Panobinostat acts synergistically with ibrutinib in diffuse large B cell lymphoma cells with MyD88 L265P mutations
Patrizia Mondello, … , Hans-Guido Wendel, Anas Younes
Patrizia Mondello, … , Hans-Guido Wendel, Anas Younes
Published November 20, 2018; First published March 23, 2017
Citation Information: JCI Insight. 2017;2(6):e90196. https://doi.org/10.1172/jci.insight.90196.
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Categories: 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 1

MyD88 confers resistance to ibrutinib in ABC DLBCL.

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MyD88 confers resistance to ibrutinib in ABC DLBCL. (A) Five DLBCL cells...
(A) Five DLBCL cells (3 ABC MyD88 mutant [TMD-8, HBL-1, and OCI-LY10], 1 ABC MyD88 WT [Ri-1] and 1 GCB [SUDHL-4]) were treated with 1 μM scramble, and MyD88 siRNA were incubated with increasing concentrations of ibrutinib (0.1, 0.25, 0.5 μM) and cell viability assessed by MTS assay after 24 hours. MyD88 siRNA + ibrutinib viability data were normalized to the effect of MyD88 siRNA alone. Error bars represent SEM of triplicate experiments. Differences between groups were calculated with the Student t test. **P < 0.005; ****P < 0.0001. (B) HBL-1, Ri-1 (ABC MyD88 mutant and WT, respectively) and SUDHL-4 (GCB) cells were infected with either tet inducible control shRNA or shRNA against MyD88, selected with puromycin, and induced with doxycycline for 2 days before treating with ibrutinib and seeding for MTS assay. After 3 days from the beginning of drug treatment, doxycyxline was washed out and ibrutinib added fresh either with or without fresh doxycycline. Error bars represent SEM of triplicate experiments. (C) Analyses of DNA sequences of a MyD88 WT and MyD88 L265P mutation, showing the replacement of reference sequence CTG with CCG. (D) Ri-1 naive cells transduced with either empty vector or activated MyD88 mutant L265P were incubated with increasing concentrations of ibrutinib (0.1, 0.25, 0.5, 0.75 μM) and cell viability assessed by MTS assay after 24 hours. MyD88 mutant + ibrutinib viability data were normalized to the effect of MyD88 mutant alone. Error bars represent SEM of triplicate experiments. Differences between groups were calculated with the Student t test. ****P < 0.0001. (E) Representative Western blot demonstrating inhibition of NF-κB pathway (pNF-κB p65, IRF4, pSTAT3 (T705]) after treatment with increasing concentrations of ibrutinib (0.1, 0.25, 0.5, 0.75 μM) for 24 hours in Ri-1 MyD88 WT but not in the Ri-1 stably expressing MyD88 mutation.