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Applying mass spectrometry to define the molecular signatures in human disease processes and accelerating target discovery in translational research.

Cellular proteolysis by the ubiquitin proteasome system UPS.  Ubiquitin (represented by small black circles) is attached to protein substrates by the E1/E2/E3 conjugating system and removed by deubiquitylating enzymes DUBs (black star / red arrow). These are critical control check points for intracellular protein degradation
Cellular proteolysis by the ubiquitin proteasome system UPS. Ubiquitin (represented by small black circles) is attached to protein substrates by the E1/E2/E3 conjugating system and removed by deubiquitylating enzymes DUBs (black star / red arrow). These are critical control check points for intracellular protein degradation

Role of ISGs in sensitizing cancer cells to ionising radiation

 Exposure of cancer cells to irradiation triggers many cellular responses including DNA damage, metabolic reprogramming and the induction of interferon pathways. The contribution of interferon stimulated genes (ISGs) observed in cancer cells in response to irradiation is not yet fully understood. In our research programme, we aim to better understand underlying mechanism as well as whether this response may be clinically beneficial or detrimental.

A model showing the interferon stimulated gene (ISGs) pathways in cancer cellsA model showing the interferon stimulated gene (ISGs) pathways in cancer cells

Altered ISG profile in cancer cells exposed to ionising radiation

These aims are aligned and integrated with goals outlined by other OIRO MRC members. In tumourigenesis, the ubiquitin-proteasome system (UPS) system has been strongly linked to DNA damage response (DDR) pathways, also in the context of radiation-based therapies 1,2. Previous work from members of the OIRO MRC Unit showed the involvement of ATAXIN3 3 (Ramadan), Fox E3 ligases 4–6 (D’Angiolella) and other proteases such as SPRTN 7,8 (Ramadan). IR also triggers an inflammatory response through interferon, including Interferon Stimulated Genes (ISGs) 9. Whether this ISG response has anti-tumour or tumour-suppressor effects is currently controversial. In addition, its contribution to protecting or aggravating IR therapy has not yet been explored, but potentially offers novel ways to increase the therapeutic index. USP18 and ISGylation modulated in cells exposed to ionising radiation. Exploring the Ubl system in IR, we noted that the deubiquitinase USP18, a cellular de-ISGylase, responsible for regulating the cellular interferon stimulated gene (ISG) response, renders cancer cells sensitive to irradiation  10. This was demonstrated in an IR dilution assay using the chronic myeloid cell line HAP-1 in which USP18 was deleted as compared to controls. As a consequence of irradiation in combination with USP18 deletion, we observed an enhanced ISG response assessed by quantitative mass spectrometry. We shall explore the role of ISGs including USP18 and components of the ISGylation conjugation machinery and their effects on cancer cells in ionising radiation in collaboration with OIRO members, in particular to test whether incapacitation of ISGs could sensitise cells to IR.

Added value brought to the OIRO Unit

(A) The MS technology platforms available through the Kessler lab and TDI (Discovery Proteomics Facility DPF – part of the Kessler group) have and will continue to provide key technology to all research programmes across the MRC Unit. The OIRO programmes of the groups Olcina, Giaccia, Humphrey, D’Angiolella, Moon and Ramadan will directly profit from this expertise. (B) Benedikt Kessler is part of the Centre for Medicines Discovery (CMD). This will facilitate the transition relevant IR molecular targets towards a drug discovery pipeline via existing links to Pfizer, Evotec (Lab282) and Dark Blue Therapeutics. (C) Considerable overlap between research interests of the Ramadan, D’Angiolella and Kessler groups by exploring targets within the ubiquitin system (e.g. E3’s and DUBs) to enhance the therapeutic index in radiation based therapies.

 References

1.  Hoeller, D., Hecker, C. M. & Dikic, I. Ubiquitin and ubiquitin-like proteins in cancer pathogenesis. in Nature Reviews Cancer (2006). doi:10.1038/nrc1994

2.  Fouad, S., Wells, O. S., Hill, M. A. & D’Angiolella, V. Cullin Ring Ubiquitin Ligases (CRLs) in Cancer: Responses to Ionizing Radiation (IR) Treatment. Frontiers in Physiology (2019). doi:10.3389/fphys.2019.01144

3.  Singh, A. N. et al.  The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8 . EMBO J. (2019). doi:10.15252/embj.2019102361

4.  Raducu, M. et al.  SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development . EMBO J. (2016). doi:10.15252/embj.201593374

5.  Burdova, K. et al.  E2F1 proteolysis via SCF ‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition . EMBO J. (2019). doi:10.15252/embj.2018101443

6.  Fung, E. et al.  FBXL 13 directs the proteolysis of CEP 192 to regulate centrosome homeostasis and cell migration . EMBO Rep. (2018). doi:10.15252/embr.201744799

7.  Fielden, J. et al. TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nat. Commun. (2020). doi:10.1038/s41467-020-15000-w

8.  Vaz, B. et al. Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair. Mol. Cell (2016). doi:10.1016/j.molcel.2016.09.032

9.  Villarroya-Beltri, C., Guerra, S. & Sánchez-Madrid, F. ISGylation - a key to lock the cell gates for preventing the spread of threats. Journal of Cell Science (2017). doi:10.1242/jcs.205468

10. Pinto-Fernandez, A. et al. Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and  radiosensitivity. Br. J. Cancer 124, 817–830 (2021).