Dual Inhibition of PKC Delta By Carfilzomib (CFZ) and Ibrutinib Results in Synergistic Activity in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models and in Primary Tumor Cells Isolated from B-Cell Lymphoma Patients
Authors: Zhang Qunling , Gu Juan J , Guo Ye , Klos Jacob , Mavis Cory , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J. 
Published: October 2019

Abstract
Introduction: A better understanding in the role of the B-cell receptor-signaling pathway in lymphomagenesis led to the clinical development of ibrutinib is an orally bioavailable Bruton's tyrosine kinase inhibitor (BTKi). Ibrutinib has clinical activity in several subtypes of B-cell lymphoma (follicular lymphoma, mantle cell lymphoma and certain diffuse large B-cell lymphoma [DLBCL] subtypes). Recently, an increasing number of ibrutinib-treated patients are developing disease progression, stressing the need to optimize its therapeutic potential. To this end, we evaluated the biological effects and interactions of ibrutinib and CFZ, a non-reversible proteasome inhibitor in several lymphoma pre-clinical models including primary tumor cells isolated from lymphoma patients.
Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines as well as primary tumor cells isolated from B-cell lymphoma patients (N=20) were exposed to ibrutinib and/or CFZ. Changes in cell viability were evaluated by Presto Blue and Titer Glo assays respectively. Coefficient of synergy was determined using the CalcuSyn software. To further study the mechanisms responsible for the biological effects of ibrutinib and CFZ, RSCL and RRCL were exposed to Ibrutinib+/- CFZ for 48hr. Changes in apoptosis, mitochondrial potential, and cell cycle distribution were evaluated by flow cytometry by AnnexinV-PI, DiOC6 or PI staining respectively. Subsequently, protein lysates were isolated from ibrutinib+/- CFZ exposed RSCL or RRCL and changes in downstream targets (PKCδ a member of diacylglycerol-dependent, calcium-independent serine/threonine-specific protein kinases PKC isoform know to be a substrate of caspase 3 and able to mediate apoptosis), Bcl-2 family members and cell cycle regulatory proteins were evaluated by Western blotting.
Results: In vitro exposure of RSCL, RRCL, and primary tumor cells to ibrutinib and CFZ resulted in synergistic anti-tumor activity. In addition, the combination of ibrutinib and CFZ resulted in a more pronounced decrease in the mitochondrial potential, higher degree of apoptosis (as demonstrated by flow cytometry and caspase 3 or PARP cleavage) and increase in G1 cell cycle arrest when compared to single agent ibrutinib or CFZ. Perhaps related to these findings, we found that exposure of lymphoma cells to Ibrutinib and CFZ resulted in an increase in caspase 3, the active form of PKCδ and BAX levels. Using caspase inhibitor Q-VD-OPh rescued the combination killing effect of ibrutinib and CFZ, as well as abrogated induction of the active form of PKCδ. In addition, Ibrutinib in combination with CFZ decreased cyclin E and CDK2 levels.
Conclusions: Together our data suggests that ibrutinib and CFZ had a strong synergistic anti-tumor activity against rituximab-chemotherapy sensitive and resistant lymphoma cells and patient samples. At the molecular level, the combination of ibrutinib and CFZ affects the balance of the apoptotic machinery and key regulatory proteins of the cell cycle that could play an important role in the synergistic effects observed. Further signaling and in vivo studies are necessary to evaluate the therapeutic role of ibrutinib in combination with carfilzomib in B-cell lymphoma (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Funds).

Disclosures
Czuczman: MorphoSys: Consultancy; Celgene: Employment; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.