Introduction: Bruton's tyrosine kinase (BTK) inhibitors (BTKi), such as ibrutinib, have become the standard of care for mantle cell lymphoma (MCL). Recent data suggest that a gold (I) triethylphosphine (AuPEt3)-containing molecule (“gold salt”), such as auranofin may synergize with ibrutinib to disrupt membrane-anchored intracellular signaling pathways and induce cell death. Membrane-anchored signaling pathways are dependent upon lipid rafts. Peroxidation of phospholipids can disrupt lipid raft organization and negatively impact these signaling pathways. Glutathione peroxidase 4 (GPx4), an antioxidant and membrane-based enzyme, contains a unique amino acid, selenocysteine, at its active site and is the sole protein responsible for detoxifying phospholipid peroxides. Auranofin has been used clinically and has been shown to inhibit selenocysteine-containing proteins by targeting the selenocysteine residue. The AuPEt3 moiety of “gold salt” compounds is bound to thiol (-SH) residues on hydrophilic molecules (e.g., thio-glucose). This localizes the active compound to aqueous environments and reduces access to more hydrophobic and amphiphilic cellular environments, like the cell membrane. In an effort to target the AuPEt3 moiety to the plasma membrane, we conjugated AuPEt3 to a thiol-containing phospholipid (DPPTE; 1-2-dipalmitoyl-sn-glycero-3-phosphothioethanol) to create a new therapeutic gold salt, DPPTE-AuPEt3. To target the delivery of DPPTE-AuPEt3 to MCL cells, we utilized an organic core (oc)-templated high-density lipoprotein (HDL)-like nanoparticle platform (ocHDL NPs). The ocHDL NPs target the high-affinity receptor for native HDLs called scavenger receptor class B type 1 (SR-B1). The ocHDL NPs have been shown to target SR-B1 and deliver therapeutic cargo to induce lymphoma cell death. We hypothesized that the targeted delivery of DPPTE-AuPEt3 by ocHDL NPs would synergize with BTKi to induce cell death in MCL.

Methods: DPPTE-AuPEt3 was synthesized by combining a thiolated phospholipid (DPPTE) with AuPEt3. The new DPPTE-AuPEt3 was characterized by NMR and mass spectrometry. ocHDL NPs were synthesized by self-assembling the HDL-defining apolipoprotein A-I (apoA-I) and phospholipids around an organic core scaffold and were characterized for size and surface charge. Four MCL cell lines were used for these studies: Jeko (TP53 null), Maver (TP53 mutant), Mino (TP53 mutant) and Rec (TP53 wild type). Cell death was measured using the MTS assay. Expression of GPx4 and SR-B1 was measured using western blot analysis and/ or flow cytometry. Accumulation of lipid peroxides was measured using the C11-BODIPY dye and flow cytometry. Synergy was determined using the Chou and Talalay method using constant ratios of DPPTE-AuPEt3 ocHDL NPs/ibrutinib of 1/1 (Rec), 1/10 (Maver, Mino) and 1/20 (Jeko).

Results: DPPTE-AuPEt3 was successfully synthesized and incorporated into ocHDL NPs, with the resultant DPPTE-AuPEt3 ocHDL NPs demonstrating a size of 13.65 ± 1.64nm in diameter and surface charge (zeta potential) of -26.6 ± 1.02 mV. SR-B1 expression was confirmed in all four MCL cell lines (Jeko, Maver-1, Rec, Mino), with Mino and Rec demonstrating the highest expression. DPPTE-AuPEt3 ocHDL NPs induced cell death in all MCL cell lines, with Mino (IC50 = 34.2 nM) and Rec (8.82nM) displaying the highest sensitivity compared with Jeko (54.4nM) and Maver (107nM). Empty ocHDL NPs had no effect. Synergy was observed between DPPTE-AuPEt3 ocHDL NPs and ibrutinib in Jeko [combination indexes (CI) for ED50 = 0.463, ED75 = 0.386, ED90 = 0.345], Mino (0.214. 0.246, 0.428) and Rec (0.897, 0.816, 0.748), and was additive in Maver cells (1.009, 1.050, 1.094).

Treatment with DPPTE-AuPEt3 ocHDL NPs increased the accumulation of phospholipid peroxides in MCL cell lines, as DPPTE-AuPEt3 ocHDL NP treatment reduced expression of GPx4. Empty ocHDL NPs and ibrutinib had no effect on GPx4 expression or phospholipid peroxide accumulation.

Conclusion: DPPTE-AuPEt3 ocHDL NPs is a novel therapeutic in MCL cell lines and was synergistic with the BTKi, ibrutinib. Synergy may be driven by gold salt inhibition of GPx4, leading to increased phospholipid peroxide accumulation. Further investigation into the mechanism of action of DPPTE-AuPEt3 ocHDL NPs in cancer is warranted.

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2025
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