We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph-) acute lymphoblastic leukemia (ALL). Two model human Ph- ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC50:5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph-ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph- ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treat-ment of patients with Ph- ALL © 2008 by The American Society of Hematology.