Büttner, Reinhard | Kondylis, Vangelis - A 03

The transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2) is an established regulator of the cellular antioxidant response to xenobiotic and oxidative stress, thereby contributing to cytoprotection. However, similar to other cell survival pathways, NRF2 activation has a dark side, namely it protects malignant cells from death and promotes cancer cell growth, metabolic reprogramming, metastasis and resistance to cancer therapies. Under basal conditions, NRF2 is bound to Kelch-like ECH-associated protein 1 (KEAP1) that orchestrates its constant ubiquitination by E3 ligase Cullin 3 and its proteasomal degradation. Conditions that increase ROS production, such as the exposure to xenobiotics, radiation or metabolic stress, lead to oxidation of a number of cysteine residues on KEAP1 and disrupt the KEAP1-NRF2 interaction. As a result, NRF2 accumulates and moves to the nucleus, where it mediates transcription of its target genes (Fig. 1A). In addition, a non-canonical pathway of NRF2 activation, which depends on Sequestosome 1 (SQSTM1)/p62, has been described. Aberrant accumulation of p62, for instance upon impaired autophagy, and its phosphorylation on S351 strongly increases the affinity of p62 for KEAP1. This leads to KEAP1 sequestration and NRF2 release and nuclear translocation (Fig. 1A). However, p62 is a multifaceted protein with described functions in selective autophagy, cell death regulation, DNA damage response and NF-κB, mTORC1, ERK and K-RAS signaling pathways (Fig. 1B).

In the era of personalized oncology and with the number of targeted treatments growing, therapeutic decisions depend on detailed molecular subtyping. Our research aims at assessing the prevalence of p62-KEAP1-NRF2 axis activation across various cancer types and whether its activation correlates with other cellular and signaling pathway markers. To find potential therapeutic targets for tumors characterized by activation of this pathway, our goal is also to identify kinase inhibitors that impair p62 phosphorylation and lead to decreased NRF2 activation.

In this project, we will address our aims with the following approaches:

• Assemble an IHC panel of markers to perform screening of patient samples from 5 common cancer types (colorectal, breast, hepatocellular, pancreatic cancer and B-cell lymphoma) for the activation of p62-KEAP1-NRF2 pathway and its potential correlation with other molecular markers (Figure 2).
• Perform a kinase inhibitor screen on a suitable cancer cell line from the cancer type with the most prominent activation of p62-KEAP1-NRF2 axis to identify drug targets that could interfere with this pathway. Further, we will characterize the effect of candidate inhibitors on the tumorigenic potential of the selected cancer cells.
• Examine the effect of glutaminolysis inhibition or ferroptosis induction in an hepatocarcinogenesis mouse model that depends on p62-KEAP1-NRF2 pathway.

Mice with hepatic impairment of autophagy have been shown to develop chronic liver injury and liver tumors in an NRF2- and p62-dependent manner. Accordingly, we have generated ATG16L1^LPC-KO mice that show autophagy inhibition in hepatocytes, as well as strong p62 accumulation and phosphorylation at S351. These mice exhibit spontaneous liver damage, inflammation, fibrosis, hepatomegaly and eventually develop liver cancer (Figure 3A-C) (Kondylis et al., 2022). As anticipated, additional p62 deficiency (p62^-/-) significantly prevented liver injury and ameliorated hepatomegaly and hepatocarcinogenesis (Figure 3B-C).
In search for potentially relevant biomarkers to be included in our IHC screening panel, we performed a quantitative proteomics analysis in liver lysates from WT, p62^-/-, ATG16L1^LPC-KO and ATG16L1^LPC-KO p62^-/- mice. We focused on proteins that are upregulated in ATG16L1^LPC-KO livers and whose expression either became normalized in the absence of p62 and NRF2 activation or remained upregulated in ATG16L1^LPC-KO p62^-/- mice (Figure 3D). In line with the previously reported correlation between the NRF2 pathway and ferroptosis, we found high expression of several ferroptosis antagonists in ATG16L1-deficient livers, suggesting that ferroptosis inducers in p62-KEAP1-NRF2-positive tumors may elicit an anti-tumor effect. Our data also showed evidence of increased glutaminolysis, a pathway that is also linked to NRF2 activation and turns glutamine into TCA metabolites to promote cancer cell metabolic adaptation. The effect of targeting these two pathways in hepatocarcinogenesis will be tested in ATG16L1^LPC-KO mice.

Development of biomarker combinations is necessary to define molecular subtypes in cancer, as this will facilitate therapeutic decisions and may improve their outcome. Our project intends to develop such an IHC profile for tumors that are characterized by NRF2 activation induced by p62 accumulation, which may result from defects in autophagy and can affect additional oncogenic signaling pathways. Besides systematically mapping the penetrance of this pathway in 5 common cancer types, we aim to identify clinically-relevant kinase inhibitors that prevent p62 phosphorylation, dampen NRF2 activation and are likely to render cancer of this molecular subtype more susceptible to cytotoxic anti-cancer treatments.