Numerous chemotherapeutic strategies have been attempted previously for patients with DIPG. The classical cytotoxic agents such as cisplatin, cyclophosphamide, carboplatin, etoposide, and vincristine were initially evaluated, but the response rates of children following the use of these drugs were lower than those after conventional radiation therapy . Temozolomide, the current standard chemotherapeutic treatment for GBM, was also attempted for DIPG patients; unfortunately, it did not improve patient outcomes compared to radiation therapy alone . Molecular-targeted therapies have also been explored—for example, the PDGF/PDGFR inhibitor imatinib, the farnesyl transferase inhibitor tipifarnib, the EGFR inhibitors erlotinib and gefitinib, and VEGFR inhibitor vandetanib —which function to block oncogenic signaling pathways. Unfortunately, these attempts at novel therapeutics have also failed to improve outcome of DIPG patients.
Recently, therapeutic focus has shifted towards targeting epigenetic alterations, the tumor microenvironment, metabolism, and immunomodulation [16, 30]. Accordingly, the epigenetic landscape altered by the H3K27M mutation has been considered as a reasonable target against DIPG, especially since alternative mutations are not found in this tumor . Reagents targeting the epigenetic mechanism, such as H3K27 demethylase inhibitors, enhancer of zeste homologue 2 inhibitors, cyclin-dependent kinase 7 inhibitors, and HDAC inhibitors, have been explored and reported . Among these, HDAC inhibitors showed the most promising effects on decreasing the viability of DIPG cells ; indeed, panobinostat and vorinostat are currently undergoing clinical trials as single agents or in combination with other drugs [33–36].
In the current study, we focused on pyroxamide based on the result of synergy screening. Pyroxamide has not been prominently investigated, possibly because it was previously reported to be less effective than other HDAC inhibitors and because it was associated with more side effects [25, 26, 37]. Erythrocytic extramedullary hematopoiesis in the spleen or bone marrow, and splenomegaly have been observed in mice treated with high doses of pyroxamide. Nevertheless, combination with other drugs may reduce the minimum dosage, and thus side effects, of pyroxamide. In our study, pyroxamide exhibited anti-tumor effects most effectively against DIPG cells in combination with the CA9/12 inhibitor, SLC-0111, although its effectiveness and toxicity need to be confirmed in vivo. This provides strong rationale for investigating pyroxamide in combination with other drugs.
The mechanism of action by which HDAC inhibitors elicit their effect is still not fully understood; however, various models have been proposed. HDAC inhibitors can affect cell cycle arrest, apoptosis, autophagy, non-coding RNA, cell differentiation via extracellular signal-regulated kinase pathways, anti-angiogenesis, and immunomodulation . In terms of HDAC inhibitors’ effects on apoptosis, the p21 and p53 genes have been the focus of prior investigations [39, 40]. Treatment with HDAC inhibitors releases HDAC1 from Sp1 (Promoter-specific RNA polymerase II transcription factor) and increases p21 expression , which mediates cell cycle arrest and apoptosis [41, 42]. HDAC inhibitors also increase the acetylation of the p53 protein, which in turn interacts with p21 ; thus, both p21 and acetylated p53 are thought to reflect the effect of HDAC inhibitors. We evaluated p21 in the current study because it is one of the few reported downstream markers of pyroxamide activity . We expect that p21 evaluation could be extrapolated as a downstream biomarker of efficacy of pyroxamide treatment in in vivo experiments. However, although the addition of SLC-0111 indeed enhanced histone acetylation, cell cycle arrest, and apoptosis, the expression of p21 decreased in three of four cell lines. The reason for this decrease may be that SLC-0111 itself has some role in weakening p21 expression, or that we did not capture p21 at the optimal time point in its arc of transient expression . Another compelling explanation might be that p21 acts as an anti-apoptotic to restore balance in the cell cycle, as has been suggested previously . Acetylated p53 may be a good indicator of HDAC inhibitors’ efficacy even when combined with SLC-0111 ; nonetheless, p21 has also been reported to inhibit the effect of p53 . Further experiments are needed to account for the changes we noted in p21 expression. Taken together, our results suggest that SLC-0111 can enhance the effect of pyroxamide in terms of histone acetylation and apoptosis (Fig. 5); however, the implications of p21 and acetylated p53 expression will need to be reconsidered when attempting to measure the effect of HDAC inhibitors, at least in combination with the CA9 inhibitor, SLC-0111.
