Supplemental Figures 1-5. Figure S1. Effect of ibrutinib treatment during in-vitro incubation of CLL cells on levels of antiapoptotic proteins MCL-1, BCL-XL, and BCL-2; Figure S2. Effect of ibrutinib in-vitro treatment on transcript levels of antiapoptotic proteins MCL-1, BCL-XL, and BCL-2; Figure S3. In vitro ABT-199-mediated cytotoxicity in lymphocytes from CLL patients treated with ibrutinib for 36 weeks; Figure S4. Changes in the expression levels of BTK pathway proteins in CLL cells from six CLL patient samples before and after ibrutinib therapy; Figure S5. Cytotoxicity elicited by ABT-199 in combination with ibrutinib in lymphocytes from a TCL1 mouse adoptive transfer model of CLL.
ARTICLE ABSTRACT
Purpose: Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes.Experimental Design: Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed.Results: The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2.Conclusions: Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. Clin Cancer Res; 21(16); 3705–15. ©2015 AACR.
