Abstract 126: Targeting PAR1 in an aggressive blood cancer: A novel strategy to eradicate leukemic stem cells

Abstract Acute Myeloid Leukemia (AML) remains a challenging hematologic malignancy with poor prognosis due to the persistence of therapy-resistant leukemic stem cells (LSCs). Current therapeutic strategies often fail to completely eradicate LSCs, leading to relapse. One promising therapeutic target is protease-activated receptor 1 (PAR1), a G-protein-coupled receptor selectively expressed in LSCs. Our research investigates the potential of targeting PAR1 to eliminate LSCs and improve treatment outcomes in AML. High PAR1 expression in a cohort of AML patients had a reduced overall survival (n=163, P<0.001). Lentiviral transduction of PAR1 knockdown and overexpression in mouse LSCs was established. PAR1 and its downstream gene and protein targets were validated utilising RT-qPCR and Western blot, respectively. PAR1 function was assessed with knockdown and PAR1 inhibitor treatment independently, which concurrently reduced colony formation, increased apoptosis, induced cell differentiation, and decreased expression of key self-renewal targets (i.e., ß-catenin and transcriptional factor Tcf7L2) and Gα subunits in LSCs. PAR1 overexpression in pre-LSCs was consistent with knockdown studies, demonstrating significantly increased colony formation and increased expression of key self-renewal genes (i.e. Tcf7l2, Ccnd1). PAR1 inhibitor showcased a dose and time-dependent early and late-stage apoptosis in LSCs, while in parallel reduced self-renewal signature markers and increased pro-apoptotic markers (i.e., cleaved caspase 3, Txnip). Murine models included intravenous transplantation of LSCs and pre-LSCs to evaluate PAR1 function in vivo. Overexpression of PAR1 in pre-LSC accelerated leukemogenesis compared to controls in vivo. Murine models demonstrated a marked reduction in leukemic burden in PAR1 knockdown mice, with prolonged survival compared to controls further enhancing LSC eradication and mitigating relapse in vivo. This innovative study is the first to establish PAR1 as a critical regulator of LSC self-renewal and survival, offering a potential breakthrough in AML therapy. These findings support the clinical development of PAR1 inhibitors as a next-generation treatment in eradicating LSCs, with the potential to overcome current therapeutic limitations and reduce relapse rates in AML patients, ultimately uniting cancer biology and therapeutic innovation. Citation Format: Nunki Hassan, Kaitlyn Trajcevski, Jenny Wang. Targeting PAR1 in an aggressive blood cancer: A novel strategy to eradicate leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 126.