Implementing RNAi Therapy Targeting FLT3 in Acute Myeloid Leukemia (AML)

Abstract

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled (Gebru et. al. J. Hematol. Oncol. 2020). RNA interference (RNAi), mediated by short interfering RNA (siRNA) presents a novel therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. The success of RNAi therapy to a great extent is dictated by the siRNA delivery vehicle employed. This study explored the use of a lipid-substituted low molecular weight polyethyleneimine (lipopolymer) for delivering FLT3 siRNA in FLT3-ITD positive AML models and assess the efficacy of combining this treatment with chemotherapeutic drugs currently used in clinic. Low molecular weight polyethyleneimine (PEI) polymers were modified with aliphatic lipids to yield lipopolymers that self-assembled into nanocomplexes with siRNA. Treatment of MV4-11 cells with lipopolymer/FLT3 siRNA nanocomplexes resulted in ~50% reduction in cell proliferation rates (p<0.001), as determined by taking direct cell counts after the siRNA treatment. Quantitative analysis of relative mRNA transcript levels conducted by qPCR revealed downregulation of FLT3 gene by 30% (p<0.01). Furthermore, a similar decline (~35%, p<0.005) in FLT3 protein levels was observed when measured by flow cytometry through immunostaining with an anti-FLT3 antibody. Evaluation of apoptosis by FITC-Annexin V/propidium iodide (PI) staining showed a 3-fold increase in percentage of cells in the late apoptosis stage after FLT3 specific siRNA treatment (p<0.005). The effect of combining lipopolymer/FLT3 siRNA treatment with daunorubucin manifested as a decline in cell proliferation rates as well as 3-fold increase in apoptosis (p<0.05). To assess the impact of combination treatment on stem cells, colony forming ability was measured by seeding treated cells in methylcellulose and the colonies generated subsequently were counted after two weeks. A notable drop in colony counts was observed in the combination relative to individual treatments. However, similar analyses with the FLT3 targeting TKI midostaurin led to only a modest augmentation of anti-leukemic activity. Subsequently, the more recently approved FLT3 targeting TKI gilteritinib was explored in combination with lipopolymer/FLT3 siRNA nanocomplexes both in vitro and in vivo. For in vivo studies, lipopolymer/FLT3 siRNA nanocomplexes were administered via tail-vein injection at a siRNA dose of 25 µg per mouse (~1 mg/kg) once the tumor volume was ≥50mm3 in AML MV4-11 subcutaneous xenograft models created in male NCG (ABL/VAF) mice. Four injections of the nanocomplexes were administered, each 72 hours apart with simultaneous measurements of the tumor volume. Gilteritinib was administered four times intraperitonially at a dose of 5 mg/kg, with a spacing of 72 hours between doses. A significant reduction in tumor volume was observed in the combination treated tumors compared to gilteritinib alone treated tumors. Refractory/relapsed tumors underwent a second round of treatment (with both nanocomplexes and gilteritinib being administered four times each). The combination therapy significantly increased median survival to 65 days as compared to 45 days with gilteritinib treatment alone (p<0.05). These findings demonstrate the promising potential of RNAi implemented with lipopolymer nanocomplexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML.