The p53/p21 axis suppresses 5FU-induced, ATM/ATR-STAT3-mediated activation of PD-L1


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Tamas Sessler1,Fiammetta Falcone2,Peter Gallagher2,Daniel Longley2,Simon McDade2
1Centre for Cancer Research & Cell Biology Queen's University Belfast, Belfast, UK, Forensic Geoscience Group Queen's University Belfast, Belfast, UK,2Queen's University Belfast, Belfast, UK

Abstract

Background

The immune system plays an important role in the response to chemotherapeutic treatments and there is increasing evidence to suggest that this is at least in part due to upregulation of PD-L1. Elevated expression of PD-L1 has been shown to promote cancer cell survival as PD-L1 is able to inactivate T-cells, inhibiting their activity, proliferation and promoting apoptosis. Given the importance of 5FU in cancer treatment, little is known about its effect on PD-L1 expression.

Method

HCT116, LoVo and RKO with/without p53 were treated with 5FU alone/in combination with oxaliplatin and FUdR for 48h. PD-L1 expression was measured at mRNA level, immunoblotting and surface staining. Cells were screened using different siRNA with 5FU treatment. Involvement of the DNA damage pathway was assessed by ATM, ATR and CHK1 inhibitors and siRNAs.

Results

5FU caused an increased S-phase arrest and a more sustained activation of the DNA damage repair pathway in the absence of p53 or p21. PD-L1 expression increased upon 5FU treatment which was more pronounced in p53-/- or p21-/- cells. By using FUdR, a metabolite of 5FU that acts through inhibition of thymidylate synthase (TS), we were able to reproduce the effect of 5FU on PD-L1 expression. Thymidine depletion can lead to stalled replication fork that in the absence of p53/p21 axis, cause elongated activation of ATR and ATM pathway. Inhibiting ATM/ATR by small molecule can reduce PD-L1 upregulation. Using siRNA we identified STAT3 as a key regulator of the 5FU-mediated PD-L1 upregulation.

Conclusion

Upon 5FU treatment, we demonstrated a marked PD-L1 upregulation in CRC models in the absence of p53 or p21. This effect is caused by inhibition of TS resulting in prolonged ATM pathway activity and in turn by STAT3 activation. This would indicate a possible therapeutic window using PD-1/PD-L1 inhibitors in p53 negative CRC with 5FU treatment.