Despite some advancements6,7,31
in the treatment of ATRT, long-term outcomes remain suboptimal. In addition, current multimodality therapy for this tumor has proven to be toxic and can be associated with late effects, most notably from intensive radiation dosing delivered to young children. Clearly, ATRT is a malignancy that requires evaluation of newer therapies to improve survival and minimize toxicity.
We have demonstrated in this study that HDI treatment of ATRT cell lines in vitro results in reduced cellular proliferation as measured by MTS and clonogenic assays. In addition, we have shown that a primary ATRT patient sample responds to HDI treatment in a similar manner when compared with established cell lines. Furthermore, we found that the HDI SNDX-275 induces apoptosis in BT-12 and BT-16 cell lines when administered at an IC50
and results in increased p21Waf1/Cip1
tumor suppressor expression. These aspects of our study correlate to previous evaluations that examined the efficacy of HDAC inhibition on rhabdoid cell lines in vitro. In Furchert et al,26
HDAC inhibition by SNDX-275 decreased proliferation of the BT-12 and BT-16 cell lines, but at higher micromolar concentrations than described in our study. In addition, 7.5 µM SNDX-275 was able to elicit an apoptotic response in BT-12 cells after 48 h of incubation. However, in our study, we observed an apoptotic effect of SNDX-275 on BT-12 cells at a much lower concentration. Jaboin et al described the efficacy of SNDX-275 on the renal rhabdoid cell line G401.24
In their study, 1 µM SNDX-275 treatment resulted in cell cycle arrest of G401 cells at 24 and 48 h after drug treatment. In addition, they found that transcription of the p21 tumor suppressor gene (CDKN1A
) was induced by SNDX-275. The HDI romidepsin has also shown in vitro activity against non-CNS rhabdoid tumor cell lines G401 and STM91-01.27
In that study, romidepsin was able to induce cell cycle arrest and restore CDKN1C
tumor suppressor gene expression. Of interest, they also found that forced expression of SMARCB1
in rhabdoid tumor cell lines was associated with increased histone H3 and H4 acetylation at the CDKN1C
promoter. In vivo data has also demonstrated that HDAC inhibition by the HDI depsipeptide results in slower ATRT tumor growth and control in a xenograft model using the BT-40 cell line.25
By evaluating differential gene expression altered by SNDX-275 treatment, we have shown that several key cellular processes are altered by HDI activity. To our knowledge, this is the first study to assess the genomic response to HDAC inhibition in rhabdoid tumor. Of note is the significant alteration of genes involved in cellular growth and proliferation, DNA replication, recombination, and repair and cell cycle. The alterations of these biologic functions correlate well to our data demonstrating the antiproliferative effects of HDAC inhibition on BT-12 and BT-16 cells. Of interest, we specifically found the known tumor suppressor genes CDKN2B
to be upregulated in response to SNDX-275 treatment. Other key biologic processes in malignancy, such as cellular development, cellular movement, and inflammatory response, were also significantly altered by HDAC inhibition. Further investigation will be required to determine these processes’ specific roles in the ATRT response to HDIs. In addition, evaluation of these pathways may prove to be valuable as biological markers of drug response in future experimentation because most current clinical studies focus on histone acetylation as the primary pharmacodynamic parameter.32–35
It has previously been shown that cells with loss of SMARCB1
are more sensitive to DNA damage,36
and we have demonstrated that ATRT is certainly a radiosensitive tumor. Because the effect of radiation can be increased in the context of preexisting DNA damage, it is particularly intriguing that pretreatment with HDIs could enhance the radioresponsiveness of ATRT. This suggests that HDIs lead to DNA damage, a hypothesis that is supported by findings of increased γH2AX foci after HDI treatment of glioma and prostate carcinoma cell lines.20
Our study has demonstrated that SNDX-275 sensitizes ATRT cells to radiation at relatively low drug concentrations and radiation doses. This is presumptively attributable to enhanced, HDI-induced DNA damage, but further studies will be required to elucidate the mechanisms behind this phenomenon. We did, however, observe that 1 of the top 10 biologic networks differentially altered by HDI treatment of BT-12 and BT-16 cells was DNA replication, recombination, and repair. In the clinical setting, the ability of HDAC inhibition to potentiate the effect of radiation in ATRT may allow for the administration of lower radiation doses without sacrificing efficacy. The lower doses could, in turn, aid in decreasing long-term effects seen in young children who receive radiation to the CNS.
HDIs are currently being evaluated in multiple adult clinical trials, including 20 currently active or completed for SNDX-275 administered alone or in combination with other agents.37
However, very few pediatric clinical studies with HDIs have been published to date. In a phase I study, depsipeptide was administered to children with recurrent or refractory solid tumors.38
Although there were no objective responses, the HDI was being used as monotherapy, and the primary goal of the trial was to evaluate dosing. Of note, no patients with ATRT were included in that study. Certainly, further investigation into combination therapy will be needed to evaluate HDIs in the pediatric population because of the in vitro evidence that HDIs frequently function optimally when administered with other treatments. Although HDIs likely have limited use as monotherapy in highly malignant tumors, we believe that our in vitro studies warrant further investigation of the use of HDIs in the context of ATRT, particularly in combination with current therapeutic modalities.