Definition of the endogenous substrates for the CRL4 CRBN E3 ligase complex and of novel substrates in c-Myc driven breast cancer

This project builds on work and insight from our understanding of the mechanism of action of thalidomide and its analogues  the so-called immunomodulating derivatives (IMiDs) such as lenalidomide and pomalidomide. Thalidomide and the IMiDs have made a major impact on the treatment of some haematological malignancies, including multiple myeloma, myelodysplasia and lymphoma [Zeldis et al, 2011]. Furthermore, there are ongoing trials of these agents in non-cancer settings such as leprosy and systemic lupus erythematosus [Zeldis et al, 2011]. Due to the pleiotropic clinical effects of these agents, there has been considerable interest over two decades in elucidating their mechanism of action. In 2010, Hiroshi Handa’s laboratory in Tokyo discovered that thalidomide binds to cereblon (CRBN) (Ito et al, 2010), a component of an E3 ubiquitin ligase complex that also contains DDB1, CUL4 and Roc1 (CRL4CRBN: cullin-ring finger ligase-4 CRBN) (Figure 1).

We and others subsequently showed that CRBN is also the target of the thalidomide analogues lenalidomide and pomalidomide, and is responsible for the immunomodulatory and antiproliferative activities of these agents in multiple myeloma (MM) [Girona-Lopez et al, 2012]. We also hypothesised that IMiDs alter the abundance, localisation and activity of CRLCRBN E3 ligase substrates [Zhu et al, 2011; Girona-Lopez et al, 2012] (Figure 1).Three studies in 2014 by the Ebert, Kaelin and Chopra groups [Gandhi et al, 2014; Krönke et al, 2014; Lu et al, 2014] showed that these changes arise from IMiDs ability to alter cereblon’s E3-ligase substrate preference, resulting in the ubiquitination and degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). The data from these and subsequent studies also demonstrated that Ikaros and Aiolos degradation was dependent on the presence of IMiDs and therefore represents drug-induced neomorphic activity, with Ikaros and Aiolos identified as neosubstrates of the cereblon E3 ligase complex [Licht et al, 2014].

We believe that the binding of IMiDs to CRBN displaces endogenous substrates, of which little is known, and promotes the recruitment of neosubstrates.

Notably, CBRN was also shown to be the protein target of thalidomide that is responsible for thalidomide-mediated teratogenesis in zebrafish [Ito et al, 2010].

We believe that the binding of IMiDs to CRBN displaces endogenous substrates, of which little is known, and promotes the recruitment of neosubstrates. Recently, the homeobox gene MEIS2, was identified as an endogenous substrate of CRBN whereby MEIS2 ubiquitination is inhibited by the IMiDs, resulting in increased protein levels. These data indicate that IMiDs modulate ubiquitination; in some instances, creating a neomorph for substrate degradation, as for Aiolos and Ikaros, and in others, competing out endogenous substrates, such as MEIS2, thereby leading to a decrease in their degradation [Fischer et al, 2014]. More recently, glutamine synthetase (GS) has also been shown to be an endogenous substrate of CRL4CRBN [Nguyen et al, 2016]. GS recognition by CRBN leads to its polyubiquitination by CRL4CRBN in response to high glutamine levels. Contrary to the case of MEIS2, IMiDs enhance GS binding to CRBN. Furthermore, two lysine resides (K11 and K14) in the N-terminus of GS are acetylated by p300/CBP in response to high glutamine concentrations. These acetylation marks serve as a protein degradation motif (degron) to allow CRBN binding and CRL4CRBN-mediated ubiquitination. These data suggest that, so far, there does not appear to be an identifiable conserved degron motif and that CRL4CRBN-mediated degradation of substrates is likely to be dependent on the cellular context and metabolic state of the cell.

Project aims:

• The postgraduate student will define the endogenous substrates of CRL4CRBN E3 ligase substrates using a novel BioID approach [Roux et al, 2012, 2013; Rees et al, 2015]. We will prepare a CRBN fusion construct with a mutant bacterial biotin protein ligase, BirA*, which will non-specifically biotinylate any protein with exposed lysine residues proximal to the fusion protein. In addition the student will use a ligase trap assay by fusing a ubiquitin-associated (UBA) domain to CRL4CRBN E3 ligase, to selectively purify the polyubiquitinated forms of E3 substrates.

• Using the assays developed in the first aim, the student will apply them to immortalised Human Mammary Epithelial cells (HMEC). These experiments will elucidate the endogenous substrates of CRBN.

• In addition these assays will be applied to HMECs engineered and passaged to gain c-Myc dependency and will be used to screen for the degradation of specific protein targets or the regulatory/synthetic lethal partners of c-Myc.