Azd4547

GF-109203X (PKC inhibitor), FR180204 (ERK inhibitor), PD0325901 (MEK inhibitor), ZSTK4547 (PI3K inhibitor), U-72122 (PLCγ inhibitor), and AZD4547 (FGFR inhibitor) were all purchased from SelleckChem. PD173074 (FGFR inhibitor) was purchased from Sigma.

All animal experiments were conducted according to protocols approved by University of California, San Diego’s
Institutional Animal Care and Use Committee (IACUC). iRFP720-labelled HK281, GSC2 or TS528 cells were injected intracranially
into immunodeficient mice, at coordinates 1 mm anterior and 2 mm lateral of the right hemisphere relative to the bregma, at a
depth of 4 mm followed by 7–10 days of tumor establishment. Tumor burden was monitored using a three-dimensional (3D)
fluorescence molecular tomographic imaging (FMT 2500, PerkinElmer). HK281 cells were first treated with or without 1 μM
FGFR inhibitor PD173074 (Selleck Chemicals) prior to 5 Gy IR and were then engrafted into immunodeficient mice at
2.0×10⁵/3 μl PBS per mice. Tumor signal intensity was detected at day 12 for control group and
day 20 for treatment group. Survival was monitored until all mice died. TS528 cells were injected at 2.5
×10⁴/1μl PBS per mouse and GSC2 cells were injected at 5.0×10⁵/3 μl PBS
per mice. At day 12 (TS528 cells) or day 18 (GSC2 cells), mice were administrated FGFR inhibitor AZD4547 (Selleck Chemicals)
(50 mg/kg by gavage) or vehicle (DMSO) 2 hr or 4 hr before 2.5 Gy irradiation. This whole treatment process was repeated for 3
consecutive days (day 12–14). FMT signal was monitored starting at day 12 until the end of the experiment and percent
of surviving mice over time was recorded.

Ependymoma cell cultures were isolated from patients and cultured on Laminin (Sigma) and in Neurobasal media (Invitrogen) consisting of: sodium pyruvate (Invitrogen), B27 (Invitrogen), Glutamine (Cleveland Clinic Media Core), human EGF (Invitrogen), human basic FGF (Invitrogen), and penicillin/streptomycin (Cleveland Clinic Media Core). Media was replenished every other day while leaving ~50% conditioned media to encourage continued cell proliferation. Cell viability assays were performed in 96 wells using an Alamar Blue stain (Invitrogen) according to manufacturer’s instructions. Drug response assays were performed by seeding cells overnight, treating the following day with increasing drug concentrations, and reading by Alamar Blue Absorption following 72 hours of treatment. AZD4547 and MK1775 were obtained from Selleck Chemicals. JQ1 was provided by the laboratory of James E. Bradner (Harvard). All cell lines were confirmed to be mycoplasma free using a PCR-based detection strategy with positive and negative controls.