Abstract 4769: Ex-vivo and in-vitro combination strategies with ibrutinib in chronic lymphocytic leukemia
Authors: Cervantes-Gomez Fabiola , Balakrishnan Kumudha , Wierda William G. , Keating Michael J. , Gandhi Varsha 
Published: May 2015

Abstract
Chronic lymphocytic leukemia (CLL) depends on the B-cell receptor (BCR) pathway. Bruton's tyrosine kinase (BTK) is an essential enzyme in BCR and is inhibited by ibrutinib. Though ibrutinib treatment resulted in impressive response rates, most patients only achieve partial remissions. In addition, treatment with ibrutinib elicits lymphocytosis (redistribution of tissue-resident CLL cells from lymph nodes into peripheral blood). We hypothesize that drugs that induce apoptosis in the mobilized CLL cells will provide combination strategy for future clinical trials. We obtained cells from blood of lymphocytosed CLL patients on ibrutinib trial and incubated ex-vivo for 24 h with different drugs currently in clinical trials including BH3 mimetics (ABT-199, ABT-737), PI3K inhibitors (GS-1101, IPI-145), alkylating agent (bendamustine), and BTK inhibitor (ibrutinib). Among these drugs, ABT-199 at 5 and 10 nM, elicited the highest cytotoxicity (35-80% range, median 53%; n = 12). ABT-737 at 5 and 10 nM resulted in a cytotoxic range of 10-67% with a median of 28% in the same 12 samples. ABT-199 and ABT-737-mediated ex-vivo cytotoxicity was compared in 5 patient samples pre- and post- (4 and 12 weeks after starting therapy) ibrutinib. Endogenous cell death prior to ibrutinib was a median 35% (range 5-57%), however after ibrutinib regimen endogenous apoptosis of lymphocytosed cells at week 4 was a median 13% (range 10-48%) and at week 12 was a median 12% (range 4-48%). This suggests that post-ibrutinib lymphocytosed CLL cells exhibit resistance compared to cells prior to ibrutinib therapy. Again, ABT-737 and specially ABT-199 showed maximum cell death; ABT-737 treatment induced a median 32% (range 13-57%) cell death in pre-ibrutinib sample and a median 29% (range 22-68%) in post-ibrutinib samples. ABT-199 treatment on the other hand resulted in 53% cell death in pre and post-ibrutinib samples with a range of 29-63% and 30-71%, respectively. This suggests that pre- and post-ibrutinib treated lymphocytes are similarly sensitive to ABT-mediated cytotoxicity. In-vitro drug combination studies in CLL samples (n = 11) suggested additive or synergistic combination of ibrutinib and ABT-199 or ABT-737 (0.5 and 1 nM) in CLL cells with or without BCR pathway stimulation by IgM. Similar to ex-vivo studies, ABT-199 was more effective than ABT-737. Immunoblots analyzing targets in the BTK signaling pathway and Bcl-2 protein family were performed in the pre- and post-ibrutinib samples after 24 h incubation with the 6 drugs. Preliminary immunoblots analysis suggests a decrease of antiapoptotic Mcl-1 and phospho-Akt Ser473 by weeks 4 and 12 after ibrutinib treatment compared to pre-ibruitinib samples. In contrast, Bcl-2, Bcl-XL, Bim, and total ERK1/2 levels remain stable in pre- and post-samples. Collectively, these data suggest that ABT-199 may be combined with ibrutinib to remove lymphocytosis and for better responses.
Citation Format: Fabiola Cervantes-Gomez, Kumudha Balakrishnan, William G. Wierda, Michael J. Keating, Varsha Gandhi. Ex-vivo and in-vitro combination strategies with ibrutinib in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4769. doi:10.1158/1538-7445.AM2014-4769