Abstract Histone acetyltransferases (HAT) catalyze the acylation of lysine side chains and are implicated in diverse human cancers as both oncogenes and non-oncogene dependencies 1 . Acetyl-CoA-competitive HAT inhibitors have garnered attention as potential cancer therapeutics and the first clinical trial for this class is ongoing ( NCT04606446 ). Despite broad enthusiasm for these targets, notably including CBP/p300 and KAT6A/B 2–5 , the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Using comparative transcriptional genomics, we found that the direct gene regulatory consequences of CBP/p300 HAT inhibition are indistinguishable in models of intrinsically hypersensitive and insensitive acute myeloid leukemia (AML). We therefore modelled acquired drug resistance using a forward genetic selection and identified dysregulation of coenzyme A (CoA) metabolism as a facile driver of resistance to HAT inhibitors. Specifically, drug resistance selected for mutations in PANK3 , a pantothenate kinase that controls the rate limiting step in CoA biosynthesis 6 . These mutations prevent negative feedback inhibition, resulting in drastically elevated concentrations of intracellular acetyl-CoA, which directly outcompetes drug-target engagement. This not only impacts the activity of structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B inhibitor that is currently in Phase-1 clinical trials. We further validated these results using a genome-scale CRISPR/Cas9 loss-of-function genetic modifier screen, which identified additional gene-drug interactions between HAT inhibitors and the CoA biosynthetic pathway. Top hits from the screen included the phosphatase, PANK4 , which negatively regulates CoA production and therefore suppresses sensitivity to HAT inhibition upon knockout 7 , as well as the pantothenate transporter, SLC5A6 8 , which enhances sensitivity. Altogether, this work uncovers CoA plasticity as an unexpected but potentially class-wide liability of anti-cancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.

Metabolic adaptations underpin resistance to histone acetyltransferase inhibition