Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.

Background: Cisplatin-induced ototoxicity is related to oxidative stress. Astaxanthin is one of the most powerful antioxidants in nature. Aims/objectives: To investigate the protective effect of astaxanthin on cisplatin-induced ototoxicity. Materials and Methods: Thirty-five Sprague Dawley female rats were divided into 5 groups: control, cisplatin, and cisplatin with 10, 20, and 40 mg/kg astaxanthin groups. Cisplatin group received a single intraperitoneal injection of 14 mg/kg cisplatin. While saline was administered in the control group, in the other 3 groups, 10, 20, and 40 mg/kg daily doses of astaxanthin were administered through orogastric cannula before administration of cisplatin. Baseline and 10th day otoacoustic emission tests were administered. An intracardiac blood sample was taken to measure total antioxidant capacity (TAC), and the cochleas of the animals were investigated histopathologically. Results: Hearing level of astaxanthin 40 mg/kg þ cisplatin group was higher at 24 kHz and 32 kHz frequencies compared to the cisplatin group. The TAC value of the cisplatin group was lower than both the control and astaxanthin þ cisplatin groups (P < .05). On histopathological examination, the other groups were deformed compared to the control group, but no statistically significant difference was observed between the astaxanthin þ cisplatin and cisplatin groups. Conclusions and significance: Astaxanthin showed protective effect at high frequencies when it was administered at high dose. Thus, astaxanthin may have protective effect against cisplatin-induced ototoxicity.

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.

Histone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer agents. Two HDACi, vorinostat (Zolinza®) and romidepsin (Istodax®), are currently FDA approved for use in cutaneous T-cell lymphoma and several others have advanced into Phase II and III clinical trials, both as single agents and in combination with cytotoxics, for a variety of malignancies. Encouraging results have been obtained in haematological malignancies. However, HDACi target many pathways and the full mechanism responsible for their anti-neoplastic activity is still far from clear. An understanding of the molecular mechanisms underlying resistance to HDACi may help to elucidate their mechanism of action and may be of relevance in an attempt to design more effective combination strategies. The purpose of this study is to understand the molecular alterations associated with resistance to vorinostat (Zolinza®). A vorinostat resistant clone (U937-VR) was derived from U937, a monocytic-like histiocytic lymphoma cell line, using a dose escalation protocol. Vorinostat-resistant cells (U937-VR) are able to grow in 2 µM vorinostat without induction of apoptosis. U937-VR cells are cross-resistant to the cytotoxic effects of similar HDACi's such as LBH589, but not to the structurally different benzamide MGCD0103, as measured by propidium iodide (PI) staining and caspase 3/7 activation. Also, resistance demonstrates a partial reversibility that could be indicative of a non-mutational mechanism of resistance. The LD50 of different chemotherapeutic drugs was evaluated by measuring apoptosis with PI staining. U937 parental and U937-VR cells have equivalent LD50 for the DNA damaging agent cisplatin and for the microtubule stabilizing agent taxol, indicating that the apoptotic machinery is intact in U937-VR cells. Interestingly, the resistant cells exhibit increased sensitivity toward chloroquine, an inhibitor of autophagy and to the proteasome inhibitors MG132 and bortezomib. This increased sensitivity correlates with an elevated accumulation of ubiquitinated proteins in U937-VR. These cells also have increased autophagic flux which can be inhibited by knock down of Beclin-1 or Lamp-2, which ultimately restores sensitivity to vorinostat. Autophagy is also activated in parental U937 cells upon treatment with vorinostat, however, in contrast to the resistant cells, its inhibition decreases sensitivity to vorinostat. We therefore propose that autophagy switches from a proapoptotic to a prosurvival signal through chronic exposure to vorinostat. The key players involved in that switch remain to be defined. These data are of importance in the design of combination strategies using inhibitors of autophagy and HDACi for the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4707. doi:1538-7445.AM2012-4707

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, as with many cancer therapies, de novoresistance is common and acquired resistance inevitably follows sensitivity. This issue is particularly difficult to resolve in HDACi therapy, as the mechanism of action is still unclear and may involve several components. Our objective was to understand the molecular mechanisms underlying resistance to HDACi in order to design better combination strategies and to identify predictive biomarkers for response to HDACi therapy. To gain insight into HDACi resistance, we developed vorinostat-resistant clones using a dose escalation protocol in the monocytic-like, histiocytic lymphoma cell line U937 and the diffuse large B-cell lymphoma SUDHL6. Indeed, resistant cells grow in 4 µM vorinostat without induction of cell death. Using a variety of targeted drugs, we evaluated the lethal dose (LD)50in the resistant cells versus their parental counterpart in order to screen for potential pathways involved in resistance to vorinostat. We found that the vorinostat-resistant cells are cross-resistant to other HDACi but not all. Interestingly, the resistant cells exhibit increased sensitivity toward bortezomib, an inhibitor of the proteasome and to chloroquine, an inhibitor of autophagy. Both these drugs target protein processing, suggesting its importance in driving resistance to HDACi. We found that, in addition to elevated autophagy, vorinostat-resistant cells exhibit marks of ER stress, such as dilated ER when visualized by electron microscopy. Moreover, resistant cells have an increased protein synthesis rate and an accumulation of ubiquitinated proteins compared to their parental counterparts. Consistent with this, we observe activation of the UPR in the resistant cells. We hypothesized that activation of UPR could be a mechanism of vorinostat resistance, because the UPR induces the upregulation of pro-survival genes and may induce autophagy. Understanding vorinostat resistance holds clinical relevance in terms of improving HDACi therapy and being able to identify subsets of patients who would benefit most from combination therapy, such as vorinostat with bortezomib. Disclosures: No relevant conflicts of interest to declare.

No abstract

No abstract

Supplementary Figure from Acquired Resistance to EZH2 Inhibitor GSK343 Promotes the Differentiation of Human DLBCL Cell Lines toward an ABC-Like Phenotype