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1079 Type I interferon regulation by USP18 is a key vulnerability in cancer
  1. Veronica Jove1,
  2. Heather Wheeler2,
  3. Chiachin Wilson Lee2,
  4. David R Healy2,
  5. Kymberly Levine2,
  6. Erik C Ralph2,
  7. Bing Yang3,
  8. Anand Giddabasappa3,
  9. Paula Loria2,
  10. Masaya Yamaguchi2,
  11. Agustin Casimiro-Garcia4,
  12. Benedikt M Kessler5,
  13. Adan Pinto-Fernandez5,
  14. Veronique Frattini1,
  15. Feng Wang2 and
  16. Paul Wes2
  1. 1Pfizer, New York, NY, USA
  2. 2Pfizer, Groton, CT, USA
  3. 3Pfizer, La Jolla, CA, USA
  4. 4Pfizer, Cambridge, MA, USA
  5. 5University of Oxford, Oxford, Oxfordshire, UK
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background USP18 is a key negative regulator of Type I interferon (IFN) signaling. USP18 cleaves ISG15, a ubiquitin-like modification, through its canonical catalytic function, and directly inhibits the Type I IFN receptor through its scaffold role. USP18 loss-of-function dramatically impacts autoimmune disease, viral susceptibility, and cancer cell survival. However, published studies have reached different conclusions as to whether catalytic or scaffold function is essential.

Methods The ability of USP18 to deconjugate ISG15 was determined using purified protein against a reporter substrate or cell lysates. Scaffolding activity was quantified using STAT1 phosphorylation upon IFN stimulation. After characterizing the impact of specific point mutations on USP18 function, mutations that selectively impair catalytic (USP18 C64S) or scaffold function (USP18 I60N) were evaluated for their effects on cancer cell vulnerability. To assess USP18 mechanism-of-action across multiple cancer cell lineages, guide RNAs that introduce the C64S mutation or knock out USP18 were introduced into cancer cells, and cells were passaged for 2 weeks in the presence or absence of IFN. IFN-dependent changes in wild-type, knockout, or C64S USP18 allelic frequency were assessed by DNA sequencing. To determine the effect of USP18 in vivo, mouse Usp18 was knocked out in CT26 colorectal cancer cells, and cells were implanted subcutaneously in wild-type or immunocompromised (NSG) mice.

Results Deletion of USP18 in a variety of human cancer cell lines of multiple lineages (blood, breast, colon, lung) rendered them vulnerable to IFN. Furthermore, Usp18 knockout in cancer cells prevented tumor formation in wild-type mice, and resulted in partial tumor growth inhibition in immunocompromised mice. Introducing the C64S mutation fully impaired catalytic activity, yet did not result in IFN sensitivity. Furthermore, preventing ISGylation by knocking out the E1 enzyme required for ISGylation, UBA7, did not rescue IFN sensitivity in USP18-deficient cells. By contrast, the I60N mutation led to partial scaffold impairment and partial cancer cell sensitivity. Finally, human USP18 exhibited minimal catalytic activity, in stark contrast to mouse USP18. Therefore, deISGylase activity of USP18 does not mediate IFN sensitivity in human cancer cells.

Conclusions Loss of USP18 creates a key vulnerability of human cancer cells to IFN. This is primarily due to loss of scaffold function, rather than loss of catalytic function (figure 1). Indeed, human USP18 does not appear to function as an enzyme under physiological conditions, unlike mouse USP18. These findings resolve the mechanistic basis for human USP18 function, paving the way to target USP18 for cancer treatment.

Ethics Approval All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.

Abstract 1079 Figure 1

The mechanistic basis for human USP18 function and cancer cell vulnerability.

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