Christa Manton

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

Specific and prolonged proteasome inhibition dictates apoptosis induction by marizomib and its analogs

Marizomib (NPI-0052) is a naturally derived irreversible proteasome inhibitor that potently induces apoptosis via a caspase-8 and ROS-dependent mechanism in leukemia cells. We aim to understand the relationship between the irreversible inhibition of the proteasome and induction of cell death in leukemia cells by using analogs of marizomib that display reversible and irreversible properties. We highlight the importance of sustained inhibition of at least two proteasome activities as being key permissive events for the induction of the apoptotic process in leukemia cells. These data provide the basis for the development of new approaches to generate more effective anti-proteasome therapies.

Efficacy of panobinostat and marizomib in acute myeloid leukemia and bortezomib-resistant models

Current relapse rates in acute myeloid leukemia (AML) highlight the need for new therapeutic strategies. Panobinostat, a novel pan-histone deacetylase inhibitor, and marizomib, a second-generation proteasome inhibitor, are emerging as valuable therapeutic options for hematological malignancies. Here we evaluated apoptotic effects of this combinatorial therapy in AML models and report earlier and higher reactive oxygen species induction and caspase-3 activation and greater caspase-8 dependence than with other combinations. In a bortezomib refractory setting, panobinostat induced high levels of DNA fragmentation, and its action was significantly augmented when combined with marizomib. These data support further study of this combination in hematological malignancies.

Oxidative Stress and the Proteasome: Mechanisms and Therapeutic Relevance

The proteasome is a key mediator of the oxidative environment in cells. Reactive oxygen species (ROS) are produced by normal cellular metabolic processes, and the proteasome can mediate ROS levels by degrading proteins that generate ROS and by controlling antioxidant turnover, as well as by clearing oxidatively damaged proteins from cells. The proteasome itself is also regulated by ROS, with certain subunits being susceptible to oxidative modification and damage, while other subunits are transcriptionally up-regulated as part of the antioxidant response. Proteasome inhibition has been shown to increase ROS in many cellular contexts, and this increase in ROS is often integral to cell death induction. Cells that have elevated basal levels of antioxidants, or that can mount a quick antioxidant response that often includes increasing proteasome subunit levels, can neutralize ROS and escape proteasome inhibitor-induced death. Many current studies are focused on overcoming this resistance by combining proteasome inhibitors with other ROS-generating agents, such as histone deacetylase inhibitors and certain kinase inhibitors, which can cause synergistic ROS induction and death.

Abstract C45: Comparison of kinetics and mechanism of cell death induction by the proteasome inhibitors bortezomib and marizomib in glioblastoma.

