Cell-Intrinsic Determinants of Ibrutinib-Induced Apoptosis in Chronic Lymphocytic Leukemia

Abstract

Purpose: Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) and CLL with del17p. Mechanistically, ibrutinib interferes with B-cell receptor (BCR) signaling as well as multiple CLL cell-to-microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted.Experimental Design: We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole-exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 to 5 μmol/L of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1, and PG-EBV cell lines and measured BCR signaling and ibrutinib responses.Results: CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib, with IC₅₀ values ranging from 0.4 to 9.7 μmol/L. Unmutated IGVH status, elevated ZAP70 expression, and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53-mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation, confirmed substantially less sensitivity to ibrutinib-induced apoptosis.Conclusions: This study identifies that CLL harboring del17p/TP53-mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild-type cells. Clin Cancer Res; 23(4); 1049-59. ©2016 AACR.

Figure 1. Ibrutinib-induced cell death in purified paired CLL cells (ibrutinib dose-response curves)

CD19+ CLL cells purified through negative selection were incubated with escalating doses of ibrutinib for 72 hours and the apoptotic and necrotic cell population measured using annexinV/PI staining and FACS. Displayed is the viable double-negative (DN) cell fraction normalized to the individual DN fraction measured in the solvent only controls. The mean of duplicate drug incubations and measurements is displayed. A: pre-treatment CLL samples; B: post-treatment (relapsed) CLL samples.

Figure 2. Ibrutinib-induced cell death is influenced by CLL cell-intrinsic traits

Groupings of ibrutinib CLL IC50 values by biomarker. The mean IC50 values are indicated by horizontal bars and a numerical value. A–D: single dichotomized biomarkers as indicated. E: Left: IGVH-UM AND ZAP70 >20% AND trisomy 12 versus right: IGVH-M AND ZAP70 <20% AND disomy 12; middle: all other cases. F: trisomy 12 cases only with or without NOTCH1 mutations.

Figure 3. Ibrutinib CLL IC50 values in paired pre-treatment and post-treatment (relapsed from chemotherapy) paired CLL samples

CLL cases with acquired del17p/TP53 mutations are marked with a red square.

Figure 4. Del17p/TP53 mutations confer partial resistance to Ibrutinib-induced cell death in CLL

A: Groupings of ibrutinib CLL IC50 values by del17/TP53 status. The mean IC50 values are indicated by horizontal bars and a numerical value. B–E: Cell fraction alive following ibrutinib treatment grouped by del17/TP53 status and by ibrutinib concentrations used.

Figure 5. A–B: Results of ex vivo B-cell receptor signaling studies in CLL

Purified CLL cells were cultured in serum-free medium for 2 hours and left untreated, or pre-treated with ibrutinib at 0 μM (DMSO only) or 0.25 μM, 0.5 μM or 1 μM for 60′ and subsequently treated with anti-IgM at 10μg/ml for 15′. Cells were pelleted, lysates made, protein fractionated and prepared for immunoblotting with antibodies to p1217-PLCy2; PLCy2; p223-BTK; BTK; p473-AKT; AKT; p202/204-ERK and ERK. Figure 5; Panel A (del17p/TP53 mutated CLL) and Figure 5; Panel B (non-del17p/TP53mutated CLL). The results for individual blots and epitopes cannot be directly quantitatively compared across different patients as exposure times for various blots are different. The IGVH mutation status for each CLL case is indicated (UM: unmutated; M: mutated). The ex vivo IC50 values to ibrutinib are indicated in brackets.

Figure 6. A–E: Effects of TP53 disruption on BCR signaling in the HG3 CLL lymphoblastoid cell line

A: Ibrutinib treatment at indicated dosing for 72 hours and annexin V/PI quantitation of cell apoptosis and death. B: p53 immunoblotting before and after 16 hours of Nutlin3a treatment; C: details of TP53 mutations induced using crispr-Cas9 targeting; D: Results of immunoblotting for control and TP53 disrupted cell clones following anti-Ig treatment; E: quantification of immunoblot results using Image J software. Displayed are ratios of p1217-PLCy2/PLCy2; p223-BTK/BTK; p473-AKT/AKT; p202/204-ERK/ERK. Ctr: un-manipulated cells; A1, A4, C7; individual TP53 disrupted cell clones.