Mechanisms of repression of the transcription factor NRF2 by KEAP1- and B-TrCP-dependent ubiquitin ligases and how the dysregulation of NRF2 contributes to lung cancer progression

Abstract

Lung cancer is the leading cause of cancer related mortality worldwide and since the discovery of the important role that NRF2 plays in regulating the expression of phase II drug metabolism genes, NRF2 has been a highly studied therapeutic target. NRF2 signalling is often elevated in lung cancer due to mutations in NFE2L2, the gene that encodes NRF2; and KEAP1, the cytoplasmic regulator of NRF2. Since it became established that elevated NRF2 signalling provides a beneficial role to the tumour through enhancing proliferation, enabling survival in highly oxidative conditions and enhancing resistance to chemotherapeutic drugs; many groups have focussed on understanding KEAP1-independent forms of NRF2 regulation.

It is now widely accepted that GSK-3 phosphorylation of the Neh6 domain of NRF2 is fundamental for SCFβ-TrCP-mediated degradation of the transcription factor. Previous work by other groups has indicated a potential role for a priming kinase to pre-phosphorylate the Neh6 domain and subsequently enhance GSK-3 mediated phosphorylation and resulting 26S proteasomal degradation. In the first results chapter of this thesis it was demonstrated using the DYRK family of isoenzymes that the presence of a priming kinase enhances GSK-3 phosphorylation and stimulates NRF2 degradation. Also, it has been shown that DYRK family members carry out priming of NRF2 through phosphorylating Ser-347, which enhances subsequent phosphorylation of Ser-342 and Ser-338 by GSK-3; regulating both NRF2 activity and stability. Additionally, it was shown that altering the phosphorylation status of Ser-347 impacts cell proliferation and chemo-sensitivity to platinum-based compounds. The ability of other kinases to phosphorylate the Neh6 domain was also revealed.

In the second results chapter of this thesis a bioinformatics style approach was utilized to analyse the impact of mutations in KEAP1, NFE2L2 and CUL3; in terms of their effect on NRF2 activity using the expression of NRF2-target genes as a read out. The following points were highlighted from this analysis; (1) KEAP1 mutations are more prevalent than NFE2L2 and CUL3 mutations in both lung cancer cell lines and tumours; (2) KEAP1 and NFE2L2 mutations vary in there associated zygosity’s, with KEAP1 mutations being predominantly homozygous and NFE2L2 mutations being predominantly heterozygous; (3) mutations in KEAP1 coexist with mutations in KRAS whereas, mutations in NFE2L2 co-exist with mutations in TP53 and (4) lung cancer cell lines and tumours harbouring mutations in KEAP1 have greater expression of NRF2-target genes than those harbouring either NFE2L2 or CUL3 mutations.

In the third and final results chapter, both a panel of commercial cell lines and an in-house generated CRISPR/Cas9 panel of cell lines have been shown in multiple experiments to validate the bioinformatics findings of the previous chapter. Also, in this chapter it was identified that KEAP1 mutant cells are more reliant on the glutathione biosynthesis pathway for survival and therefore more sensitive to alterations in this pathway.

The work demonstrated in this thesis has revealed new insights into the complex regulation of NRF2 and the importance of studying the mutations that lead to aberrant NRF2-signaling in lung cancer. Through showing the importance of a priming kinase in the regulation of KEAP1-independent degradation of NRF2, a completely novel alternative way of stimulating NRF2 degradation in the presence of a KEAP1 mutation is possible. The data displayed in the second and third results chapters highlights the importance of understanding the impact that mutations in KEAP1 and NFE2L2 have on NRF2 signalling; and suggests that theses mutations should not be grouped together in terms of the effect. Additionally, the data presented in the second and third results chapters highlights the importance of generating physiologically relevant NRF2 models. This greater understanding of the regulatory mechanisms in place to control the levels of NRF2 in the cell and the mutations that lead to aberrant NRF2-signalling in cancer will hopefully aid in the development of new therapeutic strategy to combat this disease.

Date of Award	2019
Original language	English
Sponsors	AstraZeneca
Supervisor	John Hayes (Supervisor)