Background Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. Methods We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Results Change in H3.3K27M VAF over time (“VAF delta”) correlated with prolonged PFS in both CSF and plasma samples. Non-recurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (p=0.049). Decrease in plasma VAF displayed a similar trend (p=0.085). VAF “spikes” (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression. Conclusion Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.

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Background: Pediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. We hypothesized that co-treatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib. Methods: Dose response, synergism studies, P-gp inhibition and pharmacokinetic studies were performed on in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus. Results: Dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with reduction in mTOR signaling that persists after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended survival of PPK tumor bearing mice. Pediatric patients (n=6) with glioma tolerated this combination without significant adverse events. Recurrent patients (n=4) demonstrated median overall survival of 8.5 months. Conclusion: Efficacy of dasatinib treatment of PDGFRα-driven HGG is improved with everolimus and suggests a promising route for improving targeted therapy for this patient population. Clinical Trial Registration ID #NCT03352427.

Pediatric high-grade glioma (pHGG), including both diffuse midline glioma (DMG) and non-midline tumors, continues to be one of the deadliest oncologic diagnoses (both henceforth referred to as “pHGG”). Targeted therapy options aimed at key oncogenic receptor tyrosine kinase (RTK) drivers using small-molecule RTK inhibitors has been extensively studied, but the absence of proper in vivo modeling that recapitulate pHGG biology has historically been a research challenge. Thankfully, there have been many recent advances in animal modeling, including Cre-inducible transgenic models, as well as intra-uterine electroporation (IUE) models, which closely recapitulate the salient features of human pHGG tumors. Over 20% of pHGG have been found in sequencing studies to have alterations in platelet derived growth factor-alpha (PDGFRA), making growth factor modeling and inhibition via targeted tyrosine kinases a rich vein of interest. With commonly found alterations in other growth factors, including FGFR, EGFR, VEGFR as well as RET, MET, and ALK, it is necessary to model those receptors, as well. Here we review the recent advances in murine modeling and precision targeting of the most important RTKs in their clinical context. We additionally provide a review of current work in the field with several small molecule RTK inhibitors used in pre-clinical or clinical settings for treatment of pHGG.

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Patients with H3K27M-mutated diffuse midline glioma (DMG) have no proven effective therapies beyond radiation. ONC201, a DRD2 antagonist and mitochondrial ClpP agonist, has shown promise in this population. Clinical and genetic variables associated with ONC201 response in H3K27M-mutant DMG continue to be investigated. A combined clinical and genetic study evaluated patients with H3K27M-DMG treated with single-agent ONC201 at the established phase 2 dose. Clinical outcomes of patients treated on two recently completed multi-site clinical studies (NCT03416530 and NCT03134131, n = 75) were compared with historical control data from patients with confirmed H3K27M-DMG (n = 391 total, n = 119 recurrent). Patients treated with ONC201 monotherapy following initial radiation, but prior to recurrence, demonstrated a median overall survival (OS) of 25.6 months from diagnosis and recurrent patients demonstrated a median OS of 16.2 months from recurrence, both of these more than doubling historical outcomes. Using a Cox model to correct for age, gender and tumor location, OS of ONC201-treated patients with H3K27M-mutant tumors remained significantly better than non-ONC201-treated historical controls (p = 0.0001). A survival and radiographic analysis based on tumor location, revealed stronger responses in thalamic patients. In patients with thalamic tumors treated after initial radiation (n = 16), median OS was not reached with median follow up of 22.1 months (historical control median OS of 12.5 months, n = 83, p = 0.0001). Significant correlations were found between baseline cerebral blood flow (CBF) on perfusion imaging and OS (Pearson’s r = 0.75, p = 0.003) and between nrCBF and PFS (r = 0.77, p = 0.002). Baseline tumor sequencing from treated patients (n = 20) demonstrates EGFR mutation (n = 3) and high EGFR expression as a marker of resistance and improved response in tumors with MAPK-pathway alterations (n = 5). In conclusion, ONC201 demonstrates unprecedented clinical and radiographic efficacy in H3K27M-mutant DMG with outcomes enriched in patients with thalamic tumors, treatment prior to recurrence, MAPK-pathway alterations, and patients with relatively high CBF.

