Synergistic Cytotoxicity of Ibrutinib and the BCL2 Antagonist, ABT-199(GDC-0199) in Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL): Molecular Analysis Reveals Mechanisms of Target Interactions

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. In Phase II studies of single-agent ibrutinib in MCL (Wang ML et al, NEJM 2013) and CLL (Byrd JC, et al, NEJM 2013) the overall response rate was 68% and 89% (CR, PR, and PR with lymphocytosis), respectively, with PR as the best response in the majority of patients. Thus, not all patients respond and complete responses are infrequent with single agent ibrutinib. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines using a focused drug panel (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 substantially induced apoptosis compared to each single agent alone (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 and normal donor samples, heterogeneity in response to the combination of ibrutinib and ABT-199 was seen. When evaluated by Bliss modeling, 5 of 9 CLL samples had a synergistic improvement in apoptosis with the combination with the other 4 having no change. 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. Disclosures Off Label Use: Pre-clinical data with ABT-199 for MCL and CLL, not FDA approved. Williams:Pharmacyclics, Janssen: Consultancy, Research Funding.

Pleiotropic Action of Novel Bruton's Tyrosine Kinase Inhibitor BGB-3111 in Mantle Cell Lymphoma

“…Previous studies investigating alternate therapeutic targets have suggested that MCL-1 downregulation sensitizes MCL to BCL-2 inhibition, and the combinatorial efficacy of ibrutinib and the BCL-2 inhibitor ABT-199 has clearly been demonstrated (22)(23)(24). Our proteomic profiling identified the downregulation of MCL-1 by BGB-3111 in Jeko-1 cells.…”

Protocol description of the HOVON 141/VISION trial: a prospective, multicentre, randomised phase II trial of ibrutinib plus venetoclax in patients with creatinine clearance ≥30 mL/min who have relapsed or refractory chronic lymphocytic leukaemia (RR-CLL) with or without TP53 aberrations

“…Preclinical data support the combination of the Bruton's tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma 2 inhibitor venetoclax by demonstrating synergy in a diffuse large B-cell lymphoma cell line model and in primary CLL cells, as well as in mantle cell lymphoma cell lines. 11 Furthermore, several clinical trials are currently testing IV for clinical use, with no unexpected toxicities reported so far through scientific meetings, publications or internal reports for the marketing holders. Consequently, the aim of the current trial is to evaluate if combination treatment with venetoclax+ibrutinib in patients with RR-CLL can lead to MRD negativity and induce long-lasting remission even after stopping the treatment.…”

Targeting Bruton's tyrosine kinase with ibrutinib in B‐cell malignancies

“…also demonstrated that the ibrutinib and ABT‐199 combination displayed strong synergistic effects in MCL cell lines as well as primary cells from patients with recurrent MCL. Portell et al . reported, again at the ASH 2014 meeting, the synergistic effect of ibrutinib and carfilzomib or ABT‐199 on apoptosis of circulating tumor cells from patients with MCL.…”

“…Potential ibrutinib‐containing combination therapy for CLL has also been studied preclinically. Promising candidates to combine with ibrutinib included proteasome inhibitors, Bcl‐2 inhibitors, and IRAK1/4 kinase inhibitors . These combinations were somewhat better studied in other B‐cell malignancies and are described below.…”