Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled By Functional Lipoprotein Nanoparticles and the B Cell Receptor Inhibitor Ibrutinib: A Therapeutic Strategy Dependent on Cell of Origin (COO)

Abstract

Introduction: Malignant cells, including lymphoma cells, have altered metabolism and, in some cases, an increased demand for cholesterol and cholesteryl esters. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative, exciting approach. Toward this end, we focused on DLBCL as a model because of differences in cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed bio-inspired high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol and cholesteryl ester uptake through binding to the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). We hypothesized that by inhibiting BCR signaling, and consequently cholesterol biosynthesis with the BTK inhibitor ibrutinib, we could overcome inherent resistance in cholesterol replete ABC DLBCL cell lines in vitro and in a xenograft model.Methods: HDL NPs were synthesized by surface-functionalizing a gold nanoparticle core as previously described (Thaxton et al JACS 2009) with the HDL defining apolipoprotein A-I and phospholipids, with the resultant HDL NPs having similar size, surface composition, charge and function as natural HDLs. However, owing to the gold nanoparticle core, HDL NPs are not an appreciable source of cholesterol or cholesteryl esters. GC DLBCL (SUDHL4) and ABC DBLCL (TMD8, HBL-1) cell lines were treated with HDL NPs as monotherapy or in combination with BCR signaling inhibitors (e.g. ibrutinib), and assayed for total cholesterol content and cell viability. Quantitative PCR was conducted to measure changes in expression of cholesterol biosynthesis genes. TMD8 tumor xenografts were initiated in SCID-beige mice, and following implantation, the mice were treated with vehicle, HDL NPs, ibrutinib, or a combination of the two treatments.Results: As we have previously reported (Yang et al PNAS 2013), GC DLBCL are exquisitely sensitive to HDL NP as monotherapy while ABC DLBCL are less sensitive. Here, our data demonstrate that HDL NPs, as a monotherapy, statistically significantly reduced total cellular cholesterol levels in SUDHL4 cells (7.96 ± 1.47 µg cholesterol/ 104 cells for 0nM HDL NP vs. 2.07 ± 2.07 µg cholesterol/ 104 cells for 100nM HDL NP; p=0.0007), resulting in profound cell death (HDL NP IC50 = 2nM). Further, HDL NP-induced cholesterol depletion resulted in upregulation of numerous cholesterol biosynthesis genes in SUDHL4 cells, including HMG-CoA synthase 1, 7-dehydrocholesterol reductase, lanosterol synthase, and others. In ABC DLBCL cells, the enhanced BCR signaling and resultant de novo cholesterol synthesis drastically diminishes the ability of HDL NPs to reduce cellular cholesterol (TMD8: 12.80 ± 5.08 µg cholesterol/ 104 cells for 0nM HDL NP vs. 16.35 ± 1.17 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.84; HBL-1: 29.16 ± 5.96 µg cholesterol/ 104 cells for 0nM HDL NP vs. 23.51 ± 5.65 µg cholesterol/ 104 cells for 100nM HDL NP, p=0.75) and induce cell death (TMD8 HDL NP IC50 = 69nM; HBL-1 HDL NP IC50 = 75nM). Additionally, HDL NPs had little to no effect on the expression of the cholesterol biosynthesis genes in the ABC cell lines. To reduce cholesterol biosynthesis, which depends on BCR signaling, we combined HDL NPs with the BCR signaling inhibitor ibrutinib. We found that HDL NP synergize with ibrutinib in the ABC derived TMD8 cell line in vitro and in a xenograft model. Correspondingly, HDL NPs and ibrutinib reduced the total cellular cholesterol content of TMD8 and HBL-1 cells when administered together, but had no effect when given alone. Thus, by targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis we achieved synergistic cellular cholesterol reduction and subsequent cell death, both in vitro and in the TMD8 in vivo tumor xenograft model.Conclusion: These results in DLBCL lymphoma cell lines and tumor xenografts demonstrate that depletion of cellular cholesterol, in a specifically targeted manner dependent on the HDL receptor SCARB1 and differential BCR signaling in ABC vs GCB cells, is a powerful mechanism to induce apoptosis in lymphoma and opens a new paradigm in lymphoma biology and therapy. DisclosuresThaxton:Aurasense: Other: The HDL NPs technology was licensed by Northwestern University to a biotech company (Aurasense) founded by C. Shad Thaxton . Gordon:Janssen: Other: Data Monitoring Committee.