Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

Abstract

IntroductionPosttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. ObjectivesWe aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1/-2/-3/-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. MethodsWe synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. ResultsNovel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi vorinostat. Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p<0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces a caspase-dependent processing of the autophagy proteins ULK1 and p62. ConclusionThese data reveal that HDACs are required to stabilize autophagy proteins through a suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention. HighlightsO_LINovel HDACi with nanomolar activity against leukemic cells were synthesized. C_LIO_LIHDACi of the KH-series are superior to a clinical grade HDACi. C_LIO_LIHDACi of the KH-series modulate acetylation and phosphorylation of proteins. C_LIO_LIThe new HDACi KH16 regulates cell cycle arrest, apoptosis, and autophagy. C_LIO_LIApoptosis acts upstream of autophagy in KH16-treated cells. C_LI