Transcriptomic analysis of Diffuse Intrinsic Pontine Glioma (DIPG) identifies a targetable ALDH-positive subset of highly tumorigenic cancer stem-like cells

Understanding the cancer stem-cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism and mTOR signaling in ALDH+ compared to ALDH- supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation and neurosphere formation and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacological MAPK/PI3K/mTOR targeting downregulated MYC, E2F and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. Characterization of DIPG CSCs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG. Overall design: Eight SU-DIPG-XIII samples were analyzed. Cells were maintained under normal cell culture conditions in T-75 flasks. At the time of treatments, each T-75 flask (at ~75% confluency there are 8x10^6 million cells) was centrifuged, conditioned media was saved, and cells were resuspended in 4 mL of the conditioned media and transfered to a new T-25 flask for treatment (one T-25 flask for every T-75 flask). Samples were treated with 100 nM of PD0325901 (PD-901), 100 nM of GSK2126458 (GSK-458), 50 nM PD-901 + 50 nM GSK-458 (combination) or equimolar DMSO for two hours prior to ALDEFLUOR Assay staining and FACS sorting into ALDH+ and ALDH- conditions. ALDH+ DMSO treated and ALDH- DMSO treated samples were used as controls for comparison.