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BACKGROUND The cyclin D-CDK4/6-Rb pathway is activated in most glioblastomas. Abemaciclib is a potent CDK4/6 inhibitor with good brain penetration approved for ER/PR/HER2- breast cancer. In order to efficiently evaluate the potential impact of abemaciclib on overall survival (OS) in newly diagnosed glioblastoma and to simultaneously develop information regarding potential genomic biomarker associations, abemaciclib was included as an arm on the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) trial. INSIGhT is a phase II platform trial using response adaptive randomization and deep genomic profiling to more efficiently test experimental agents in MGMT unmethylated glioblastoma and potentially accelerate identification of novel therapies for phase III testing. Initial randomization was equal between abemaciclib, control, and two other experimental arms but subsequent randomization was adapted based on efficacy as determined by progression-free survival (PFS). Ineffective arms were discontinued and new arms added by protocol amendment. We report preliminary results for the abemaciclib arm which has completed accrual. METHODS Patients with newly diagnosed MGMT-unmethylated glioblastoma were randomized to receive either radiotherapy with concomitant and adjuvant temozolomide at standard doses or standard radiochemotherapy followed by adjuvant abemaciclib (150–200 mg orally BID) without temozolomide. Treatment continued until progression or development of unacceptable toxicities. The primary endpoint was OS. Association between abemaciclib efficacy and cyclin D-CDK4/6-Rb pathway genomic alterations was also investigated. RESULTS There were 123 patients (50 control; 73 treated with abemaciclib). Abemaciclib was generally well-tolerated with no new toxicity signals identified. PFS was significantly longer (p=0.03, logrank test) with abemaciclib (median 6.31 months 95% CI [5.29, 8.18]) compared to the control arm (5.16 months 95% CI [4.37, 6.28]). 28/50 control and 36/73 abemaciclib patients remain alive. CONCLUSION Preliminary analysis suggests that abemaciclib increases PFS compared to control. Updated toxicity, PFS and survival data and potential genomic biomarker associations will be presented.

BACKGROUND CC-115 is an oral, CNS-penetrant, selective inhibitor of mammalian target of rapamycin kinase (mTOR) and deoxyribonucleic acid-dependent protein kinase (DNA-PK). Both targets are important in glioblastoma; PI3K/Akt/mTOR signaling is hyperactive in most glioblastomas, and DNA-PK is integral to repair of radiotherapy-mediated DNA damage. To investigate CC-115 in newly diagnosed glioblastoma and explore potential genomic biomarker associations, CC-115 was evaluated in the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) trial, an adaptive platform trial designed to efficiently test experimental agents. METHODS Adults with newly diagnosed MGMT-unmethylated glioblastoma, with genomic data available, are eligible for this ongoing trial. Patients are adaptively randomized to one of several experimental arms or the control arm: standard radiotherapy with concurrent and adjuvant temozolomide. The primary endpoint is overall survival (OS). Patients randomized to CC-115 (10mg po BID) received it concurrently with radiotherapy and as adjuvant monotherapy. As the first in-human use of CC-115 with radiation, a safety lead-in 3 + 3 design was used. RESULTS Twelve patients were randomized to CC-115; seven patients had possible treatment-related CTCAE grade > 3 toxicity, including four pre-specified dose-limiting toxicities: liver function abnormality (n=1), hyperlipidemia (n=1), lipase elevation (n=1) and cerebral edema (n=1). There was no significant difference in progression-free survival (PFS, median 4.2 months [CC-115] vs. 5.2 months, p=0.9) or OS (median 10.1 months [CC-115] vs. 14.5 months, p=0.9) compared to the 50 patients randomized to the control arm. Based on early PFS results, randomization probability to CC-115 decreased from 25% to < 10% at time of the trial arm closure. CONCLUSION Concurrent and adjuvant CC-115 was associated with toxicity and failed to improve PFS or OS. The INSIGhT trial design allowed for more efficient testing of CC-115, decreasing patients and resources allocated to a therapy that was discontinued due to concerns about toxicity and unfavorable risk-to-benefit ratio.

