Journal: BMC Cancer

Article Title: Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

doi: 10.1186/s12885-024-12534-w

Figure Lengend Snippet: The DHODH inhibitor represses the growth of human DHL cells. ( A ) FISH analysis of DB and SU-DHL4 cells confirmed c-MYC and BCL2 translocation. Separate probes were used, and the split points were labeled by yellow arrows. ( B ) Dose-dependent effects of brequinar (BRQ) on the viabilities of DB and SU-DHL-4 cells. The cells were treated with DMSO as the control or 1 ∼ 10,000 nM BRQ for 24 to 72 h. The cell viability was calculated by the formula: Cell viability (%) = [OD (drug+) - OD (Blank)] / [OD (drug-) - OD (Blank)] × 100% at 24 h, 48 h and 72 h. Data was normalized as 100% by the formula: OD (drug-) - OD (Blank). The x-axis log 0 means 1 nM BRQ. ( C ) Cell proliferation assay of DB and SU-DHL4 cells treated with DMSO or 5 µM BRQ with or without uridine at the indicated time points. Significance was achieved by one-way ANOVA followed by Dunnett’s test. *, P < 0.05, **, P < 0.01, ***, P < 0.001. ( D ) Flow cytometry cell apoptosis assays were used in DB and SU-DHL4 cells with DMSO (con), 2.5 µM, or 5 µM BRQ and 5 µM BRQ plus 1000 µM uridine (rescue) for 24 and 48 h. Significance was achieved by two-way ANOVA followed by Bonferroni post-hoc analysis. *, P < 0.05, **, P < 0.01, ***, P < 0.001. ( E ) Cell cycle analysis in BRQ-treated DLBCL cells by flow cytometry using propidium iodide (PI). DB and SU-DHL-4 cells were treated with DMSO or 5 µM BRQ for 6, 12, and 24 h and 5 µM BRQ plus 1000 µM uridine for 24 h. The percentage of cells in G2/M phases is indicated in the histogram. ***, P < 0.001. ( F ) The total protein levels of P53, P21, caspase3, caspase9, and PARP and the cleaved versions of caspase proteins in DB and SU-DHL4 cells were detected by western blotting after treatment with DMSO, 5 µM BRQ, and 5 µM BRQ plus 1000 µM uridine for 48 h. Data are shown as means ± standard error (SE). All experiments were performed in triplicate

Article Snippet: Brequinar (HY-108,325) and venetoclax (HY-15,531) were purchased from MCE (USA).

Techniques: Translocation Assay, Labeling, Control, Proliferation Assay, Flow Cytometry, Cell Cycle Assay, Western Blot

Journal: BMC Cancer

Article Title: Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

doi: 10.1186/s12885-024-12534-w

Figure Lengend Snippet: The combination of brequinar and venetoclax affects DHL cells through complementary pathways. ( A ) The protein levels of c-MYC, BCL-2, BAX, Mcl-1, and BCL-XL in DB and SU-DHL4 cells treated with DMSO (control), 20 nM venetoclax (VEN), 500 nM BRQ, and 20 nM venetoclax plus 500 nM BRQ (combination) for 48 h. ( B ) Gene expression of c-MYC and MCL-1 from DB and SU-DHL4 cells treated with single or combined venetoclax and BRQ for 48 h. The values were calculated from 6 independent experiments. Significance was achieved by one-way ANOVA followed by Turkey’s test.****, P < 0.0001, ***, P < 0.001, **, P < 0.01, *, P < 0.05. ( C ) The protein levels of c-MYC and BCL-2 in SU-DHL2 cells treated with DMSO (control), 20 nM venetoclax (VEN), 500 nM BRQ, and 20 nM venetoclax plus 500 nM BRQ (combination) for 48 h. ( D ) Gene expression of MCL-1 downregulated by BRQ could be rescued by uridine. DB and SU-DHL4 cells were DMSO, 5 µM BRQ, and 5 µM BRQ plus 1000 µM uridine for 48 h. The values are shown as the mean ± SE of 3 independent experiments. Significance was achieved by one-way ANOVA followed by Dunnett’s test. **, P < 0.01, ***, P < 0.001. ( E ) The protein level of MCL-1 in DB and SU-DHL4 cells treated with DMSO or 5 µM BRQ at the indicated time points (hours). ( F ) Gene expression of MCL-1 in MYC-NC and MYC-OE DB and SU-DHL4 cells. The values are shown as the mean ± SE of 3 independent experiments. ( G ) Protein-protein interactions of BCL-2/BIM in SU-DHL4 and DB cells. The cells were treated with DMSO, 20 nM venetoclax, 500 nM BRQ, and 20 nM venetoclax plus 500 nM BRQ for 48 hours

Article Snippet: Brequinar (HY-108,325) and venetoclax (HY-15,531) were purchased from MCE (USA).

