Abstract 7549: Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1
Authors: Bayraktar Emine , Rodriguez-Aguayo Cristian , Stur Elaine , Kumar Sudhir , Mangala Lingegowda S. , Bayram Nazende Nur , Hanjra Pahul , Corvigno Sara , Baylin Stephen , Lopez-Berestein Gabriel , Sood Anil K. 
Published: March 2024

DNA methylation inhibitors such as 5-azacytidine (Aza) and 5-aza-2′-deoxycytidine have been extensively used in patients with hematologic disorders and other neoplasms since their approval by the FDA. However, global DNA demethylating agents can result in intolerable toxicities; therefore, targeted epigenetic modulation is needed. Here, we searched for epigenetic changes with adaptive resistance to anti-VEGF antibody (AVA) in the TME and a novel strategy for targeted demethylation.

Methods: We examined methylation and transcriptional changes in orthotopic ovarian cancer models with emergence of AVA resistance. We developed a targeted demethylation strategy for BARD1 to enhance the effectiveness of AVA. We used a novel dCas9-CRISPR-based system for targeted BARD1 CpG demethylation (derived by fusing the deactivated Cas9 [dCas9] with the TET1 catalytic domain) delivered in vivo using a nanoliposomal carrier.

Results: We identified BARD1 methylation and reduced expression as lead candidate with AVA resistance; multiple CpG island sites in the BARD1 gene from adaptive AVA-resistant tumors were methylated. Next, we investigated the biological effects of global and targeted DNA methylation. Azacytidine along with AVA therapy was tested in a series of models (fibrosarcoma, PDX and orthotopic ovarian cancer models) and resulted in reduced tumor burden compared with the control and monotherapy groups (p&lt;0.01). Next, to determine whether targeted demethylated BARD1 would have antitumor effects in vivo, we used an orthotopic SKOV3ip1luc ovarian cancer mouse model. Mice were treated with DOPC-dCas9-TET1-sgcontrol- (control) and DOPC-dCas9-TET1-sgBARD1 (sgBARD1). Tumor weight in the sgBARD1-3 group was significantly (p&lt;0.01) reduced compared with the control group with no significant change in mouse body weight. We found that BARD1 expression was significantly increased in sgBARD1-treated group compared with control (p&lt;0.001). Next, to determine whether demethylated BARD1 would combine well with AVA therapy in the SKOV3ip1 model, we combined sgBARD1-3 with bevacizumab (bev); sgBARD1-3 alone led to a significant decrease in tumor burden compared with control groups. Moreover, tumor burden was significantly reduced in the sgBARD1-3 and bevacizumab combination group compared with the sgcontrol group and bev alone group. BARD1 expression was significantly increased in sgBARD1-3-treated and in combination tumor samples compared with controls. To check for potential off-target effects, we tested the efficacy of another sgBARD1 and found that tumor burden in the sgBARD1-1 group was significantly reduced compared with control group.

Conclusion: Our novel targeted approach results in robust antitumor effects when combined with AVA therapy in multiple cancer models. Collectively, our findings provide a novel approach for gene targeted demethylation and could have implications for epigenetic therapies.

Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Elaine Stur, Sudhir Kumar, Lingegowda S. Mangala, Nazende Nur Bayram, Pahul Hanjra, Sara Corvigno, Stephen Baylin, Gabriel Lopez-Berestein, Anil K. Sood. Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7549.