Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer
Jun Fang, Jeremy M. Simon, Tao Wang, Yunpeng Gao, Xianju Bi, Lianxin Hu, Chengheng Liao, Cheng Zhang, Yayoi Adachi, Jin Zhou, Hongyi Liu, Qian Liang, James A. Nathan, Ram Mani, James Brugarolas, Qing Zhang
Jun Fang, Jeremy M. Simon, Tao Wang, Yunpeng Gao, Xianju Bi, Lianxin Hu, Chengheng Liao, Cheng Zhang, Yayoi Adachi, Jin Zhou, Hongyi Liu, Qian Liang, James A. Nathan, Ram Mani, James Brugarolas, Qing Zhang
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Research Article Genetics Oncology

Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer

Abstract

Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel–Lindau–deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α–dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy–driven enhancer activation, this work defines a trans-acting cytokine–JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.

Authors

Jun Fang, Jeremy M. Simon, Tao Wang, Yunpeng Gao, Xianju Bi, Lianxin Hu, Chengheng Liao, Cheng Zhang, Yayoi Adachi, Jin Zhou, Hongyi Liu, Qian Liang, James A. Nathan, Ram Mani, James Brugarolas, Qing Zhang

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Figure 1

FACS-based whole-genome CRISPR screen identifies HIF-2α positive regulators.

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FACS-based whole-genome CRISPR screen identifies HIF-2α positive regulat...
(A) Schematic of the HRE- HIF-2α ODD mCherry reporter construct. (B) Diagram of the loss-of-function, genome-wide screen using the human lentiviral sgRNA library (Brunello) in the HIF-2α reporter 786-O cell line. Ten percent of cells with either low or high signal intensity were sorted for NGS to identify factors that regulate HIF-2α activity. (C) Top genes identified for positive regulation of HIF-2α activity versus their log10 robust rank aggregation (RRA) scores in the genome-wide loss-of-function screen. Red circles are regulators reported before and green circles are genes related to the mRNA export pathway. (D) Distribution of sgRNA log fold change (LFC) comparing the mCherry-low group with the presorted group (positive regulators). Red bars represent 4 individual sgRNAs for the indicated genes. (E–H) Immunoblot analysis of cells with TREX component KOs. (E and F) 786-O and A498 ccRCC cell lines. (G and H) HKC and HK2 epithelial cell lines were treated with 10 μM MG132 for 6 hours prior to harvesting. (I and J) Representative soft agar growth (I)FACS-based analysis of HIF-2α activity in 786-O HIF-2α reporter cells following KO of RNA export pathway components. Each gene was targeted with 2 independent sgRNAs, and HIF-2α sgRNAs were used as positive controls. (J and K) Representative soft agar growth (J) and corresponding quantification (n = 3) (K) of 786-O, A498, and HKC cells expressing control (sgCtrl) or sgRNAs targeting the TREX components THOC2 and ALYREF. Scale bars: 1 mm. (L and M) Representative soft agar growth (L) and corresponding quantification (n = 3) (M) of 786-O cells with EV or HIF-2α overexpression that were then transduced with sgRNAs targeting TREX components or control. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA followed by Tukey’s multiple-comparison test (J) or multiple, unpaired t test (L). Data show the mean ± SEM.