The CDK7 inhibitor THZ1 overcomes venetoclax resistance in acute myeloid leukemia through dual regulation of p53 signaling and transcriptional networks

Abstract Background: Venetoclax (Ven), a selective BCL-2 inhibitor, has shown clinical efficacy in AML, but the mechanisms underlying resistance remain incompletely understood. Emerging evidence suggests that dysregulated transcriptional programs—particularly involving MYC, E2F, and cell cycle regulators—may contribute to resistance in certain contexts. CDK7, a central regulator of transcription, has become an attractive therapeutic target, and its inhibitor THZ1 has demonstrated broad anti-tumor activity across multiple cancer types, especially in transcriptionally addicted malignancies. Methods: We transduced the human AML cell line MOLM-13 with the genome-wide CRISPR/Cas9 TKOv3 library and treated the cells with the CDK7 inhibitor THZ1. Key regulators of THZ1 sensitivity were identified using the MAGeCK algorithm. The top-ranked gene was further validated via single-gene CRISPR/Cas9 knockout. To characterize transcriptional changes upon THZ1 treatment, we performed bulk RNA sequencing and quantitative PCR (qPCR), and confirmed protein-level alterations by Western blotting. To investigate the effects of THZ1 in Venetoclax-resistant AML, we established a Ven-resistant (VenR) MOLM-13 cell line. THZ1 responsiveness was evaluated using flow cytometry-based apoptosis assays and CCK-8 cell viability assays. Transcriptional reprogramming in VenR cells was assessed via bulk RNA-seq and qPCR, while key protein expression changes were validated by Western blot. Finally, the in vivo efficacy of THZ1 was evaluated using a cell line-derived xenograft (CDX) mouse model generated from VenR cells. Results: CRISPR screening analyzed via MAGeCK identified TP53 as the top gene significantly associated with THZ1 sensitivity (RRA score = 6.18E-23). TP53 knockout reduced cellular sensitivity to THZ1, while THZ1 treatment led to early induction of p53 protein expression, supporting a critical role for p53 in mediating THZ1 response. MAGeCK-based pathway enrichment analysis further revealed significant enrichment of gene sets related to MYC signaling, E2F targets, G2/M checkpoint, and DNA damage repair following THZ1 treatment. These findings were validated across multiple platforms, including bulk RNA-seq, qPCR, and Western blotting. Notably, Venetoclax-resistant (VenR) AML cells exhibited heightened sensitivity to THZ1-induced apoptosis compared to parental cells (IC50: VenR = 8.11 nM vs. parental = 15.82 nM). Transcriptomic profiling of VenR cells revealed extensive transcriptional reprogramming, characterized by increased activity of MYC/E2F-associated pathways and metabolic rewiring toward glycolysis. These oncogenic programs were significantly suppressed by THZ1 treatment, as confirmed by qPCR and protein-level validation through Western blot. In vivo, low-dose THZ1 (5 mg/kg, administered every other day) significantly inhibited leukemia progression in VenR cell-derived xenograft (CDX) mouse models. Conclusion: Our study demonstrates that THZ1 effectively overcomes Venetoclax resistance in AML by targeting dual vulnerabilities: reactivating p53 signaling and suppressing MYC/E2F-driven transcriptional and metabolic programs. The identification of TP53 as a key determinant of THZ1 sensitivity highlights the importance of functional p53 in mediating therapeutic response. In Venetoclax-resistant AML cells, THZ1 not only induces apoptosis more potently but also reverses transcriptional reprogramming associated with resistance. These findings support CDK7 inhibition as a promising therapeutic strategy for Venetoclax-resistant AML and provide a rationale for clinical translation.