Panbinostat decreases cFLIP and enhances killing of cancer cells by immunotoxin LMB-100 by stimulating the extrinsic apoptotic pathway

// Xiu-Fen Liu 1 , Qi Zhou 1 , Raffit Hassan 2 and Ira Pastan 1 1 Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 2 Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Correspondence to: Ira Pastan, email: pastani@mail.nih.gov Keywords: combination therapy, apoptosis, cFLIP, pancreatic cancer, mesothelin Abbreviations: CI, combination index; EF2, elongation factor 2; eEF2, eukaryotic EF2; rITs, recombinant immunotoxins Received: May 31, 2017      Accepted: July 14, 2017      Published: August 14, 2017 ABSTRACT LMB-100 (RG7787) is a recombinant immunotoxin, which kills mesothelin-expressing cancer cells and now being evaluated in phase 1 trials. To enhance the anti-tumor activity of LMB-100, we have searched for agents, already approved for cancer therapy, that can be combined with LMB-100 to increase its efficacy. Panbinostat is a pan-histone deacetylase inhibitor that is used to treat multiple myeloma. We incubated different types of cancer cells with panbinostat and LMB-100 and found that they interacted synergistically to cause cell death. We found that panbinostat and the combination increased levels of mRNAs encoding TNF/TNFR family members, as well as BNIP3L and CASP-9 , and markedly decreased mRNA levels for c-FLIP and BID . Western blots confirmed a fall in levels of cFLIP protein and a rise in BNIP3L and caspase-9. The combination also increased levels of cleaved BID (t-BID), cleaved-capsase-3 and -8 and PARP. To assess the importance of the fall in cFLIP levels, we treated cells with the cFLIP inhibitor, Rocaglamide, and found it also enhanced killing of tumor cells by LMB-100. LMB-100, which activates the intrinsic pathway of apoptosis, and panbinostat, which activates the extrinsic pathway, work in a synergistic manner to kill cancer cell lines.