Abstract B028: Familial CTR9 mutation reprograms Wilms Tumor cells to a blastemal state and confers vulnerability to CDK9 inhibition

Abstract Blastemal Wilms tumor (WT) is a less differentiated subtype of WT with poor prognosis. To study this aggressive form, we previously generated a partially reprogrammed cell model (WiT49-PRC) by introducing pluripotency factors into WiT49, a mixed-type of WT cell line, which faithfully recapitulates blastemal-predominant WTs. WT is believed to originate from nephron progenitor cells (NPCs) that fail to undergo proper differentiation. Developmental regulators such as SIX1, SIX2, and EYA1 are frequently mutated or dysregulated in WTs, with their promoters subject to tight transcriptional control. CTR9 is a scaffold protein within the PAF1 complex, essential for transcriptional elongation through direct interaction with RNA polymerase II (RNAPII). Low-frequency CTR9 mutations have been observed in familial cases, often causing exon9 skipping. During transcription, RNAPII initiates and rapidly enters a paused state, which is released by P-TEFb (CDK9–Cyclin T2) mediated phosphorylation to enable productive elongation. The molecular mechanism underlying the causal effect of CTR9 exon 9 deletion on RNAPII-mediated transcriptional elongation and development of blastemal WTs remains unknown. Using CRISPR/Cas9, we generated CTR9Δexon9 WiT49 cells modeling familial WT mutations. Remarkably, CTR9Δexon9 cells exhibited enhanced colony formation and accelerated tumor growth compared to both parental and CTR9 knockout WiT49 cells, revealing a gain-of-function effect. Comparing the transcriptional signatures of CTR9Δexon9 cells with WiT49-PRCs revealed enrichment of somatic stem cell signaling pathways in both cell lines, confirmed by increased SIX2 and EYA1 expression measured by qPCR. These findings were validated by SIX2 and NCAM1 immunohistochemistry staining of tumor samples from kidneys of mice injected with WiT49, CTR9Δexon9 WiT49, and WiT49-PRCs. These findings prompted us to assess whether CTR9Δexon9 WiT49 cells exhibit increased selectivity to selective CDK9 inhibitors. Cell viability assays demonstrated that CTR9Δexon9 cells were more sensitive to selective CDK9 inhibitors, including NVP-2, MC180295, and Enitociclib, while no differences were observed with pan-CDK inhibitors, Dinaciclib, indicating specific vulnerability to CDK9-targeted therapy. Together, our study uncovered that CTR9Δexon9 mutation causes aberrant activation of stem/blastemal genes and promotes CDK9/ RNAPII-mediated elongation, leading to a less differentiated, blastemal-like WT phenotype. The increased vulnerability of CTR9Δexon9 cells to selective CDK9 inhibitors provides a strong rationale for developing CDK9-targeted therapies as precision treatments for children with CTR9 mutations and aggressive, blastema-like Wilms tumors. Citation Format: Gui Ma, Ang Gao, Mingshan Hu, Peng Liu, Yidan Wang, Wei Xu. Familial CTR9 mutation reprograms Wilms Tumor cells to a blastemal state and confers vulnerability to CDK9 inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr B028.