HDAC inhibitors induce cell cycle arrest, activate the apoptotic extrinsic pathway and synergize with a novel PIM inhibitor in Hodgkin lymphoma‐derived cell lines (2010)

Molecular studies have shown that the tumoural component of classical Hodgkin lymphoma (cHL), Hodgkin and Reed-Sternberg (HRS) cells, shows a profound disturbance of the cell-cycle and apoptosis regulation (Küppers, 2009). Although cHL is often curable, there remains a group of patients who do not achieve remission with initial therapy and about 30% patients will die of the disease. Therefore, novel targeted agents need to be developed to reduce treatment-related toxicity and to further patient outcome. Histone deacetylase inhibitors (HDACi) are new agents with demonstrable preclinical antitumour activity in a wide range of malignancies. However, their exact mechanism of action remains to be fully elucidated. The rationale for evaluating HDACi in cHL is based on observations implicating epigenetics in the HRS-phenotype, on high levels of HDACs in approximately 80% cHL cases (Gloghini et al, 2009), and on the deregulation in cHL of pathways that can be restored by HDACi in other cancer types (Garcia et al, 2003). In order to test drugs therapeutically useful in cHL, we used the Connectivity Map (Cmap) program and a gene expression signature from poor-prognosis cHL-patients, (Sanchez-Aguilera et al, 2006). Cmap revealed two HDACi, Trichostatin A and SAHA, to be the most specific drugs able to reverse the cHL-chemoresistance signature, suggesting that HDACi might be very efficient in cHL-treatment (Fig S1A). To demonstrate HDACi efficiency as antitumoral agents in cHL, four cHL-derived cell lines were treated with two HDACi (suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB)). HDACi significantly reduced cell viability in all cHL cell lines in a dose-dependent manner (Fig 1A), with different effects on cell-cycle in each cell line. Both HDACi induced a time-dependent G2/M phase arrest in L428 and KMH2 cell lines, similar to recent findings with SAHA (Buglio et al, 2008) and depsipeptide (Hartlapp et al, 2009); while HDLM2 and L1236 cell lines did not experience cell-cycle arrest (Figs 1B and S1B[link]). Moreover, a significant and time-dependent increase in apoptosis in all cell lines was observed after HDACi-treatment (Fig 1C). Effect of HDACi on cHL-derived cell lines. L428, KMH2, HDLM2 and L1236 cell lines were treated with SB or SAHA at several doses and times, and (A) cell viability, (B) cell cycle, (C) apoptosis, and (D–E) gene expression changes were determined. (A) Cell viability was measured by ATP intracellular content and calculated as the percentage of DMSO-treated cells; 50% inhibitory concentration (IC50) values for 48 h are shown. (B) L428 and KMH2 cells were arrested at G2/M phase and underwent cell death in a time-dependent manner. HDLM2 and L1236 cells were not arrested, but cell death was induced after long treatments. Numbers indicate the percentage of HDACi-treated cells minus percentage of DMSO-treated cells (mean ± SEM) (*indicates statistically significant differences from the control, P < 0·05). (C) Apoptosis was calculated as the percentage of non-viable cells (AnnexinV+/Propidium Iodide- and AnnexinV+/Propidium Iodide+ cells) in HDACi-treated cells minus the percentage of non-viable cells in DMSO-treated cells (mean ± SEM) (*indicates statistically significant differences from the control, P < 0·05). (D) Altered pathways in L428 and HDLM2 cell lines after treatment with 1 mmol/l SB and 2 μmol/l SAHA at the indicated times (FDR < 0·25). Red, green and white indicate upregulation, downregulation and no significant deregulation, respectively. Intensity of colours indicates the degree of significance (dark: FDR < 0·05; intermediate: 0·05 < FDR < 0·1; light: 0·1 < FDR < 0·25). (E) Hierarchical clustering with all 1946 significantly deregulated genes (FDR < 0·1) in L428 and HDLM2 cell lines after treatment with 1 mmol/l SB and 2 μmol/l SAHA at the indicated times. Data were normalized with respect to untreated control cells. Highlighted genes indicate the mechanism of action of HDACi in cHL cell lines. As L428 and KMH2 seemed to respond similarly to HDACi (G2/M arrest), while the response of HDLM2 and L1236 was different (no cell-cycle arrest), L428 was used as the sensitive cell line and HDLM2 as the resistant