Targeting PUMA as a Novel Fertility Preservation Strategy to Prevent Chemotherapy-Induced Ovarian Damage

Lauren R. Alesi^1,2; Jessica M Stringer¹; Roseanne Rosario3; Jasveena Kaur^4,5; Thierry Jardé^1,2,5; Richard A Anderson³; Amy L Winship^1,2; and Karla J Hutt¹.

1. Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University (Clayton, VIC, Australia)


2. Cancer Program, Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University (Clayton, VIC, Australia)


3. MRC Centre for Reproductive Health, Queens Medical Research Institute, University of Edinburgh (Edinburgh, UK)


4. Department of Surgery, Cabrini Health, Cabrini Monash University (Malvern, VIC, Australia)


5. Monash Biomedicine Discovery Institute Organoid Program, Monash University (Clayton, VIC, Australia)

Cancer therapies inflict irreversible ovarian damage and deplete the finite reserve of primordial follicles, often leading to infertility and premature menopause in female survivors. Unfortunately, current fertility preservation options have significant drawbacks, with no strategies available to protect both fertility and long-term endocrine function. In landmark studies, our team revealed chemotherapy directly damages oocyte DNA, inducing apoptosis. The apoptotic protein PUMA is chiefly responsible for triggering this, as genetic loss of Puma confers striking oocyte protection and preserves fertility in mice post-chemotherapy. Excitingly, a small-molecule PUMA inhibitor (PUMAi) is now available, making targeting PUMA for fertility preservation a real therapeutic possibility. This research investigated whether blocking PUMA prevents chemotherapy-induced primordial follicle depletion; is safe, and does not compromise chemotherapy efficacy; and protects oocyte quality and offspring health long-term.

To establish whether blocking PUMA prevents oocyte apoptosis during chemotherapy exposure, human ovarian cortical pieces (n=5 patients) were cultured with the commonly-used and highly gonadotoxic cyclophosphamide derivative 4-HC (2µM) ± PUMAi (200µM). Whilst 4-HC alone decreased primordial follicles (p < 0.0001); remarkably, PUMAi treatment restored this significantly (p < 0.05). Next, mice (n=6/group) received a fertility-damaging cyclophosphamide dose (150mg/kg) ± PUMAi (10mg/kg) 2h before/20h after; a regimen that prevents chemotherapy-induced gut toxicity. Cyclophosphamide alone reduced primordial follicles by 75% (p < 0.01); however, PUMAi co-treatment rescued 25% of follicles (p < 0.05). This is extremely promising, as protecting just 12% of follicles in Puma^-/- mice sustains long-term fertility, without compromising offspring health.

To ensure that blocking PUMA-mediated apoptosis does not inadvertently increase tumour cell survival, human triple-negative breast cancer cells (MDA-MB-231) were incubated with increasing concentrations of PUMAi (0-512µM) for 30 minutes, followed by an IC₅₀ concentration of 4-HC (5µM). Cell viability was assessed 48h later via an MTT assay. As anticipated, 4-HC alone significantly reduced MDA-MB-231 viability to 54% (p < 0.0001). Encouragingly however, co-treatment with PUMAi did not significantly increase cancer cell survival, with viability remaining at approximately 50% across all concentrations assayed. Next, patient-derived triple negative breast cancer organoids (HBC14) were exposed to multiple increasing concentrations of PUMAi (0-100µM) for 30 minutes, followed by an IC₅₀ concentration of 4-HC (5µM) on days 1, 3 and 5. On day 7, 48h after the final 4-HC treatment, organoid viability was assessed via a Presto Blue assay. As expected, HBC14 organoid viability was significantly reduced to 56% with 4-HC alone (p < 0.0001). However, similarly, co-treatment with PUMAi did not significantly alter organoid survival, with viability remaining at approximately 50% across all conditions, indicating that blocking PUMA does not compromise chemotherapy efficacy in vitro.

To examine if PUMAi preserves fertility and offspring health, mice (n=10/group) received cyclophosphamide ± PUMAi (as above), and were mated with unexposed males for 3 litters. Though average litter sizes were similar, cyclophosphamide alone dramatically impacted offspring health, with only 28% of pups surviving past PN5 (p < 0.001) versus 69% from control-treated dams. PUMAi significantly improved survival to control levels (54%; p< 0.05), suggesting oocyte quality and offspring health are effectively preserved.

Together, these data demonstrate PUMA blockade is a promising oncological fertility preservation avenue. Further studies are underway to investigate the efficacy of PUMAi in preventing ovarian damage from other chemotherapies such as cisplatin and doxorubicin; to ensure PUMAi does not impact chemotherapy efficacy in vivo using tumour xenograft mouse models; and to determine whether multi-organ protection is conferred, beyond the reproductive tract.