RESUMO
The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.
Assuntos
Lipossomos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Interferente Pequeno/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Técnicas de Silenciamento de Genes , Ordem dos Genes , Terapia Genética , Humanos , Mediadores da Inflamação , Masculino , Proteínas de Fusão Oncogênica/administração & dosagem , Proteínas de Fusão Oncogênica/química , Plasmídeos/administração & dosagem , Plasmídeos/genética , Proteína Proto-Oncogênica c-fli-1/administração & dosagem , Proteína Proto-Oncogênica c-fli-1/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Proteína EWS de Ligação a RNA/administração & dosagem , Proteína EWS de Ligação a RNA/química , Sarcoma de Ewing/mortalidade , Sarcoma de Ewing/patologia , Sarcoma de Ewing/terapia , Reparo Gênico Alvo-Dirigido , Transfecção , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.
