Fig. 3.

Inhibition of EP300/CBP potentiates BETi-mediated silencing of oncogenic KRAS signaling. (A) An equal number of Panc1 cells were seeded in 24-well plates and treated with either a DMSO vehicle, 1 μM JQ-1, JQ-1 and 1 μM SGC-CBP30, 1 μM XP-524, or XP-524 and SGC-CBP30. Cell growth was evaluated every 4 h until the control group reached 100% confluence (n = 4 per group). (B) Panc1 cells were incubated with increasing concentrations of either JQ-1 or XP-524, each with or without a fixed 1-μM dose of SGC-CBP30. After 48 h, cell viability was evaluated by MTT assay. (C) Panc1 cells were incubated with either a DMSO vehicle, 1 μM JQ-1, JQ-1, and 1 μM SGC-CBP30, or 1 μM XP-524 and subjected to RNA sequencing. (D) Focused heatmap showing select, significantly altered genes in the cell cycle pathway using a false discovery rate (FDR)–adjusted P value of <0.05. (E) Focused heatmap showing select, significantly altered genes in the KRAS signaling pathway using an FDR-adjusted P value of <0.05. (F–H) Panc1, MiaPaCa2 (MP2), and ASPC1 cells were treated with 1 μM XP-524 and KRAS mRNA evaluated by qPCR. Values are presented as fold change compared to DMSO control (C)-treated samples. (I) Panc1 cells were again treated with 1 μM JQ-1, JQ-1 and 1 μM SGC-CBP30, or 1 μM XP-524 and the interaction between BRD4 and EP300 evaluated by immunoprecipitation (IP) followed by Western blot (IB). (J and K) Panc1 and KPC-105 cells were treated as described and evaluated by Western blot for pRB, H3K27 acetylation, as well as KRAS expression and downstream activation of the MEK/ERK pathway. (L) MiaPaCa2 (MP2) and ASPC1 cells and KRAS expression and downstream MEK/ERK activation evaluated by Western blot. (M–P) Excisional biopsies from three PDAC patients undergoing survival resection were cored, sectioned at 250-μm interval, and cultured ex vivo either in a control PBS vehicle or 5 μM XP-524. After 72 h, slice cultures were formalin fixed, paraffin embedded, and stained with H&E or by IHC for pERK or CK19 and PCNA.