Abstract 4051: Optimizing breast cancer therapy by modulating oxygen consumption

Abstract

Abstract Immune checkpoint blockade (ICB) has achieved great clinical responses in multiple cancers. However, only a subset of patients with triple negative breast cancer (TNBC), a highly hypoxic tumor, respond to ICB. In TNBC, the immune checkpoints PD-1/PD-L1 and adenosine receptor (A2aR) are often activated to escape anti-tumor responses. These two immune checkpoints are sensitive to hypoxia. We propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will improve the efficacy of PD-1/PD-L1 and A2aR blockade. We generated two mouse breast cancer cell lines (4T1 and E0771) expressing green fluorescent protein (GFP) fused to Firefly luciferase under the control of the hypoxia reporter element (HRE). This construct, which we called HRE/TGL, serves as a hypoxia reporter, with GFP expression and luciferase activity increasing in hypoxic conditions. To induce hypoxia in vitro, we used two approaches: 1) we incubated cells in a hypoxia chamber (3% oxygen), and 2) treated cells with cobalt chloride, which chemically mimics hypoxia by inducing the expression of the hypoxia master regulator HIF1α. Both 4T1-HRE/TGL and E0771-HRE/TGL cells exhibited increased GFP expression and luciferase activity within hypoxia conditions. We orthotopically implanted these cells into mice. We measured luciferase activity in vivo using the IVIS spectrum imaging system overtime after tumor implantation. We observed high luciferase activity in 4T1-HRE/TGL tumors. Next, we examined the effects of DFP, Phen and Met on cell hypoxic responses by performing immunoblotting against HIF1α and phosphorylated AMPK (p-AMPK) on 4T1 and E0771 cells. We observed that, unlike Phen or Met, DFP increased HIF1α and p-AMPK expression. Binding of HIF1 proteins to HREs is known to activate the expression of the HRE promoter. To confirm our results, we treated 4T1-HRE/TGL and E0771-HRE/TGL cells with low (10μM) or high (100μM) doses of the drugs in vitro. We observed that, while DFP increased GFP expression and luciferase activity, Phen had no consistent measurable effect. We observed similar results in vivo. Finally, we examined the effect of DFP, Phen and Met either alone or in combination with anti-PD-1 on tumor progression and survival in mice bearing 4T1 or E0771 tumors. Contrary to what expected, we did not observe better tumor control and survival after treatment with these drugs. In addition, DFP, Phen and Met failed to improve the anti-tumor effect of anti-PD-1 treatment in both TNBC models. Future experiments will focus on charactering the adenosine pathway in vivo in 4T1 and E0771 tumors and examine how drugs targeting A2aR and oxygen consumption (DFP, Phen and Met) influence the activation of immune cells within the tumor microenvironment and potentially combinate these two strategies to improve their anti-tumor efficacy. Citation Format: Anais Assouvie, Sadna Budhu, Mayuresh Mane, Mamadou Bah, Juan Zurita, Inna Serganova, Soe Min, Jason Koutcher, Vladimir Ponomarev, Jedd Wolchok, Taha Merghoub. Optimizing breast cancer therapy by modulating oxygen consumption [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 4051.