Figure 4.

In Vitro and In Vivo Anti-tumor Effects of BMI1 Inhibition in DIPGs

(A) Growth inhibitory IC₅₀ values of BMI1 small-molecule inhibitors (i) in DIPG cell lines using PTC209 and PTC028 and (ii) in H3K27M-mutant-modified lines (+H3K27M is mutant transduced, and H3K27M-KO is the CRISPR-CAS9 KO of the mutant) using PTC028. See also Figure S5B.

(B) Neurosphere size was measured by live-cell imaging with DIPG cells treated with PTC drugs, using Incucyte at day 12 after treatment. By ANOVA, DMSO versus PTC (three groups), p = 0.0034 (SU-DIPGXIII) and p = 0.0048 (SF8628). Pairwise comparison; ^∗p < 0.01, ^∗∗p < 0.002, ^∗∗∗p < 0.0001; DMSO versus PTC treatments by Student’s t test. See also Figure S5C. Data represent mean ± SEM

(C) Cell viability measured using a primary DIPG patient-derived cell, UPN1285, treated with 100 nM of PTC028 for 3 days. Pairwise comparison; ^∗∗∗p < 0.004; ^∗DMSO versus PTC treatments by Student’s t test. Data represent mean ± SEM.

(D) (i) Tumor implant and treatment protocol, (ii) Kaplan-Meier survival plot of the animals treated with either vehicle or PTC028 (n = 7 each group), and (iii) IHC analysis of p16, p21, and GLB1 expression of the treated tumor tissue. See also Figure S5D.

(E) Volcano plot, with a heatmap adjacent to it, showing the differentially expressed genes detected by RNA-seq in PTC028-treated SF8628 cells compared with DMSO control. Genes highlighted in orange are expressed with an FDR = 0.05 and are associated with cell proliferation, differentiation, or tumor-suppressor pathways. See also Figure S5E.

(F) GSEA from the RNA-seq data obtained from SF8628 cells treated with PTC028 (IC₅₀) compared with DMSO-treated cells annotated to multiple signaling pathway gene sets. See Figure S5F.