Acute myeloid leukemia (AML) is a heterogeneous disease of the hematopoietic progenitor cell driven by the subsequent acquisition of genetic alterations. Approximately 20% of AML patients show strong expression of CD56 (neural cell adhesion molecule; NCAM). Expression of NCAM is associated with poor overall survival; however, the functional role of aberrant NCAM expression has not been investigated to date. The goal of this study is to examine the biological role of NCAM in AML and to explore whether NCAM represents a potential therapeutic target.
In order to evaluate the clinical significance of elevated NCAM expression in AML, we screened a panel of human cell lines for CD56 expression. Most cell lines were positive and cell surface expression correlated with mRNA levels. Knockdown of NCAM with three different doxycycline-inducible shRNAs suppressed cell growth and MTT activity in all positive cell lines. Propidium iodide staining demonstrated an increase in G1 arrest. Importantly, there was a marked apoptosis after NCAM suppression and this effect was proportional to the knockdown efficiency. Survival of NOD-SCIDgamma chain mice in a leukemia engraftment model was significantly prolonged upon NCAM knockdown. Suppression of NCAM sensitized leukemic blasts to treatment with Ara-C or Daunorubicin in vitro and in xenotransplantation experiments.
To test the consequences of NCAM overexpression in negative leukemic cell lines we transduced the NCAM transcript from Nomo-1 into HL60 and K562 cells. HL60 cells had a significantly lower sensitivity towards Ara-C or Daunorubicin treatment. IC50 for the BCR-ABL inhibitor Dasatinib in K562 cells increased from 0.95 nM (EV, empty vector) to 2.2 nM in NCAM overexpressing cells.
To dissect possible upstream regulation mechanisms of NCAM expression we performed DNAseI hypersensitivity assays coupled to qRT-PCR mapping of known putative sites in the NCAM promoter and observed open chromatin for the binding sites of Meis1, Mef2 and Stat1. shRNA-mediated knockdown of MEIS1, MEF2c and MLL-AF9 resulted in significant suppression of NCAM cell surface expression, suggesting an upstream regulatory role for MLL-AF9.
To gain insights into the mechanisms underlying the NCAM function in AML we performed gene expression comparisons of the 30 highest versus 30 lowest expressing samples in the GSE8043 dataset. Fifty-seven Biocharta pathways were differentially expressed between NCAMhigh and NCAMlow samples, while expression changes predicted abnormal cell-cycle regulation, stress and DNA damage response, cell survival, renewal and adhesion. Western blot, protein array and qRT-PCR analyses of candidate downstream signaling pathways upon knockdown of NCAM demonstrated enhanced degradation of BetaCatenin, decreased expression of BCL-2 and increased levels of p21 and p27.
The upstream regulation mechanism described above revealed MLL-AF9 (M/A9) as a top candidate for NCAM regulation. Subsequent analysis of M/A9 L-GMPs (Lin- cKit+ CD34+ FcgR+) demonstrated strong surface expression of NCAM, whereas normal HSCs (Lin-cKit+ Sca1+) were NCAM-negative. This could be validated by gene expression analyses of M/A9 L-GMPs compared with normal HSCs.
In order to elucidate the role of NCAM on leukemic cell function in a mouse model, NCAM-/- and control wildtype (WT) bone marrow cells were transformed with a retroviral construct of M/A9 and transplanted into lethally irradiated littermates. Recipients of NCAM-/- M/A9 cells developed acute leukemia with prolonged disease latency. NCAM-/- M/A9 cells had lower CD117 and Gr-1 expression, but higher expression of Mac-1 and, in some samples, aberrant B220 co-expression. Importantly, there was a reduced representation of L-GMPs in the NCAM-/- M/A9 group and limited dilution retransplantation assays revealed a significantly prolonged survival of NCAM-/- M/A9 mice. Replating activity in methylcellulose was diminished and could be eradicated with sublethal doses of Cytarabine.
Targeting aberrant expression of NCAM demonstrated strong antileukemic activity in vitro and sensitized leukemic blasts to genotoxic stress. In vivo, depletion of NCAM resulted in prolonged disease survival in syngeneic and xenotransplantation experiments and diminished self-renewal capacities. Our data suggest that NCAM represents a promising therapeutic strategy and likely targets AML cells at the LSC level.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.