BACKGROUND We report phase I data examining pharmacokinetics, safety and preliminary efficacy of the dimeric, pan-RAF inhibitor DAY101 (formerly TAK-580/MLN2480) in pediatric patients with radiographically recurrent/progressive LGGs harboring MEK/ERK pathway alterations. METHODS Oral DAY101 was administered weekly to patients < 18 years of age with radiographically recurrent/progressive LGG for 4-week cycles up to a maximum of 2 years. The starting DAY101 dosage was 280 mg/m2. Dose limiting toxicities were determined after one cycle. RESULTS We treated nine eligible patients at 280, 350, and 420 mg/m2. Seven patients had KIAA1549:BRAF fusions, one a novel SRGAP3-RAF1 gene fusion and one with NF1 mutation. PK parameters following weekly administration of DAY101 in children mirrored the adult data, with dose-proportional increases in Cmax and AUC observed. There were no DLTs. The most common toxicity was skin rash followed by achromotrichia and nevi formation. There was only one grade 3 elevation is CPK and no grade 4 adverse events. A 2.2-fold mg/kg exposure difference was observed with respect to weight-based dosing and suggested a correlation to independent, centrally reviewed best radiographic RANO responses of 2 complete responses, 2 partial responses, 3 stable disease, and 2 progressive disease. Median time to response was 10.5 weeks (range: 8–32 weeks). CONCLUSIONS These phase 1 data provide initial pharmacokinetic parameters outlining oral weekly dosing of DAY101 in pediatric patients with radiographically recurrent and progressive LGG. Plasma exposures of DAY101 are similar in children and adults. Oral weekly DAY101 is well-tolerated and shows anti-tumor activity. The amended protocol explores differential dosing to achieve similar responses across a variety of body surface areas.

BACKGROUND Hypermutation is an emerging biomarker for predicting response to immunotherapy in cancer patients, however its clinical value in gliomas is not established. We sought to assess the determinants of hypermutation in gliomas, and its value for predicting response to standard of care and immune checkpoint blockade (ICB). METHODS We performed comprehensive genomic characterization of 2,420 clinically annotated gliomas. We assessed the clinical and molecular characteristics associated with hypermutation and relationships between hypermutation and response to cancer treatments. RESULTS Hypermutation occurred predominantly as an adaptive resistance mechanism to temozolomide in gliomas and was most prevalent in recurrent gliomas with MGMTpromoter methylation (33.8%), IDH1/2mutation (41.0%) or 1p/19q co-deletion (59.5%). Hypermutation was almost always associated with molecular defects in DNA mismatch repair (MMR), and was associated with shorter survival after its appearance based on multivariate analysis (hazard ratio 1.91; 95% CI 1.24–2.94; P=0.004). The molecular mechanisms whereby gliomas undergo hypermutation during therapy with alkylating agents were dissected using patient-derived glioma models in vitro and in vivo. Outcomes of hypermutated gliomas treated with immune checkpoint blockade or with standard of care agents will be presented at the conference. CONCLUSIONS Using the largest set of hypermutated gliomas described to date, this study establishes that mutational burden and mutation signatures are clinically and biologically significant biomarkers that can be used to predict therapy response and guide treatment decisions in gliomas

New approaches to the treatment of diffuse intrinsic pontine gliomas (DIPGs) are desperately needed. DNA damage response is essential for cells to maintain genome integrity as DNA is damaged by both endogenous and exogenous stressors. Many cancer cells exhibit hyper-dependency on specific DNA repair pathways due to either defects in DNA repair mechanisms and/or high levels of endogenous stress leading to accumulation of DNA damage lesions. Identification of DIPG-specific DNA repair deficiencies and resultant dependencies may establish novel therapeutic strategies for DIPGs. METHODS To identify pathways critical for DIPG cell survival, genome wide CRISPR-Cas9 screen was performed on patient derived DIPG cell lines followed by gene set enrichment analyses. To monitor the effects of pathway inhibition on survival, apoptosis, DNA damage and repair, assays were performed to measure cell proliferation, cleaved-caspase3, gamma-H2AX and reporter based-DNA repair efficiency. RESULTS Our unbiased CRISPR approach to uncover vulnerabilities in DIPGs identified DNA double strand break (DSBs) repair pathways as essential for DIPG cell proliferation and survival. Further studies revealed high basal DSBs in DIPG cells compared to neural stem cells and primary astrocytes that suggest dependence of DIPG cell survival on specific DSB repair pathways. We confirmed the intrinsic reliance of DIPG cells on the specific DSB repair pathway of mutagenic end-joining, and defined a key role for DNA repair in suppressing endogenous DNA damage-induced apoptotic cell death. CONCLUSION DIPG cells have high endogenous DNA damage levels and escape catastrophic genomic instability and cell death by engaging DNA repair pathways, in particular the mutagenic end-joining DNA repair pathway. Inhibition of this specific DNA repair pathway represents a promising new avenue for the treatment of DIPGs.

