Leukemic Stem Cell-Targeted Liposomal Nanoimmunotherapy Reverses Immune Evasion and Inhibits Fusion Oncoprotein-Driven Acute Myeloid Leukemia by Silencing B-Cell Lymphoma 2.

Abstract

Acute Myeloid Leukemia (AML) is a prevalent and aggressive hematologic cancer driven by leukemic stem cells and immune evasion. Among its subtypes, Mixed Lineage Leukemia-AF9 (MLL-AF9)- rearranged AML is exceptionally lethal, with B-cell lymphoma 2 (Bcl2), a key anti-apoptotic protein, supporting leukemic survival and immune dysfunction. Targeting Bcl2 in this context is crucial for dismantling oncogenic circuits and restoring immune activity. Here, we have synthesized, de novo, IL-3 receptor alpha chain (IL-3Rα, commonly known as cluster of differentiation 123 or CD123) targeted aptamer-tethered liposomal nanocarriers (CD123-si-Bcl2@LNPs) encapsulating Bcl2 siRNA (CD123-si-Bcl2@LNPs) for selective AML targeting, survival disruption, and immune restoration. Mechanistic studies established a previously uncharacterized signaling axis wherein the MLL-AF9 fusion oncoprotein induces the overexpression of the transcription factor c-Myb. This induced c-Myb binds directly to the Bcl2 promoter, upregulating Bcl2 expression and enhancing both STAT3 expression and its Ser727 autophosphorylation. Consequently, this pathway amplifies IL-6-driven immunosuppression and dampens NK and CD8⁺ T cell responses. Nanocomposite-mediated disruption of this specific axis successfully reversed these oncogenic and immunosuppressive signals. Therapeutic efficacy was evaluated using a patient-derived xenograft (PDX) model, generated by retroviral MLL-AF9 overexpression in CD34⁺/CD38⁻ sorted leukemic stem cells from AML patients and engraftment into NOD/SCID mice. CD123-si-Bcl2@LNPs significantly reduced leukemic burden in vivo. Bcl2 siRNA encapsulated liposomal nanoparticles demonstrated robust Bcl2 knockdown, apoptosis induction, and impaired leukemic self-renewal across in vitro and in vivo models. Post-treatment immune profiling revealed enhanced NK cell (CD56⁺) expression and reversal of T cell exhaustion, with CD8⁺ T cell activation marked by downregulation of CTLA-4. Differentiation marker analysis revealed decreased CD45+ and c-KIT+ expression, accompanied by an upregulation of the myeloid monocyte differentiation marker CD11b, indicating myeloid maturation and a reduction in leukemic stemness. This dual-action therapeutic strategy simultaneously targets survival and immune evasion pathways in MLL-AF9-positive AML cells by the treatment of CD123-si-Bcl2@LNPs, offering a promising, precision-guided nano-immunotherapeutic strategy for future translational potential for MLL-AF9-positive acute myeloid leukemia. STATEMENT OF SIGNIFICANCE: •The CD123 aptamer-targeted liposomal nanocarrier facilitates precise siRNA delivery in acute myeloid leukemia (AML), significantly enhancing the transfection efficiency and stability of Bcl2 siRNA. •siRNA nanotherapy reveals a novel immune-evasion axis involving c-Myb, Bcl2, and STAT3 axis which is critical for the progression of AML. •Targeted silencing of Bcl2 disrupts the survival networks of leukemic stem cells. •Nano-immunotherapy revitalizes the antitumor activity of natural killer (NK) cells and CD8⁺ T-cells. •Liposomal siRNA therapy effectively suppresses MLL-AF9 leukemia in NOD/SCID patient-derived xenograft (PDX) mouse models.