Abstract PR010: Deciphering the mechanistic basis of HIFα paralog target selectivity in kidney cancer

Abstract Clear cell renal cell carcinoma (ccRCC) is driven by loss of VHL, leading to stabilization of HIF1α and HIF2α. While HIF1α suppresses ccRCC, HIF2α promotes tumorigenesis, yet the mechanistic basis of their paralog-specific transcriptional programs remains unclear. Some studies attribute these differences to paralog-specific DNA binding, whereas others implicate their transactivation domains (TADs). To address these conflicting reports, first, I used CRISPR-HDR to introduce a FLAG-HA epitope tag into endogenous HIF1α and EPAS1 (HIF2α) loci, followed by ChIP-Seq with a FLAG antibody. HIF1α and HIF2α largely occupy the same genomic sites, indicating DNA binding alone does not explain their paralog-specific effects. Next, to assess the contribution of TADs, I performed domain-swapping experiments in which chimeric HIFα proteins were generated by fusing the N-terminal DNA-binding/dimerization domains (HIFα-NT) of one paralog to the TAD (HIFα-TAD) of the other. Chimeras containing HIF1α-NT fused to HIF2α-TAD restored HIF2α target gene expression and proliferation in PT2399-treated cells, whereas the reciprocal chimera failed to do so, even with a PT2399-resistance mutation in HIF2α-NT. These results, consistent with previous studies, support that paralog-specific target selectivity of HIFs is driven by distinct regulators of TAD activity. To identify such regulators in an unbiased, high-throughput manner, I developed a HIFα paralog-specific transcriptional activation reporter system. Here, the tetO-binding protein TetR is fused to either the HIF1α or HIF2α TAD to drive expression of the suicide gene DCK*. DCK* converts the BVdU into a toxic compound, so cells die when the HIFα TAD is active. Loss of transactivation regulators allows survival, enabling positive-selection screens with high signal-to-noise ratios. Using this system, I performed whole-genome CRISPR knockout screens, which revealed novel paralog-specific regulators, including ZNF658B for HIF1α and THO complex subunits for HIF2α. In parallel, to identify proteins that interact dynamically and transiently with HIFα TADs through their intrinsically disordered regions, I performed proximity labeling by fusing the promiscuous biotin ligase TurboID to full-length HIF1α, HIF2α, and HIFα chimeras. Biotinylated proteins were captured with streptavidin beads and analyzed by mass spectrometry. The HIF2α TAD specifically interacts with proteins involved in post-transcriptional RNA regulation, including AGO2/3 and TNRC6A/B, suggesting HIF2α TAD function is modulated by RNA processing regulators. Belzutifan, an inhibitor that blocks HIF2α-ARNT dimerization, is FDA-approved for ccRCC treatment, but resistance mutations preventing drug binding or HIF2α-ARNT disruption have been reported. In conclusion, the findings from this study reveal an unexpected mechanism in which post-transcriptional RNA-abundance regulators contribute to HIF2α-specific transcription, highlighting new therapeutic avenues for small-molecule HIF2α inhibition and for overcoming resistance to current HIF2α-targeted therapies. Citation Format: Nitin H. Shirole, John G. Doench, William G. Kaelin Jr. . Deciphering the mechanistic basis of HIFα paralog target selectivity in kidney cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr PR010.