Selectively targeting CCR2(+) macrophage with BET inhibitor overcomes resistance to anti-VEGF therapy in ovarian cancer (218)

Abstract

<b>Objectives:</b> The diversity of tumor-associated macrophages (TAMs) is known to contribute to adaptive resistance to anti-VEGF therapy in ovarian cancer. <i>CCR2</i>⁺ macrophages are thought to have immunosuppressive effects and contribute to immune evasion through the <i>CCL2-CCR2</i> axis. MSMP (a novel <i>CCR2</i> ligand)/<i>CCR2</i> axis plays an important role in promoting adaptive resistance in ovarian cancer models. Here, we examined the effects of BET inhibitors (BETis) (e.g., ABBV-075, CPI 203, and JQ1) on MSMP/<i>CCR2</i> axis and <i>CCR2</i>⁺ TAMs in ovarian cancer resistant to anti-VEGF therapy. <b>Methods:</b> We used the TIMER and CIBERSORT analytical tools for estimating the associations between macrophages and <i>CCR2</i> or clinical associations in ovarian cancer. We also carried out a series of <i>in vitro</i> (chemotaxis, apoptosis, flow cytometry, and RNA sequencing) and <i>in vivo</i> (adaptive resistant model of SKOV3ip1 ovarian cancer) experiments to determine the effects of BETis on the MSMP/<i>CCR2</i> axis and overcoming resistance to bevacizumab in ovarian cancer. <b>Results:</b> CIBERSORT database revealed that high M1-like macrophages (tumoricidal) were associated with better overall survival of patients with ovarian cancer, while high M2-like macrophages (pro-tumoral) were associated with poor overall survival (p<0.01). TIMER analysis further revealed that <i>CCR2</i> expression was significantly associated with monocyte and macrophage infiltration in ovarian cancer (p<0.01). To search druggable transcriptional factors of MSMP and <i>CCR2</i>, we used the transcriptomic database and western blot analyses and identified that BETi decreased MSMP expression in cancer cells and <i>CCR2</i> in monocytes/macrophages, likely in a BRD4-dependent manner. <i>In vitro</i> chemotaxis assays demonstrated that rMSMP increased monocyte migration (p< 0.001), and BETi treatment decreased migration of monocytes. Notably, treatment with the ABBV-075 selectively induced greater apoptosis in M2-like macrophages than in M1-like macrophages and ovarian cancer cells (p <0.01). M1-like macrophages (CD68⁺CD80⁺) were significantly increased after treatment with low dose ABBV-075 (p <0.05), while CD68⁺<i>CCR2</i>⁺ macrophages were significantly decreased. Furthermore, using RNA seq analysis, we found that ABBV-075 selectively downregulated chemokines, such as <i>CCL2/CCR2</i>, <i>CCR1</i>, and <i>CCR5</i> in macrophages and matrix metalloproteinases (MMP19) and cyclin-dependent kinases (<i>CDK15</i>) in cancer cells. Finally, utilizing the SKOV3ip1 adaptive resistant ovarian cancer model, the combination of ABBV-075 and bevacizumab resulted in a 95% lower tumor weight compared to control (p<0.05). The combination also resulted in a 62.2% and 78.3% decrease in F4/80+ and ARG+ macrophages, respectively, compared to control (p<0.01). <b>Conclusions:</b> Our findings indicate a previously unrecognized role for BET inhibitors in selectively targeting <i>CCR2</i>⁺ macrophages and repolarizing the macrophages into M1-like phenotype. These findings may have implications for new combinations to overcome resistance to bevacizumab in ovarian cancer.