Page 67 - I. Chemical biology and drug discovery

Reversing the recurrent loss of STING via negative feedback blockade for
efficient cancer immunotherapy

1)
1)
Wansang Cho , Yoona Choi , Seung Bum Park* 1)2)
1) Center for Chemical Proteomics, Department of Chemistry, Seoul National University, Seoul 08826, Korea
2) Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea
BACKGROUND AIM

As cancer immunotherapy has been emerged as a new pillar of cancer therapy, diverse strategies are In this study, we demonstrated new strategy that could reverse
suggested based on modulating host immunity for cancer treatment. STING, which is at the center of
anti-DNA virus immunity, is one of well-studied target for cancer immunotherapy. STING is activated by the recurrent loss STING for efficient cancer immunotherapy. Aim
small molecule-like innate ligand, cyclic dinucleotide (CDN). CDN analogs are suggested as new cancer of this study is to discover novel small molecule that inhibits the
immunotherapy agents that activates host immune system. Recent phase I trial of CDN analogs, MK-1454
by Merck, against solid tumors and lymphoma patients co-treated with Pembrolizumab showed robust interaction between STING and ubiquitin E3 ligase, thereby
antitumor efficacy. However, patients treated with MK-1454 alone showed limited antitumor response. blocking the degradation of STING.
Moreover, recent findings indicate that there are complex negative feedback mechanisms that restrict
the activity, or reduce the expression of STING.
METHODS

To discover the desired small molecule, we designed high-throughput screening system based on luciferase complementation assay to monitor the protein-
protein interaction between STING and ubiquitin E3 ligase. From the screening, we successfully discovered SB24001 that inhibits STING-E3 ligase interaction.
We conducted surface plasmon resonance (SPR) experiment to find the binding partner of SB24001. We then validated if SB24001 induces cellular
accumulation of STING. Moreover, we checked if SB24001 augments CDN-mediated immunity. Intratumoral injection of CDN augments local immune
response inside tumor microenvironment, which consists of various immune cells including antigen presenting cells (APC). Activated APCs recruit and present
tumor antigen to adaptive immune cells, which will selectively eliminate the cancer cell. Therefore, we examined if blocking STING and ubiquitin E3 ligase
interaction with our discovered small molecule would enhance the immune response.
RESULTS

Figure 1. Development of luciferase complementation assay-based
PPI modulator screening. A. Selection of NanoBiT-tagging pair. Each
plasmid was transfected in HEK293T cell line. NL, N-term tagged LgBiT; NS,
N-term tagged SmBiT; CL, C-term tagged LgBiT; CS, C-term tagged SmBiT.
B. Validation of luminescence signal with negative PPI pair, HaloTag-SmBiT.
Each plasmid was transfected in HEK293T cell line. Represented data
shows relative luminescence signal after luciferase substrate addition. ****,
p < 0.0001. C. Dose-dependent inhibition of STING-E3 ligase interaction
upon SB24001 treatment. D-E. WST assay of SB24001 in Raw264.7 and
A431 cell line. SB24001 was treated in a dose-dependent manner for 24 h.
F. Surface plasmon resonance (SPR) sensorgram. STING C-terminal
domain (AA 138~379) was immobilized on a CM5 chip, and SB24001 was
treated in a dose-dependent manner.

Figure 2. SB24001 increases cellular STING expression level through modulating
ubiquitin-proteasome system. A. Western blot after time-dependent treatment of
SB24001 treatment in A431 cell line. B. Western blot after co-treatment of Cycloheximide
and Actionmycin with SB24001 for 18 h in A431 cell line. Cycloheximide and Actinomycin Figure 3. SB24001 enhances cGAMP-mediated STING immunity. A. Western blot of
D was co-treated with SB24001 for 18 h in A431 cell line. CHX, Cycloheximide; ActD, STING downstream signaling pathway upon co-treatment of SB24001 and cGAMP in
Actinomycin D. C. Western blot after co-treatment of MG132 with SB24001 for 18 h in timecourse manner, in Raw264.7 cell line. B. qPCR of inflammatory cytokines upon co-
A431 cell line. SB24001 was treated in a dose-dependent manner, from 1.25 μM to 20 μM. treatment of SB24001 and cGAMP for 6 h in Raw264.7 cell line.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

SB24001 induced cellular accumulation of • Acta Pharm. Sin. B. 2020; In press. This work was supported by the Creative Research Initiative
STING, which was not affected by transcription Grant, the Bio & Medical Technology Development Program,
or translational regulation. Upon co-treatment • Annu. Rev. Cancer Biol. 2019; 3, 323– and NRF-Fostering Core Leaders of the Future Basic
with proteasome inhibitor, MG132, STING Science Program/Global Ph.D. Fellowship Program through
accumulation by SB24001 was desensitized, 344. the NRF, funded by Ministry of Science & ICT, Korea.
which clearly demonstrates that SB24001
accumulates STING through blocking ubiquitin- • Cancer Res. 2012; 72, 3125–3130.
proteasome pathway. Contact information
• Cancer Res. 2016; 76, 6747–6759.
Upon co-treatment with CDN, our small Corresponding Author: Prof. Seung Bum Park
molecule augmented cytokine expression and • Nat. Rev. Mol. Cell Biol. 2020; 21,
STING downstream signaling. From our findings, Lead contact: sbpark@snu.ac.kr
we anticipate that our strategy with new small 501–521.
molecule could enable efficient and enhanced 1 Author: dhkstkd123@snu.ac.kr
st
CDN cancer immunotherapy.