Hematopoietic cells orchestrate waves of transcriptomic networks to guide essential processes such as differentiation, growth or response to stress. However, how these transcriptional networks are silenced to restore homeostasis is poorly defined. Nonsense Mediated Decay (NMD) is a highly conserved quality control pathway to target aberrant mRNA for degradation but also functions to fine tune normal gene expression. Poison exon (PE) sequences are ultra-conserved intronic elements containing premature in-frame stop codons which when spliced in, activate NMD-mediated RNA degradation. Cell intrinsic PE retention is enriched in specific genes encoding splicing factors, RNA-binding proteins and chromatin remodeling genes. However, it is unknown whether PE retention plays a regulatory role in guiding hematopoiesis nor the specific genes that harbor these elements.

To address these questions, we utilized VoyageR Engine, an AI-driven platform leveraging over 17,000 RNA sequencing experiments across 54 tissues allows in silico prediction of de novo cassette exons derived from alternative splicing events. VoyageR then pairs this analysis to an AI-designed, novel and diverse RNA splicing compound library to identify lead splicing compounds capable of promoting retention of specific intronic sequences. To discover potential PEs that regulate hematopoiesis, we employed a pharmacogenomic screen in human HUDEP erythroid precursors to identify genes susceptible to alternative splicing induced degradation using branaplam (BRN). BRN is a pyridazine derivative splicing modulator stabilizing U1 snRNP complex binding to enhance alternative splicing events.

We hypothesized that down regulated mRNA after BRN treatment may be due to induced PE retention in specific genes. Using the AI-assisted VoyageR platform, we identified a novel, highly conserved candidate PE and confirmed an intronic, cryptic U1snRNP recognition site in STAT1. STAT family proteins represent a vital hub for signaling to integrate inflammation, proliferation and self-renewal in HSCs. Furthermore, STAT1 activation plays a key role in regulation of transcriptional networks involved in immune responses, definitive erythroid development and myeloproliferative neoplasms. However, currently there is a paucity of targeted agents that selectively repress STAT1.

We found that STAT1 is highly sensitive to BRN treatment resulting in retention of a PE sequence and downregulation of STAT1 mRNA and protein, but not STAT3, 5A and 5B, in a dose-dependent manner. Retention of the PE sequence is dependent upon NMD as BRN-induced STAT1 degradation is reversed with NMD inhibition. SpliceAI analysis of the PE splice donor sequence of the endogenous STAT1 gene in HUDEP cells found this sequence to be a weak donor. Remarkably, disruption of three nucleotides at the PE splice donor led to constitutive PE retention and ablation (1000x reduction) of STAT1 protein expression.

Although BRN is an effective tool compound, off-target effects of BRN have limited its clinical use. To identify compounds with higher selectivity, we utilized VoyageR to identify a potent small molecule splicing modulator that is a specific regulator of STAT1 PE retention, SRE109. SRE109 promotes STAT1 poison exon retention with an IC50 of 30nM (10-fold higher potency than BRN), rapid induction of PE retention within 2 hours and reduction of mRNA and protein by >70% within 24hrs. SRE109 blocks IFN-g induced transcriptomic and cytokine responses in models of benign erythropoiesis, Myeloproliferative Neoplasms and Acute Myelogenous Leukemia.

These data illustrate how this new AI-driven platform VoyageR has the potential to identify new mechanisms by which hematopoiesis is regulated and identification of compounds that modulate these pathways. Here, we provide evidence for a novel therapeutic strategy and several candidate therapeutic agents to downregulate STAT1-driven processes through targeted splicing modulators to direct gene expression.

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2025
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