RESUMO
Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Genes ras , Células-Tronco Hematopoéticas/citologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/genética , Inibidores de Proteínas Quinases/química , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/citologia , TransgenesRESUMO
'Pre-leukaemic' mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukaemia. Here we show that a single allele of oncogenic Nras(G12D) increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukaemia initiation. Nras(G12D) also confers long-term self-renewal potential to multipotent progenitors. To explore the mechanism by which Nras(G12D) promotes HSC proliferation and self-renewal, we assessed cell-cycle kinetics using H2B-GFP label retention and 5-bromodeoxyuridine (BrdU) incorporation. Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide. This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not. Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness and self-renewal through bimodal effects on HSC gene expression, cycling and reconstituting potential.
