Jason Fangusaro

Focal radiation therapy has long been and remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing radiation therapy (RT). Since chemotherapy does not provide significant outcome improvement, it is crucial to find a suitable radiosensitizer. Our research has shown that the JMJD3 demethylase inhibitor, GSK-J4, exerts potent anti-tumor activity on DIPG cells while restoring methylation. Our aim is to We hypothesized that GSK-J4 may inhibit radiation-induced DNA repair, making it a potential radiosensitizer. RNA sequencing (RNA-Seq) was used to analyze gene expression changes by GSK-J4 in DIPG cells. ATAC-seq was conducted to determine chromatin accessibility in DIPG cells treated with GSK-J4. We evaluated DNA damage repair using DNA repair assay and immunocytochemistry of DSB markers γH2AX and 53BP1. Western blotting and quantitative PCR (qPCR) were conducted to evaluate differential expression of mRNA and proteins involved in DNA DSB repair. In vivo response to radiation monotherapy and combination of RT + GSK-J4 were measured by animal survival studies. RNA-Seq and qPCR show that GSK-J4 significantly reduces DNA DSB repair genes in DIPG cells. ATAC-seq results reveal that GSK-J4 modifies DNA accessibility to regulate expression of DNA repair genes. Immunocytochemistry results support that GSK- J4 sustains high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair. DNA repair assay demonstrate that GSK-J4 inhibits DNA damage repair via the homologous recombination pathway. Western blotting revealed that GSK-J4 causes a sustained level of phosphorylated Rad50 and gH2AX in irradiated DIPG cells. In vivo studies revealed increased survival of animals treated with combination therapy compared to monotherapy. These results highlight GSK-J4 as a potential radiosensitizer in DIPG treatment.

Children with diffuse intrinsic pontine glioma (DIPG) have an extremely poor survival. This underscores the desperate need for novel, biology-driven therapies. Here, we investigated the involvement of RE1 Silencing Transcription Factor (REST), a chromatin remodeler and regulator of brain development, in DIPG pathology. We discovered that when compared to normal controls, REST protein is significantly elevated in DIPG patient samples with H3K27M mutation. Compared to isogenic DIPG cell lines with endogenous REST expression, cells engineered to express higher levels of human REST transgene exhibited enhanced tumor growth. Surprisingly, REST elevation also caused an increase in tumor vasculature. In vitro, REST loss in DIPG cells caused a decrease in tube formation by human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), indicating that REST regulates the DIPG microenvironment. Mechanistically, we identified a bone morphogenic factor (BMP) signaling antagonist and ligand for VEGFR2, Gremlin-1 (GREM-1), as a downstream effector of REST-dependent changes in tumor vasculature. REST-dependent GREM-1 secretion promoted activation of AKT signaling in HUVECs and HBMECs. VEGFA-C levels were not significantly altered between normal and H3K27M mutant DIPGs and did not correlate with REST expression. In summary, our study is the first to demonstrate both tumor intrinsic and tumor extrinsic functions for REST in DIPG. The latter is of particular significance because it opens the door for re-evaluation of anti-angiogenic therapies targeting GREM-1, in DIPGs with REST elevation, possibly in combination with standard of care.