cell line in further experiments. To ensure that HDACi were able to target HDACs we confirmed histones H3 and H4 hyperacetylation upon HDACi-treatment (Fig S2A). The differential sensitivity observed in cHL-cell lines after HDACi-treatment could be explained by differences in basal status of acetylated histones and gene expression profile (Fig S2A,B), because the HDLM2 cell line, with a poor-prognosis molecular profile compared with L428, and a inactivating TP53 mutation (Feuerborn et al, 2006), is more resistant to HDACi-apoptosis. Moreover, overexpression of antiapoptotic proteins, such as Bcl2, is known to be involved in resistance mechanisms to HDACi. Since histone-acetylation is a well-known mechanism of gene expression regulation, we evaluated genes that showed modified expression after HDACi-treatment. Data obtained from three independent hybridizations for each time point were analysed by a t-test. This approach revealed deregulation of <5% of the whole genome in each HDACi-treated cHL-cell line (false discovery rate [FDR]<0·1) in agreement with most of studies performed in HDACi-treated cancer cell lines. Gene Set Enrichment Analysis (GSEA) identified several pathways altered by HDACi (Fig 1D). The t-test found 1946 differentially expressed genes (FDR < 0·1) from 0 h to at least one subsequent time point in both cHL-cell lines after treatment (Fig 1E). In accordance with the functional analysis, a number of cell cycle (CDKN1A, CDKN2D) and apoptosis genes (TNFSF9, TNFSF10, TNFRSF12A, BCL2L11 and BCL2L12) were significantly deregulated. A number of genes associated with chemoresistance in cHL were consistently downregulated by HDACi (Fig 1E). This supports that HDACi could be added to current chemotherapy protocols, given that a number of evidences have indicated the benefits of the combined treatment (Bhalla et al, 2009). The complete list of significant genes is available in Table S1. GSEA analyses identified apoptosis as one of the most important HDACi-modified pathways. To confirm these alterations, we measured the key proteins involved in apoptosis. Consistent with the results of our GSEA, we observed subtle HDACi-effects on Bax levels (intrinsic apoptosis) and a release of Cytochrome-c (Fig S2C). Moreover, there was a decrease in ProCaspase-8 and Fas levels, indicating extrinsic apoptotic pathway activation. Surprisingly and in contrast with many other tumour entities, HRS cells strongly expressed Fas, activating nuclear factor (NF)-kB transcription factor (Mathas et al, 2004), and suggesting the importance of death receptor apoptosis signalling in HRS cells. Furthermore, the extrinsic apoptotic pathway, alone or in conjunction with the intrinsic pathway, has already been reported to be induced by SAHA in other cell types (Gillenwater et al, 2007). Unexpectedly, we found a strong upregulation of the proto-oncogene PIM2 in both HDACi-treated cHL-cell lines (1, 2). Moreover, we have previously observed significant upregulation of PIM1 (FDR = 0·03) and PIM2 (FDR = 0·001) in a series of 29 cHL-patients (Fig S3A) (published data from Sanchez-Aguilera et al, 2006).These findings prompted us to hypothesize that HDACi effects could benefit from PIM2 inhibition. We performed cell viability assays with a novel PIM inhibitor (PIMi) and found it to be efficient in four cHL-cell lines (Fig 2B). PIMi efficiency was checked by measuring its target pBAD(Ser112) (Fig S3B). HDACi have a synergistic effect with a PIM inhibitor. (A) After treatment with HDACi, a significant upregulation of PIM2 was observed in L428 and HDLM2 cell lines. The expression value of PIM2 was normalized with respect to control untreated cells. (B) Four cHL-derived cell lines were treated with a novel PIMi for 48 h, and IC50 values were calculated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. (C) Cell lines were treated simultaneously with the PIMi and with HDACi (SB and SAHA) for 48 h, using IC50 values of each drug in each cell line, and cell viability was measured by MTT assay. Data are representative of two independent experiments. Finally, we performed experiments with HDACi and PIMi in cHL-cell lines. The combination SB+PIMi was as efficient as each single drug in all cell lines but HDLM2, where the effect was synergistic. However, the combination SAHA+PIMi enhanced the reduction in cell viability in a synergistic