Inhibition of ARNT suppresses growth of primary human kidney cancer cells

Background: Hypoxia‑inducible factor (HIF) signaling drives clear cell renal cell carcinoma (ccRCC). ARNT (HIF‑1beta) dimerizes with HIF‑1alpha and HIF‑2alpha to enable gene transcription. Belzutifan is a small-molecule drug that blocks HIF-2alpha, which is approved for clinical use. It's been proposed that increased signaling through HIF-1alpha represents a belzutifan-resistance mechanism. Blocking ARNT should attenuate both HIF programs. We assessed an ARNT‑selective small‑molecule inhibitor (ARNTi) in renal cancer models. Methods: 786‑O cells were cultured under hypoxia. Cells received belzutifan, ARNTi, or vehicle control (DMSO). After treatment, cells were fixed, and the interaction between HIF‑2alpha and ARNT was quantified using a proximity ligation assay. Primary cultures were generated from patients who underwent nephrectomy. Under hypoxia, both normal and ccRCC primary cell cultures were exposed to ARNTi or vehicle and then processed for proliferation assessment and qPCR of HIF target genes. Primary papillary renal cell carcinoma cultures were evaluated using the same workflow. Results: ARNTi lowered the proximity ligation signal between HIF‑2alpha and ARNT. The extent of inhibition matched the effect of belzutifan. In patient‑derived ccRCC cultures (n=5), ARNTi produced significant growth inhibition under hypoxia compared with vehicle. The magnitude of the effect was similar to belzutifan. Expression of hypoxia‑responsive target genes, such as GLUT1, NDRG1, and VEGFa, decreased after ARNTi treatment. ARNTi also suppressed growth in papillary renal cell carcinoma cultures (n=3), indicating activity across histologic subtypes. Conclusions: Inhibition of ARNT reduces the formation of the HIF‑2alpha and ARNT complex and suppresses proliferation in primary human kidney cancer cells. These effects coincide with reduced expression of HIF target genes under hypoxia. These results support the development of ARNT‑directed candidates for the treatment of kidney cancer.