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Deciphering the role of E3 ubiquitin ligases in the pathogenesis and radiotherapy response of cancers.

Ubiquitin laddering visualised by Western blot. The image represents the structure of ubiquitin overlayed on a Western blot to visualise the typical laddering of bands obtained by the modification of multiple ubiquitin molecules on target proteins.

RESEARCH:

The Ubiquitin Proteasome System (UPS) operates the transfer of ubiquitin molecules to lysine residues of target substrates using a cascade of 3 enzymes named E1, E2 and E3. In the initial step, a ubiquitin molecule is activated by ATP and transferred from an E1 enzyme to the E2s. E3s represent a large family of enzymes that recruit the E2s to the substrates and manage the formation of ubiquitin chains. The polyubiquitin chains formed on proteins are recognised by the proteasome and proteins are degraded. A significant breakthrough in the ubiquitin field occurred in 2014 with the discovery that lenalidomide acts as a molecular glue through the recruitment of transcription factors to E3 ubiquitin ligases for degradation (1). The observation led to the emergence of a large field of investigation by universities and drug companies to engage E3 ubiquitin ligases with molecular glues and PROTACs (Proteolysis Targeting Chimaera). Alterations of E3 ubiquitin ligases play a crucial role in tumorigenesis and E3s are amenable to chemical inhibition and activation, however, although the human genome codes for over 700 E3 ubiquitin ligases, the biological function and molecular mechanisms of catalysis are unknown for the majority of E3s. The mission of the D’Angiolella laboratory is to decipher the role of E3 ubiquitin ligases in the pathogenesis and radiotherapy response of cancers. The laboratory focuses on the investigation of a class of multi-subunit E3s called Cullin Ring ubiquitin Ligases (CRLs). The CRLs modular structure and assembly is outlined below and their role in IR has been reviewed here (2,3):

Figure 1. The modular architectures of Cullin-RING E3 Ligases (CRLs). (A) CRL1 ubiquitin ligase complexes are perhaps the best-known examples and bind their substrates via an F-box containing subunit. D'Angiolella discovered the function of cyclin F (FBXO1), the founding member of the F-box family of Cullin-RING ubiquitin ligases. (B) BTB-Kelch proteins, such as KBTBD4, assemble into CRL3 ubiquitin ligase complexes in which the BTB domain binds to Cullin3 and the Kelch domain forms the substrate recognition domain. (C) Some Cullin-RING ubiquitin ligase subunits, such as Cereblon are bound by “molecular glue” drugs (e.g. lenalidomide) that enable the recruitment of neo-substrates (e.g. Ikaros) for ubiquitylation.Figure 1. The modular architectures of Cullin-RING E3 Ligases (CRLs). (A) CRL1 ubiquitin ligase complexes are perhaps the best-known examples and bind their substrates via an F-box containing subunit. D'Angiolella discovered the function of cyclin F (FBXO1), the founding member of the F-box family of Cullin-RING ubiquitin ligases. (B) BTB-Kelch proteins, such as KBTBD4, assemble into CRL3 ubiquitin ligase complexes in which the BTB domain binds to Cullin3 and the Kelch domain forms the substrate recognition domain. (C) Some Cullin-RING ubiquitin ligase subunits, such as Cereblon are bound by “molecular glue” drugs (e.g. lenalidomide) that enable the recruitment of neo-substrates (e.g. Ikaros) for ubiquitylation.

 

In the last quinquennium we have investigated novel aspects of CRLs mechanisms of action and regulation (4,5,6,7). In addition to the published findings, we completed a high resolution focused CRISPR screen of the ubiquitin system role in the response of cancer to IR, which will form the basis of our next quinquennium programme. 

IMPACT:

The ubiquitin system is crucial for the cellular response to IR. Given that the system is targetable with a range of classical and novel compounds (PROTACs and molecular glues), the study of the ubiquitin system in IR responses hold significant promises for the development of drugs which could enter in the clinic.  We have defined novel properties and regulation of E3s highlighting targets for cancer treatment (4,5,6,7). After our CRISPR screen, we have now a comprehensive view of the E3s in IR responses and we will focus on studying their biological role and mechanisms of action. In collaboration with structural biologists, we plan to define the details of their molecular interactions, to initiate development of compounds to improve the therapeutic index of radiotherapy.

REFERENCES:

1. Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, Wong KK, Bradner JE, Kaelin WG Jr. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 2014 Jan 17;343 (6168):305-9. doi:10.1126/science.1244917.

2. Fouad S, Wells OS, Hill MA, D'Angiolella V. Cullin Ring Ubiquitin Ligases (CRLs) in Cancer: Responses to Ionizing Radiation (IR) Treatment. Front Physiol. 2019 Oct 1;10:1144. doi: 10.3389/fphys.2019.01144. 

3. Humphreys LM, Smith P, Chen Z, Fouad S, D'Angiolella V. The role of E3 ubiquitin ligases in the development and progression of glioblastoma. Cell Death Differ. 2021 Feb;28(2):522-537. doi: 10.1038/s41418-020-00696-6.

4. Fung E, Richter C, Yang HB, Schäffer I, Fischer R, Kessler BM, Bassermann F,D'Angiolella V. FBXL13 directs the proteolysis of CEP192 to regulate centrosomehomeostasis and cell migration. EMBO Rep. 2018 Mar;19(3): e44799. doi:10.15252/embr.201744799.

5. Mavrommati I, Faedda R, Galasso G, Li J, Burdova K, Fischer R, Kessler BM, Carrero ZI, Guardavaccaro D, Pagano M, D'Angiolella V. β-TrCP- and Casein Kinase II-Mediated Degradation of Cyclin F Controls Timely Mitotic Progression. Cell Rep. 2018 Sep 25;24(13):3404-3412.

6. D'Angiolella V, Donato V, Forrester FM, Jeong YT, Pellacani C, Kudo Y, Saraf A, Florens L, Washburn MP, Pagano M. Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair. Cell. 2012 May 25;149(5):1023-34.

7. Burdova K, Yang H, Faedda R, Hume S, Chauhan J, Ebner D, Kessler BM, Vendrell I, Drewry DH, Wells CI, Hatch SB, Dianov GL, Buffa FM, D'Angiolella V. E2F1 proteolysis via SCF-cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition. EMBO J. 2019 Oct 15;38(20):e101443. 

 

In the last quinquennium we have investigated novel aspects of CRLs mechanisms of action and regulation (4,5,6,7). In addition to the published findings, we completed a high resolution focused CRISPR screen of the ubiquitin system role in the response of cancer to IR, which will form the basis of our next quinquennium programme.