<div>Abstract<p>Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor with a median survival of less than 1 year. No effective therapy is currently available, and no therapeutic advances have been made in several decades. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of histone 3 subtype. In the present study, we show that the modulation of BMI-1 leads to DNA damage, M phase cell-cycle arrest, chromosome scattering, and cell death. Interestingly, EZH2 inhibition did not alter these effects. Furthermore, modulation of BMI-1 sensitizes DIPG patient-derived stem-like cells to ionizing radiation (IR). Treatment of DIPG stem-like cells with PTC596, a BMI-1 modulator, and IR impairs the kinetics of DNA damage response (DDR). Both DDR foci formation and resolution were delayed, resulting in further reduction in cell viability compared with either treatment alone. <i>In vivo</i>, treatment of mice bearing DIPG xenografts with PTC596 leads to decreased tumor volume and growth kinetics, increased intratumoral apoptosis, and sustained animal survival benefit. Gene expression analysis indicates that <i>BMI-1</i> expression correlates positively with DIPG stemness and BMI-1 signature. At the single-cell level, the analysis reveals that BMI-1 pathway is upregulated in undifferentiated cells and positively correlates with stemness in DIPG tumors.</p>Implications:<p>Together, our findings indicate that BMI-1 modulation is associated with mitotic abnormalities, impaired DDR, and cell death, supporting the combination of BMI-1 modulation and radiation as a promising novel therapy for children with DIPG.</p></div>
BACKGROUND: High-grade gliomas (HGGs) in children consistently demonstrate poor outcome despite multi-modal therapy. ACNS0822 was a feasibility and phase II trial in newly-diagnosed pediatric HGG. METHODS: In the feasibility study, patients received vorinostat 230 mg/m2/dose daily during radiation therapy (RT). In the phase II study, patients were randomized to one of three radiosensitizing agents during RT: Arm A [vorinostat 230 mg/m2/dose on Days 1-5 weekly], Arm B [temozolomide (TMZ) 90 mg/m2/dose daily], and Arm C [bevacizumab (BEV) 10 mg/kg/dose on Days 22 and 36]. All patients received BEV (10 mg/kg/dose every 2 weeks) + TMZ (200 mg/m2/dose on Days 1-5 q 28 days) for maintenance therapy × 12 cycles. The primary objectives were to determine 1) the feasibility of administering vorinostat during RT and 2) the 1-year event-free survival (EFS) of vorinostat or BEV with RT versus TMZ with RT as the historical control followed by maintenance chemotherapy. RESULTS: Six eligible patients were enrolled on the feasibility study; none experienced a dose limiting toxicity. The 95 patients enrolled (92 eligible) on the phase II study, were randomized to Arm A (vorinostat, n = 31), Arm B (TMZ, n = 29), and Arm C (BEV, n = 32). One-year EFS for Arms A, B, and C were 36.1 ± 10.4%, 54.8± 10.2%, 38.5 ± 10.1%, respectively. All chemoradiotherapy arms, and the maintenance arm were well-tolerated. The most common grade 3/4 toxicities in maintenance therapy included thrombocytopenia (9.6%), neutropenia (7%), and leukopenia (3.6%). Intracranial hemorrhage possibly attributable to BEV was reported in 2 patients in maintenance cycles 2 [grade 3] and 7 [grade 2]. There were no therapy-related deaths. CONCLUSION: Vorinostat (230 mg/m2/dose weekly) was tolerable during RT, as was maintenance therapy with BEV + TMZ. However, neither vorinostat, nor BEV proved superior to TMZ as a radiosensitizer in pediatric HGG.
<p>B, CCHMC-DIPG-1 cells treated with PTC596 (100 nM) were quantified for the number of γH2AX foci/cell at the indicated time points. C, Representative IF images and cell cycle analysis (with the indicated % of cells) of CCHMC-DIPG-2 cells treated with PTC596 (100 nM) for 24 hrs and 48 hrs. DNA damage (γH2AX in green) and apoptosis (cleaved caspase-3 in red) were evaluated and their respective quantification. DAPI (blue) represent nuclei. Error bars represent the SD from 4 different fields (n=150-400 cells/field). D, Immunoblot analysis of H3 S10-P in DIPG cells treated with indicated doses of PTC596 for 24 hrs. Total H3 served as loading control.</p>
Abstract Diffuse intrinsic pontine glioma (DIPG) remains an incurable disease with median overall survival <12 months despite decades of clinical trials investigating multimodal therapies. Immunotherapy represents a promising treatment paradigm which has been successfully used in other cancers. An adequate understanding of the tumor microenvironment and immunologic profile is essential to identify potential immunotherapeutic targets to inform immunotherapy design. Previous studies have shown that most DIPG tumors harbor low mutational burden compared to adult cancers and are characterized by a non-inflammatory microenvironment, limiting the development of immunotherapies in this disease. Our team’s prior work similarly demonstrated that most DIPG tumors have an immunologically “cold” microenvironment, but a subset of tumors harbors a more inflammatory gene expression profile and/or higher mutational burden, with trends toward improved survival and favorable radiographic response to radiation. Here, we applied a deconvolution analysis using CIBERSORTx on bulk RNA-seq data from 28 DIPG patients’ tumors paired with matched normal tissue specimens, to profile the immune microenvironment of DIPG and evaluate immune-related gene expression to determine percentages of different types of immune cells. Our results indicate that DIPGs have very limited lymphocyte infiltration. However, the infiltration of macrophages “M2-like” type cells and CD4 memory resting T cells were significantly higher in tumors compared to normal tissue samples. Similar results were found using single-cell RNA sequencing performed on biopsy and autopsy tissue, with less than 5% of total cells identified as immune cells. MHC I components were widely expressed in DIPGs with no significant difference between tumor and normal tissue. Expression of CD11B and CD68 were higher in tumor compared to normal tissue, suggesting enrichment of myeloid cells. Overall, deconvolution analysis of bulk RNA-seq can be used to profile DIPG tumors’ immune microenvironment to aid in the thoughtful design of effective immunotherapeutic strategies for this disease.
Brain tumors are the most common solid tumor in children and the leading cause of cancer-related deaths. Over the last few years, improvements have been made in the diagnosis and treatment of children with Central Nervous System tumors. Unfortunately, for many patients with high-grade tumors, the overall prognosis remains poor. Lower survival rates are partly attributed to the lack of efficacious therapies. The advent and success of immune checkpoint inhibitors (ICIs) in adults have sparked interest in investigating the utility of these therapies alone or in combination with other drug treatments in pediatric patients. However, to achieve improved clinical outcomes, the establishment and selection of relevant and robust preclinical pediatric high-grade brain tumor models is imperative. Here, we review the information that influenced our model selection as we embarked on an international collaborative study to test ICIs in combination with epigenetic modifying agents to enhance adaptive immunity to treat pediatric brain tumors. We also share challenges that we faced and potential solutions.
