Nuclear protein of the testis (NUT) midline carcinoma (NMC), is a rare and highly aggressive form of undifferentiated squamous cell carcinoma. NMC is molecularly characterized by chromosomal rearrangement of the NUT gene to another gene, most commonly the bromodomain and extraterminal domain (BET) gene BRD4, forming the BRD4-NUT fusion oncogene. Therefore, inhibiting BRD4-NUT oncogenic function directly by BET inhibitors represents an attractive therapeutic approach but toxicity may limit the use of pan-BET inhibitors treating this cancer. We thus performed a drug screening approach using a library consisting of epigenetic compounds and ‘Donated Chemical Probes’ collated by the Structural Genomics Consortium (SGC) and identified the p300/CBP HAT inhibitor A-485, in addition to the well-known BET inhibitor JQ1, to be the most active candidate for NMC treatment. In contrast to JQ1, A-485 was selectively potent in NMC compared to other cell lines tested. Mechanistically, A-485 inhibited p300-mediated histone acetylation, leading to disruption of BRD4-NUT binding to hyperacetylated megadomains. Consistently, BRD4-NUT megadomain-associated genes MYC, CCAT1 and TP63 were downregulated by A-485. A-485 strongly induced squamous differentiation, cell cycle arrest and apoptosis. Combined inhibition of p300/CBP and BET showed synergistic effects. In summary, we identified the p300/CBP HAT domain as a putative therapeutic target in highly therapy-resistant NMC.