Study Design and Drug Selection

Four epigenetic drugs were selected from the 64 listed in the Human Epigenetic Drug Database (HEDD) (15) because they have been reported to have antitumor activity and have passed clinical phase II trials to prove their safety. The four drugs chosen were azacitidine (AZA) and HDZ, both DNMTi, and VAL and suberanilohydroxamic acid (SAHA), both HDACi. These four candidate epigenetic drugs were preliminarily tested for their cytotoxicity. HDZ and VAL showed higher cellular toxicity, no matter whether carrying the JAK2V617F mutation or not ( Figure 1 ). They were, therefore, selected for retrospective cohort studies for association with incidence reduction of all subgroups of HNs. To study the dose effect, the dose range was stratified according to the defined daily dose (DDD).

Flowchart showing steps for screening candidate drugs, from in silico and in vitro to ex vivo. (A) In-silico screening led to four candidate drugs AZA, HDZ, VAL, and SAHA. (B–E) JAK2V617F mutation may affect the therapeutic efficacy of AZA and SAHA, but not of HDZ-VAL. The cell survival of four MPN cell lines treated with HDZ-VAL combination. (F, G) The cell survival rate of four MPN cell lines treated with AZA or SAHA. (H–K) The inhibitory efficacy of cell survival rate after HDZ-VAL combinational treatment on the leukocytes of PV or ET patients.