Investigations of Redox Resistance in Aggressive Lymphomas with Cholesterol Modulating Lipid Nanoparticles

Introduction: High grade B cell lymphoma (HGBCL), characterized by C-MYC and BCL-2 rearrangements, are particularly aggressive with poor response to standard therapy and decreased overall survival (OS). We previously demonstrated that B-cell lymphomas, such as germinal center (GC) diffuse large B-cell lymphomas (DLBCL), are addicted to cholesterol uptake and that bioinspired gold core lipid nanoparticles (Au-LNPs) antagonize lipid uptake through the cell surface receptor called scavenger receptor type B-1 (SR-B1) and lead to cholesterol depletion in several lymphoma cell lines (Rink et al JBC 2020). Au-LNP treatment leads to lymphoma cell death by ferroptosis, through reduction of glutathione peroxidase (GPX4) and subsequent accumulation of oxidized lipids. The antioxidant Nrf2 pathway is known to resist oxidative stress and high levels of Nrf2 proteins predict poor response to chemotherapy in hematologic malignancies. We therefore hypothesized that 1) HGBCLs would be more resistant to Au-LNP and 2) antioxidant pathways might mediate that resistance.

Methods: The GC DLBCL cell line (SUDHL-4) cells and HGBCL cell lines (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were used. Cells were treated with PBS (control) or Au-LNPs for 72h. Au-LNPs were synthesized as previously described (Rink JBC 2022). Cell viability was measured by MTS assay. Total RNA in RC and SUDHL-4 cells were extracted and used to construct cDNA libraries for RNA sequencing (RNAseq) by HiSeq 4000 with single-end 50bp reads and 20-25 million reads per sample in the NUSeq Core of Northwestern University. Metascape was used for functional analysis of identified differentially expressed genes (DEGs) and DEGs related to Nrf2 survival signaling, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 were confirmed in control and Au-LNP treated SUDHL-4, RC, OCI-ly1, OCI-ly8 and OCI-ly18 cells by RT-qPCR and Western blot analysis.

Results: HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were more resistant to treatment with Au-LNPs (IC50 = 31.42 nM, 27.22 nM, 58.24 nM, 26.06 nM, respectively) compared with SUDHL-4 (IC50 8.14 nM). RNAseq analysis identified 320 upregulated and 162 downregulated genes in SUDHL-4 cells and 346 upregulated and 182 downregulated genes in RC cells after treatment with Au-LNPs. Of these DEGs, 62 upregulated and 8 downregulated DEGs appeared in both cell lines after treatment. The top 5 upregulated common genes include ABHD4, DUSP5, JUN, RGS1 and SQSTM1. The top 5 downregulated common genes include ABCG1, DDN, GPX1, GPX4 and IKZF1. There were 284 upregulated and 174 downregulated DEGs only identified in Au-LNP treated RC cells. Among 284 upregulated DEGs the top 2 enriched clusters were Nrf2 survival signaling and nuclear receptors meta-pathways. Au-LNP treatment in RC cells showed log2 fold increase of NQO1 (4.6), FTH1 (1.28), FTL (2.1), and TXNRD1 (1.78), genes in the Nrf2 survival signaling pathway. In SUDHL-4 cells, only NQO1 and FTL1 showed significant differential expression with log2 fold increase of 0.7 and 0.45 after Au-LNP treatment. To confirm RNAseq results, we measured the expression of Nrf2 survival signaling genes, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 with or without Au-LNP in SUDHL4, RC, OCI-ly1, OCI-ly8, and OCI-ly18 cells by RT-qPCR. In SUDHL4 cells, compared with PBS control, Au-LNP treatment led to mild increase in expression of NQO1 (4.2 ± 0.6 fold). By contrast, in HGBCL cells, Au-LNPs increased NQO1 expression 19.9 ± 0.2 fold for RC cells, 9.0 ± 0.3 fold for OCI-ly1 cells and 36.5 ± 2.3 fold for OCI-ly8 cells compared with PBS control. Higher NQO1 and FTH1 protein expression in treated RC cells was also confirmed by western blot analysis. These data suggest that 1) HGBCL cell lines RC, OCI-ly1, OCI-ly8 and OCI-ly18 are more resistant to cell death by cholesterol depletion and that 2) this phenomenon is mediated by upregulation of genes in the Nrf2 pathway and NQO1, in particular.

Conclusion: The redox related Nrf2 survival signaling pathway, especially the NQO1 gene, is upregulated after treatment with Au-LNPs. This was observed to a greater extent in the more resistant HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) than the SUDHL4 cells. Strategies to target the Nrf2 pathway may reverse resistance to cholesterol depletion in lymphoma.

McMahon:Zylem Biosciences.: Other: founder. Gordon:BMS: Research Funding; Ono Pharmaceuticals: Consultancy; Zylem: Current equity holder in private company, Current equity holder in publicly-traded company, Patents & Royalties: Patent on nanoparticles for lymphoma therapy; Janssen: Other: DSMB.