Two proteasome inhibitors, bortezomib (BTZ) and carfilzomib (CFZ), are FDA-approved for treatment of specific liquid tumors, validating the proteasome as a target for cancer therapy. Marizomib (MRZ) is another inhibitor studied in Phase I and II settings. These drugs have different inhibitory profiles that lead to differences in efficacy and toxicity. In leukemia, MRZ causes longer lasting proteasome inhibition and greater cell death than BTZ. These same studies found that MRZ is more dependent on the caspase-8 pathway of apoptosis and production of reactive oxygen species (ROS), while BTZ is more equally dependent on caspases-8 and -9. Given their success in liquid tumors, we were interested in applying these drugs to the solid tumor glioblastoma (GBM), a deadly brain tumor with a median survival time of 15 months. To determine which proteasome inhibitor (PI) was the most potent for GBM, we examined the mechanism of apoptosis induced by BTZ and MRZ as well as the potential for these drugs to cross the blood brain barrier (BBB) to be delivered to brain tumors. We find that BTZ causes longer lasting proteasome inhibition and greater cell death than MRZ in GBM cell lines. This is unexpected, as it is opposite of what was seen in leukemia. Examination of initiating caspase cleavage revealed an early cleavage of caspase-2 (4 h) followed by a later cleavage of caspases-8 and -9 (8-12 h). Early activation of caspase-2 was confirmed using a bimolecular fluorescence complementation model, where caspase-2 induced proximity by recruitment to its activation platforms gives off a fluorescent signal. Furthermore, MRZ induced stronger activation of caspases than BTZ, suggesting that MRZ treatment leads to up-regulation of a resistance mechanism that prevents it from carrying out apoptosis downstream of caspase activation. We also examined the role of ROS in PI-mediated cell death. We treated cells with N-acetyl cysteine (NAC), which increases synthesis of the antioxidant glutathione (GSH). NAC inhibited caspase activation and strongly protected against both BTZ- and MRZ-induced apoptosis. However, raising GSH levels by introducing glutathione ethyl ester did not have the same protective effect. Ongoing studies indicate that NAC is acting independently from its impact on GSH, as NAC still protects cells from proteasome inhibitors when GSH production is inhibited by treatment with buthionine sulfoximine. These data provide insight into mechanisms of PI resistance, perhaps through modulation of the cellular cysteine pool. Though BTZ is more potent in vitro, the question of delivery to brain tumors is an important clinical factor. Using an intracranial, orthotopic model of GBM we find that marizomib causes greater accumulation of the proteasome substrate p27 in brains and tumors of these mice. All together, this research aims to understand the mechanism of these inhibitors to determine the most promising therapeutic regimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C45. Citation Format: Christa A. Manton, Blake Johnson, Lisa Bouchier-Hayes, Joya Chandra. Comparison of kinetics and mechanism of cell death induction by the proteasome inhibitors bortezomib and marizomib in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C45.

Abstract 5429: Inhibition of LSD1 and HDACs causes synergistic cell death in glioblastoma cells

Glioblastoma multiforme is a particularly aggressive brain tumor and remains a clinically devastating disease. More than 90% of children diagnosed with glioblastoma die within 2 years making it among the leading causes of cancer related morbidity and mortality. Despite innovative therapies for the treatment of glioblastoma, there has not been a significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations have become popular targets for cancer therapy due to their ability to control cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACi) have been developed and are in clinical use for cancer therapy. Histone methylation can be modulated by HDACi and here we propose to examine the effect of dually inhibiting deacetylases and demethylases. Specifically, we focused on LSD1, lysine specific demethylase 1, a demethylase known to be in complex with HDAC1/2 and implicated in several high risk cancers. We evaluated apoptosis induction upon knockdown of LSD1 in combination with HDACi in glioblastoma cell lines. Our results demonstrate a greater than two-fold increase in apoptosis in LSD1 knockdown cells treated with increasing doses (1-5 μM) of HDACi for 48 hours as compared to control as measured by DNA fragmentation by propidium iodide staining and analysis by flow cytometry. Furthermore, pretreatment of LSD1 knockdown cells with the general caspase inhibitor zVAD-fmk prior to treatment with vorinostat only partially protects against DNA fragmentation suggesting that cell death proceeds through both caspase-dependent and -independent mechanisms. Similar results were obtained by pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine. Treatment of LN-18 cells with combinations of tranylcypromine (100-1000 μM) and vorinostat (1-5 μM) for 72 hours demonstrate that these two agents synergize to promote cell death with 1000 μM tranylcypromine and 5 μM vorinostat showing the most synergistic effect (CI 0.313). Surprisingly, the increase in apoptosis observed upon LSD1 inhibition, by knockdown and pharmacological inhibitors, and HDACi also correlates with an increase in intracellular superoxide as measured by hydroethidum staining and analysis by flow cytometry. These data suggest that reactive oxygen species may play a role in the apoptosis induced by the combination of LSD1 and HDAC inhibition. Further studies aimed at understanding the mechanism by which LSD1 and HDAC inhibition leads to enhanced tumor cell death will substantially increase our knowledge of how epigenetic modifiers interact to control cell survival and may suggest novel options for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5429.