Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Abstract

High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34 ؉ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that downmodulation of BMI1 in cord blood CD34 ؉ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34 ؉ cells, down-modulation of BMI1 impaired long-term expansion, whereby selfrenewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and selfrenewal of normal and leukemic stem and progenitor cells, and that expression of IntroductionBMI1 is a member of the Polycomb group (PcG) genes, which are transcriptional repressors that play essential roles in the maintenance of appropriate gene expression during development. [1][2][3][4][5] Two distinct multiprotein PcG complexes have been identified, the Polycomb repression complex (PRC) 1 and PRC2. 1 The PRC2 complex is involved in initiation of silencing and contains histone deacetylases and methyltransferases that can methylate H3 lysine 9 and 27. 6 Deletion of PRC2 genes in mice results in embryonic lethality, emphasizing their importance in development. [7][8][9] PRC1 is implicated in stable maintenance of gene repression and recognizes the methylation marks set by PRC2. 10,11 Mice mutant for most PRC1 genes survive until birth as a result of partial functional redundancy provided by their homologues, but developmental defects do arise thereafter, as is, for example, the case in the hematopoietic compartment after deletion of BMI1. [12][13][14] Targeted deletion of Bmi1 has shown that although the numbers of fetal liver-derived hematopoietic stem cells (HSCs) are normal in these mice, their proliferative and self-renewal capacity is severely impaired. 15,16 In adult Bmi1-deficient mice, the HSCs are less frequent and display an impaired competitive repopulation capacity. 16,17 Gain-of-function studies demonstrated enhanced selfrenewal of murine HSCs and with a shift in balance toward more symmetric stem cell divisions. 17 We have demonstrated that constitutive expression of BMI1 in human cord blood (CB) cells results in prolonged maintenance of the stem cell pool and enhances self-renewal of human stem and progenitor cells. 18 BMI1 is a potent negative regulator of the IN...

Leukemia-associated antigens and their relevance to the immunotherapy of acute myeloid leukemia

“…36 Apoptosis and proliferation LAAs can tilt the balance between AML cell apoptosis and proliferation in favor of the latter, either by preventing apoptosis or by stimulating cell proliferation. 36 Several of the hitherto characterized LAAs are indeed known to possess potent antiapoptotic properties, including Bcl-2, 36 Bax inhibitor-1 (BI-1), 37 BMI1, 38 Mcl-1, 39 proteinase 3 (ref. 36) and survivin.…”

“…For example, BMI1 and proteinase 3 negatively affect the expression levels of the cell cycle regulators p16 INK4a and p21 WAF1 , respectively, resulting in an acceleration of the G1/S-phase transition of the cell cycle. 38,43,44 BMI1 also represses the expression of p19 ARF and its critical downstream target p53, resulting in a loss of proliferation control. 44 In addition to antiapoptotic genes, WT1 directly modulates the expression of genes that encode components of the cell cycle apparatus, such as cyclin E, which co-regulates the G1/S-phase transition.…”

“…These studies, including WT1 peptide and WT1-targeted dendritic cell vaccine trials, have unequivocally established that CD8 þ T-cell immune responses specific to WT1 can be induced by active immunization in a substantial number of AML patients. 14 In those patients, multiple WT1 CTL epitopes have been recognized as immunogenic (including WT1 [37][38][39][40][41][42][43][44][45] , WT1 [126][127][128][129][130][131][132][133][134] , WT1 [187][188][189][190][191][192][193][194][195] and WT1 [235][236][237][238][239][240][241][242][243] ), demonstrating the excellent in vivo immunostimulatory potential of WT1 (Table 1). 14,72 This is further supported by the capacity to induce WT1-specific CD4 þ helper T-cell immunity in patients with AML through active immunization.…”

“…87 These findings were corroborated by the later work of Rezvani et al, 73 who observed spontaneous T-cell responses to PR1 and WT1 [126][127][128][129][130][131][132][133][134] in two out of eight patients before immunization with a combined PR1/WT1 peptide vaccine. 73 In a separate study, the same group found that such CTL responses were not limited to the immunodominant WT1 [126][127][128][129][130][131][132][133][134] epitope, but could also be directed against other HLA-A*0201-restricted WT1-derived epitopes (WT1 [37][38][39][40][41][42][43][44][45] , WT1 [187][188][189][190][191][192][193][194][195] and WT1 [235][236][237][238][239][240][241][242][243] ). 88 A similar observation was made regarding PRAME, which is known to contain at least four different HLA-A*0201-restricted epitopes that can be naturally recognized (PRA …”

BMI-1 is important in bufalin-induced apoptosis of K562 cells

“…Leukemia stem and progenitor cells lacking BMI-1 eventually undergo proliferation arrest and exhibit signs of differentiation and apoptosis, leading to transplant failure of the leukemia (20). Rizo et al (68) suggested that repression of BMI-1 in cord blood CD34 + cells impaired their long-term expansion and progenitor-forming capacity, in cytokine-driven liquid cultures and in bone marrow stromal co-cultures. In addition, the long-term culture-initiating cell frequencies were markedly decreased upon knockdown of BMI-1, indicating an impaired maintenance of stem and progenitor cells.…”