DIPG and HGG lead to the majority of pediatric cancer-related deaths. Despite multimodal treatment, the 2-year survival rates for DIPG and HGG are less than 10% and 20%, respectively. Anti-PD-1 therapy combined with radiotherapy has demonstrated synergistic anti-tumor effects. Cemiplimab, a human PD-1 monoclonal antibody, has demonstrated safety and efficacy in patients with advanced malignancies. Combination of cemiplimab with radiation has the potential to be an effective treatment for pediatric patients with DIPG or HGG. This is a multicenter, Phase 1 and early efficacy study (NCT03690869). Phase 1 will enroll pediatric patients with R/R solid or CNS tumors (N≥30) into two age cohorts (0 to < 12 and 12 to < 18 years). Cemiplimab will be administered intravenously every 2 weeks as monotherapy. Primary objectives of Phase 1 are to confirm safety and assess the pharmacokinetics (PK) of cemiplimab to recommend a Phase 2 dose (RP2D) for clinical efficacy assessment. The Efficacy phase will enroll patients with newly diagnosed DIPG or HGG (dose escalation: N≥12 patients each; dose expansion: N≤40 patients each), or recurrent HGG (dose escalation: N≥6 patients; dose expansion: ≤20 patients) into two age cohorts (≥3 to < 12 years and 12 to ≤25 years). There will be two treatment arms for patients with newly diagnosed DIPG or HGG: cemiplimab in combination with conventionally fractionated (Arm 1) or hypofractionated (Arm 2) radiotherapy. Patients with recurrent HGG will receive cemiplimab concomitantly with reirradiation. All patients will continue cemiplimab as monotherapy at completion of combination therapy. Primary objectives of Efficacy Phase are to confirm safety and anticipated RP2D, assess PK, and determine survival and antitumor activity at 12 months (overall survival for newly diagnosed DIPG and recurrent HGG; progression-free survival for newly diagnosed HGG) of cemiplimab when given concomitantly with radiation. This study is currently recruiting patients across multiple sites.

Pilocytic astrocytoma (PA) is the most common pediatric low-grade glioma. While PAs exhibit a favorable prognosis compared to higher-grade gliomas, treatment morbidity and tumor recurrence still represent major challenges for some PA patients. PAs frequently harbor rearrangements resulting in expression of oncogenic KIAA1549-BRAF fusion transcripts. We performed scRNAseq of both tumor and non-tumor cells in newly-diagnosed PAs that contained KIAA1549-BRAF rearrangements. To confidently distinguish tumor cells from nontumor cells, we sorted cells by glial progenitor marker A2B5 status and profiled KIAA1549-BRAF fusion status of cells using a quantitative PCR-based assay. Results were validated using RNA in situ hybridization and compared to bulk expression data from 151 pediatric low grade gliomas. When compared to higher-grade gliomas, a higher proportion of the PA tumor cells exhibited a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibited a progenitor-like phenotype with evidence of proliferation. These progenitor-like tumor cells expressed a mitogen-activated protein kinase (MAPK) gene program that was absent from higher-grade gliomas. Similar patterns of expression of genes associated with the astrocyte-like and MAPK gene programs were also seen in formalin fixed, paraffin embedded PA tissues using RNA in situ hybridization. Immune cells, especially microglia, comprised almost half of all cells in the PAs and accounted for many differences seen in bulk pediatric low grade glioma expression profiles. These single cell transcriptional data reveal a transcriptional developmental hierarchy in a pediatric low-grade glioma that is skewed towards more mature brain cells compared to higher-grade gliomas. The results indicate that future analyses of bulk PA tissues should attempt to account for considerable infiltration by nontumor cells. Finally, the finding that a MAPK gene program is not uniformly expressed in PA tumor cells has implications for ongoing clinical investigations of therapies directed at the MAPK pathway for the treatment of PA.

