Supplementary Table 1. Primers used for qRT-PCR Supplementary Fig. 1. Heterozygous IDH1R132H/WT enhanced therapy-induced senescence in glioma cells. Supplementary Fig. 2. a. Schematic graph of genome editing strategy with two guide RNAs to knock out human ATRX. b. The ATRX gene sequence of two clones (C4, C10) after genome editing with CRISPR/Cas9 and two ARTX guide RNAs. c. Transwell assays of cell migration showing ATRX knockout in mIDH1 WT cells decreased cell migration. Supplementary Fig. 3. ATRX knockout in SVG IDH1 WT cells did not affect cell growth and migration. Supplementary Fig. 4. a, b. BrdU incorporation assays. c. Cell cycle analysis with PI and flow cytometry. Cells were synchronized by serum withdrawing for 24 h, followed by serum replenishing for 18 h. d. Western blot analyses of Y-H2AX and DNA damage response pathways in cells. Quantification of protein level changes was from the average of three independent experiments. Supplementary Fig. 5. IDH1R132H/WT and ATRX knockout did not induce ALT and alter chromosome numbers in SVG astroglial cells. Supplementary Fig. 6. Effect of AGI-5198 (a) and 5-Aza (b) on SASP expression. Supplementary Fig. 7. M0 human primary macrophages responses to LPS and IL4. Supplementary Fig. 8. ABT-263 decreased SA-beta-gal staining.
ARTICLE ABSTRACTHeterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2–M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non–cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies.
The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant–mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.