TTK inhibitor OSU13 promotes immunotherapy responses by activating tumor STING
Vijaya Bharti, Amrendra Kumar, Yinchong Wang, Nikhil Roychowdhury, Daniel de Lima Bellan, Beimnet B. Kassaye, Reese Watkins, Marina Capece, Catherine G. Chung, Gerard Hilinski, Anna E. Vilgelm
Vijaya Bharti, Amrendra Kumar, Yinchong Wang, Nikhil Roychowdhury, Daniel de Lima Bellan, Beimnet B. Kassaye, Reese Watkins, Marina Capece, Catherine G. Chung, Gerard Hilinski, Anna E. Vilgelm
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Research Article Oncology

TTK inhibitor OSU13 promotes immunotherapy responses by activating tumor STING

Abstract

TTK spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the antitumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent antitumor activity of OSU13 in melanoma, colon and breast cancer cells, organoids derived from patients with melanoma, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS/STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a low-toxicity dose of OSU13 with anti–PD-1 checkpoint blockade resulted in prominent STING- and CD8+ T cell–dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.

Authors

Vijaya Bharti, Amrendra Kumar, Yinchong Wang, Nikhil Roychowdhury, Daniel de Lima Bellan, Beimnet B. Kassaye, Reese Watkins, Marina Capece, Catherine G. Chung, Gerard Hilinski, Anna E. Vilgelm

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Figure 1

OSU13 inhibits tumor cells by inducing apoptosis, cell cycle arrest, and senescence.

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OSU13 inhibits tumor cells by inducing apoptosis, cell cycle arrest, and...
(A) Representative images of colon (HCT116), melanoma (A375), and breast (MCF7) tumor cells treated with vehicle or 1 μM OSU13 for 5 days and stained with crystal violet. Scale bar: 150 μm. (B) Cell numbers in indicated cultured cells treated with 0.3 or 1 μM OSU13 or vehicle. Cells were imaged at 1, 3, 5, and 7 days after treatment. N = 9 microscopic fields from 3 wells in a 6-well plate per condition. Statistics were performed using 2-way ANOVA with Dunnett’s post test. (C) Representative flow cytometry plots of MCF7 cells treated with vehicle control or 0.3 μM OSU13 for 5 days and stained with fluorescent antibodies recognizing cleaved PARP, BRDU, and γH2AX. BRDU was added to the cell culture media 4 hours before cell collection at the concentration of 25 μg/mL. (D) Quantified data from flow cytometry analysis shown in C in indicated cancer cell lines. N = 3 replicates per condition. Statistical analyses using multiple unpaired t tests. (E) Representative results of cell cycle phase gating in A375 cells analyzed as described in C. (F) Quantified cell cycle distribution data from analysis shown in E across 3 indicated cancer cell lines. N = 3 biological replicates per condition. (G) Western blot analysis of cell cycle–related proteins in A375, HCT-116, and MCF7 cells treated with 0.3 μM OSU13 or vehicle for 5 days. (H) Representative microphotographs of MCF7 cells treated for 3 days with 0.3 or 1 μM OSU13 or vehicle and stained for the activity of senescence-associated β-galactosidase (SA-β-Gal). Scale bar: 150 μm. (I) Percentages of SA-β-Gal+ cells quantified from the experiment shown in H. Three indicated cancer cell lines were used. N = 12–15 microscopic fields per condition (across 3 wells in a 6-well plate). Statistics using 1-way ANOVA with Dunnett’s multiple comparisons tests. **P < 0.01; ***P < 0.001; ****P < 0.0001.