19/IGF2 ICR. The degree of ICR hypermethylation correlated with vorinostat resistance in patient-derived lung tumors and in patients with hematologic malignancies.Conclusions: DNMT1-mediated transcriptional upregulation of IGF2 is a novel mechanism of resistance to HDIs, highlighting the role of epigenetic deregulation of IGF2 in HDI resistance and the potential value of the H19/IGF2 ICR hypermethylation and DNMT1 expression as predictive biomarkers in HDI-based anticancer therapies. Clin Cancer Res; 23(5); 1299-311. ©2016 AACR.

ZEB1 Mediates Drug Resistance and EMT in p300-Deficient CRC.

We discuss the hypothesis that ZEB1-Wnt-p300 signaling integrates epithelial to mesenchymal transition (EMT) and resistance to histone deacetylase inhibitors (HDACis) in colorectal cancer (CRC) cells. The HDACi butyrate, derived from dietary fiber, has been linked to CRC prevention, and other HDACis have been proposed as therapeutic agents against CRC. We have previously discussed that resistance to butyrate likely contributes to colonic carcinogenesis, and we have demonstrated that butyrate resistance leads to cross-resistance to cancer therapeutic HDACis. Deregulated Wnt signaling is the major initiating event in most CRC cases. One mechanism whereby butyrate and other HDACis exert their anti-CRC effects is via Wnt signaling hyperactivation, which promotes CRC cell apoptosis. The histone acetylases (HATs) CBP and p300 are mediators of Wnt transcriptional activity, and play divergent roles in the downstream consequences of Wnt signaling. CBP-mediated Wnt signaling is associated with cell proliferation and stem cell maintenance; whereas, p300-mediated Wnt activity is associated with differentiation. We have found that CBP and p300 differentially affect the ability of butyrate to influence Wnt signaling, apoptosis, and proliferation. ZEB1 is a Wnt signaling-targeted gene, whose product is a transcription factor expressed at the invasive front of carcinomas where it promotes malignant progression and EMT. ZEB1 is typically a transcriptional repressor; however, when associated with p300, ZEB1 enhances transcription. These changes in ZEB1 activity likely affect the cancer cell phenotype. ZEB1 has been shown to promote resistance to chemotherapeutic agents, and expression of ZEB1 is upregulated in butyrate-resistant CRC cells that lack p300 expression. Since the expression of ZEB1 correlates with poor outcomes in cancer, ZEB represents a relevant therapeutic target. Here we propose that targeting the signaling network established by ZEB1, Wnt signaling, and p300 signaling can reverse HDACi resistance and inhibit EMT.

Abstract 275: Combining low dose microtubule targeting agents with belinostat potentiates cytotoxic response in HDACi resistant diffuse large b-cell lymphoma

Abstract Patients diagnosed with Diffuse Large B-cell lymphoma have an overall 60% five-year survival rate. New therapeutic approaches are needed to effectively treat aggressive forms of DLBCL that are refractory to the standard treatment or that relapse within two years of treatment. Histone deacetylase inhibitors (HDACi) are novel therapeutics that are well tolerated in humans and are being extensively evaluated in combination with other therapeutics against hematologic malignancies. Rational selection of companion therapeutics has been difficult due to the cell type-specific mechanisms of HDACi action. In order to address this, we have developed a pre-clinical model system of sensitivity and resistance to HDACi-induced cytotoxicity in DLBCL cell lines that share characteristics with aggressive DLBCL tumors. We previously reported that HDACi resistance is associated with reversible arrest in G1 that involves sustained up-regulation of cyclin-dependent kinase inhibitors. In the current study we demonstrate that HDACi-sensitive cell lines undergo mitotic arrest prior to anaphase in response to treatment with the approved HDACi, belinostat, consistent with activation of the spindle assembly checkpoint (SAC). In contrast, HDACi-resistant cell lines are capable of completing mitosis in the presence of belinostat. To force SAC activation in HDACi resistant cell lines, we used low dose microtubule targeting agents (MTA) vincristine and paclitaxel to induce maximal mitotic arrest with minimal cytotoxicity. The combination of these low dose MTAs and belinostat efficiently caused SAC failure, mitotic slippage, and apoptosis in a synergistic manner. A key mechanism associated with resistance to MTAs is their ability to induce aneuploid cell populations that survive and undergo endoreduplication. The addition of belinostat eliminated the accumulation of a vincristine-induced aneuploid population. Pan-caspase inhibition in conjunction with belinostat and vincristine co-treatment resulted in a sustained survival of the aneuploid population. Thus belinostat triggers apoptosis in the aneuploid cells to enhance the cytotoxic effects of vincristine. Our study identifies the use of low dose MTA/HDACi combination as a potential therapeutic strategy for treatment of relapsed or refractory DLBCL. Citation Format: Aaron P. Havas, Anvi Bhakta, Kameron Rodrigues, Catharine L. Smith. Combining low dose microtubule targeting agents with belinostat potentiates cytotoxic response in HDACi resistant diffuse large b-cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 275.

