Inhibition of CBP synergizes with the RNA-dependent mechanisms of azacitidine by limiting protein synthesis

The nucleotide analogue azacitidine interferes with RNA and DNA metabolism and is currently the best treatment option for a subset of patients with high-risk myelodysplastic syndromes. However, only half of treated patients respond and almost all patients that initially respond eventually relapse. We performed an optimized loss-of-function shRNA screen in combination with azacitidine treatment in an MDS-derived acute myeloid leukemia cell line to identify chromatin regulators affecting drug response. We identified CBP, as a major regulator of azacitidine sensitivity. Compounds inhibiting the enzymatic activity of CBP synergistically reduced viability of MDS-derived AML cell lines when combined with AZA. Surprisingly, this effect was specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is limited to DNA incorporation. The identification of immediate target genes suggested that the effect of CBP inhibition is mediated by downregulation of genes encoding the translational machinery, which could be confirmed in proteomic analysis of nascent proteins. Furthermore, proteins most affected by CBP inhibition include key drivers of cell cycle progression. Overall design: Nascent mRNA transcripts in the sAML cell line MOLM-13 in response to C646 treatment (2h, 10µM) identified by SLAM-seq MOLM-13 cells were either treated with 10 µM C646 or DMSO (3 replicates each) for 1 hour, labelled with 100 µM 4sU for another hour and SLAM-seq performed using the SLAMseq Anabolic Kinetics Kit (Lexogen Gmbh, Austria) followed by library preparation using the QuantSeq 3'mRNA-Seq for Illumina (FWD) and PCR Add-on Kit for Illumina (Lexogen Gmbh, Austria) according to the manufacturer's instructions. The subsequent data analysis was performed using the software SLAM-Dunk and the R package DEseq2.