manner in all four of the cell lines (Combination Index = 0·19, 0·57, 0·73 and 0·83 for HDLM2, L428, KMH2 and L1236, respectively) (Fig 2C). This finding highlights the importance of SAHA+PIMi combination in cHL therapeutics. Further studies will be needed to fully understand why PIM2 is upregulated after HDACi-treatment, the underlying mechanisms of this drug combination, and the role of PIM1 and PIM2 in cHL. In conclusion, we have demonstrated that HDACi exert antiproliferative and proapoptotic effects on cHL-cell lines by altering the expression of cell-cycle and apoptosis-genes, with common and different (cell line-dependent) effects. Additionally, HDACi can downregulate the expression of genes associated with chemoresistance in cHL-patients. Importantly, SAHA acts in a highly synergistic manner with a novel PIMi in cHL-cell lines. Collectively, our results provide the proof-of-principle that justifies the investigation of HDACi in combination with PIM inhibitors for cHL-treatment. The authors would like to thank to Antonia Alvarez (Flow Cytometry and Confocal Microscopy Service, University of the Basque Country) for her technical assistance with the flow cytometer; and to Gonzalo Gomez (Bioinformatics Unit, Spanish National Cancer Research Centre) for his bioinformatic help. The authors declare no competing financial interests. This work was supported by grants from RTICC (RD/06/0020/0048), the Basque Government (GIC07/61-IT-463-07), Ministerio de Sanidad y Consumo (FIS, PI08/1985) y Fundación Mutua Madrileña. EMS is supported by a grant from the Department of Education, Universities and Research of the Basque Government (BFI08·207). EMS performed experiments, analysed and interpreted the data and wrote the manuscript; MSB interpreted the data and revised the manuscript; MER and CGA performed experiments; BSE analysed data; JRB contributed vital reagents for the development of the novel PIM inhibitor; MAP revised the manuscript; AGO designed the research and revised the manuscript; JFG designed the research, interpreted the data and revised the manuscript. Figure S1. (A) Output from Connectivity Map (Cmap). Top ten pharmaceutical perturbagens identified through the Cmap that reverse cHL-chemoresistance signature (with negative enrichment score) in the panel of cell lines included in the software. Trichostatin A and SAHA, also known as vorinostat, belong to the HDACi family. (B) Cell-cycle changes induced by HDACi in cHL-derived cell lines. HDACi induced a time- and dose-dependent G2/M arrest and cell death in L428 and KMH2 cells, while no cell cycle arrest but cell death after high doses/long treatments was induced in HDLM2 and L1236 cells. Numbers indicate mean ± SEM of at least three independent experiments. Figure S2. Molecular alterations induced by HDACi in cHL-derived cell lines. (A) Effect of HDACi on histone acetylation: L428 and HDLM2 cell lines were treated with SB (1 mmol/l) and SAHA (2 μmol/l) at the indicated times, and levels of acetylated and total histones H3 and H4 were measured by western blot. Numbers represent the ratio of acetylated/total histones. (B) Basal differentially expressed genes between L428 and HDLM2 cell lines (FDR < 0.05). Highlighted genes could explain the different sensitivity and response to HDACi treatment. (C) Effect of HDACi on key proteins involved in apoptosis: L428 and HDLM2 cell lines were treated with SB (1 mmol/l) and SAHA (2 μmol/l) at the indicated times, and levels of whole-cell lysed proteins were measured by western blot. Numbers represent the ratio of measurements of each protein relative to α-tubulin levels. Figure S3. (A) PIM1 and PIM2 expression levels in cHL. Two sorted centroblasts samples, five lymph nodes and a series of 29 cHL cases (14 with favorable outcome and 15 with unfavorable outcome) were profiled for PIM1 and PIM2 expression levels. (B) Levels of pBAD(Ser112) and BAD were measured after treatment with IC50 values of PIMi in cHL-cell lines. Table S1. Expression values normalized with respect to untreated control cHL cell lines and a brief description extracted from Feature Extraction software of all 1946 significantly deregulated genes identified by a t-test (FDR < 0.1) in L428 and HDLM2 cell lines after treatment with 1 mmol/l SB and 2 μmol/l SAHA at the indicated times. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.