Essential role of insulin-like growth factor 2 in resistance to histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDIs) are promising anticancer therapies and have been clinically used for the treatment of hematological malignancy. However, their efficacy in solid tumors is marginal and drug resistance hampers their further clinical utility. To develop novel strategies for the HDI-based anticancer therapeutics in non-small cell lung cancer (NSCLC), in the present study, we investigated the mechanisms underlying resistance to HDI treatment in NSCLC cells. We show the STAT3-mediated IGF2/IGF-1R signaling cascade as a key modulator for both acquired and primary HDI resistance. Treatment with HDI up-regulated IGF2 transcription in NSCLC cells carrying intrinsic or acquired drug resistance via direct binding of STAT3 in IGF2 P3 and P4 promoters. Acetylated STAT3 emerged upon HDAC inhibition was protected from the proteasome-mediated degradation of STAT3 and functioned as a direct transcription factor for IGF2 expression. Genomic or pharmacological strategies targeting STAT3 diminished the HDI-induced IGF2 mRNA expression and overcame the resistance to HDI treatment in HDI-resistant NSCLC- or patient-derived tumor xenograft models. These findings provide new insights into the role of acetylated STAT3-mediated activation of IGF2 transcription in HDI resistance, suggesting IGF2 or STAT3 as novel targets to overcome HDI resistance in NSCLC.

A genome scale RNAi screen identifies GLI1 as a novel gene regulating vorinostat sensitivity.

Vorinostat is an FDA-approved histone deacetylase inhibitor (HDACi) that has proven clinical success in some patients; however, it remains unclear why certain patients remain unresponsive to this agent and other HDACis. Constitutive STAT (signal transducer and activator of transcription) activation, overexpression of prosurvival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance; however, none have yet been used to aid the clinical utility of HDACi. Herein, we aimed to further elucidate vorinostat-resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitized cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome protein-coding RNAi library was used to identify genes that when knocked down cooperated with vorinostat to induce tumor cell apoptosis in otherwise resistant cells. Through iterative screening, we identified 10 vorinostat-resistance candidate genes that sensitized specifically to vorinostat. One of these vorinostat-resistance genes was GLI1, an oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI1 small-molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitized tumor cells to vorinostat-induced apoptosis is at least in part through interactions with vorinostat to alter gene expression in a manner that favored apoptosis. Upon GLI1 knockdown and vorinostat treatment, BCL2L1 expression was repressed and overexpression of BCL2L1 inhibited GLI1-knockdown-mediated vorinostat sensitization. Taken together, we present the identification and characterization of GLI1 as a new HDACi resistance gene, providing a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy.

Over-Expression of Beclin-1 Facilitates Acquired Resistance to Histone Deacetylase Inhibitor-Induced Apoptosis

Apoptotic cell death plays a predominant role in histone deacetylase (HDAC) inhibitor-induced cytotoxicity. Nuclear morphological changes and activation of apoptotic executors are involved in CTS203-induced cell death. However, emerging issues of HDAC inhibitor-resistance have been observed in patients. Herein, MCF-7 cells were continuously exposed to CTS203 until the derived cells could proliferate normally in its presence. The newly obtained CTS203-resistant cells were nominated as MCF-7/203R. Compared to MCF-7 original cells, the MCF-7/203R cells were less sensitive to CTS203-induced apoptosis, with a minimal 6-fold higher IC50 value. In contrast, the expression of Beclin-1 was dramatically up-regulated, positively correlated to the acquisition of CTS203-resistance. Our results revealed the participation of autophagy in acquired HDAC inhibitor-resistance and further identified Beclin-1 as a promising target for anti-drug resistance.