Targeting enzymes involved in epigenetic regulation of gene expression has become increasingly popular as a cancer therapy. The success of the HDACi vorinostat, the first HDACi to have been approved by the FDA for treatment of cutaneous T-cell lymphoma, prompted the development of other small molecule HDACis for clinical use. Although HDACis show promise in many cancer types, they are not as effective against solid tumors as monotherapies.7,10
Therefore, the identification of agents that sensitize tumor cells to HDACis is highly clinically relevant. We evaluated the ability of 2 broad-spectrum HDACi, vorinostat and PCI-24781, to cause changes in histone acetylation and methylation in GBM cell lines. We found that both of these HDACis led to increased levels of histone H3 acetylation and H3K4 methylation, a modification removed by the LSD1 demethylase. On the basis of these results, we sought to determine whether targeting LSD1 and HDACs simultaneously caused enhanced GBM tumor cell death. We found that shRNA knockdown of LSD1 in GBM cells led to sensitization to the broad-spectrum HDACis vorinostat and PCI-24781. The cell death observed by the combination treatment was partially blocked (~50%) by pretreatment with the general caspase inhibitor zVAD-fmk, suggesting that cell death occurs partially through the apoptotic pathway, but other caspase-independent pathways of cell death are also likely to contribute to our observations. Supporting these data are several reports of vorinostat inducing autophagy in a variety of cell types.35
We also obtained similar results when HDACis were combined with chemical inhibition of LSD1 with the monoamine oxidase inhibitor, tranylcypromine. Importantly, we demonstrated that combined inhibition of LSD1 and HDACs is selective for GBM tumor cell lines versus immortalized human astrocytes, an observation that may be due to elevated levels of LSD1 in GBM cell lines and patient samples (Fig. ). Overall, these data offer a novel therapeutic option for the treatment of GBM.
It has been demonstrated that each HDACi has a different ability to inhibit various HDAC enzymes, some more specifically than others. The targets of each HDAC enzyme, both histone and nonhistone proteins, and their contribution to GBM is still unclear. In addition, most HDACis target the active site of the HDAC and function through moieties that chelate zinc. Therefore, other proteins that require zinc for their function may also be affected by very high doses of HDACis. However, efforts to improve HDACis have yielded compounds with chemical modifications that provide some selectivity against other zinc-containing enzymes. Our studies were performed with doses that have previously proven to be effective in inducing tumor cell death in vitro and in vivo32–34
and are clinically relevant. The maximum plasma concentration for vorinostat in patients is 2.5 μM for oral administration of 400 mg per day and 9 μM for 300 mg/m2
per day for intravenous administration,37
which is well within the range of doses used in the current study.
Histone tails are modified by a variety of posttranslational modifications, including phosphorylation, acetylation, methylation, and ubiquitination. An emerging hypothesis is that these modifications do not act alone but instead influence one another.38,39
Our data indicate that, in GBM cell lines, histone acetylation influences the demethylation of H3K4 (Fig. ). Consistent with our results, inhibition of HDACs in HEK293 cells also influences methylation on nucleosomes in vivo and acetylation of nucleosomes decreases demethylation by LSD1 in vitro.22
One mechanism proposed to explain the interplay between histone acetylation and methylation is the physical association of HDAC1 and LSD1 by which each enzyme influences the activity of the other.22
More recently, Huang et al40
provided an alternative mechanism and demonstrated that treatment of prostate cancer cells with HDACi leads to the suppression of histone demethylase enzymes, including LSD1, through transcriptional repression of Sp1. These data indicate that there is a biological link between acetylation and H3K4 methylation and suggest targeting LSD1 as a possible therapeutic strategy. Similar to HDACs, LSD1 has also been reported to target proteins other than histones, which may play a role in the enhanced apoptosis observed on combined LSD1 and HDAC inhibition. For example, p53 is a substrate of LSD1.41,42
Demethylation of lysine 370 of p53 inhibits association of the p53-binding protein 1, leading to the down-regulation of p53-regulated genes, such as the pro-apoptotic p21 protein.41
In addition to regulating methylation of p53, LSD1 is also recruited by p53 to the α-fetoprotein gene during hepatic development, in which loss of p53 leads to decreased occupancy of LSD1, an increase in H3K4 methylation, and a loss of gene repression.42
To determine the contribution of p53 to the synergy observed with HDACis and tranylcypromine, we used GBM cell lines that contain wild-type p53 (U87) and mutant p53 (LN-18) in which there is a mutation in the DNA binding domain. We found no difference in the ability of tranylcypromine and HDACis to cause synergistic cell death in these 2 cell lines, suggesting that the cell death caused by these agents is p53-independent, although additional studies are required in isogenic cell lines to prove this concept. Additional studies aimed at determining the nonhistone targets of LSD1 will potentially reveal alternative pathways that contribute to the synergistic cell death observed in our studies.
Because of the homology of LSD1 with the amine oxidase family of enzymes, several mono- and polyamine oxidase inhibitors have been evaluated as inhibitors of LSD1.19,23,24
Inhibition of LSD1 in vivo leads to the reexpression of aberrantly silenced genes.23,26,27
These inhibitors have been used in mouse xenograft models with success,27,28
providing evidence for the use of these agents in the treatment of cancer. Our data demonstrate that the MAOi tranylcypromine enhances the cytotoxic effect of HDACis. Although tranylcypromine is a good candidate for use in combination studies for the treatment of GBM, because it is already clinically used for the treatment of depression, it is often associated with unfavorable side effects due to interactions with certain foods and medications.43
Moreover, the specificity of tranylcypromine for MAOs is much higher than that for LSD1,44
which may induce unwanted adverse effects when used for cancer chemotherapy. Several groups have developed new compounds that are more specific LSD1 inhibitors and are being evaluated preclinically.27,44–46
Our studies suggest that these new compounds could be evaluated, alone and in combination with HDACis, in GBM.
In conclusion, our work demonstrates that inhibition of LSD1 in combination with HDACis results in synergistic cell death. These data provide proof of principle experiments that indicate that combined inhibition of HDACs and LSD1 leads to enhanced GBM cell death. Future studies are focused on evaluating the combination of vorinostat and tranylcypromine in the intracranial glioblastoma mouse model described by Lal et al47
and understanding the mechanism(s) by which HDAC and LSD1 inhibition enhances tumor cell death in vitro and in vivo. One possible explanation is that HDACs and LSD1 cooperate to regulate the expression of genes involved in apoptosis. In fact, treatment of LSD1-knockdown cells with vorinostat yields changes in several genes known to be involved in apoptosis, including Bid, p53, and p73; several tumor necrosis factor receptors; and caspases (unpublished data). Overall, these studies are designed to make substantial contributions to the understanding of GBM biology, which may provide molecular explanations for resistance and identify novel targets for therapy.
Conflict of interest statement. None declared.