PTEN deficiency confers sensitivity to ATR inhibitor–based treatment in high-grade serous ovarian cancer
Jie Hao, Bose Kochupurakkal, Timothy B. Branigan, Ozge Sezin Somuncu, Renyan Liu, Heta Jadhav, Alexandre André B. A. da Costa, Yuqing Jiao, Jenny Z. Yu, David B. Martignetti, Golbahar Sadatrezaei, Sirisha Mukkavalli, Prafulla C. Gokhale, Su-Chun Cheng, Steven J. Skates, Dimitrios Nasioudis, Panagiotis A. Konstantinopoulos, Joyce F. Liu, Stephanie Gaillard, Robert L. Giuntoli II, Lainie Martin, Janos Tanyi, Nawar Latif, Ian Heller, Fiona Simpkins, Kalindi Parmar, Alan D. D’Andrea, Geoffrey I. Shapiro
Jie Hao, Bose Kochupurakkal, Timothy B. Branigan, Ozge Sezin Somuncu, Renyan Liu, Heta Jadhav, Alexandre André B. A. da Costa, Yuqing Jiao, Jenny Z. Yu, David B. Martignetti, Golbahar Sadatrezaei, Sirisha Mukkavalli, Prafulla C. Gokhale, Su-Chun Cheng, Steven J. Skates, Dimitrios Nasioudis, Panagiotis A. Konstantinopoulos, Joyce F. Liu, Stephanie Gaillard, Robert L. Giuntoli II, Lainie Martin, Janos Tanyi, Nawar Latif, Ian Heller, Fiona Simpkins, Kalindi Parmar, Alan D. D’Andrea, Geoffrey I. Shapiro
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Research Article Cell biology Clinical Research Oncology

PTEN deficiency confers sensitivity to ATR inhibitor–based treatment in high-grade serous ovarian cancer

Abstract

Ataxia telangiectasia and Rad3-related (ATR) inhibition is under evaluation for the treatment of high-grade serous ovarian cancer (HGSOC) to reverse acquired resistance to poly (ADP-ribose) polymerase (PARP) inhibition and to exacerbate chemotherapy-induced replicative stress. Here, we define PTEN deficiency as a predictive biomarker for the response to ATR inhibition, as monotherapy and in combination with PARP inhibition or gemcitabine. In response to ATR inhibition and compared with PTEN-proficient cells, PTEN-deficient cells are prone to (a) uncoupling of DNA polymerase and helicase activities, leading to excessive ssDNA and replication stress; (b) cytoplasmic sequestration of checkpoint kinase 1 (CHK1), compromising cell-cycle checkpoint control with reduced compensatory effects by ataxia-telangiectasia mutated (ATM) and DNA–dependent proteinase K (DNA-PK), leading to mitotic catastrophe; and (c) reduced DNA repair protein RAD51 homolog 1 (RAD51) recruitment, exacerbating replication fork instability, also leading to lethality. Retrospective analyses revealed that patients with HGSOC who expressed low PTEN levels experienced greater clinical benefit on ATR inhibitor–based trials than did those with high PTEN levels. These results justify prospective trials evaluating ATR inhibition as a therapeutic strategy for PTEN-deficient tumors.

Authors

Jie Hao, Bose Kochupurakkal, Timothy B. Branigan, Ozge Sezin Somuncu, Renyan Liu, Heta Jadhav, Alexandre André B. A. da Costa, Yuqing Jiao, Jenny Z. Yu, David B. Martignetti, Golbahar Sadatrezaei, Sirisha Mukkavalli, Prafulla C. Gokhale, Su-Chun Cheng, Steven J. Skates, Dimitrios Nasioudis, Panagiotis A. Konstantinopoulos, Joyce F. Liu, Stephanie Gaillard, Robert L. Giuntoli II, Lainie Martin, Janos Tanyi, Nawar Latif, Ian Heller, Fiona Simpkins, Kalindi Parmar, Alan D. D’Andrea, Geoffrey I. Shapiro

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Figure 2

Loss of PTEN leads to uncoupling of helicase and polymerase activities at the replication fork after ATR inhibition, resulting in the accumulation of ssDNA and replication stress.

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Loss of PTEN leads to uncoupling of helicase and polymerase activities a...
(A) Western blot analysis of chromatin-bound fractions from WT and PTEN-KO cells after ATR inhibitor treatment, showing the expression of pMCM2 and Ub-PCNA. (B and C) Cells were labeled with BrdU (10 μM) for 48 hours, followed by treatment with DMSO or tuvusertib (1 μM) for 16 hours. Cells were then pre-extracted and subjected to native BrdU immunostaining to detect ssDNA. Samples were costained separately with antibodies against either pMCM2 S41 or pMCM2 S108. Original magnification, ×63. Scale bars: 10 μm. Fluorescence intensities were quantified using CellProfiler. (D) Immunofluorescence staining of RPA and γH2AX in OVCAR8 PTEN WT and PTEN-KO cells treated with DMSO or tuvusertib (1 μM) for 24 hours, following pre-extraction. Increased RS and DNA damage were observed in PTEN-deficient cells upon ATR inhibition. Scale bars: 100 μm. (E and F) Quantification of RPA and γH2AX fluorescence intensities using CellProfiler. Each dot represents an individual cell from 3 independent experiments, with at least 200 cells analyzed per experiment. Symbols represent the mean of each biological replicate. (G) Western blot analysis of lysates from OVCAR8 and OVCAR8-PTEN-KO cells treated with tuvusertib (1 μM) for 24 hours. ATR inhibition induces increased replication stress and DNA damage in PTEN-deficient cells. Data are presented as the mean ± SD (n = 3 independent experiments per group). Statistical significance was assessed using biological replicate means by 1-way ANOVA with Tukey’s multiple-comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).