BACKGROUNDL Diffuse intrinsic pontine gliomas (DIPGs) are fatal childhood gliomas and radiotherapy is the only modality that prolongs survival; however, all patients progress with no long term survivors, indicating an urgent need to develop new treatment approaches. METHODS: Genome wide CRISPR screen was used to identify the cellular pathway(s) critical for DIPG cell survival and doxycycline inducible shRNA were used for temporal knockdown of gene expression. Colony formation assays were performed to monitor effects on cell proliferation, comet assay and immunoflourescence based gamma-H2AX foci detection were used for DNA damage assessment, and caspase3/7 activity measured apoptosis. RESULTS: Our preliminary studies to understand radio- and chemo-resistance mechanisms in DIPGs uncovered high levels of persistent basal DNA double strand breaks (DSBs) in the majority of analyzed DIPG cell lines compared to neural stem cells and astrocytes, a phenomenon that promotes oncogenic potential. The basal increase in DSBs was independent of H3.3K27M mutational status. To pinpoint the molecular mechanism(s) that support proliferation and survival of DIPG cells with highly damaged DNA, we performed unbiased CRISPR screens in unperturbed cells and preliminary results suggest an addiction of DIPG cells to specific DSB repair factors. Further analyses confirmed this intrinsic reliance of DIPG cells on specific DSB repair pathways and the key role of DNA repair mechanisms in suppressing DNA damage induced apoptotic cell death. CONCLUSION: DIPG cells exhibit persistent DNA damage. To evade catastrophic genomic instability and cell death, DIPG cells hijack DNA repair pathways. Therefore, a promising therapy against DIPG would include inhibition of specific DNA damage response pathways that allow DIPGs to survive persistent DNA damage.

Diffuse midline gliomas (DMG) are aggressive and lethal pediatric brain tumors that cannot be cured by conventional therapeutic modalities. Using a genome wide CRISPR screen we identified the de novo pyrimidine biosynthesis pathway as a metaboilic vulnerability in DMGs. BAY2402234 is a small molecule inhibitor of DHODH -a rate liminting enzyme in the de novo pyrimidine biosynthesis pathway. BAY2402234 induces cell death in DMG cells at low nanomolar concentrations while sparing adult glioblastoma cells and normal astrocytes. Further investigations revealed drammatic reduction in cellular UMP pools, the precursor for all pyrimidine nucleotides, after DHODH inhibition, specifically in DMG cells. Cytotoxicity of DHODH inhibition in DMG cells is rescued by exogenous uridine, supporting UMP depletion as the mechanism underlying DMG cell death and also showing that cell death is an “on target” response to BAY2402234. Cell death induced by BAY2402234 is a consequence of replication fork stalling as evident by accumulation of chromatin-bound RPA foci and g-H2AX. Stalled replication forks eventually collapse, resulting in replication catastrophy and apoptosis. Cytotoxic effects of DHODH inhibition are further exacerbated by inhibition of the intra-S checkpoint protein, ATR. Combined treatment of DMG cells with DHODH and ATR inhibitors resulted in enhanced accumulation of chromatin-bound RPA, g-H2AX, replication fork collapse and apoptosis. Importantly, in vivo studies verify that both BAY2402234 (DHODHi), and BAY1895344 (ATRi), cross the blood-brain barrier, accumulate in the brain at therapeutically relevant concentrations, and induce DNA damage in intracranial DMG xenografts in mice. Taken together, our studies have identified DHODH inhibition as a DMG-specific vulnerability resulting in cell death; the mechanism of DHODHi-induced cell death led us to identify combined inhibition of DHODH and ATR as a synergistic therapy against DMG tumors.

BACKGROUND COG ACNS 1123 tested reduced radiotherapy (RT) for non-metastatic, non-germinomatous germ cell tumor (NGGCT) patients. The impact of central review on quality of RT and pattern of failure for NGGCT patients is evaluated. METHODS Patients who achieved a complete response (CR) or partial response (PR) to induction chemotherapy were eligible for reduced dose and field RT of 30.6 Gy whole ventricular field (WVI) and 54 Gy tumor-bed total dose. An online contouring atlas was available. Within three days of RT start, WVI plans were submitted for rapid central review. Within one week of RT completion, the complete RT record was submitted. Brain and spine MRIs of relapsed patients were centrally reviewed. RESULTS Between 5/2012–9/2016, 107 eligible patients were accrued and 70 met reduced RT criteria. Rapid RT review was performed for 49 (70%) of 70 patients. Forty-four (89.8%) required no modification. All modifications were completed and plans became compliant. Final central review was performed for 66 evaluable patients: 62 (94%) were per protocol; there were 2 major (1 dose and 1 target) and 2 minor deviations. Eight patients progressed; none had deviations. Median time to progression was 3.54 months (range: 1.7–19.1) from RT start. All failures had a spine component; two also had cranial component: one local progression (within the RT boost volume) and one leptomeningeal disease. CONCLUSION Providing an online contouring atlas and performing a rapid central review lead to high quality radiotherapy on this prospective trial. The deviations did not contribute to the pattern of failure.

