The Kinase Inhibitor, PCI-32765, Demonstrates Activity In Chronic Lymphocytic Leukemia Cells Independent of Microenvironmental Survival Signals

Abstract

AbstractAbstract 1385Bruton tyrosine kinase (Btk), a member of the Tec family kinases, has a well-characterized role in B-cell antigen receptor (BCR) signaling and B-cell activation. Btk is activate by Src family kinases which lead to downstream activation of essential cell survival pathways such as NF-κB and MAP kinase. Although Btk is expressed in multiple hematopoietic cells, the primary defect in Btk knockout mice is B-cell specific, suggesting a more selective B-cell function consistent with its role in the BCR signaling pathway. Btk was identified in an unbiased screen as an essential signaling kinase for survival of certain lymphomas including diffuse large B-cell lymphoma, but no information is available about its role in CLL cell survival. Inhibition of Btk by PCI-32765, a selective irreversible Btk inhibitor, has demonstrated promising clinical activity in an ongoing phase 1 study in B-cell non-Hodgkin lymphoma. We therefore hypothesized that inhibition of Btk would induce cytotoxicity in B-CLL cells. BTK protein expression was observed in CLL cells, but not NK or T-cells as previously reported. Surprisingly, significant variability in BTK expression was noted among different CLL patient samples. Treatment of primary CLL cells with PCI-32765 at concentrations ranging from 0.1–10μM resulted in significant cell killing compared to untreated control. At a 10μM concentration, PCI-32765 resulted in a median 73% viable cells at 48 hours as detected by annexin V/propidium iodide flow cytometry (n=55). Although again, considerable variability was observed among patient samples (range 93.5–5.5%). We found no correlation between response to PCI-32765 and prognostic factors such as interphase cytogenetics or IgVH gene mutational status, suggesting a potential clinical benefit to even high-risk CLL patients. To determine the selectivity of PCI-32765, we evaluated cytotoxicity of lymphocytes derived from healthy volunteers. At 10μM, PCI-32765 resulted in a median 90% viable normal B-cells with little variability (n=5), showing substantially lower cytotoxicity in normal B-cells relative to CLL tumor cells. The cytotoxicity observed with PCI-32765 in CLL cells was accompanied by PARP cleavage and the induction of caspase 3 activity, and PCI-32765-mediated cell death was blocked by the pan-caspase inhibitor z-VAD-fmk (n=3). These results indicate that PCI-32765 is dependent on caspase activation for its ability to induce apoptosis in CLL cells. Further studies to determine the apoptotic mechanism showed that PCI-32765 treatment (10μM) partially reversed CD40L- or CpG-induced phosphorylation of ERK1/2 and also caused an increase in endoplasmic reticulum-derived intracellular calcium flux in CLL cells (n=8). Microenvironmental factors such as CD40L, TNF-α, IL-6 and BAFF can induce activation of CLL and/or decrease spontaneous apoptosis. Treatment of CLL cells with PCI-32765 prevented the protective anti-apoptotic effect promoted by these cytokines, suggesting that this agent can overcome the protection induced by microenvironmental stimuli. Similarly, co-culture of CLL cells on the human stromal cell line HS-5 significantly decreases the spontaneous apoptosis of CLL cells. PCI-32765 treatment was able to overcome this stromal protection, and induced apoptosis comparable to cells cultured in suspension (median cytotoxicity 86% vs. 84%, respectively; 10μM PCI-32765 at 48 hr, n=5). This suggests that the cytotoxic effect elicited by PCI-32765 will not be significantly diminished by the presence of an in vivo microenvironment. Taken together, our results suggest that PCI-32765 acts directly on CLL cells to induce apoptosis and helps to block microenvironment-derived protection. These investigations demonstrate the efficacy of targeting Btk in CLL and provide preclinical validation for ongoing Phase 1/2 clinical trials for the treatment of this disease.This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation and The OSU Leukemia SPORE grant funded by the NCI. Disclosures:Buggy:Pharmacyclics, Inc.: Employment. Hamdy:Pharmacyclics, Inc.: Employment.