14 Evaluation of the Effects of the HIF-2α Inhibitor Belzutifan on T-cell–Mediated Cytotoxicity in Renal Cell Carcinoma

Abstract

Abstract Background Renal cell carcinoma (RCC) is characterized by a hypoxic tumor microenvironment that recruits immunosuppressive macrophages and regulatory T cells to suppress anti-tumor immunity. Belzutifan, a hypoxia-inducible factor 2 alpha (HIF-2α) inhibitor recently approved by the FDA, significantly improves progression-free survival in patients with previously treated, advanced RCC (LITESPARK-005 trial). Belzutifan is now being evaluated in combination with immune checkpoint inhibitors (ICI) in multiple phase III trials. However, preclinical murine data suggests that hypoxia signaling is important for effector T-cell function (Doedens, et al. Nat Immunol. 2013), raising the concern that pharmacologic HIF-2α inhibition might impair anti-tumor immunity. It remains unclear whether belzutifan has an impact (either positive or negative) on human T cell function, particularly anti-tumor cytotoxic activity. Therefore, we established an in vitro RCC antigen model system to evaluate the impact of belzutifan on T-cell cytotoxicity. Methods T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs). HIF2α knockout (KO) T cells were generated using a CRISPR-Cas9 RNP electroporation, and they were stimulated with anti-CD3/CD28 beads for 48 hours under normoxic or hypoxic (1% O2) conditions. RT-qPCR and bulk RNA sequencing were used for transcriptomic analysis of the T cells. PBMCs were also genetically modified to express an HLA-A*02:01-restricted NY-ESO-1–specific T-cell receptor, thereby generating antigen-specific T cells for the cytotoxicity assay. These T cells were pretreated with belzutifan for 48 hours under hypoxia. The human VHL-mutated RCC cell line 786-O (parental HLA-A03:01) was utilized as the target cell population. Endogenous HLA class I expression was knocked out using the CRISPR–Cas9 system. Subsequently, the cells were transduced with a lentiviral vector encoding HLA-A02:01, CTAG1B (NY-ESO-1), and HIF-2α (either wild-type or the belzutifan-resistant G323E variant), generating the 786-O-WT and 786-O-G323E cell lines. As the G323E substitution interferes with belzutifan binding to HIF-2α, any differences observed in cytotoxicity assays can be attributed to effects of belzutifan on T cells rather than direct cytotoxic effects on 786-O-G323E tumor cells. RCC target cells were labeled with CellTrace Far Red and subsequently co-cultured with the T cells at effector-to-target (E:T) ratios of 0:1 or 1:1 for 12 hours. Cytotoxic activity was assessed by flow cytometry using Annexin V and propidium iodide staining, with analysis restricted to doublet-excluded, CellTrace-positive target tumor cells. Specific lysis (%) was calculated according to the formula: (1 − [viability at E:T = 1:1] / [viability at E:T = 0:1]) × 100. Statistical significance was evaluated using two-sided paired t-tests. Results HIF2α mRNA expression markedly increased in T cells upon stimulation under hypoxic conditions. Differential gene expression analysis of bulk RNA sequencing revealed that IL-2 and IFNG were among the most significantly downregulated genes in HIF2α knockout T cells, with log2 fold changes of –4.08 and –2.13, and adjusted p-values of 3.1 × 10⁻¹¹ and 5.5 × 10⁻²0, respectively. In contrast to genetic KO, pharmacologic HIF2a inhibition with belzutifan did not impair T-cell function. In fact, T cells preconditioned with belzutifan demonstrated modestly increased specific lysis of both 786-O-G323E and 786-O-WT target cells compared with DMSO-treated controls (p = 0.0385 and p = 0.0256, respectively). These findings indicate that belzutifan may augment T-cell–mediated cytotoxic activity. Conclusions Although HIF2α is strongly upregulated under hypoxia and T cell stimulation, and its genetic deletion reduces cytokine gene expression, short-term pharmacologic inhibition does not impair antigen-specific T-cell cytotoxicity against RCC target cells within a hypoxia-modeled antigen system. These data support the biological plausibility of combining belzutifan with immune checkpoint inhibitors (ICIs) as a therapeutic strategy in RCC.