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C. Ciardullo et al.
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Figure 3. Apoptosis-related gene expression signature induced by RG7388 in primary chronic lymphocytic leukemia cells. Cells from patients with chronic lympho- cytic leukemia (CLL) with functional p53 (n=24) were exposed ex vivo to RG7388 for 6 h. mRNA expression of genes relating to intrinsic apoptosis (BAX, FDXR, PUMA, TP53INP1), extrinsic apoptosis (FAS, TNFRSF10B), cell cycle arrest (CDKN1A, ZMAT3, GADDA45A), and p53-negative autoregulation (MDM2, WIP1) was measured in response to RG7388 relative to treatment with the dimethylsulfoxide (DMSO) solvent control. Genes induced above the cut-off of 2-fold were considered upregu- lated by the treatment. (A) Expression of p53-target genes in 24 p53-functional samples exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 6 h. Gene induction occurred in a concentration-dependent manner. (B) Expression of p53-target genes in two p53-non-functional samples exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 6 h. No genes were significantly induced by the treatment. (C) Scatter plot showing significant mean induction of PUMA (8.5-fold), MDM2 (5.1-fold), BAX (3.8-fold), TNFRSF10B (2.7-fold), FAS (2.6-fold), and WIP1 (2.2-fold) in p53-functional CLL samples treated with 1 μM RG7388 for 6 h. A slight upregulation of CDKN1A (1.6-fold) was observed. Data are presented as mean ± standard error of mean (SEM). (D) Scatter plot of real-time reverse transcriptase polymerase chain reaction (qRT-PCR) Ct values (cycle number to reach the critical threshold) for anti-apoptotic genes MCL1, BCL2 and BCL-XL, plus additional pro-apoptotic genes NOXA and BIM, in comparison with PUMA for patients’ CLL samples (n=7), showing no significant change in Ct values and hence mRNA expression between RG7388-treated and untreated (DMSO control) samples except for PUMA; Change in Ct for PUMA untreated vs. PUMA treated at 6 h P=0.0001, at 24 h P=0.0066 (paired t-test, n=7).
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haematologica | 2019; 104(12)