Purpose To explore the efficacy and define mechanisms of action of co-administration of the PI3K/mTOR inhibitor BEZ235 and pan-HDAC inhibitor panobinostat in DLBCL cells. Experimental Design 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. Results Panobinostat and BEZ235 interacted synergistically in ABC-, GC-, and double-hit DLBCL cells, and MCL cells, but not normal CD34+ cells. Synergism was associated with pronounced AKT dephosphorylation, GSK3 dephosphorylation/activation, Mcl-1 downregulation, Bim up-regulation and 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 p21CIP1 induction. BEZ235/panobinostat lethality was not susceptible to stromal/microenvironmental forms of resistance. Genetic strategies confirmed significant functional roles for AKT inactivation, Mcl-1 down-regulation, Bim up-regulation, and Bax/Bak in synergism. Finally, co-administration 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 increased animal survival compared to single agents. Conclusions BEZ235/panobinostat exhibits potent anti-DLBCL activity, including in poor-prognosis ABC- and double-hit sub-types, but not in normal CD34+ cells. Synergism is most likely multi-factorial, involving AKT inactivation/GSK3 activation, Bim up-regulation, Mcl-1 down-regulation, enhanced DNA damage, and is operative in vivo. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL.

<div>Abstract<p><b>Purpose:</b> 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.</p><p><b>Experimental Design:</b> 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.</p><p><b>Results:</b> 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 <i>in vitro</i>, and combined treatment increased animal survival compared with single agents.</p><p><b>Conclusions:</b> 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 <i>in vivo</i>. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL. <i>Clin Cancer Res; 20(18); 4849–60. ©2014 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> 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.</p><p><b>Experimental Design:</b> 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.</p><p><b>Results:</b> 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 <i>in vitro</i>, and combined treatment increased animal survival compared with single agents.</p><p><b>Conclusions:</b> 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 <i>in vivo</i>. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL. <i>Clin Cancer Res; 20(18); 4849–60. ©2014 AACR</i>.</p></div>

<p>Cell lines and primary antibodies.</p>

<p>Supplementary Fig. 1: Ectopic expression of constitutively active AKT diminished panobinostat toxicity. Supplementary Fig. 2: BEZ235 and panobinostat interact synergistically to kill NHL cell lines. Supplementary Fig. 3: Effects of BEZ235 and panobinostat coadministration on histone acetylation, DNA damage and p21CIP1 expression. Supplementary Fig. 4: Role of Bim in BEZ235/panobinostat toxicity. Supplementary Fig. 5: Effects of BEZ235/panobinostat on Bax, Bak, or Bim binding to Bcl-2, Bcl-xL or Mcl-1 proteins. Supplementary Fig. 6: BEZ235/panobinostat toxicity involves Bax and Bak Supplementary Fig. 7: BEZ235/panobinostat activity in the presence of HS-5-conditioned media. Supplementary Fig. 8: Effect of BEZ235/panobinostat on animal weight</p>