De-novo purine biosynthesis is a major driver of chemoresistance in glioblastoma

Abstract

This year nearly 20,000 lives will be lost to Glioblastoma (GBM), a treatmentresistant primary brain cancer. In this study, we identified a molecular circuit driven by epigenetic regulation that regulates the expression of ciliary protein ALR13B. We also demonstrated that ARL13B subsequently interacts with purine biosynthetic enzyme IMPDH2. Removal of ARL13B enhanced TMZ-induced DNA damage by reducing denovo purine biosynthesis and forcing GBM cells to rely on the purine salvage pathway.Furthermore, targeting can be achieved by using an FDA-approved drug, Mycophenolate Moefitil. Our results suggest a clinical evaluation of MMF in combination with TMZ treatment in glioma patients. standard of care for newly diagnosed GBM, is associated with EZH2/PRC2 regulated ARL13B. Further, we demonstrated an interaction between ARL13B and IMPDH2, a ratelimiting enzyme of purine biosynthesis, that impacts GBM's adaptive response to TMZ by inhibiting purine salvaging. Disruption of IMPDH2 activity by using an FDA approved compound Mycophenolate Moefitil (MMF) significantly increased the therapeutic efficacy of TMZ. MMF extends the survival of patient-derived xenograft (PDX) models of mice across all GBM subtypes. Our study, therefore, provides evidence of a rapidly clinically translatable opportunity to enhance the efficacy of alkylating agents in GBM. 5 Materials and MethodsAnimal studies: Athymic nude mice (NU(NCr)-Foxn1nu; Charles River Laboratory) were maintained according to all Institutional Animal Care and Use Committee guidelines. In compliance with all applicable federal and state statutes governing animal use in biomedical research, the mice were housed before and during the study in a temperatureand humidity-controlled room following a strict 12-hour light/dark cycle. Cell culture: Patient-derived xenograft (PDX) glioblastoma specimens GBM5, GBM6, GBM43, and GBM52, were obtained from Dr. C. David James (Northwestern University) and maintained for in vitro experiments in DMEM (Thermo Fischer Scientific) supplemented with 1% fetal bovine serum (FBS; Atlanta Biologicals) and 1% Antibiotic Antimycotic Solution (Corning) according to established protocols with slight alterations 23 . For the generation of shRNA knockdown lines, lentivirus particles were made using HEK293 cells (ATCC) transfected with 2nd generation packaging/envelope plasmids (Dr. Yasuhiro Ikeda, Mayo Clinic) and shRNA clones (GeneCopoeia). U251 cells were obtained from American Type Cell Culture and maintained for in vitro experiments in DMEM supplemented with 10% FBS and 1% Antibiotic Antimycotic Solution. CRISPR knockout of U251 cells was created by direct transfection with Cas9 nuclease and sgRNA targeting ARL13B (Dharmacon). All cells were passaged by washing one time with phosphate-buffered saline solution (PBS; Gibco) and detached using 0.25% trypsin/2.21mM EDTA (Corning).Flow cytometry: Flow cytometric analysis was performed on homogenized tumor tissue after HLA-based isolation from murine brains and adherent PDX cells after trypsinization.Cells we...

Reuse of Molecules for Glioblastoma Therapy

“…Importantly, these studies showed that MMF treatment or genetic inhibition of IMPDH significantly decrease GBM growth in mouse models. Furthermore, MMF treatment sensitizes GBM cells to chemotherapy and radiotherapy [ 46 , 47 ].…”

“…Currently, there is a phase 0–1 Trial (NCT04477200) looking at the effects of MMF combined with radiation, based on the observation that MMF increases the efficacy of radiotherapy against GBM [ 47 ]. This is a dose-escalation study to determine the maximum tolerated dose of MMF when administered with radiation, in patients with recurrent GBM or recurrent gliosarcoma.…”

Ribonucleotide Reductase Regulatory Subunit M2 as a Driver of Glioblastoma TMZ-Resistance through Modulation of dNTP Production

“…For complete metabolomic profiling, samples were analyzed by high-performance liquid chromatography, high-resolution mass spectrometry and tandem mass spectrometry (HPLC-MS/MS) that our lab previously has performed 19 . The system consisted of a Thermo Q Exactive TM with an electrospray source, and an Ultimate 3000 (Thermo) series HPLC which consisted of a binary pump, degasser, and auto-sampler outfitted with an Xbridge Amide column (Waters; dimensions of 4.6 mm 100 mm and a 3.5 mm particle size).…”

“…To measure the relative abundances of specific intracellular metabolites, extracts were prepared and analyzed by LC-MS/MS, as described previously 19,20 . For targeted steady-state samples, metabolites were washed with 4-mL PBS and extracted on dry ice with 4-mL 80% methanol (−80°C), as described previously (Weinberg et al, 2019;Yuan et al, 2019).…”

“…Treat cells were labelled with 1 μCi of either 3H-uridine or 3H-guanine as previously described 19 .…”