Intracranial germ cell tumors represent 3-5% of pediatric CNS tumors. Non-germinomatous germ cell tumors (NGGCT) have worse outcomes compared to germinomas, however, improved survivals were achieved on COG ACNS0122 utilizing a combination of chemotherapy and craniospinal irradiation (CSI). Since CSI is associated with significant late effects, a Phase 2 study was undertaken to determine whether irradiation could be reduced without impacting survival in a subgroup of NGGCT patients. Patients with localized disease who achieved a complete (CR) or partial response (PR) to chemotherapy were eligible for reduced irradiation to 30.6Gy whole ventricular field (WVI) and 54Gy tumor-bed boost as compared to 36Gy CSI plus tumor-bed boost as used on ACNS0122. Between 5/2012 and 11/2016, 107 eligible patients were accrued. Median age was 11 years (range: 4-22) and 75% were male. Tumor location was pineal in 58, suprasellar in 37, ventricular in 6 and bifocal in 6 patients. Sixty-six patients achieved CR/PR post-induction and received reduced irradiation. The 2-year progression-free survival (PFS) was 89% (95% CI: 81%-97%) and overall survival was 92% (95% CI: 86%- 99%). Eight patients progressed; seven had a distant relapse (outside the irradiation field) and one patient had a local plus distant relapse. Alpha-fetoprotein and beta-human chorionic gonadotropin levels were not associated with PFS. There were no unexpected treatment-related adverse events or deaths. Although these survival data are encouraging, distant relapses noted in all of the patients who progressed are concerning. Longer follow-up of the ACNS1123 cohort may better inform our recommendations in the future.

Pineoblastoma (PB) is a rare but aggressive pediatric brain tumour arising from the pineal gland. Outcomes remain dismal with long-term survival rates between 10–40% despite intensive treatment regimens. Although germline RB1 and DICER1 alterations have been reported in a small proportion of PB, the clinical significance of such alterations and the biology of sporadic cases remains unknown. We undertook global methylation profiling of 75 PB cases, and discovered that PBs comprise four molecular sub-types, designated groups 1 to 4 with characteristic copy number alterations and mutational patterns. Molecular sub-groups of PB also exhibited distinct clinical features and survival outcomes. While PBs group 1–3 arose in older children (median ages 5.2–12.6 years), group 4 PB was restricted to much younger children (median age 1.4 years). Group 4 PB exhibited the highest incidence of metastases (53%) and had the worst 5-year event-free survival (EFS) and overall survival (OS), respectively 7.7% and 16.7%. In contrast, group 2 patients had a 5-year EFS and OS of 100%, while group 1 and 3 had intermediate outcomes (68.1%, 53.3% respectively). These findings demonstrate significant clinical and molecular heterogeneity in PB and underscore the critical need to define mechanisms underlying PB, so that precise patient stratification and selection of biology-informed therapies can be undertaken in future clinical trials.

Focal radiation therapy remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing radiation therapy (RT). Since chemotherapy does not provide significant outcome improvement, it is crucial to find a suitable radiosensitizer. Our research has shown that the JMJD3 demethylase inhibitor, GSK-J4, exerts potent anti-tumor activity on DIPG cells while restoring methylation. We hypothesized that GSK-J4 may inhibit radiation-induced DNA repair, making it a potential radiosensitizer. RNA seq was conducted to analyze gene expression changes by GSK-J4 in DIPG cells. We evaluated DNA damage repair by immunocytochemistry of DSB markers γH2AX and 53BP1 and DNA repair assay. Western blotting and quantitative PCR (qPCR) were conducted to evaluate differential expression of mRNA and proteins involved in DNA DSB repair. In vivo response to radiation monotherapy and combination of RT + GSK-J4 were measured by animal survival studies. RNA seq and qPCR showed that GSK-J4 significantly reduces DNA DSB repair genes in DIPG cells. Immunocytochemistry results support that GSK- J4 sustains high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair. DNA repair assay showed that GSK-J4 inhibits DNA damage repair through the homologous recombination pathway. Western blotting revealed that GSK-J4 causes a sustained level of phosphorylated Rad50 and gH2AX in irradiated DIPG cells. In vivo studies revealed increased survival of animals treated with combination therapy compared to monotherapy. These results highlight GSK-J4 as a potential radiosensitizer in DIPG treatment.