Techniques: Control, Gene Expression, Protein-Protein interactions

Journal: BMC Cancer

Article Title: Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

doi: 10.1186/s12885-024-12534-w

Figure Lengend Snippet: Venetoclax and BRQ treatments lead to distinct gene expression profiles. ( A ) Differentially expressed genes involved in ribosome biosynthesis, DNA repair, and damage pathway in SU-DHL4 cells after treatment with venetoclax and/or BRQ for 48 h. ( B ) Genes reported to be mutated in DLBCL cells differentially expressed in SU-DHL4 cells. ( C ) Genes regulating the function of mature B cells differentially expressed in SU-DHL4 cells. ( A-C ) Significantly dysregulated genes are represented in the heatmap. The color scale bar represents higher (red) to lower (green) expression. Gene expression levels are expressed as FPKM values, and differences are shown on a color scale after Z-score transformation. FPKM, fragments per kilobase of exon per million fragments mapped. ( D ) GO enrichment analysis of SU-DHL4 cells treated with the BRQ and venetoclax combination compared with DMSO (control). ( E ) Gene set enrichment analysis enrichment plot of ribosome biosynthesis genes from data obtained 48 h after stimulation as indicated. VEN, venetoclax; BRQ, brequinar; CON, Control

Article Snippet: Brequinar (HY-108,325) and venetoclax (HY-15,531) were purchased from MCE (USA).

Techniques: Gene Expression, Expressing, Transformation Assay, Control

Journal: BMC Cancer

Article Title: Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

doi: 10.1186/s12885-024-12534-w

Figure Lengend Snippet: BRQ downregulates ribosome biosynthesis genes and the combination effect in DHL xenografts. ( A ) Gene expression of RPL26, RPS27, and MRPS6 in SU-DHL4 cells incubated with DMSO (Control) or 20 nM venetoclax (VEN), 500 nM BRQ, or 20 nM venetoclax plus 500 nM BRQ (Combination) for 48 h. The values are shown as the mean ± SE of 6 independent experiments. Significance was achieved by one-way ANOVA followed by Turkey’s test. *, P < 0.05, **, P < 0.01, ***, P < 0.001. ( B ) Protein levels of RPL-26, RPS-27 and MRPS-6 from DB and SU-DHL4 cells treated with DMSO, 20 nM venetoclax, 500 nM BRQ, and 20 nM venetoclax plus 500 nM BRQ for 48 h. ( C ) Images of the tumor-bearing NOD-SCID mice treated with vehicles(control), venetoclax, brequinar, venetoclax and brequinar combination at 20 days after inoculation. ( D ) Comparison of tumor volumes in tumor-bearing NOD-SCID mice treated with venetoclax, brequinar, or venetoclax and brequinar combination to vehicles(control) at the end of treatment. The values are presented as the mean ± SD ( n = 4). Significance was achieved by one-way ANOVA followed by Dunnett’s test. ***, P < 0.001, **, P < 0.01, *, P < 0.05, ns, non-significance. ( E ) Tumor growth curves in tumor-bearing NOD-SCID mice after SU-DHL4 cells inoculation. ( F ) Survival analysis of the DHL xenograft mice was performed using a log-rank test for comparison. ( G ) Stripped tumor tissue images from xenograft tumors with the indicated treatments at day22 post incubation. ( H ) Representative HE and immunochemical images from xenograft tumors with the indicated treatments at day 22 post incubation. ( I ) A schematic diagram showing the synergistic mechanism of DHODH and BCL-2 inhibitors

Article Snippet: Brequinar (HY-108,325) and venetoclax (HY-15,531) were purchased from MCE (USA).

Techniques: Gene Expression, Incubation, Control, Comparison