Inhibition of Pro-Survival Pathways in DLBCL Cells By Functional Lipoprotein-like Nanoparticles
Authors: Rink Jonathan , Yang Shuo , Cen Osman , Karmali Reem , Thaxton Colby Shad , Gordon Leo I. 
Published: October 2019

Abstract
Introduction: Diffuse large B cell lymphoma (DLBCL) cells have an increased demand for cholesterol and cholesteryl esters to maintain membrane-anchored pro-survival signaling pathways, such as B cell receptor (BCR) signaling. In addition to BCR-mediated stimulation of de novo cholesterol synthesis, another mechanism employed by lymphoma cells to maintain cholesterol and cholesteryl ester homeostasis is by binding cholesterol-rich HDLs via the high-affinity receptor, scavenger receptor type B-1 (SCARB1). We previously reported that, unlike normal B cells, DLBCL cells highly express SCARB1, and that synthetic, cholesterol-poor high-density lipoprotein (HDL)-like nanoparticles (HDL NPs) exquisitely target SCARB1, deplete cellular cholesterol, and induce lymphoma cell death in vitro and in vivo. Given the critical role of cholesterol in maintaining proper membrane organization for BCR-mediated intracellular signaling, we hypothesized that HDL NP binding to SCARB1 and subsequent cellular cholesterol depletion modulates the organization of the cell membrane, reduces intracellular signaling downstream of membrane-anchored pathways like the BCR, and modulates the expression of genes relevant to cholesterol synthesis. Collectively, these mechanisms potently induce lymphoma cell death.
Methods: We focused on germinal center (GC) DLBCL and Burkitt's Lymphoma (BL), due to the previously reported sensitivity of the SUDHL4 (GC DLBCL) and Ramos (BL) cell lines to HDL NP-induced cell death. In addition to SUDHL4 and Ramos, we tested the GC DLBCL cell line SUDHL6 and the BL cell lines Raji, Daudi and Namalwa. HDL NPs were synthesized, purified and characterized using standard protocols. Cell viability was quantified using the MTS assay, and total cholesterol levels were measured using the Amplex Red cholesterol assay. A blocking antibody against SCARB1 was used to inhibit binding of HDL NPs to the receptor. Confocal microscopy was used to visualize changes in membrane organization of SUDHL4 and Ramos cells following HDL NP treatment. Changes in the phosphorylation of signaling kinases (e.g. AKT, ERK1/2) following HDL NP treatment was measured using phospho-kinase arrays, phos-flow analysis, and western blot assays. RNA was harvested from cells treated with the HDL NPs for 48 hours and analyzed using Illumina's HT-12 microarray and RT-qPCR. Protein expression changes were measured using western blot assays.
Results: HDL NPs potently induced cell death in all of the GC DBLCL and BL cell lines, all of which expressed SCARB1, with HDL NP IC50 values between 1 ~ 5 nM. As expected, HDL NP-induced cell death correlated with reduced total cellular cholesterol levels. Inhibition of HDL NP binding to SCARB1 by antibody blockade protected cells from HDL NP-induced cell death. HDL NP treatment induced a reorganization of the plasma membrane in SUDHL4 and Ramos cells, resulting in clustering of SCARB1, a phenomenon not seen in cholesterol-rich human HDL or saline controls. HDL NP treatment led to a decrease in the phosphorylation of a number of kinases downstream of BCR signaling, including AKT, ERK1/2, LCK and LYN over time. HDL NPs demonstrated synergy with small molecule inhibitors of various signaling kinases, such as ATK (inhibitor = GDC-0068) and SYK (inhibitor = R406), in Ramos and SUDHL4 cells. By microarray analysis and RT-qPCR, HDL NPs up-regulated a number of cholesterol biosynthesis genes, as well as the cell cycle inhibitor p21 and the pro-apoptotic protein APOPT1.
Conclusions: These data demonstrate that HDL NPs bind SCARB1, altering the organization of the plasma membrane and reducing cellular cholesterol levels. Collectively, this results in decreased membrane-anchored pro-survival signaling, changes in gene expression pathways, and, ultimately, lymphoma cell death. Enhancing cholesterol depletion strategies, such as the combination of the targeted cholesterol depletion agent HDL NP with small molecule inhibitors of signaling kinases (e.g. AKT and SYK inhibitors), represents a novel and targeted therapeutic strategy for DLBCL and may be broadly applicable to other malignancies dependent on cholesterol homeostasis and membrane-anchored signaling pathways.

Disclosures
Karmali: Gilead: Speakers Bureau; AstraZeneca: Speakers Bureau. Thaxton:AuraSense: Other: Co-founder of the biotech company AuraSense.