To evaluate predictors of neuropsychological outcomes for pediatric brain tumor patients undergoing x-ray radiotherapy (XRT) or proton radiotherapy (PRT). 125 patients received treatment for tumors located in the supratentorial (17.6%), midline (28.8%) or posterior fossa (53.6%) compartments. All received age-appropriate neuropsychological assessment of intelligence quotient (IQ), processing speed (PS), visual motor integration (VMI), executive function, memory and parent-reported function. Median age at diagnosis was 7.0 years; median time from treatment to last assessment was 4.0 years. Patients receiving PRT had higher socioeconomic status (SES), differing distributions of race and tumor locations, as well shorter median follow-up time compared to XRT (9.5 vs. 5.0 years, p<0.001). Univariate and multivariate analyses with tests for interaction of treatment group and follow-up identified higher verbal IQ (β=0.84 points/year, p=0.02) and full-scale IQ (β=1.03, p=0.01) for older patients; lower PS (β=-15.9 points, p=0.04) and VMI (β=-14.0, p=0.006) following CSI; and higher full-scale IQ (β=10.6 points, p=0.048), PS (β=12.6, p=0.02), and parent-reported practical function (β=13.8, p=0.049) following PRT relative to XRT. VMI was higher in those with higher SES (β=1.2 points/$10,000 household income, p=0.04), but lower following receipt of vincristine chemotherapy (β=-16.6, p=0.01). Parent-reported practical function was lower in those with posterior fossa tumors (β=-10.8, p=0.048). Neuropsychological outcomes of pediatric brain tumor patients are impacted by age, SES, receipt of CSI or vincristine chemotherapy, and tumor location. Relative to XRT, PRT is associated with favorable neuropsychological outcomes in terms of full-scale IQ, PS, and parent-reported practical function.

Medulloblastoma (MB) is a malignant pediatric brain tumor characterized by poor neuronal differentiation. The RE1 Silencing Transcription Factor (REST), which negatively regulates neurogenesis by promoting repressive chromatin remodeling via epigenetic modifications, is overexpressed in human MBs. Analyses of a publically available gene expression array data set revealed association of elevated REST expression in patients with Sonic Hedgehog (SHH) medulloblastoma with poor prognosis. Upon closer evaluation, we identified a subset of highly undifferentiated SHHα tumors with elevated REST expression and activity as determined by expression of REST target genes. These patients had an overall survival rate of 50% at 5 years. To understand the underlying mechanisms, we generated a novel mouse model expressing human REST transgene in a conditional manner in granule neural progenitors (GNPs), the cells of origin of SHH driven MB. Mice with constitutive activation of Shh signaling and REST elevation (Ptch+/-/RESTTG) in their GNPs developed tumors with a significant decrease in latency and increase in penetrance. We observed that 100% of Ptch+/-/RESTTG animals developed heterogeneously proliferative and undifferentiated tumors compared to tumor-bearing Ptch+/- animals. Mechanistic studies revealed deregulation of the Shh signaling pathway by REST-dependent G9a activity, accompanied by loss of Ptch1 heterozygosity in Ptch+/-/RESTTG tumors. Thus, our studies suggest that REST contributes to mis-regulation of SHH activity and drives an aggressive disease course by deregulating proliferation and differentiation. Ours is also the first study to implicate G9a, a repressive chromatin remodeler, in medulloblastoma pathogenesis and as a potential therapeutic target.

Ependymoma is the third most common brain tumor in children, but there is a paucity of large studies with more than 10 years of follow-up examining the long-term survival and recurrence patterns of this disease. We conducted a retrospective chart review of 103 pediatric patients with WHO Grades II/III intracranial ependymoma, who were treated at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and Chicago’s Ann & Robert H. Lurie Children’s Hospital between 1985 and 2008, and an additional 360 ependymoma patients identified from the Surveillance Epidemiology and End Results (SEER) database. For the institutional cohort, we evaluated clinical and histopathological prognostic factors of overall survival (OS) and progression-free survival (PFS) using the log-rank test, and univariate and multivariate Cox proportional-hazards models. Overall survival rates were compared to those of the SEER cohort. Median follow-up time was 11 years. Ten-year OS and PFS were 50 ± 5% and 29 ± 5%, respectively. Findings were validated in the independent SEER cohort, with 10-year OS rates of 52 ± 3%. GTR and grade II pathology were associated with significantly improved OS. However, GTR was not curative for all children. Ten-year OS for patients treated with a GTR was 61 ± 7% and PFS was 36 ± 6%. Pathological examination confirmed most recurrent tumors to be ependymoma, and 74% occurred at the primary tumor site. Current treatment paradigms are not sufficient to provide long-term cure for children with ependymoma. Our findings highlight the urgent need to develop novel treatment approaches for this devastating disease.

