2.26 Clopidogrel (Plavix) Induces In Vivo Reduction of Normal B Cells and In Vitro Apoptosis of B-Cell Chronic Lymphocytic Leukemia Cells

Abstract

using human stroma. We then explore the hypothesis that the deltaisoform specific PI3K inhibitor CAL-101 is able to both effectively kill stroma-exposed CLL cells and sensitize them to treatment with other agents, by releasing the cells from stroma and/or increasing their level of mitochondrial apoptotic priming. Methods: CLL cells were co-cultured with or without drug treament for 24 hours in the absence of stroma and with either the human stromal cell line StromaNKTert or with nurse-like cells (NLC) grown from primary CLL patient samples as previously described. CLL cell adhesion to stroma in the presence or absence of CAL-101 was visualized using a Calcein-based adhesion assay, and quantified by fluorimetry. CLL cell viability was assessed by Annexin/PI analysis. BH3 profiling was performed by exposing malignant CD19 B cells from CLL patients to a panel of BH3-domain peptides, and mitochondrial apoptotic priming was quantified by JC-1-based FACS to assess mitchondrial outer membrane permeabilization, as previously described. Results: CLL cells exposed to human stroma were resistant to treatment with the purine analog fludarabine and the BH3-mimetic ABT-263. CAL-101 was able to partially overcome this resistance, and the combination of CAL-101 and fludarabine led to a marked increase in sensitivity of stroma-exposed CLL cells. To investigate the mechanism of this increased sensitivity in the presence of CAL-101, we labelled CLL cells with calcein, and co-cultured them with the StromaNKTert cell line. We found that the CLL cells were highly adherent to stroma after only one hour of co-culture, and were significantly less adherent in the presence of CAL-101. Also, a significant number of calcein-labelled CLL cells co-cultured with stroma for 24 hours were released from stroma within one hour after the addition of CAL-101. To determine the effect of the release of CLL cells from stroma on apoptotic priming, we performed BH3 profiling on CLL cells co-cultured in the presence or absence of stroma. CLL cells cultured in the absence of stroma were significantly more primed for apoptosis than CLL cells co-cultured with either the StromaNKTert cell line or primary human NLC. The addition of CAL-101, with or without fludarabine, to CLL cells in culture with stroma, led to increased apoptotic priming compared to CLL cells in culture that were untreated or treated with fludarabine alone. Conclusions: Taken together, these results suggest that the sensitivity of stromaexposed CLL cells to CAL-101 may be related to the increased mitochondrial apoptotic priming that is facilitated by release of these cells from the stroma. These data may partially explain the clinical observation that, unlike traditional chemotherapy agents, CAL-101 has been efficacious in CLL despite causing an increase in the white blood cell count.