Abstract
Acute myeloid leukemia (AML) is a genetically heterogeneous myeloid malignancy that accounts for approximately 20% of pediatric leukemia cases. Despite intensive therapy, long-term remission remains suboptimal, with relapse occurring in 25–35% of patients, particularly among those with high-risk molecular subtypes. The transcription factor SP1 has been implicated in leukemogenesis, yet its precise role in AML progression remains incompletely understood. Analysis of publicly available transcriptomic datasets revealed that SP1 is significantly upregulated in AML compared to normal hematopoietic cells, and higher SP1 levels are associated with worse clinical outcomes.
To elucidate the functional relevance of SP1 in AML, we performed loss-of-function studies using SP1-targeting shRNAs. Knockdown of SP1 markedly impaired AML cell viability, proliferation, stemness, and in vivo progression in cell line–derived xenograft models. Notably, microscopy revealed the formation of distinct nuclear condensates by endogenous SP1 in AML cells. Using fluorescence recovery after photobleaching, we confirmed that SP1 undergoes liquid–liquid phase separation (LLPS). PSPhunter analysis identified three intrinsically disordered regions (IDRs) in SP1 that may mediate LLPS. To functionally dissect their role, we generated a series of rescue constructs: wild-type SP1, SP1 harboring LLPS-disrupting mutations, and LLPS-deficient SP1 fused to heterologous IDRs (from FUS or hnRNP). Rescue with wild-type SP1 restored AML cell growth and stemness, whereas the LLPS-deficient SP1 mutant failed to do so. Notably, chimeric constructs containing IDRs from FUS or hnRNP partially restored function, highlighting that the ability of SP1 to form condensates is essential for its oncogenic activity.
CUT&RUN profiling using SP1 and H3K27ac antibodies revealed that SP1 preferentially occupies super-enhancer (SE) regions enriched in H3K27ac, suggesting a role for SP1 in enhancer-driven transcriptional regulation. Compared to controls, SP1 knockdown altered H3K27ac patterns, leading to the identification of 15 SE-associated genes co-regulated by SP1. Integrating expression profiles and survival data, we identified five SP1-SE target genes highly expressed in AML and associated with poor prognosis. Among them, heterogeneous nuclear ribonucleoprotein U-like 1 (HNRNPUL1) emerged as a critical target, with its expression tightly dependent on SP1 condensate integrity. Reporter assays and ChIP-qPCR validated SP1 occupancy at the HNRNPUL1 SE region, and knockdown or pharmacologic disruption of SP1 significantly suppressed HNRNPUL1 expression.
To therapeutically target SP1-driven SE activity, we employed B026, a selective p300/CBP histone acetyltransferase inhibitor. B026 treatment disrupted SP1 condensates, decreased H3K27ac enrichment at SP1-bound SEs, downregulated HNRNPUL1, and impaired AML cell viability in vitro. These effects phenocopied those of SP1 depletion and highlight the therapeutic vulnerability of SE-dependent transcriptional programs maintained by SP1 condensates.
In summary, our study reveals that SP1 promotes AML progression through LLPS-mediated formation of nuclear condensates that serve as functional hubs for super-enhancer activation and oncogene transcription. The SE-regulated gene HNRNPUL1 is a key effector in this pathway. Disruption of SP1 phase separation, either genetically or pharmacologically, abrogates this transcriptional program and diminishes leukemic potential. These findings identify SP1 condensates as novel regulators of AML pathogenesis and highlight LLPS interference as a promising therapeutic strategy, particularly for high-risk and relapsed pediatric AML.