OBJECTIVE To estimate the 6-month Progression Free Survival (PFS6) associated with everolimus for progressive/recurrent pLGGs and to determine if activated PI3K/Akt/mTOR pathway as measured by positive phosphorylated-ribosomal protein S6 (p-RPS6) status was associated with response. METHOD: Patients 3–21 years of age with recurrent or progressive pLGG were enrolled. Everolimus was administered orally at 5 mg/m2 daily. Tissue availability for molecular analysis was mandatory. Immunohistochemistry (IHC) for p-RPS6 was performed centrally. An adaptive Simon two-stage design was employed based on p-RPS6 status. Based on results of the first stage, enrollment in the second stage was either limited to pathway activated patients or open to all subjects. RESULTS From December 2012 to July 2019 a total of 65 subjects enrolled [median age 9 years (range 3–19); 43% female]. As of December 15, 2019 median number of treatment cycle is 8 (range 1–24); 7 patients remain on treatment. Toxicity profile is similar to published reports with rash and elevated lipid profiles as most common adverse events. PFS6 for the entire cohort is 63%; PFS6 is 64% for the activated and 61% for the non-activated patients. Central imaging review (n=52) revealed 1 partial response, 1 complete response, 33 stable disease, and 17 progressive disease at the end of study treatment. Initial molecular analysis identified BRAF alterations in 35/65 patients. CONCLUSION Everolimus is well tolerated and active in a subset of pLGGs. Ongoing analyses will assess predictive biomarkers of response and will be reported at the meeting.

OBJECTIVES: Children with diffuse intrinsic pontine glioma (DIPG) continue to have a dismal outcome and median survival remains stagnant at 9 months for decades. AZD1775 is an orally available inhibitor of Wee1 kinase, a key G2-M checkpoint regulator that has been shown to cross the blood brain barrier and has demonstrated efficacy in preclinical DIPG studies. METHODS: AZD1775 was administered orally in newly diagnosed children with DIPG, only on days of radiation therapy. The protocol assessed 6 dose levels starting from 50 mg/m(2)/dose and escalated up to 200mg/m(2)/dose. Dose escalation occurred first by the number of days on which AZD1775 was administered and then by an increase of the actual dose. The entire length of radiation therapy constituted the dose limiting toxicity period. Correlative studies included pharmacokinetic (PK) analyses as well as determination of expression of p-CDC2, p-HH3 and g-H2AX in peripheral blood mononuclear cells (PBMC). In the late breaking abstract we will present results of this phase 1 study including PK analyses as well as results of the correlative studies. Results of this study will lay the groundwork for subsequent clinical trials using AZD1775 in pediatric brain tumors.

Background Neuroblastoma is the most common pediatric extracranial solid tumor. Within conventional risk groups, there is considerable heterogeneity in outcomes, indicating the need for improved risk stratification. Methods In this study we analyzed the somatic mutational burden of 515 primary, untreated neuroblastoma tumors from three independent cohorts. Mutations in coding regions were determined by whole-exome/genome sequencing of tumor samples compared to matched blood leukocytes. Survival data for 459 patients were available for analysis of 5-year overall survival using the Kaplan–Meier method and log-rank test. All statistical tests were two-sided. Results Despite a low overall somatic mutational burden (mean = 3, range = 0–56), 107 patients were considered to have high mutational burden (>3 mutations). Unfavorable histology and age 18 months and older were associated with high mutational burden. Patients with high mutational burden had inferior 5-year overall survival (29.0%, 95% confidence interval [CI] = 17.2 to 41.8%) vs those with three or fewer somatic mutations (76.2%, 95% CI = 71.5 to 80.3%) (log-rank P Conclusions This study demonstrates that mutational burden of primary neuroblastoma may be useful in combination with conventional risk factors to optimize risk stratification and guide treatment decisions, pending prospective validation.

