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 3

SOCS3 overexpression suppresses HIF-2α levels and inhibits ccRCC tumorigenesis.

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SOCS3 overexpression suppresses HIF-2α levels and inhibits ccRCC tumorig...
(A and B) Immunoblots of ccRCC cell lines 786-O (A) and A498 (B) expressing DOX-inducible EV or SOCS3, treated with 200 ng/mL DOX. iSOCS3-rep, independent biological replicates of DOX-induced SOCS3 expression. (C and D) RT-qPCR quantification of HIF-2α mRNA levels in 786-O (C) and A498 (D) cells with EV or SOCS3 expression induced by 200 ng/mL DOX. (E–G) Immunoblots of 786-O (E), A498 (F), and UMRC2 (G) cells expressing EV or DOX-induced SOCS3 and coexpressing SOCS3 and GFP or HIF-2α. (H–J) Soft agar growth quantification of 786-O (H), A498 (I), and UMRC2 (J) cells expressing EV or DOX-induced SOCS3, and coexpressing SOCS3 and GFP or HIF-2α. (K) Representative soft agar growth of 786-O, A498, and UMRC2 cells expressing EV or DOX-induced SOCS3 and coexpressing SOCS3 and GFP or HIF-2α (n = 3). Scale bars: 1 mm. (L–O) 786-O cells transduced with DOX-inducible SOCS3 or EV subcutaneous injection. Image of tumors after dissection (scale bar: 1 cm) (L), tumor weights (M), subcutaneous tumor growth (N), and immunoblots of tumor lysates (O). (P–V) 786-O cell lines with SOCS3-inducible expression were injected orthotopically into the renal capsule of NSG mice. Representative bioluminescence images at 0 weeks and 10 weeks after DOX treatment (P), corresponding quantification data (Q), Image of kidney orthotopic tumors (scale bar: 1 cm) (R), tumor weights (S), immunoblots of representative tumor samples (T), representative lung ex vivo bioluminescence images (U), and quantification of ex vivo imaging (V). Data show the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparison test (H–J) or unpaired Student’s t test (C, D, M, N, Q, S, and V).