Abstract 6173: AKT1 prevents aberrant R-loops accumulation via regulating DHX9 and suppresses transcription-replication collision in PARP inhibitor-resistant ovarian cancer

Abstract

Abstract Background: Acquired resistance to PARP inhibitors (PARPis) is a pressing problem in high-grade serous ovarian cancer (HGSC), highlighting the development of novel therapeutic options. To address this unmet need, we conducted high-throughput drug combination screens in PARPi-resistant HGSC cells using ATR inhibitor (ATRi), given that ATR/CHK1 signaling activation is one of the main mechanisms of PARPi resistance. In the screens, PI3K/AKT pathway inhibitors were synergistic with ATRi. Mechanistically, ATR signaling plays a major role in R-loop dynamics and replication stress (RS) response pathways, but the role of AKT is unclear. Here, we aim to investigate AKT’s role in R-loop-mediated RS in PARPi-resistant HGSC cells. We also hypothesize that AKT inhibitor (AKTi) would augment ATRi-induced R-loop formation by reducing the expression of helicases. Methods: We used PARPi-resistant variants and parental PEO1 as a negative control. ATRi (ceralasertib) and AKTi (capivasertib) were used for further mechanistic studies. Induction of RS, DNA double-strand break, and R-loop were evaluated using immunofluorescence staining for markers pRPA, γH2AX, and S9.6, respectively. We measured multiple nuclear helicases and in situ DHX9-R-loop interaction by western blot and proximity ligation assay. DHX9-AKT1 interaction was confirmed by co-immunoprecipitation. All data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P < 0.05 was considered statistically significant. Results: The ATRi and AKTi combination induced greater RS, as evidenced by a 1.7-12.0-fold increase in pRPA+/γH2AX+ population relative to each drug alone. Adding AKTi to ATRi significantly increased R-loops compared to monotherapy alone (signal intensity 14-19 x 105 vs. 9.9-14.6 x 105, P < 0.001), suggesting that AKT also contributes to R-loop dissolution. Moreover, the combination showed a higher percentage of cells with γH2AX and R-loops relative to monotherapy (54.7-75.4% vs. 33.3-49.0%, P < 0.001) in the presence of pan-caspase inhibitor, indicating the increased DNA damage is unlikely caused by increased apoptosis but by R-loop accumulation. Also, AKTi addition to ATRi significantly reduced the nuclear DHX9 among other helicases. Moreover, we found that AKT1 directly binds with DHX9 via its kinase domain and couples with DHX9 to resolve R-loops. Conclusion: Our data suggest ATRi and AKTi combination could be a new therapeutic combination in HGSC with PARPi resistance. Mechanistically, our data reveal a previously unknown direct role of AKT1 and its interaction with DHX9 in R-loop resolution besides ATR. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Jayakumar R. Nair, Chi-Ting Shih, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. AKT1 prevents aberrant R-loops accumulation via regulating DHX9 and suppresses transcription-replication collision in PARP inhibitor-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6173.