Activation of NF-Kappa B-p62-NRF2 Signaling Supports the Survival of CLL Cells That Express High Levels of ROR1
Authors: Sanchez-Lopez Elsa , Ghia Emanuela M. , Antonucci Laura , Rassenti Laura Z. , Chen Yun , Kipps Thomas J. , Karin Michael 
Published: October 2019

Abstract
Recent studies have linked the autophagy adaptor p62/SQSTM1 to tumorigenesis via NRF2 signaling. Increased accumulation p62 is associated with disease progression in many cancers, however its expression in CLL has yet to be investigated. Importantly, p62, encoded by the SQSTM1 gene, mRNA expression and protein accumulation is regulated by nuclear factor-kappa B (NF-κB), a key transcription factor for CLL survival. In addition, CLL cells express receptor tyrosine kinase-like orphan receptor 1 (ROR1), an oncoembryonic protein that also is expressed on cancer cells of numerous malignancies, including those reported to show accumulation of p62. As in solid-tumor malignancies, high-level expression ROR1 in CLL is associated with enhanced disease-progression and shorter overall survival (Cui B, et al, Blood 25:2931, 2016). Furthermore, recent studies found that activation of Wnt5a-ROR1 cascade induces NF-κB activation and increased expression of NF-κB target genes in CLL (Chen, Y et al, submitted ASH Abstract, 2018). Additionally, CLL cells exhibit increased basal activation of nuclear factor erythroid 2-related factor 2 (NRF2), which supports antioxidant and detoxifying responses that enhance resistance to cytotoxic drugs. We hypothesized that high-level expression of ROR1 in CLL may influence the accumulation of p62, which in turn could lead to activation of NRF2-signaling to enhance CLL-cell survival and resistance to therapy. We found that CLL cells with high-level surface expression of ROR1 (ROR1HIGH) had significantly greater accumulation of p62 than ROR1LOW CLL cells (p=0.003). Elevated p62 accumulation was accompanied by enhanced protein and mRNA expression of NRF2 targets, including NQO1 (p=0.009), GPX2 (p=0.019), GSTM1 (p=0.001), SOD1 (p=0.001) and MDM2 (p=0.001) (n=6 ROR1LOW and n=13 ROR1HIGH). We analyzed published gene expression data (GSE13204, Kohlmann A, et al BJH 142:802, 2008), of 448 CLL cases. We designated CLL cells with expression levels of ROR1 above the medium (n=224) as ROR1HI and cases with ROR1 expression below the medium value as ROR1LO (n=224), We confirmed that SQSTM1 (p=0.01), NQO1 (p=0.0001) and HMOX1 (p=0.002) were significantly overexpressed in ROR1HI CLL patients. In addition, we segregated GSE13204 dataset into two groups: ROR1>90% (n=45), representing the 10% of patients who had CLL cells with highest levels of ROR1 mRNA, and ROR1<10% (n=45), representing the 10% of patients who had CLL cells with lowest levels of ROR1 mRNA. Consistently, SQSTM1 (p=0.009), NQO1 (p=0.02) and HMOX1 (p=0.008) were significantly overexpressed in ROR1>90%. We performed gene set enrichment analysis (GSEA) to investigate whether NRF2 inducible genes (Malhotra D, et al, Nucleic Acids Research 38(17):5718, 2010) were expressed at higher levels in ROR1HI or ROR1>90% compared with ROR1LO or ROR1<10% CLL. In both comparisons, the GSEA revealed that NRF2 inducible genes were enriched in CLL cells with high ROR1 mRNA (FDR q of 0.01 and 0.06, respectively). Collectively, these data indicate that p62-NRF2 cascade is upregulated in CLL cells with high expression of ROR1 and suggest that it may play a role in the enhanced proliferation observed in ROR1HIGH CLL cells. Similarly, ROR1HIGH CLL cells and MEC1-ROR1 CLL cell line that express high protein level of NQO1 were resistant to ABT-199 (p<0.05 and p<0.001 respectively). Such agent induces the accumulation of reactive oxygen species (ROS), which are detoxified by products of the NRF2-activated antioxidant response. The role of NQO1 in protection from cell death was confirmed using a specific pro-drug, designated as 29h, which becomes active after being metabolized by NQO1 (p<0.05). Furthermore, treatment with 29h overcame NQO1-mediated resistance to ABT-199 (venetoclax) (p<0.05), resulting in PARP cleavage and accumulation of cytoplasmic cytochrome C, markers of apoptosis. This study illustrates a previously unknown and intricate signaling network through which ROR1-NF-κB-p62-NRF2 cascade may enhance disease progression and resistance to therapy in CLL B cells that express high levels of ROR1.

Disclosures
Kipps: Genentech Inc: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.