Redox Redux: Nrf2 Mediates Resistance to Ferroptosis and Apoptosis in High Grade B-Cell Lymphoma

Introduction: High grade B cell lymphomas (HGBCL) with c-MYC and BCL-2 rearrangements are particularly aggressive, respond poorly to standard chemotherapy and patients have poor outcomes. We previously demonstrated that in diffuse large B-cell lymphomas (DLBCL), treatment with gold core high-density lipoprotein-like nanoparticles (Au-HDL NPs) targeting scavenger receptor class B type 1 (SR-B1) induces ferroptosis and apoptosis (Rink et al JBC 2020). Subsequently, we showed that HGBCL cell lines are more resistant to Au-HDL NPs, and found that in these cells, the redox related Nrf2 survival pathway, especially the NQO1 gene, is upregulated after treatment with Au-HDL NPs (Yang et al ASH 2022). Based on these data, we hypothesized that Nrf2 mediates resistance mechanisms in HGBCL. Here we describe knockout (KO) studies of the Nrf2 gene in the high grade OCI-ly1 cell line and show that we restore sensitivity to Au-HDL NPs in all Nrf2 KO (Nrf2 ^-/-) clones.

Methods: The GC DLBCL cell line SUDHL-4 and HGBCL cell lines RC, OCI-ly1, OCI-ly8 and OCI-ly18 were used. The Au-HDL NPs were synthesized as previously described (Rink et al JBC 2020). Cell viability was measured by MTS assay after treatment with Au-HDL NPs or PBS control for 72h. Nrf2 ^-/- OCI-ly1 cells were generated using CRISPR/Cas9 genome editing with two Nrf2 single guide RNAs (sgRNA) targeting Nrf2 exon 2. The knockout of Nrf2 was confirmed by detection of the insert or deletion of Nrf2 DNA sequences spanning the sgRNA by Sanger sequencing and the absence of Nrf2 protein was confirmed by Western Blot. Single cell clones of Nrf2^-/- OCI-ly1 cells were obtained by limited dilution and the knockout of Nrf2 in single cell clones was further validated by Sanger sequencing and Western Blot. After Nrf2^-/- OCI-ly1 cells were obtained, apoptosis (by Annexin V-FITC) and ferroptosis (by C11-BODIPY +/- ferroptosis inhibitor, ferrostatin-1) were detected and compared with WT OCI-ly1 cells after exposure to Au-HDL NPs.

Results: HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were more resistant to treatment with Au-HDL NPs (IC₅₀31.42 nM, 27.22 nM, 58.24 nM, 26.06 nM, respectively) compared with SUDHL4 (IC₅₀ 8.14 nM). Several Nrf2^-/- OCI-ly1 single cell clones were obtained by limited dilution from the Nrf2^-/- OCI-ly1 pool. Single clone (SC) 46 showed 6 nucleotides (nt) deletion, SC49 showed 21 nt deletion and SC53 showed 13 nt deletion in exon 2 spanning the sgRNA 1 and 2 sites. Nrf2 protein was undetectable by Western blot in all Nrf2^-/- OCI-ly1 clones under the same conditions as with WT, which confirms that Nrf2 was completely knocked out in all Nrf2^-/- OCI-ly1 clones. We examined cell death induced by Au-HDL NPs in WT and Nrf2^-/- clones by MTS assay. We found that in WT cells, after exposure to 20 nM of Au-HDL NPs for 72h, cell viability was 64±8% %. However, in the Nrf2^-/- cells, cell viability was 28±0.5, 26±0.1 and 24±0.1 % for SC46, 49 and 53 respectively. We next explored the potential cell death pathways by which Nrf2 mediates resistance of HGBCL to Au-HDL NPs. After 20nM Au-HDL NPs treatment, there was a marked increase in apoptosis as measured by Annexin V in Nrf2 ^-/- cells (52±3, 65±2 and 63±1% in SC46, 49 and 53 respectively) compared with WT (35±0.5%). Au-HDL NPs-induced cell death in Nrf2^-/- cells could be completely rescued by ferrostatin-1 and partially rescued by Z-VAD-FMK and deferoxamine. Exposure of Nrf2 KO cells to Au-HDL NP did not increase NQO-1 and FTH1 protein expression.

Conclusion: Taken together, these data point to Nrf2 as a critical resistance pathway in HGBCL to Au-HDL NP exposure by blocking ferroptosis and apoptosis, as well as blocking expression of downstream antioxidant genes. These studies have implications for targeting resistance in HGBCL.

Lin:Genmab: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau. Thaxton:Zylem: Current equity holder in private company, Patents & Royalties: nanoparticles as cancer therapy . Gordon:nanoparticles for cancer therapy (HDL NP As Inducers of Ferroptosis in Cancer, PCT/US2020/051549; b) Nanostructures for Treating Cancer and Other Conditions, PCT/UlllllLS2013/027431): Patents & Royalties: nanoparticles for cancer therapy (HDL NP As Inducers of Ferroptosis in Cancer, PCT/US2020/051549; b) Nanostructures for Treating Cancer and Other Conditions, PCT/UlllllLS2013/027431); Janssen: Other: data and safety monitoring board ; Ono Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb, Kite Pharmaceuticals: Other: Advisory board.