Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Genetic silencing of leukemia-associated proteins with small interfering RNAs (siRNAs) is a straightforward way to delineate their functions. It can be very challenging to deliver siRNAs to leukemia-derived cells with high transfection efficiency and without compromising their viability. This protocol describes an efficient approach to silence oncogenic feline McDonough sarcoma (FMS)-like tyrosine kinase-3 in leukemia cells using siRNAs that are delivered by electroporation. The protocol maintains high cell viability and is generally useful to decrease RNAs encoding proteins of interest.