RESUMO
Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage-depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1-overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI-resistant CML stem cells in patients.
Assuntos
Movimento Celular , Proliferação de Células , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Apoptose , Western Blotting , Imunoprecipitação da Cromatina , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Persistent activation of the Abl tyrosine kinase in the BCR-ABL fusion protein is the major cause of chronic myeloid leukemia (CML). Among many other substrates BCR-ABL phosphorylates STAT5 and Src family kinases (SFK). Activated pSTAT5 is essential for initial transformation and maintenance of the disease. Cytokine-induced phosphorylation on tyrosine 694 typically leads to nuclear accumulation of pSTAT5 and target gene expression. We verified that in BCR-ABL-positive progenitor cells from a CML patient and in K562 cells pSTAT5 is cytoplasmic. However, upon ectopic expression of BCR-ABL p210 in non-myeloid cells, co-transfected STAT5A is phosphorylated on Y694 and localized in the nucleus arguing for an additional factor mediating cytoplasmic retention in CML cells. Expression of the SFK v-Src, Hck or Lyn together with STAT5A results in phosphorylation on Y694 and cytoplasmic retention. Upon coexpression of BCR-ABL and individual SFK the cytoplasmic retention of activated STAT5A mediated by v-Src and Hck but not Lyn is dominant over nuclear translocation induced by BCR-ABL. Cytoplasmic retention depends on the kinase activity of SFK and is mediated through the interaction of the SH2 domain of STAT5A with the SFK. Interestingly, nuclear accumulation of STAT5A as a result of activation by FLT3-ITD, an oncogene found in acute myeloid leukemia, cannot be prevented by coexpression of SFK. Importantly, inhibition of SFK in K562 cells restored nuclear accumulation of pSTAT5A, enhanced STAT5 target gene expression and increased colony formation. Thus, SFK mediate cytoplasmic retention of pSTAT5A in BCR-ABL-positive cells. Cytoplasmic pSTAT5A in CML cells might balance the controversial functions of STAT5 in cellular senescence and differentiation versus G1/S progression and survival.