Pediatric high-grade gliomas (pHGGs) and diffuse intrinsic pontine gliomas (DIPGs), portend a poor prognosis and are particularly recalcitrant to standard treatment. Aberrant chromatin remodeling and activation of the phosphatidylinositol 3-kinase (PI3K) pathway have been identified as important mediators of pHGG and DIPG pathogenesis. As inhibition of these pathways are promising therapeutic avenues and radiation is the only modality to prolong survival of DIPG patients, we sought to explore radiosensitizing functions of such inhibition and explore mechanisms of action of such agents. Here, we demonstrate that treatment with CUDC-907, a novel, first-in-class dual inhibitor of histone deacetylases (HDACs) and PI3K, in combination with radiotherapy evokes a synergistic cytotoxic response in pHGG and DIPG models in vitro and in vivo. We identify a novel mechanism of CUDC-907 in modulating DNA damage response by inhibiting radiation-induced DNA repair pathways, including homologous recombination and non-homologous end joining. The radiosensitizing effects of CUDC-907 were mediated by decreasing NFκB/Forkhead box M1 (FOXM1) recruitment to promoters of genes involved in the DNA damage response, and exogenous expression of NFκB/FOXM1 rescued CUDC-907-induced cytotoxicity. Together, these findings reveal CUDC-907 as a novel radiosensitizer with potent anti-tumor activity in pHGG and DIPG, and provide a preclinical rationale for the combination of CUDC-907 with radiotherapy as a novel therapeutic strategy for pHGG and DIPG. More globally, we have identified NFκB and FOXM1, and their downstream transcriptional elements, as critical targets for new treatments for pHGG and DIPG.

INTRODUCTION: We sought rational targeted agents that exhibit single agent activity and sensitize pediatric high-grade gliomas (pHGG) to radiation (RT). We investigated a novel dual inhibitor of class I and II histone deacetylases and class I PI3Ks in combination with RT. METHODS: pHGG cells including DIPGs were treated with CUDC-907 alone or in combination with radiation and effects on cell viability, cell cycle, apoptosis, DNA damage, DNA damage repair pathways were determined. Homologous recombination (HR) and non-homologous end joining (NHEJ) were assessed by functional repair assays and loss of formation/resolution of RAD51 (HR) and phospho-DNA-PKCs (NHEJ) foci, respectively. In vivo evaluation of CUDC-907 efficacy was performed using SF8628 (DIPG xenograft) cells implanted in mice brainstems and randomized to: (1) control, (2) RT (0.5 Gy x 6, M-W-F), (3) CUDC-907 (100 mg/kg, oral gavage, daily x 2 weeks), (4) CUDC907+concurrent RT. RESULTS: G1 cell cycle arrest, induction of apoptosis, and accumulation of double strand DNA breaks (DSB) as measured by g-H2AX contribute to CUDC-907-induced radiosensitization. CUDC-907 attenuates radiation-induced DNA damage responses and repressed DSB repair. Specifically, CUDC-907 impairs the key DSB repair pathways of HR and NHEJ through reduced expression of RAD51 and Ku proteins, respectively. CUDC-907-mediated down-regulation of DNA damage responses is partly transcriptional and is driven by RelB-p50-based NFkB complex and FOXM1, activators critical for expression of key DSB repair genes. Following irradiation, RelB-p50-FOXM1 recruitment on the promoters of RAD51 and KU70/80 is enhanced to induce DSB repair, and CUDC-907 reduces RelB-p50-FOXM1 binding at the promoters thus interfering with DSB repair. Radiosensitization by CUDC-907 is also observed in vivo in mice-bearing DIPG xenografts where CUDC907+RT combination treatment significantly prolongs survival compared to monotherapy (p

Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations most children are still treated with standard chemotherapy protocols as first line therapy. Alteration of the BRAF/MEK/MAPK pathway is the hallmark of PLGG and mTOR activation has been documented in the majority of these tumors. This study investigates combinations of MEK1/2, BRAFV600E and mTOR inhibition in PLGGs carrying specific genetic alterations of the MAPK pathway. We used human glioma cell lines containing BRAFV600E (AM38, DBTRG, BT40), or wild-type BRAF (SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. In BRAFV600E tumors, the combination of MEK inhibitor with either mTOR or BRAFV600E specific inhibitor is superior to monotherapy and to combination of BRAF and mTOR inhibitors in reducing cell viability reduction and arresting cell cycle progression. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition alone. In vivo studies in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244 + PLX4720 or AZD6244 + everolimus compared with respective monotherapies (p < 0.01).

