Highlights d CBL0137 inhibits DIPG tumor growth and restores H3K27me3 through FACT inhibition d Co-administration of CBL0137 and panobinostat enhances survival in DIPG xenografts d CBL0137 and panobinostat synergistically inhibit the Rb/ E2F1 pathway and restore H3K27me3

Diffuse midline gliomas (DMGs) are invariably fatal pediatric brain tumours that are inherently resistant to conventional therapy. In recent years our understanding of the underlying molecular mechanisms of DMG tumorigenicity has resulted in the identification of novel targets and the development of a range of potential therapies, with multiple agents now being progressed to clinical translation to test their therapeutic efficacy. Here, we provide an overview of the current therapies aimed at epigenetic and mutational drivers, cellular pathway aberrations and tumor microenvironment mechanisms in DMGs in order to aid therapy development and facilitate a holistic approach to patient treatment.

Diffuse Intrinsic Pontine Glioma (DIPG) are devastating paediatric brainstem tumours. Loss of function mutations in DIPG decrease genetic stability and impair DNA damage response pathways promoting tumourigenesis. Polo-like Kinase 1 (PLK1) is a pivotal controller of cell growth, regulating key intermediaries of DNA replication, homologous repair, the cell cycle and cell division. We have found DIPG cultures consistently overexpress PLK1 with inhibition resulting in decreased tumour cell growth, heightened cell cycle arrest and apoptosis. Single agent treatment using PLK1 inhibitors unprecedentedly doubled the median survival of animals harbouring DIPG tumours. Through gene expression analysis, we’ve showed PLK1 inhibition affected multiple pathways which control the cell cycle, cell death regulation, microtubule organization and regulation of cell migration. We found these pathways of differentially expressed genes were significantly enriched for known targets of both E2F1 and E2F4. Analysis of gene expression and proteomic studies also revealed PLK1 inhibition decreased the activation and expression of key tumour promoting mediators within multiple phases of the cell cycle, decreased expression of tumour promoters including MYC and the PI3K/mTOR pathway and reactivated tumour suppressors p53 and PTEN. Assessing these changes in the treated transcriptome and proteome, we aim to develop multiple potentially translatable combination treatment strategies for DIPG. We have performed mechanistic studies and identified synergism with PLK1 inhibitors and the epigenetic regulator panobinostat, bet/bromodomain inhibitor JQ1, dual PI3K/mTOR inhibitor bimiralisib and PI3K inhibitor BKM120. Finally, we found PLK1 inhibitors act as potent radiosensitizers, enhancing the therapeutic effects of radiotherapy in vitro and in vivo.

DIPG is an aggressive and incurable childhood brain tumour for which new treatments are needed. A high throughput drug screen of 3500 pharmaceutical compounds identified anti-malarials, including quinacrine as having potent activity against DIPG neurospheres. CBL0137, a compound modelled on quinacrine, is a novel anti-cancer compound which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. CBL0137 effectively crosses the blood-brain barrier and has recently completed Phase I testing in adult patients. CBL0137 induced apoptosis in DIPG neurospheres and had profound cytotoxic activity against a panel of DIPG cultures. In a DIPG orthotopic model, treatment with CBL0137 significantly improved survival. We found that treatment with CBL0137 up-regulated TP53 and increased histone H3.3 acetylation and tri-methylation in DIPG cells. We therefore examined the interaction between CBL0137 and the histone deacetylase (HDAC) inhibitor panobinostat. In vitro experiments showed that the two agents had profound synergistic activity against DIPG neurospheres in clonogenic assays and enhanced caspase activation and apoptosis. The FACT subunit SSRP1 was found to directly interact with H3.3K27M and treatment with CBL0137 targeted this epigenetic defect, restoring histone H3.3 trimethylation and leading to tumor cell death. Transcriptomic analysis and immunoblotting indicated that combination treatment activated signalling pathways controlled by Retinoblastoma (RB)/E2F1 and subsequently increased phosphorylation and enzymatic activity of enhancer of zeste homolog 2 (EZH2). Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged survival in two independent orthotopic models of DIPG, while histological analysis showed restoration of H3K27me3 and decreased Ki67 positive cells. In addition to panobinostat, CBL0137 has been found to combine synergistically in vitro and in vivo with PARP and BET inhibitors. Given these promising results, a paediatric trial of CBL0137 will open through the Children’s Oncology Group with an expansion cohort for DIPG patients.