Bruton's tyrosine kinase (BTK) is a member of the TEC family of non-receptor tyrosine kinases and is predominantly expressed in hematopoietic cells, except in T cells. BTK plays a prominent role in B cell receptor (BCR) signaling and several other pathways, including CXCR4 signaling, which is essential for lymphocyte homing. BTK activation downstream of the BCR leads to proliferation, differentiation, and survival of B cells. Functional BTK is necessary for normal B cell development, defective BTK result in a primary immunodeficiency called X-linked agammaglobulinemia (XLA). Because of the restricted expression and the B cell phenotype in BTK-deficient mice and XLA patients, BTK has become a promising therapeutic target in mature B cell malignancies. Ibrutinib is a selective, orally bioavailable, covalent BTK inhibitor currently studied in clinical trials in patients with Chronic Lymphocytic Leukemia (CLL) and other mature B cell malignancies. The importance of BTK in B-ALL is controversial. Initial study in childhood B-ALL revealed normal BTK protein levels, and gene expression profiling data from the St. Jude's group revealed BTK expression in B-ALL, but not in T-ALL. Other studies revealed abnormally spliced BTK mRNA and truncated BTK protein lacking kinase activity in some B-ALL samples. To explore the pre-clinical activity of ibrutinib in B-ALL, we used a panel of 14 B-ALL cell lines, representing pro-B (CD10+/−, TdT+, cyt Igμ-), pre-B (CD10+, TdT+, cyt Igμ+) and mature (CD10+/−, TdT-, surf IgM+) phenotypes. The panel included 4 Ph+/BCR-ABL1+ cell lines (Z-119, NALM-20, NALM-21, TOM-1). Western blot analysis revealed BTK expression in 12 out of 14 lines, while phospho-BTK (Y223) was present only in half of the cases. Effects of ibrutinib on B-ALL proliferation were tested in XTT assays, using increasing concentrations of ibrutinib (0.5 – 5 μM). All B-ALL cells responded to ibrutinib except for BTK-negative TANOUE cells. All other B-ALL cells displayed decreased proliferation with variable half maximal inhibitory concentrations of ibrutinib (IC50). The most sensitive cell lines (RCH-ACV, SMS-SB) had IC50 of < 1 μM; all BCR-ABL1+ cells showed IC50 < 3.5 μM (see Figure). We also analyzed inhibitory effects of ibrutinib on B-ALL cell proliferation by serial automated cell counting, which confirmed the XTT assay data. B-ALL cells viability was determined after incubation with ibrutinib, which induced only minor decreases after 3 days of incubation. Preliminary data with primary B-ALL samples revealed BTK protein expression in 5 out of 5 samples. In co-culture with KUSA-H1 stromal cells, primary B-ALL cells showed moderate levels of apoptosis, ranging from 10 – 25% after 3 days of incubation with 1 μM ibrutinib, which is similar to levels of ibrutinib-induced apoptosis in CLL. Collectively, these data demonstrate that the BTK inhibitor ibrutinib can interfere with B-ALL cell proliferation and survival, providing a rationale for clinical testing of this novel, well-tolerated targeted agent in patients with relapsed B-ALL.
O'Brien:Pharmacyclics: Research support Other. Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.