(291) Deciphering the Mechanistic Interaction between JNK and TAp63α to Elucidate the Mechanisms of Chemotherapy-Induced Primary Ovarian Insufficiency

Connie Mitra^1,2; Wenlong Zhao^1,2; Jiyang Zhang^1,2; Qiang Zhang³; So-Youn Kim⁴, Shuo Xiao^1,2

Abstract Text:

The advances of cancer therapy greatly enhance the awareness of the side effects of anti-cancer agents. One major concern in young female cancer patients is off-target effect in the ovary, resulting in primary ovarian insufficiency (POI) and associated early menopause and infertility. Primordial follicles establish the ovarian reserve and are highly susceptible to DNA-damaging chemotherapeutic drugs, including doxorubicin (DOXO), cisplatin (CDDP) and cyclophosphamide (CPA). Prior research revealed that hyperphosphorylation of TAp63α, a member of p53 tumor suppressor family transcription factors highly expressed in oocytes of primordial follicles, is a key step contributing to chemotherapy-induced oocyte apoptosis and POI. Recently, our lab discovered that the activation of c-Jun N-terminal kinase (JNK) is required for chemotherapy-induced apoptosis of primordial follicle oocytes, and pharmacological inhibition of JNK prevents chemotherapy-induced hyperphosphorylation of TAp63α in oocytes from primordial follicles and mitigates POI. In the current study, we tested the hypothesis that JNK critically contributes to the function of TAp63a in oocyte death induced by DNA-damaging chemotherapeutic drugs. We expressed Flag-tagged JNK1/2/3 and/or Myc-tagged TAp63α in H1299 lung carcinoma cells followed by DOXO treatment for 6 hours. The results of co-immunoprecipitation (Co-IP) revealed that Myc-TAp63α was detected by Western blotting only in the group with co-transfection of Flag-JNK and Myc-TAp63α, but not in the control group with Myc-TAp63α transfection alone, indicating the interaction between JNK and TAp63α. Because JNK has been shown to bind to the p53 DNA-Binding Domain (DBD) via basic and aromatic amino acids, we next used the NCBI-BLASTp program to identify the regions in the DBD of TAp63α aligned with the DBD of p53 with the similar basic and aromatic amino acids. UCSF ChimeraX v1.9 software was further applied to investigate whether the DBD of TAp63α contains specific binding sites for JNK regulating its transcription factor activity. The computational analysis identified five potential binding sites within the DBD of TAp63α, including amino acid regions of 165-183, 188-207, 230-243, 251-266 and 318-362. Ongoing studies include the generation of site-directed mutagenesis of the candidate binding sites of TAp63α to determine whether the mutation of binding sites causes the loss of binding specificity of TAp63α for JNK by Co-IP assay. In summary, our study indicates JNK may critically contribute to the function of TAp63α in oocyte death of primordial follicles in response to the treatment of DNA-damaging anti-cancer agents, which provides insights into the development of fertoprotectant for young female cancer patients. (This work is supported by NIH/NICHD R01HD115810)