Characterisation of HIF2A targets identifies MYC as a key determinant of HIF2A dependency in ccRCC
Despite advancements in ccRCC treatment, the prognosis for metastatic disease remains poor with a 5-year survival of approximately 10%. VHL loss is an early driver event in the vast majority of ccRCC cases and results in constitutive activation of HIF2A, making HIF2A an important clinical target. While HIF2A inhibitors are showing promising results in clinical trials, many patients do not respond and ultimately most patients progress. To improve our understanding of HIF2A dependency in metastatic ccRCC, I developed and thoroughly validated a comprehensive approach to identify HIF2A transcriptional targets *in vivo*. I demonstrated that HIF2A can be acutely degraded in a xenograft model of ccRCC using AID2 technology, and that combining this with RNA-seq uncouples direct targets from downstream signalling cascades. This unbiased method enabled the shortlisting of clinically relevant candidate HIF2A direct target genes which could then be functionally tested by *in vivo* CRISPR/Cas9 screening and subsequent rescue experiments. CRISPR/Cas9 screening revealed that *MYC* and *CCND1* are key dependencies in ccRCC *in vivo*, in addition to highlighting key differences between *in vitro* and *in vivo* dependencies. By leveraging a doxycycline-inducible HIF2A expression system, combined with exogenous constitutive expression of *MYC* or *CCND1*, I showed that MYC, but not CCND1, is sufficient to rescue tumour maintenance upon HIF2A loss. While both cell cycle-associated genes drive proliferative signalling, I demonstrated that only MYC protects from apoptosis, providing mechanistic insights into the role of HIF2A-dependent *MYC* expression in ccRCC. The results presented here provide a workflow for unbiased, *in vivo* characterisation of oncogenic transcription factors, and novel insights into HIF2A dependency in ccRCC.
acute protein degradation, AID2, Cancer, ccRCC, CRISPR/Cas9, Genetics, HIF2A, Oncology, Renal cancer, Transcriptomics
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