10543 Background: CNS malignancies are the most common solid tumors among children. Novel therapies are needed to help improve the survival outcomes in children with recurrent disease. Pomalidomide is an immunomodulatory agent thought to also function through a combination of anti-angiogenic, anti-inflammatory and cytotoixic activity making it a good candidate to explore in pediatric CNS tumors. Methods: A Phase I trial of pomalidomide was conducted among children ≥ 3 to < 21 years old with recurrent, progressive/refractory CNS tumors using the “rolling 6” dose escalation design. The primary objective was to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) when given orally once daily for 21 consecutive days of a 28-day course. Once the MTD was established, 12 additional patients were enrolled on expansion cohorts based on age and steroid use. Results: 29 children were enrolled and 25 were evaluable for dose limiting toxicity (DLT) evaluation. The MTD was 2.6 mg/m² (Dose level 2). DLTs observed at dose level 3 (3.4 mg/m²) included diarrhea (n = 1), thrombocytopenia (n = 1) and lung infection (n = 1), all grade 3. The most common toxicities were grade 1 lymphopenia (55%), leukopenia (62%) and thrombocytopenia (38%). There were no obvious differences in tolerability based on age or steroid use. Pharmacokinetics were similar to those observed in adults and increased in a dose-dependent manner. At the RP2D of 2.6 mg/m², the C_max was 97.4 ng/mL and the t½ was 4.1 hours. The median number of treatment cycles was 1.6 (0.2-12.3). Two patients, one with an oligodendroglioma and one with anaplastic pleomorphic xanthoastrocytoma, had long term stable disease for 9 and 18+ cycles. No objective responses were observed. Twelve month progression-free and overall-survivals were 5.2+/-3.6% and 12.8+/-8.5%, respectively. Immunologic correlate analyses are ongoing. Conclusions: The RP2D of pomalidomide is 2.6 mg/m² in children with recurrent brain tumors. Further prospective evaluation of this agent alone or in combination will be necessary to better understand its efficacy in specific pediatric CNS tumor populations. Clinical trial information: NCT02415153.

Background. There is no proven medical therapy for plexiform neurofibromas (PNs). We undertook a phase II trial of pegylated interferon (PI) to evaluate response and time to progression (TTP). Methods. PI was administered as a subcutaneous injection to patients with neurofibromatosis type 1‒related PN, stratified by the presence of symptoms (asymptomatic: stratum 1, symptomatic: stratum 2) or documented imaging progression (stratum 3). Patients in strata 1 and 2 received PI for up to one year if stable, 2 years for those with clinical (stratum 2) or imaging response (≥20% decrease in volume). Patients on stratum 3 continued PI until progression. PI was considered active in stratum 3 if TTP doubled compared with the placebo arm of a previous randomized trial using tipifarnib. Results. Enrolled were 82 evaluable patients (median age 10 y; range 1.6 to 21.4). Fatigue and/or worsening of behavioral issues were the most common toxicities requiring dose modification. Across all strata, imaging responses were seen in 4 patients (5%). Three of 26 symptomatic patients on stratum 2 met the criteria for clinical response without corresponding imaging changes. In stratum 3, median TTP was 29.4 months versus 11.8 for the placebo arm of the previous trial (P=.031). The slope of tumor growth on PI slowed significantly compared with the slope before starting PI (P=.044). Conclusions. In patients with active PN, PI results in more than doubling of the TTP compared with placebo. Imaging changes in symptomatic patients were not associated with changes in clinical status.