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Background: Pediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. Treatment of PDGFRA-driven HGG with targeted agents, such as the tyrosine kinase inhibitor dasatinib, has failed in the clinic. We hypothesized that co-treatment with everolimus may improve the efficacy of dasatinib in PDGFRA-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. De novo tumors were generated in mice using intra-uterine electroporation (IUE) by injecting PB plasmids of TP53 mutation, PDGFRA mutation and H3K27M mutation (PPK) in the lateral vertical of mice embryos. Two children with recurrent PDGFRA-driven HGG were treated with dasatinib and everolimus with correlate CSF analysis. Results: Dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a dose-dependent reduction of PDGFRA and pPDGFRA. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended survival of PPK tumor bearing mice. Two children with recurrent PDGFRA-driven HGG treated with dasatinib and everolimus survived six months and nine months after progression. A paired CSF sample from the patient with PDGFRA-amplified HGG showed 50% increase in CSF dasatinib levels after the addition of everolimus. Conclusion: Efficacy of dasatinib treatment of PDGFRA-driven HGG is improved with everolimus and suggests a promising route for improving targeted therapy for this patient population. Clinical Trial Registration ID # NCT03352427 .

Figure S1. Serial dilution of K27M mutant oligonucleotide in constant background of wild-type DNA demonstrates consistent detection down to at least 2% VAF under typical experimental conditions, with the possibility of detection at even lower VAF under ideal conditions. One such dilution series is shown above, with (a) showing number of droplets positive for mutant or wild-type H3F3A sequence and (b) showing the corresponding VAF values. Figure S2. Plot of droplets (blue â positive mutant H3F3A K27M, green â positive wildtype H3F3A, grey â negative droplets) from ddPCR performed on (a) non-tumor human CSF spiked with synthetic K27M mutant sequence oligonucleotide and (b) non-tumor human CSF alone. (DOCX 268 kb)

Supplemental Information. Detailed methods and H3F3A K27M assay design. (DOCX 28 kb)

Supplemental Information. Detailed methods and H3F3A K27M assay design. (DOCX 28 kb)

Figure S1. Serial dilution of K27M mutant oligonucleotide in constant background of wild-type DNA demonstrates consistent detection down to at least 2% VAF under typical experimental conditions, with the possibility of detection at even lower VAF under ideal conditions. One such dilution series is shown above, with (a) showing number of droplets positive for mutant or wild-type H3F3A sequence and (b) showing the corresponding VAF values. Figure S2. Plot of droplets (blue â positive mutant H3F3A K27M, green â positive wildtype H3F3A, grey â negative droplets) from ddPCR performed on (a) non-tumor human CSF spiked with synthetic K27M mutant sequence oligonucleotide and (b) non-tumor human CSF alone. (DOCX 268 kb)