Synthetic High-Density Lipoprotein-like Nanoparticles (HDL NP) Cause Apoptosis and Enhance Killing By B-Cell Receptor and BCL-2 Inhibitors in Chronic Lymphocytic Leukemia (CLL)

Introduction:

Chronic lymphocytic leukemia is the most frequent adult leukemia that remains incurable, despite the use of innovative compounds targeting crucial signaling pathways of the disease. Additional non-toxic, targeted therapies are urgently needed in order to improve the curability of CLL as well as to increase the therapeutic index of existing drugs through rational combinations. Biomimetic high-density lipoprotein-like nanoparticles (HDL NP) have comparable size, shape, and surface chemistry as natural, spherical HDLs. HDL NPs modulate cellular cholesterol homeostasis, and potently induce apoptotic cell death in B cell lymphoma cell lines after tightly binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SR-B1). Based on these data, we investigated SR-B1 expression and tested the nanoparticles' effect on primary human CLL with the hypothesis that HDL NP binding to SR-B1 and modulation of cellular cholesterol homeostasis in the leukemic cells would produce a therapeutic response. In addition, we explored the possibility of enhancing its therapeutic effect through the combination of the HDL NP with BCL2-inhibitor ABT-199 and the BTK-inhibitor, ibrutinib, in a series of fresh leukemia cells from patients with CLL.

Methods:

Fresh human CLL cells from peripheral blood were obtained from patients with CLL (N = 20) after signing an informed consent as part of a investigational study approved by the IRB. Samples were also obtained from healthy volunteers. Normal and leukemic B cells were negatively purified using a B-lymphocyte enrichment kit (RosetteSep; StemCell Technologies) and cell lysate was utilized to evaluate SR-B1 expression by Western blotting utilizing a monoclonal antibody (AbCam). HDL NPs were added to the samples at a concentration of 30nM and 100nM and allowed to incubate for 72 hours in vitro. In parallel experiments, ABT-199 (0.003µM) and Ibrutinib (1µM) were added either alone or in the presence of the HDL NP to the cell culture. Flow cytometry was performed using Annexin/PI staining to measure cell death.

Results:

Among the 11 CLL patients' samples tested, 8 (72%) expressed the SR-B1 receptor as detected by WB. In this series, SR-B1 expression was independent of any known biological features, including IGHV gene mutational status. When purified leukemic cells (n=9) were incubated for 72 hours in the presence of HDL NPs, a dose response pro-apoptotic effect was evident with a higher percentage of Annexin V positive cells compared to untreated controls at a concentration of 30nM (52.6% ± 6.7% range vs 28.6% ± 2.6%), peaking at 100nM (74.2% ± 4.6%) (p<0.01). Interestingly, when total mononuclear cells from CLL patients were cultured in the presence of the particles, the pro-apoptotic effect at 30nM was abrogated (19.8% ± 5.0% vs 17.3% ± 3.5% while it remained significant (p<0.05) at the higher concentration of 100nM (41.7% ± 7.7%). This might be explained either by a protective effect by the microenvironment, as shown also with other drugs, or by a dilution effect due to the uptake of HDL NPs by other by-stander cells. Viability of neither B nor T lymphocytes from healthy donors was affected by the highest concentration of the particles. The combination of HDL NP with ABT-199 and Ibrutinib significantly enhanced the killing of CLL cells by the nanoparticles in particular when utilized at low concentrations (30nM) on unpurified mononuclear cells.

Conclusions:

Human primary CLL cells express the SR-B1 receptor. HDL NPs are a potential non-toxic therapy for human CLL. A protective effect of the microenvironment is also apparent that can be overcome by increasing the concentration of the particles, without increasing toxicity on normal lymphocytes. In addition, an enhanced response was achieved upon co-treatment of human CLL cells with HDL NPs and ABT-199, improving the efficacy of low doses of the particles, achievable in vivo. Taken together, these data argue for the continued development of the HDL NP platform as a treatment for B cell malignancies.