SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance. [ChIPseq and Cut&Tag]

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells. Overall design: RMC2C cells were modified to integrate a doxycyline-inducible copy of either SMARCB1 or mCHERRY control. 48Hrs after doxycyline treatment, roughly 10¹° cells were either crosslinked using 0,4% PFA for fixed ChIP-seq (MYC and H3K27ac) or simply harvested for native ChIP-seq (BRG1). 100Ug of extracted chromatin was used for IP before DNA extraction and sequencing. For Cut&Tag (BRG1 and SMARCB1), 24hrs after doxycycline treatment, roughly 500,000 cells were harvested in native conditions and handled using the Active Motif Cut&Tag kit.