CNSC-07 - Using CRISPR-Cas9 screening to identify biomarkers for MMF sensitivity in glioblastoma multiforme

Glioblastoma (GBM) the most common malignant brain tumor with a median survival of 21 months. Recurrent GBM remains resistant to available therapies. Our phase I clinical trial investigates combining mycophenolate mofetil (MMF) and temozolomide (TMZ) to target de novo purine biosynthesis in GBM. Previously, our lab revealed that GBM cells upregulate purine biosynthesis and inhibit purine salvage through the ARL13B-IMPDH2 axis during TMZ therapy. Targeting IMPDH2 with MMF/TMZ extended survival in GBM patient-derived xenograft models. In preparation for Phase II of our clinical trial, we aim to identify a predictive biomarker for MMF+ TMZ therapy. In a CRISPR-Cas9-mediated knockout screen, we identified ten genes, including SLC1A5, showing enrichment (differential β score mean values > 60), indicating sensitivity to MMF/TMZ. SLC1A5 displays a wide distribution of expression in Glioviz patient datasets and encodes the glutamine transporter ASCT2, which is crucial for de novo purine biosynthesis. RNA-seq and protein expression analysis in six glioma cell lines confirmed that high SLC1A5 expression appears to correlate with sensitivity to MMF/TMZ (IC₅₀ 0.58-21.30 μM), while low expression led to resistance (IC₅₀ 73.43-1148 μM). Knockdown of SLC1A5 in a high-expression cell line demonstrated reduced sensitivity to MMF/TMZ (IC₅₀ 2.099 vs IC₅₀ 1007 μM). To further validate our findings, we plan to perform SLC1A5 overexpression in low-expression glioma cell lines. We hypothesize that GBM patients with higher SLC1A5 expression may respond favorably to MMF/TMZ therapy due to the reliance of their tumors on de novo purine biosynthesis. We aim to combine this data with analysis of our Phase I clinical samples to retrospectively establish SLC1A5 as a biomarker for MMF/TMZ sensitivity and investigate its role in tumorigenic purine metabolism. Identifying predictive markers will enhance personalized treatment strategies for GBM, offering hope for improved outcomes.