Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Figure 1

Specific knockdown of BTK impairs survival in CLL cells. (A) Knockdown of BTK by siRNA. Western blot analysis of BTK protein levels 72 hours after nucleofection with control or BTK-specific siRNA shows knockdown of BTK. This is representative of 37 patient samples. (B) CLL cell viability 72 hours after nucleofection with control or BTK siRNA. Following BTK knockdown, cells were cultured for 72 hours, and viability was determined at that time by percentage of cells negative for PI and Annexin V. Cells transfected with BTK siRNA had diminished viability compared with those treated with control siRNA. (C) Viability of CLL cells after transfection with BTK or control siRNA and coincubation with stromal cells or stimulation with IgM. BTK knockout reduced CLL cell viability even in the presence of stromal cells or after IgM stimulation.

Figure 2

Kinase-functional BTK is necessary for the development and expansion of TCL1 leukemia. (A) TCL1 protein expression is retained after the cross with the XID mouse. Splenic lymphocytes from a pool of 3 1-month-old female XID/TCL1 mice and 1-month-old female B6/TCL1 mice were purified and B cell selected using the EasySep Mouse B Cell Enrichment Kit (Stem Cell Technologies) and then lysed; 50 μg of protein from each mouse was used for western blot analysis of TCL1 protein expression. TCL1 is present in both the B6/TCL1 and the XID/TCL1 mice. (B) WT/TCL1 mice have a higher percentage of leukemic lymphocytes in the peripheral blood compared with XID/TCL1 mice. Peripheral blood flow cytometry for CD5 and CD19 was performed on 59 WT/TCL1 and 61 XID/TCL1 mice. XID/TCL1 mice had a lower percentage of leukemic CD5/CD19 coexpressing cells than did WT/TCL1 mice. (C) OS is improved for XID/TCL1 mice compared with WT/TCL1 mice. All male XID/TCL1 and WT/TCL1 mice born within a 1-year time period, which included 65 XID/TCL1 and 78 WT/TCL1 mice, were followed for survival. OS is significantly prolonged in the XID/TCL1 cohort (P < .0001).

Figure 3

Ibrutinib improves OS in mice transplanted with TCL1 leukemia. After transplantation with leukemic TCL1 spleen lymphocytes, SCID mice were followed for leukemia development, and once leukemic, randomized to treatment with ibrutinib or vehicle. Mice treated with ibrutinib (n = 8) survived significantly longer than those treated with vehicle (n = 9; P = .003).

Figure 4

Ibrutinib improves outcomes in continuously-treated TCL1 mice. (A) Continuous administration of ibrutinib inhibits the development of CLL in TCL1 mice. TCL1 mice were randomized at 1 month of age to treatment with vehicle drinking water (n = 24) or ibrutinib drinking water (n = 24), and monitored for leukemia development by monthly peripheral blood flow cytometry. Ibrutinib significantly impairs leukemia development (P < .0001). (B) Continuous administration of ibrutinib improves survival in TCL1 mice. TCL1 mice were randomized at 1 month of age to treatment with vehicle drinking water (n = 24) or ibrutinib drinking water (n = 24) and followed for survival. Survival was significantly improved by ibrutinib administration compared with vehicle (P < .0001).