ONC201, the first bitopic DRD2 antagonist for clinical oncology, has shown efficacy in H3 K27M-mutant glioma. We performed an integrated preclinical and clinical analysis of ONC201 in thalamic H3 K27M-mutant glioma. ONC201 was effective in mouse intra-uterine electroporation (IUE)-generated H3 K27M-mutant gliomas, with an in vitro IC50 of 500 nM and 50% prolongation of median survival in vivo (p=0.02, n=14). Elevated DRD2 expression was found in the thalamus of non-malignant brain tissue, leading to the hypothesis that thalamic tumors may be a particularly ONC201-sensitive sub-group. We analyzed thalamic H3 K27M-mutant glioma patients treated with ONC201 as of the 05/22/2019 cutoff date, which included patients who had recurrent disease prior to initiating ONC201 (n=20; 15–73 years old) and post-radiation non-recurrent patients (n=11; 5–19 years old). As of 5/22/2019, 10 of 20 recurrent patients and 9 of 11 non-recurrent patients remain on-treatment. Median PFS has not been reached for either cohort: median follow-up of 2.2 months (range: 0.6–37.9) for recurrent patients and 10.6 months (range: 4.3–20.5) from diagnosis for non-recurrent patients. Best response so far by RANO includes 1 CR, 2 PR, 7 SD, 9 PD, 1 NE for recurrent patients and 1 PR, 7 SD, 3 PD for non-recurrent patients. Additionally, 3 recurrent (-66%, -47%, -34%) and 2 non-recurrent (-40%, -10%) patients experienced regressions but are not yet confirmed PRs. For recurrent patients, median onset of response is 3.5 months (range: 2.2–3.8) and median duration of response has not been reached with a median follow-up of 12.5 months (range: 8.1–32.8). Preliminary analyses demonstrated a strong correlation of cell-free tumor DNA in plasma and CSF with MRI response. In summary, ONC201 demonstrates promising clinical efficacy in thalamic H3 K27M-mutant glioma patients, regardless of age. Micro-environmental DRD2 expression may enhance the overall ONC201 response and extend its therapeutic utility beyond H3 K27M-mutant glioma.

Diffuse midline glioma (DMG) is a high-grade glioma with a median overall survival of 9-11 months. Radiotherapy is the only recognized treatment. The DMG tumor microenvironment (TME) contains few, if any, tumor infiltrating lymphocytes (TILs) or inflammatory cytokines, thus is distinctive of an ‘immunologically cold’ tumor/TME.1 DMG lack the expression of immunosuppressive immune checkpoint proteins, likely explaining the failure of immune checkpoint inhibitors (ICI) tested under clinical trials for DMG patients, and suggestive of an alternative mechanism underpinning the cold TME. 1 Glioblastomas also harbor a cold TME, which can be somewhat explained by T cell lymphopenia, influenced by the sequestration of T cells in the bone marrow (through Beta-arrestin-induced internalization of Sphingosine-1-phosphate receptor 1 [S1PR1]). 2 Dopaminergic activation of Beta-arrestin and hence S1PR1 internalization, is potentially regulated through dopaminergic peripheral nerves in primary and secondary lymphoid organs, regulated by the Dopamine receptor D2 (DRD2), that is highly expressed on T cells. ONC201 is a potent DRD2 antagonist, currently in phase I-III clinical trials for DMG patients, alone and in combination with radiotherapy and the PI3K/AKT inhibitor paxalisib (NCT05009992). Proteomic profiling of DMG patient-derived cells +/-ONC201 showed increased expression of several antigen presenting pathway proteins, including Beta-2-microglobulin (B2M) and HLA class I histocompatibility antigen, A alpha chain (HLA-A). This was confirmed in vivo using SU-DIPG-VI patient-derived xenograft mouse model tissues +/-ONC201 alone, and together with paxalisib. Excitingly, this combination (given orally) promoted the recruitment of TILs to the tumor, revealing novel immunomodulatory effects. In vivo, ONC201 promoted the expression of EMILIN-3, a TGF-β antagonist that is known to inhibit HLA-A/B2M expression, possibly explaining the increased MHC-I activity. This study uncovers a novel link between treatment of DMG with ONC201 and paxalisib and the role dopaminergic peripheral nerves signaling may play on the sequestration of T cells within lymphoid organs and lymphopenia.