Histone Deacetylase Inhibitors Induce Cell Growth Inhibition and Apoptosis in NPM1-Mutated AML Cells: A Possible Role for Epigenetic Therapies in AML Carrying NPM1 Gene Mutations

Abstract

Abstract 2621Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations account for about one-third of adult AML, show distinctive biological and clinical features and have been included as a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. As for other types of cancer, evidence is emerging that, besides the genomic DNA alterations, epigenetic dysregulation may play a role in AML pathogenesis. We investigated the effects of sodium butyrate, a short-chain fatty acid which has long been known to be a histone deacetylase inhibitor (HDACi) able to induce maturation in normal and tumor cells, in cellular models of NPM1-mutated AML in vitro: i) the OCI/AML3 cell line, previously identified as a human AML cell line carrying cytoplasmic mutated NPM1 in the absence of FLT3-ITD; ii) primary AML cells originated from a patient with NPM1-mutated AML bearing FLT3-ITD mutation (MONT1) and propagated as cell line in NOD/SCID mice; and iii) primary AML cells from NPM1-mutated AML patients at diagnosis. In either cell lines or patients’ primary AML cells carrying NPM1 mutation, but not in the U937 or OCI/AML2 cell lines (not harboring NPM1 gene mutation) used as control, growth arrest, cell cycle arrest (G0-G1 phase) and pro-apoptotic effects were evident after 24 hrs and marked after 48 hrs of treatment with doses of drug of 0.5–1 mM. No signs of differentiation were evident at morphological and flow cytometric examinations of treated cells. Western blot analysis with specific antibodies showed that levels of either NPM1 mutant or wild-type protein did not appear significantly affected by treatment with sodium butyrate. Interestingly, induction of apoptosis was associated with marked activation of caspase-8, suggesting involvement of the death cell receptors pathway. Indeed, flow cytometric analysis showed 2-fold increased expression of TRAIL-receptor DR5 upon drug treatment at 48 hrs. Moreover, concomitant treatment with a specific caspase-8 inhibitor prevented sodium butyrate induced-cell growth arrest and markedly reduced apoptosis in OCI/AML3 cell line. Derivatives of butyrate, which are capable of acting as HDAC inhibitors, are currently being investigated in clinical trials; preliminary results suggest their potential applicability in cancer treatment. Here we investigated the effect of panobinostat (LBH589), a pan HDACi, in human AML cells lines and show results similar to those observed upon sodium butyrate treatment in OCI/AML3 cells. In particular, we show that panobinostat induces dose-dependent cell growth arrest, with G0-G1 block, associated with p21 protein induction, and apoptosis, associated with TRAIL-receptor DR5 upregulation and activation of caspase 8, in AML cells harboring NPM1 gene mutation. However, unlike what was observed with sodium butyrate, panobinostat induced also dowregulation of NPM1 mutant (and to a lesser extent, wild type) protein. Mechanisms underlying this phenomenon are under investigation. In particular, the possible role of acetylation of the heat shock protein Hsp90 (as a consequence of panobinostat-mediated HDAC6 inhibition) with disruption of its chaperone functions followed by instability of its client proteins, including cytoplasmic NPM1, is explored. Disclosures:Falini:Xenomics: Patents & Royalties.