Leed, A. | Search bioRxiv

bioRxiv2025

Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

Translocation renal cell carcinoma (tRCC) is an aggressive kidney cancer driven by gene fusions of the TFE3 transcription factor. TFE3 is essential in tRCC but dispensable in normal cells, presenting an attractive but pharmacologically challenging therapeutic target. We show that the basic helix-loop-helix (bHLH) domain of TFE3 is crucial for chromatin binding and transcriptional function. Via a phenotypic screen of 25,000 compounds, we identified molecules that either displace or retain chromatin-bound TFE3. BRD6866, a compound trapping TFE3 on chromatin, emerged as a pan-CDK inhibitor. Mechanistically, its inhibition of CDK9 - a key regulator of transcriptional elongation - was linked to impaired TFE3 fusion activity. These effects were recapitulated by the CDK9-selective inhibitor enitociclib, which downregulated TFE3 targets and suppressed tRCC cell growth. Our findings nominate CDK9 inhibition as a therapeutic strategy in tRCC and demonstrate the utility of mechanism-informed phenotypic screening for challenging targets.

cancer biology↗

bioRxiv2025

Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

Src homology 3 (SH3) proteins regulate numerous fibroproliferative pathophenotypes including pulmonary arterial hypertension (PAH) but are challenging to target therapeutically. We innovated a peptidomimetic that occupies the canonical focal adhesion kinase (FAK) binding site on the SH3 domain of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) protein, a pro-PAH regulator. Peptidomimetic derivatization with a bromoacetamide group alkylated a NEDD9 cysteine positioned uniquely among SH3 domains (Cys18), which stabilized the RT loop, prevented FAK binding, and inhibited human pulmonary artery endothelial cell (HPAEC) migration. When linked to a thalidomide moiety, the peptide showed degrader activity of NEDD9 protein and, therefore, we next investigated therapeutic application of NEDD9 inhibition. In HPAECs, si-NEDD9 downregulated sulfatase-1, which increased podosome rosette formation and cell migration via 6-O-desulfation of glycocalyx-forming heparan sulfate proteoglycans, and reversed vascular remodeling and PAH in vivo. Whereas sulfatase-1 overexpression decreased pulmonary endothelial podosome formation, cell migration, and tube formation and increased collagen III synthesis, sulfatase-1 knockdown prevented fibroproliferative remodeling and pulmonary hypertension in PAH in vivo. These data leverage cysteinyl thiol reactivity to establish an SH3 domain-targeting structure-validated covalent peptide and identify two convergent mechanisms through NEDD9 that control endothelial phenotype, including reverse remodeling via sulfatase-1 transcriptional control. Overall, this study advances an SH3-specific therapeutic approach with relevance to PAH and other fibroproliferative pathophenotypes.

molecular biology↗