INTRODUCTION: First generation allosteric inhibitors of mTORC1 have shown clinical benefit for the treatment of pediatric low-grade gliomas (PLGG). MEK inhibition has shown great promise in early phase clinical trials for PLGG with the hypothesis that tumors carrying the KIAA1549:BRAF are particularly sensitive to such treatment. MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2. Herein we investigate the effects of MLN0128 monotherapy as well as in combination with MEK inhibition in models of PLGG. METHODS: We used human glioma cell lines containing BRAFV600E (AM38), wild-type BRAF (LN229, TN98, SF188) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells. Signaling inhibitors included MLN0128, everolimus, BRAFV600E specific inhibitor PLX4720 and MEK specific inhibitors AZD6244 and GSK1120212. Cell proliferation was determined using an ATP-based assay. Biochemical effects were assessed using western blot analysis. In vivo activity of these inhibitors are ongoing using the BT40 PLGG xenograft mouse model. RESULTS: MLN0128 monotherapy shows stronger anti-proliferative effects compared to everolimus across cell lines independent of BRAF status. As expected, treatment with MLN0128 leads to down-regulation of p-AKT and p-4EBP1, whereas treatment with everolimus only reduces expression of p-S6. MEK inhibition with AZD6244 effectively decreases p-ERK expression in all lines; however, the addition of MLN0128 to AZD6244 causes a rebound in p-ERK expression in BRAFWT gliomas but not in gliomas expression BRAFV600E or KIAA1549:BRAF alterations. In cells carrying either the BRAFV600E or KIAA1549:BRAF alteration, combined treatment with MLN0128 and GSK1120212 or AZD6244 leads to synergistic anti-proliferative effects whereas such synergy is not present in BRAFWT gliomas. In vivo studies in the PLGG xenograft model BT40 are ongoing. CONCLUSION: MLN0128 has synergistic effects with MEK inhibition in gliomas bearing BRAFV600E or KIAA1549:BRAF. In BRAF wild-type cells such synergy is not present, perhaps due to rebound in p-ERK when MLN0128 is combined with AZD6244.

INTRODUCTION: Activating BRAF-fusion mutations occur frequently in pediatric low-grade gliomas (PLGGs). We have previously shown targeting of KIAA1549-BRAF fusion by PLX4720, the research analog of vemurafenib, results in paradoxical activation of the mitogen activated protein kinase (MAPK) signal cascade. We hypothesized that BRAF-fusions could be targeted by downstream MEK inhibitors (MEKi), and that combinatorial targeting of BRAF-fusions could also prevent acquired resistance to single agent therapy. METHODS: BRAF-fusions were screened with a selection of clinically relevant MEKi and with PLX8394, a second-generation RAF inhibitor, in vitro in cell culture and soft agar. BRAF V600E and RAS mutant cells were also screened for comparison. MEKi/PLX8394 resistant BRAF-fusion cell lines were generated in vitro and interrogated by sequencing, immunoblotting, and RNA-seq analysis to identify potential co-targets. Combinatorial therapy was then tested in vitro and in vivo to confirm efficacy. RESULTS: BRAF-fusions in PLGGs can be targeted by MEKi and by the pan-RAF inhibitor PLX8394. AZD6244, MEK162, GDC0623, and GDC0973 all inhibited downstream ERK phosphorylation; however, GSK1120212 more robustly inhibited MAPK activation in the BRAF-fusion, BRAF V600E, and RAS mutant cells. Interestingly, the MEKi/PLX8394 resistant BRAF-fusion cells all showed activation of phosphatidylinositol 3-kinase (PI3K) pathway. BRAF-fusion parental cells were screened with RAD001, a targeted mTOR inhibitor, resulting in modest downstream inhibition. Co-targeting of the parental BRAF-fusions with GSK1120212 and RAD001 resulted in robust inhibition of down-stream signaling and in significant tumor inhibition, and prevented acquired resistance to targeted inhibition. CONCLUSION: Our work demonstrates that BRAF-fusion oncogenes can be effectively targeted with GSK1120212 as single agent but even more robustly with GSK1120212 and RAD001 co-targeting. This provides preclinical rationale for treating PLGGs harboring BRAF-fusion oncogenes with combinatorial MEK and mTOR inhibitors in clinical trial.