Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Transcriptome-guided high-throughput drug screening identifies SN-38 as a potent topoisomerase I inhibitor, inducing cell cycle arrest and apoptosis in TP53 wild-type DIPG. (A) Transcriptomic analysis of 98 brainstem glioma tissue samples (31 DIPG, 67 non-DIPG) revealed distinct pathway activation patterns. (B-C) Pathway correlation network and Circos diagram illustrate relationships among enriched pathways, highlighting a strong association between TP53 signaling and cell cycle regulation. (D-E) Drug screening schematic: patient-derived DIPG cell lines (150630, 150714, 170720, 190326, 190313) and primary pons progenitor cells (PPCs) were treated with 950 cell cycle inhibitors (1 μM) in 384-well plates for 72 h. Among 597 compounds with ≤10% cytotoxicity to PPCs, 18 topoisomerase I (TOP1) inhibitors selectively inhibited TP53 wild-type DIPG lines. (D) Heatmap shows relative viability inhibition by 18 TOP1 inhibitors across DIPG lines and PPCs. Isogenic TP53 or PPM1D knockdown lines were included for comparison. Viability assessed by CellTiter-Glo, normalized to 0.1% DMSO or water control ( n = 3). (E and F) 150714 cells treated with 18 TOP1 inhibitors (100 nM, 72 h) in 96-well plates showed significant viability reduction (mean ± SD, n = 3; P < .0001, two-tailed unpaired t-test). (G) Dose-response of SN-38 in PPCs and 150714 cells at 15, 30, and 60 nM over 24, 48, and 72 h ( n = 3). Statistical significance: **** P < .0001, *** P < .001, NS: P > .05. (H) Six patient-derived DIPG cell lines and PPCs treated with SN-38 at multiple concentrations for 72 h; viability normalized to control ( n = 3). (I) Caspase 3/7 activity after SN-38 treatment (10 or 20 nM, 48 h) increased apoptosis in TP53 wild-type DIPG lines compared to controls ( n = 3). (J) Flow cytometry cell cycle analysis of DIPG lines treated with 10 nM SN-38 for 48 h; PPCs used as controls.

Article Snippet: Cell cycle distribution was assessed using the Cell Cycle and Apoptosis Analysis Kit (#C1052 Beyotime).

Techniques: High Throughput Screening Assay, Drug discovery, Activation Assay, Derivative Assay, Inhibition, Knockdown, Comparison, Control, Two Tailed Test, Activity Assay, Flow Cytometry, Cell Cycle Assay

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: SN-38 activates the TP53 signaling pathway and promotes apoptosis in TP53 wild-type DIPG cells while showing enhanced effects under PPM1D knockdown conditions. (A) KEGG enrichment analysis of RNA-seq data from TP53 wild-type DIPG cells (150714, 150630) treated with SN-38 (10 nM, 72 h) showed significant activation of TP53 and cell cycle pathways (adjusted P < .05). (B and C) Heatmaps of differentially expressed genes in 150714 and 150630 cells treated with SN-38 (10 nM, 72 h), based on 2 biological replicates. (D) Western blot analysis of 150714 (TP53 WT) and 190326 (TP53 mutant) cells after SN-38 treatment (10 or 20 nM, 72 h). In TP53 WT cells, SN-38 increased P53 and BAX, decreased BCL2, and elevated cleaved PARP1, while 190326 cells showed no cleaved PARP1 activation. (E-F) Western blot analysis of 150714 cells following 72-h treatment with 10 nM SN-38. TP53 knockdown reduced p53 protein levels and impaired downstream apoptotic signaling, as evidenced by diminished BAX upregulation, reduced BCL2 downregulation, and absence of cleaved PARP induction. In TP53 wild-type cells, SN-38 treatment triggered robust apoptotic responses, including decreased total PARP, whereas these effects were abrogated in TP53 KD cells. (E). PPM1D knockdown ( PPM1D KD) enhanced P53 expression (F). (G-J) TP53 knockdown in 150714 and 150630 cells treated with 10 nM SN-38 for 72 h (G and H). PPM1D knockdown in TP53 wild-type DIPG cells treated with 10 nM SN-38 for 72 h (I and J). Cell viability was assessed using the CellTiter-Glo assay. Data are presented as mean ± SD of 3 independent experiments. Statistical significance was determined by two-tailed unpaired t -test (* P < .05, ** P < .01, *** P < .001, **** P < .0001).

Article Snippet: Cell cycle distribution was assessed using the Cell Cycle and Apoptosis Analysis Kit (#C1052 Beyotime).

Techniques: Knockdown, RNA Sequencing, Activation Assay, Western Blot, Mutagenesis, Expressing, Glo Assay, Two Tailed Test

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Synergistic anti-tumor effects of AZ20 and SN-38 in TP53 -mutant DIPG cells through inhibition of ATR pathway signaling and induction of apoptosis. (A) Twenty-one ATR pathway inhibitors were screened in combination with SN-38 (1 μM each) in TP53-mutant DIPG cells. Viability was measured by CellTiter-Glo ( n = 3) analyzed by a two-tailed unpaired t -test. (B-D) Synergy analysis using the BLISS model confirmed a robust synergistic interaction between SN-38 and AZ20 in 190326 cells (D). In contrast, this synergistic effect was not observed in TP53 wild-type DIPG cells (150714, DIPG17) (B and C). (E-G) Cell viability was measured after 24, 48, and 72 h of treatment with DMSO, SN-38 (10 nM), AZ20 (10 nM), or both in 190326, 150714, and DIPG17 cells. Combination significantly reduced viability in 190326 (**** P < .0001). (H) Western blot analysis of protein expression in 190326 cells following 72 h of treatment with DMSO (vehicle control), SN-38 (10 nM), AZ20 (10 nM), or their combination. SN-38 monotherapy activated ATR and its downstream targets, CHK1 and WEE1, while combination treatment with SN-38 and AZ20 suppressed ATR activation and downregulated CHK1 and WEE1 expression. The combination treatment also induced apoptosis, as evidenced by increased levels of cleaved PARP1. (I) Chou-Talalay-based combination index (CI) heatmap for SN-38 and AZ20 in TP53-mutant DIPG cell line 190326. Combination index values were calculated from a 72-h viability assay using fixed-ratio matrix combinations of SN-38 and AZ20. CI < 1 indicates synergy, CI = 1 indicates additivity, and CI > 1 indicates antagonism. (J) 190326 cells transfected with siATR and treated with SN-38 or AZ20 showed reduced viability in SN-38 + siATR and AZ20 + SN-38 + siATR groups (**** P < .0001).

Article Snippet: Cell cycle distribution was assessed using the Cell Cycle and Apoptosis Analysis Kit (#C1052 Beyotime).

Techniques: Mutagenesis, Inhibition, Two Tailed Test, Western Blot, Expressing, Control, Activation Assay, Viability Assay, Transfection