Abstract 6001: Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer
Authors: Bayraktar Emine , Rodriguez-Aguayo Cristian , Kumar Sudhir , Stur Elaine , Mangala Lingegowda S. , Baylin Stephen , Lopez-Berestein Gabriel , Pradeep Sunila , Sood Anil K. 
Published: April 2023

Introduction: Vascular endothelial growth factor (VEGF) is a key promoter of angiogenesis and disease progression in epithelial ovarian cancer (OC). Bevacizumab, a humanized anti-VEGF monoclonal antibody, is approved for treatment of patients with ovarian and other cancers. Despite the wide use of anti-VEGF therapy for cancer treatment, most patients develop resistant disease. Therefore, new combinations/strategies are needed to reduce adaptive resistance to anti-VEGF antibody (AVA) therapy. Our aim is to determine the potential role of epigenetic changes in AVA-adaptive resistance. We identified and characterized epigenetic changes in BARD1, a tumor suppressor, at the emergence of AVA resistance.

Methods: To define epigenetic changes during AVA therapy induced resistance, we performed series of in vitro (methylation array, RNA sequencing) and in vivo (orthotopic OC (PDX-MDA-OVCA1, immunocompetent-IG10, and immunocompromised-SKOV3) and fibrosarcoma-HT1080, an inherently VEGF resistant model) experiments. The biological effects of DNA methylation agent (5`-Azacytidine; 5`-Aza) with B20 (murine anti-VEGF antibody) treatment were subsequently tested using in vivo models.

Results: To determine whether 5`-Aza can enhance the efficacy of AVA therapy and overcome resistance to AVA in OC, we established AVA-resistant SKOV3 model. In AVA-resistant mice treated with B20 + 5`-Aza, there was a statistically significant reduction in tumor weight (p=0.03). Next, we performed a Human 450k methylation and mRNA arrays from xenografts of SKOV3 tumors. Based on both analyses, we demonstrated that BARD1 was one of the top candidate which was highly expressed (p=0.02) and less methylated (p&lt;0.05) in the combination of B20 and 5`-Aza group compared to B20 alone. Our data also showed that BARD1 expression was restored in the combination therapy. The sequential treatment with 5`-Aza reverted the resistance towards the AVA therapy in SKOV3 model. Furthermore, the combination of B20 and 5`-Aza significantly decreased tumor weight in SKOV3 (p=0.005), IG10 (p&lt;0.05), MDA-OVCA1 (p=0.01), and HT1080 (p&lt;0.05). BARD1 expression levels were substantially increased in combination therapy compared to the B20 alone group (&gt;4-fold, p=0.006). Tumor weight and BARD1 expression were inversely correlated. To determine the biological significance of BARD1, we knockdown BARD1 using Tet-inducible shRNA system. Our data showed a significant reduction in tumor weight in the BARD1 knockdown group in SKOV3 model (p=0.021). RNA-sequencing of BARD1 knockdown showed an enrichment of the angiogenesis pathway. Moreover, BARD1 expression levels were decreased in hypoxic conditions both in vitro and in vivo.

Conclusion: Our findings indicate a previously unrecognized role for BARD1 in mediating resistance to AVA therapy. Use of epigenetic drugs to control the expression of tumor suppressor genes may enhance the efficacy of AVA therapy in OC.

Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Sudhir Kumar, Elaine Stur, Lingegowda S. Mangala, Stephen Baylin, Gabriel Lopez-Berestein, Sunila Pradeep, Anil K. Sood. Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6001.