Abstract B40: Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions.
Authors: Portell Craig A. , Axelrod Mark J. , Brett Laura Kyle , Gordon Vicki L. , Capaldo Brian , Xing Jeffrey , Bekiranov Stefan , Williams Michael E. , Weber Michael J. 
Published: August 2015

Abstract
Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK, but not all patients respond and complete responses are infrequent with single agent ibrutinib. We hypothesize that additional agents used in combination could enhance the breadth, depth and duration of responses. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy.
Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 displayed synergistic cytotoxicity (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL cells from 9 patients, 5 displayed synergistic cytotoxicity and 4 did not, indicating substantial patient heterogeneity in response to the combination, presumably due to variations in genetic landscape. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors.
Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy.
In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned.

Citation Format: Craig A. Portell, Mark J. Axelrod, Laura Kyle Brett, Vicki L. Gordon, Brian Capaldo, Jeffrey Xing, Stefan Bekiranov, Michael E. Williams, Michael J. Weber. Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B40.