Overcoming leukemia stem cells via targeting AKT-wnt/β-catenin by AI-powered mRNA-delivery of peptides

Abstract Leukemia stem cells (LSCs) are key drivers of chemoradiotherapy resistance, immune evasion, and frequent relapse of leukemia. Despite achieving initial remission, 30-50% of acute myeloid leukemia (AML) patients eventually relapse, even after allogeneic stem cell transplantation. Relapse samples show a 10- to 90-fold increase in LSC frequency, further implicating LSC's role in leukemia recurrence. The Wnt/β-catenin and PI3K/AKT pathways are frequently dysregulated in many cancers, and are crucial for LSCs' self-renewal and immune escape. β-catenin directly regulates the expression of multiple immune checkpoint genes. Additionally, AKT promote Wnt/β-catenin by phosphorylating and inhibiting GSK-3β to promote β-catenin accumulation, and directly phosphorylating β-catenin at Ser552 and Ser675 to enhance the nuclear transcriptional activity of β-catenin. Thus, hyperactivating the AKT-Wnt/β-catenin signaling plays a critical role in supporting LSCs' self-renewal and survival. The intricate relationship between the AKT-Wnt/β-catenin pathway and the mechanisms of immune escape and cancer recurrence underscores the urgent need for targeted interventions. We previously demonstrated that low-dose doxorubicin inhibits AKT-mediated Ser552 phosphorylation of β-catenin, and reduces LSCs in both T-cell acute lymphoblastic leukemia (T-ALL) mouse models and in a pilot clinical trial in refractory AML patients. However, due to topoisomerase-II binding, doxorubicin's cardiotoxicity limits its clinical utility. Over 20% of pediatric ALL survivors develop grade II cardiotoxicity post-chemoradiotreatment. Peripheral neuropathy occurs in approximately 38% of patients receiving neurotoxic chemotherapies, causing long-term sensory and motor deficits. Hence, there is an urgent need for safer LSC-targeting strategies. To address these challenges, we developed Artificial intelligence-powered, mRNA-delivery of Peptides (ARP), an integrated approach that combines AI-guided peptide designing with lipid-nanoparticle (LNP)-facilitated mRNA delivery for precise intracellular targeting of the AKT-β-catenin interaction. Leveraging AlphaFold-simulated docking in silico, and reporter assays in HEK293-AKT-β-catenin-TOPFlash-luciferase cells in vitro, we validated several highly potent peptide inhibitors, including β-catenin fragment 529-581. Next, we used RFdiffusion to generate 2,000 de novo binders targeting two hydrophobic hotspots within the AKT kinase-pocket. Following a streamlined de novo binder screening pipeline in silico, including competitive binding assay, immunogenicity and toxicity filters, we selected the top 35 candidates. Meanwhile, we created a library of 416 β-catenin fragments centered on the AKT phosphorylation site at Ser552. We then trained a machine-learning model by integrating (1) reporter assay data from these fragments in HEK293-AKT-β-catenin-TOPFlash-dGFP cells and (2) six structural and energetic features obtained from their AlphaFold3 docking conformations. Through this model, we ranked the last 35 candidates and predicted the top-ranked de novo peptide, Dnv, demonstrated its superior binding and stability in silico, potent inhibition in reporter cells, and enhanced stability relative to 529-581 in vitro. ARPs were then tested in SclCreER;Ptenfl/f;Ctnnb1Dex/+3(β-catenin) T-ALL mouse model. Linear or circular RNA encoding β-catenin fragments or Dnv was packaged in LNP and intravenously administered. ARP treatments were well tolerated and significantly reduced LSCs, partially restored hematopoietic stem/progenitor cells without obvious cardiotoxicity or hepatotoxicity. Competitive transplantation assays revealed that unlike control, ARP-treated bone marrow cells didn't drive leukemogenesis. ScRNA-seq of bone marrow cells confirmed a significant decrease in LSCs. Moreover, we found a pronounced shift of CD8⁺ T-cells that β-catenin fragments or Dnv treatment decreased stem-like CD8+ T memory cells, which are induced by elevated Wnt/β-catenin signaling. Effector and effector memory cells expanded, and their expressions of anti-tumor cytotoxic genes were elevated, accompanied by downregulation of exhaustion markers. Together, these results establish ARP as a versatile strategy to target intracellular AKT-Wnt/β-catenin signaling. By simultaneously disrupting LSCs' maintenance and reprogramming anti-tumor immunity, ARP offers a promising avenue for overcoming relapse and enhancing therapeutic responsiveness in leukemia.