Chek2 Signaling Is the Key Regulator of Oocyte Survival After Chemotherapy

Abstract

Radiation and chemotherapy can damage the primordial follicle reserve in female cancer patients leading to ovarian failure and infertility. Preservation of ovarian function requires treatment strategies that prevent loss of immature oocytes in primordial follicles during cancer therapy. Checkpoint kinase 2 (CHEK2) inhibition prevents loss of primordial oocytes caused by DNA damage and thus is a promising target for ovoprotective treatment against genotoxic agents. To determine which cancer treatments could benefit from ovoprotective activity of CHEK2 inhibition we investigated oocyte survival in Chek2-/- mice exposed to different chemotherapy drugs. Here, we show that loss of CHEK2 function prevents elimination of primordial oocytes damaged by cisplatin, cyclophosphamide, mafosfamide, doxorubicin, and etoposide, suggesting it could be used to reduce ovarian damage caused by wide range of drugs. Using genetic knockouts we reveal a critical role for TRP53 in oocyte response to chemotherapy drugs and show that both targets of CHEK2, TAp63 and TRP53, are activated by cisplatin and cyclophosphamide. Furthermore, we show that checkpoint kinase inhibitor and radiation- and chemotherapy sensitizer AZD7762 reduces oocyte elimination after radiation and chemotherapy treatments, despite its cytotoxic effect on ovarian somatic cells. Altogether, these findings demonstrate the role for CHEK2 as the master regulator of primordial oocyte survival or death and credential its targeting for ovoprotective treatments.

Single-cell and bulk transcriptional profiling of mouse ovaries reveals novel genes and pathways associated with DNA damage response in oocytes

“…Phosphorylated p53 (S15) was detected in oocytes, pre-granulosa cells, and other cell types in irradiated ovaries ( Figure 1F ). TAp63 is a direct target of CHEK2 phosphorylation and the key proapoptotic factor in oocytes (11,12,20). Therefore, activation of p53 in oocytes after IR suggests that p53 may still play a role in the DDR and potentially regulates other cellular responses in oocytes.…”

“…Interestingly, novel ovary-RRGs such as Cbr2, Ankrd65, Fermt1 and Fermt3 , were all confirmed to be regulated by either p53 or p63 ( Figure 4A, B ). As the ReMap dataset does not include ovarian tissue, we performed RT-qPCR analysis on irradiated ovaries from wildtype, Chek2-/-, Trp63A/A (phosphomutant S621>A (12)), and double mutants lacking both active TAp63 and p53 ( Trp63A/A; Trp53-/- ) to test whether these novel ovary-RRGs are activated by p53 or TAp63. The upregulation of Cdkn1a , a known p53-specific target involved in cell cycle arrest, was observed in wildtype and TAp63 phosphomutant ovaries, but not in ovaries lacking active p53 or CHEK2.…”

“…Although p53 is activated by DNA damage in primordial oocytes, and p53-regulated genes are upregulated in response to IR in oocytes, much less is known about the role of p53 in oocyte DDR compared to TAp63. Our previous work has shown that alkylating agents induce more DNA damage in oocytes, which leads to activation of p53-dependent apoptosis in oocytes, indicating that specialized mechanisms may regulate p53 activity in oocytes (12). A better understanding of the DDR responses driven by p53-that may be beneficial to oocyte's survival but are made ineffective by excessive (or precautious) TAp63-driven apoptosis-will be critical for elucidating the mechanisms of ovarian aging, and ovariotoxic effects of therapeutic or environmental genotoxic exposures.…”

“…For ovaries, it has been shown that IR-and chemotherapy-induced DNA damage causes accelerated depletion of the ovarian follicle reserve (9,10). Multiple studies have highlighted the critical role of checkpoint kinase 2 (CHEK2) in the establishment and maintenance of the ovarian follicle reserve (11)(12)(13)(14)(15). Moreover, CHEK2 loss-of-function variants in humans correlate with females having a larger ovarian follicle reserve and later onset of menopause (16,17).…”

“…In contrast to somatic cells, which rely mainly on p53 activity, oocytes express a related protein TRP63 (p63) (19,20). TA isoform of p63 (henceforth TAp63) expressed exclusively in oocytes in the ovary has been shown to trigger oocyte apoptosis after activation by CHEK2 in response to DNA damage (11,12,21). Animal studies show that inactivation of CHEK2 or TAp63 but not p53, prevents apoptotic elimination of oocytes exposed to low doses of IR (0.2 - 0.5 Gy) (11,19,20).…”