Panobinostat acts synergistically with ibrutinib in diffuse large B cell lymphoma cells with MyD88 L265P mutations
Patrizia Mondello, Elliott J. Brea, Elisa De Stanchina, Eneda Toska, Aaron Y. Chang, Myles Fennell, Venkatraman Seshan, Ralph Garippa, David A. Scheinberg, José Baselga, Hans-Guido Wendel, Anas Younes
Patrizia Mondello, Elliott J. Brea, Elisa De Stanchina, Eneda Toska, Aaron Y. Chang, Myles Fennell, Venkatraman Seshan, Ralph Garippa, David A. Scheinberg, José Baselga, Hans-Guido Wendel, Anas Younes
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Research Article Hematology Oncology

Panobinostat acts synergistically with ibrutinib in diffuse large B cell lymphoma cells with MyD88 L265P mutations

Abstract

Diffuse large B cell lymphoma (DLBCL) frequently harbors genetic alterations that activate the B cell receptor (BCR) and TLR pathways, which converge to activate NF-κB. While selective inhibition of BTK with ibrutinib causes clinical responses in relapsed DLBCL patients, most responses are partial and of a short duration. Here, we demonstrated that MyD88 silencing enhanced ibrutinib efficacy in DLBCL cells harboring MyD88 L265P mutations. Chemical downregulation of MyD88 expression with HDAC inhibitors also synergized with ibrutinib. We demonstrate that HDAC inhibitor regulation of MyD88 expression is mediated by STAT3. In turn, STAT3 silencing caused a decrease in MyD88 mRNA and protein levels, and enhanced the ibrutinib antilymphoma effect in MyD88 mutant DLBCL cells. Induced mutations in the STAT3 binding site in the MyD88 promotor region was associated with a decrease in MyD88 transcriptional activity. We also demonstrate that treatment with the HDAC inhibitor panobinostat decreased phosphorylated STAT3 binding to the MyD88 promotor. Accordingly, combined treatment with panobinostat and ibrutinib resulted in enhanced inhibition of NF-κB activity and caused regression of DLBCL xenografts. Our data provide a mechanistic rationale for combining HDAC inhibitors and ibrutinib for the treatment of DLBCL.

Authors

Patrizia Mondello, Elliott J. Brea, Elisa De Stanchina, Eneda Toska, Aaron Y. Chang, Myles Fennell, Venkatraman Seshan, Ralph Garippa, David A. Scheinberg, José Baselga, Hans-Guido Wendel, Anas Younes

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Figure 4

Panobinostat synergizes with ibrutinib in killing ABC DLBCL harboring MyD88 mutation.

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Panobinostat synergizes with ibrutinib in killing ABC DLBCL harboring My...
(A) MTS assay confirming enhanced antiproliferative effects of panobinostat in combination with ibrutinib in ABC cells harboring MyD88 mutation (HBL-1, TMD-8, and OCI-LY-10) but not in ABC cells with MyD88 WT (Ri-1 and U-2932). Cells were incubated with increasing concentrations of panobinostat (0.01, 0.025, 0.05, 0.075 μM) and ibrutinib (0.01, 0.025, 0.05, 0.075 μM), and viability was assessed after 24 hours. Error bars represent SEM of 3 independent experiments. Differences between groups were calculated with 2-way ANOVA with Bonferroni’s test. ***P < 0.0005; ****P < 0.0001. (B) Scatterplots summarizing the results of panobinostat + ibrutinib combinations in our cell line panel after 24 hours of incubation. Cells were incubated with increasing concentrations of panobinostat (0.01, 0.025, 0.05, and 0.075 μM) and ibrutinib (0.01, 0.025, 0.05, and 0.075 μM), and combination index was calculated according to the Chou-Talalay method. All the experiments were repeated 3 times. (C) MTS assay showing enhanced antiproliferative effects of panobinostat in combination with ibrutinib in Ri-1 stably expressing MyD88 mutation but not in Ri-1 with MyD88 WT. Cells were incubated with increasing concentrations of panobinostat (0.01, 0.025, 0.05, and 0.075 μM) and ibrutinib (0.01, 0.025, 0.05, and 0.075 μM), and viability was assessed after 24 hours. Error bars represent SEM of 3 independent experiments. Differences between groups were calculated with 2-way ANOVA with Bonferroni’s test. ***P < 0.0005; ****P < 0.0001. (D) Relative NF-κB-luciferase activity in 2 representative ABC (HBL-1 and TMD-8) cell lines. Cells were treated for 16 hours with indicated concentration of either panobinostat, ibrutinib, or the combination of the 2 drugs. Cells were incubated with 2 ng/ml of TNF-α as positive control for NF-κB activation. Error bars represent SEM of triplicates. Differences between groups were calculated with 2-way ANOVA with Bonferroni’s test. **P < 0.005; ***P < 0.0005; ****P < 0.0001. (E) IL-10 level change in 3 ABC DLBCL cell lines harboring MyD88 mutation (HBL-1, TMD-8, and OCI-LY-10). Cells were treated for 12 hours with indicated concentration of either panobinostat, ibrutinib, or the combination of the 2 drugs. Error bars represent SEM of triplicates. Differences between groups were calculated with 2-way ANOVA with Bonferroni’s test. **P < 0.005; ****P < 0.0001.