PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM- and MCL-1-dependent mechanisms in vitro and in vivo.

The aim of this study is to explore the efficacy and define mechanisms of action of coadministration of the PI3K/mTOR inhibitor BEZ235 and pan-HDAC inhibitor panobinostat in diffuse large B-cell lymphoma (DLBCL) cells. Various DLBCL cells were exposed to panobinostat and BEZ235 alone or together after which apoptosis and signaling/survival pathway perturbations were monitored by flow cytometry and Western blot analysis. Genetic strategies defined the functional significance of such changes, and xenograft mouse models were used to assess tumor growth and animal survival. Panobinostat and BEZ235 interacted synergistically in ABC-, GC-, and double-hit DLBCL cells and MCL cells but not in normal CD34(+) cells. Synergism was associated with pronounced AKT dephosphorylation, GSK3 dephosphorylation/activation, Mcl-1 downregulation, Bim upregulation, increased Bcl-2/Bcl-xL binding, diminished Bax/Bak binding to Bcl-2/Bcl-xL/Mcl-1, increased γH2A.X phosphorylation and histone H3/H4 acetylation, and abrogation of p21(CIP1) induction. BEZ235/panobinostat lethality was not susceptible to stromal/microenvironmental forms of resistance. Genetic strategies confirmed significant functional roles for AKT inactivation, Mcl-1 downregulation, Bim upregulation, and Bax/Bak in synergism. Finally, coadministration of BEZ235 with panobinostat in immunocompromised mice bearing SU-DHL4-derived tumors significantly reduced tumor growth in association with similar signaling changes observed in vitro, and combined treatment increased animal survival compared with single agents. BEZ235/panobinostat exhibits potent anti-DLBCL activity, including in poor-prognosis ABC- and double-hit subtypes, but not in normal CD34(+) cells. Synergism is most likely multifactorial, involving AKT inactivation/GSK3 activation, Bim upregulation, Mcl-1 downregulation, enhanced DNA damage, and is operative in vivo. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL.