Abstract 6273: TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination
Authors: Benjamin Chandler, Leah Moubadder, Cassandra Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea Pesch, Anna Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar Niknafs, Sofia Merajver, Dafydd Thomas, Powel Brown, Theodore Lawrence, Shyam Nyati, Lori Pierce, Arul Chinnaiyan, Corey Speers
Journal: Cancer Research (2020)

Abstract Background: Basal-like breast cancer (BC) has the highest rates of local recurrence despite the use of radiation therapy. Therefore, approaches for radiosensitization are critically needed for patients with this subtype of BC. Methods: Four independent datasets were used to correlate gene expression with local recurrence (LR) and Kaplan-Meier analysis validated the impact of TTK expression on LR. The METABRIC dataset was used to determine TTK expression in BC subtypes. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mouse models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using Rad51 foci formation assays. Rescue experiments were performed using wild-type (WT) and kinase-dead (KD) TTK plasmids in combination with siRNA targeting the UTR region of TTK. Results: Ten genes were found to significantly correlate with early LR (≤3 years) after surgery and radiation across 4 independent datasets (N=896 pts), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in multiple cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p&amp;lt;0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like BC. Using inducible shRNA, the combination of TTK knockdown and RT increases radiosensitivity in multiple basal-like BC cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in multiple cell lines (rER 1.10-2.27). In vivo, TTKi, using shRNA or B909, in combination with RT led to delayed tumor growth and a significant increase in time to tumor tripling (Placebo: 9 days vs. B909+RT: undefined [&amp;gt;35 days], p&amp;lt;0.0001) in both cell line and PDX models. Increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. The efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, Rad51 foci formation was reduced by TTKi after RT compared to RT alone. Reintroduction of WT TTK, after knockdown of endogenous TTK, rescued radioresistance and HR efficiency, however, reintroduction of kinase-dead (KD) TTK was unable to do so in multiple cell lines. WT TTK also rescued Rad51 foci formation after knockdown of endogenous TTK while KD TTK did not. Conclusion: These data support TTKi as a radiosensitizing strategy for clinical development in basal-like BC patients and that radiosensitization is mediated, at least in part, through impaired HR repair. Citation Format: Benjamin Chandler, Leah Moubadder, Cassandra Ritter, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Amanda Zhang, Andrea Pesch, Anna Michmerhuizen, Nicole Hirsh, Marlie Androsiglio, Tanner Ward, Eric Olsen, Yashar Niknafs, Sofia Merajver, Dafydd Thomas, Powel Brown, Theodore Lawrence, Shyam Nyati, Lori Pierce, Arul Chinnaiyan, Corey Speers. TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6273.