Inhibition of oogenic JNK prevents chemotherapy-induced premature ovarian insufficiency and infertility in mice

Abstract Authors: Wenlong Zhao¹, Jiyang Zhang¹, Yingzheng Wang², Yamei Zuo¹, Ege Bostanci¹, Emily Au¹, Qiang Zhang³, So-Youn Kim⁴, Shuo Xiao¹
1. Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Environmental and Occupational Health Science Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA
2. Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, 14853, USA
3. Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA, 30322, USA
4. Olson Center for Women's Health, Department of Obstetrics and Gynecology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA

Abstract Text:

The remarkable advances in cancer survival rates greatly increase the awareness of the side effects of cancer therapy and long-term life quality after cancer treatment. A major concern in child, adolescent, and young adult female cancer survivors is primary ovarian insufficiency (POI) caused by gonadotoxic anti-cancer agents and the related early menopause, infertility, and endocrine disorders. Chemotherapy often activates the DNA damage response (DDR) signaling pathway in oocytes of primordial follicles. The hyperphosphorylation of TAp63a, a member of the p53 family of transcription factor, triggers oocyte apoptosis, leading to primordial follicle atresia and POI. However, the precise molecular mechanisms underlying this complex DDR-involved process remain elusive, and there is a lack of promising druggable targets to develop fertoprotectants that preserve ovarian reserve and fertility without compromising the efficacy of chemotherapy. In this study, we employed multiple research models and approaches to investigate the molecular mechanisms of chemotherapy-induced POI. Five-day-old CD-1 female mice received a single intraperitoneal injection of vehicle or 10 mg/kg doxorubicin (DOXO), a widely used chemotherapeutic drug. Granulosa cells and oocytes from primordial follicles were isolated 6 hours post-injection for single-cell SMART-seq2 RNA sequencing (RNA-seq). Bioinformatic analysis revealed distinct transcriptomic changes in oocytes and granulosa cells following DOXO treatment, including several differential expressed genes (DEGs) in oocytes related to the activation of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family. The results of RT-qPCR and immunostaining showed that all three JNK isoforms (JNK1, JNK2, and JNK3) were present in oocytes of primordial follicles, and oogenic JNK was phosphorylated following treatment of DOXO, as well as cisplatin (CDDP) and cyclophosphamide (CPA), two other gonadotoxic chemotherapeutic agents. Moreover, administering a selective JNK inhibitor two hours prior to chemo-drug treatment prevented TAp63a hyperphosphorylation, primordial follicle oocyte apoptosis, and POI. A follow-up study further demonstrated that pharmacological inhibition of JNK preserved mouse ovarian reserve, reproductive cycles, and fertility long-term post-treatment of all three gonadotoxic anti-cancer agents described above. To further validate these findings, we generated genetically modified mouse models with oocyte-specific deletion of one or more JNK isoforms. The results showed that mice lacking all three isoforms were resistant to chemotherapy-induced primordial follicle oocyte apoptosis and POI. Mechanistically, both pharmacological inhibition and genetic deletion of JNK prevented TAp63α hyperphosphorylation and suppressed the transcriptional induction of downstream apoptosis effector genes such as Puma and Noxa. Co-immunoprecipitation (Co-IP) using H1299 lung carcinoma cells confirmed the interaction between JNK and TAp63α and potential binding sites on TAp63α. A breast cancer-bearing mouse model was further established to verify that pharmacological inhibition of JNK protected mouse ovarian reserve without affecting the anti-cancer efficacy of chemotherapy. In conclusion, our study establishes oogenic JNK as a key regulator of DDR-TAp63α driven apoptosis in oocytes of primordial follicles following chemotherapy, suggesting JNK inhibition is a promising ovarian protective strategy for preserving ovarian reserve, fertility, and ovarian endocrine functions in young female cancer survivors undergoing gonadotoxic chemotherapy. (This work is supported by NIH/NICHD R01HD115810)