Therapeutic targeting of ADAR1 p150 splicing activity impairs CD44 + TNBC cell populations in preclinical studies

Background: Triple-negative breast cancer (TNBC) remains clinically challenging due to the lack of targeted therapies and the presence of therapy-resistant tumor-propagating cells. CD44 and ADAR1 contribute to TNBC progression and therapeutic resistance. Rebecsinib is a small-molecule inhibitor designed to block splicing-mediated activation of ADAR1 (adenosine deaminase acting on RNA 1). This study evaluates its efficacy in selectively targeting CD44 + and ADAR1 + cells in preclinical humanized TNBC models. Methods: MDA-MB-231 TNBC cell line-derived xenograft (CDX) models were established in Rag2 -/- gammac -/- and NSG-SGM3 mice. MDA-MB-231 ADAR-nanoluciferase-GFP expressing cells enable tracking of ADAR1 activity by IVIS imaging. Engrafted mice received vehicle, Rebecsinib IV (10 mg/kg), or Rebecsinib PO (15mg/kg), twice a week for two weeks. Tumor burden was measured by IVIS and single-cell suspensions from peripheral blood, and tissues (lung, liver, spleen, and bone marrow) were analyzed by flow cytometry to quantify CD44 + and ADAR1 + cells. Results: Rebecsinib significantly reduced CD44 + cells in peripheral blood ( p < 0.05), lung ( p < 0.01), liver ( p < 0.01), and spleen ( p < 0.05) in Rag2 -/- gammac -/- models (Student t test). Corresponding decreases in ADAR1 + cells was observed in the lung (p < 0.05), liver (p < 0.01), and spleen (p < 0.01) in NSG-SGM3 mouse models ( S tudent t test). IVIS imaging demonstrated tumor bioluminescence in Rebecsinib-treated groups ( p = 0.02, student t test). Combination with Fedratinib further suppressed tumor growth, indicating a synergistic effect ( p < 0.05, student t test). Conclusion: Rebecsinib selectively reduces CD44 + and ADAR1 + TNBC cell populations and inhibits tumor progression in humanized preclinical models. These findings support further evaluation of Rebecsinib alone and in combination, as a targeted therapeutic approach for TNBC. Keywords: TNBC, ADAR1, CD44, Rebecsinib, targeted therapy, preclinical models.