The expected result of HDAC inhibitor treatment is to restore the function of the histone. However, HDAC inhibitors can affect a variety of pathways as described above . Due to the universal importance of histone acetylation in normal physiology, it might be challenging to eliminate the possibility of off-target effects . Moreover, HDAC inhibitors have shown anti-tumor effects against H3 wild-type DIPG cells as well as H3K27M cells . This may suggest that the effectiveness of HDAC inhibitors is not specific to the epigenetic landscape of DIPG. Regardless, the efficacy of HDAC inhibitors against DIPG cells has been corroborated in several preclinical studies [32, 46]. Therefore, we still consider HDAC inhibitors to be promising anti-cancer drugs. To fully uncover the potential of HDAC inhibitors, it is important to further elucidate the complex nature of histones themselves, as well as to complete clinical trials that are currently underway .
DIPG presents an additional challenge in terms of drug delivery. Unlike other high-grade gliomas such as GBM, many DIPGs do not show gadolinium extravasation under magnetic resonance imaging indicating that the blood-brain barrier (BBB) remains intact in DIPG . The BBB prevents many drugs from reaching the central nervous system, thus making it difficult to treat brain tumors effectively with chemotherapeutic agents. To overcome this problem, molecular, cellular, and physical strategies, including focused ultrasound and convection-enhanced delivery, are being investigated [23, 48]. Despite the promise shown by combination therapy with CA9 and HDAC inhibitors, this drug regimen may need to be augmented with transient focal BBB opening to elicit sufficient effect and avoid systemic toxicity.
CA9 is overexpressed in solid tumors like breast and lung cancer . In brain tumors, upregulation of CA9 has been reported in astrocytoma, oligodendroglioma, meningioma, hemangioblastoma, choroid plexus tumors, and in pediatric brain tumors, ependymoma, medulloblastoma, and primitive neuroectodermal tumors . Importantly, our study is the first report describing CA9 expression in DIPG. Through our RNA sequencing data analysis, we found CA9 to be significantly upregulated in DIPG compared to normal brain tissue. Moreover, the pathways of hypoxia, EMT, and glycolysis, in which CA9 has a major role, were all activated. Although the CA9 inhibitor SLC-0111 did not suppress the proliferation of DIPG cell lines, SLC-0111 single treatment inhibited cell invasion and migration, as has been shown for other tumor types such as breast and pancreatic cancers [9, 51], supporting the rationale for targeting the CA9 gene.
Additionally, our pHi measurements indicate that pyroxamide may also enhance the primary effect of SLC-0111—namely, a lowering of pHi. SLC-0111 did not decrease pHi in our studies possibly because the exposure time to each reagent (15 min) might have been insufficient to see an effect. HDAC inhibitors may indirectly help reduce pHi by suppressing angiogenic pathways and the hypoxia-inducible factor 1 pathway as postulated previously . In summary, CA9 is a valid and encouraging therapeutic target in DIPG. Additionally, pyroxamide and SLC-0111 may mutually reinforce one another (Fig. 5), making the combination of these two drugs a promising strategy against DIPG. Since no other studies focus on carbonic anhydrase inhibition in the context of DIPG, we hope that our findings herein will encourage future investigation into the DIPG microenvironment and hypoxia, ultimately spurring creative new interventions for this devastating disease.
The CA family in mammals is divided into four broad subgroups comprised of several isoforms . Another potentially important finding of note from our study is the significant downregulation of expression of the CA4,7 and 11 in DIPG relative to normal brain. The tissue specific functional roles of these CA isoforms are not well understood, but their significant downregulation may provide clues about DIPG pathophysiology and requires further investigation.
Future studies in this field should evaluate drug response in DIPG samples that have low or no expression of CA9 protein. Our RNA seq data analysis showed high expression of CA9 in DIPG, but on the other hand, there are indeed samples that did not express CA9. Therefore, any proposed benefit described in this study can only be applied to CA9-positive DIPG, and should be further validated in CA9-negative samples. Our study does not take into account the differences in synergy effect that may be seen with SLC-0111 and other HDAC inhibitors.