NCAM1 expression confers a drug-resistance phenotype. (A) Cell lines were exposed to Dox for 4 days and different concentrations of Ara-C (0.1- 8 µM) for 48 hours. Cell proliferation was assessed by MTT absorbance at day 4. Error bars represent mean of 3 measurements ± SD of mean. (B) MOLM-14 cells were exposed to Dox for 4 days and either DMSO (vehicle), 25 nM PKC412, Ara-C (1 μM) or daunorubicin (DNR; 30 nM) for 48 hours. Cell death was assessed by flow cytometry–based measurement of the propidium iodide–positive population and compared by 2-tailed Student t test. Error bars represent mean of 3 measurements ± SD of mean. (C-E) The NCAM1⁻ cell lines K562 and HL60 were transduced with lentiviral particles either expressing the NCAM1_140 isoform (NCAM_140) or empty vector (EV) control. (C) Expression of NCAM1 was analyzed by flow cytometry and shown as MFI values relative to IgG isotype control. HL60 cells were exposed to DMSO (vehicle), Ara-C, or daunorubicin as indicated. (D) Apoptosis was assessed by flow cytometry–based analysis of Annexin V/7-AAD double-positive population after treatment of 24 hours as indicated and compared by 2-tailed Student t test. Error bars represent mean of 3 measurements ± SD of mean. (E) Differentiation was explored upon treatment with 2 μM ATRA or vehicle (DMSO) for 48 hours. Left, CD11b cell surface expression analysis by flow cytometry; right, images of cells stained with Max-Grünwald-Giemsa (scale bar, 10 μM; the enlarged cells in the corners are 4× enhanced). Shown are representative examples of 3 independent experiments. (F) EV- or NCAM_140-overexpressing K562 cells were treated for 48 hours as indicated. Apoptosis was assessed by flow cytometry-based analysis of Annexin V/7-AAD double-positive population and compared by 2-tailed Student t test (left). Error bars represent mean of 3 measurements ± SD of mean. Right, K562 cells were treated with different concentrations of dasatinib and inhibition of proliferation was assessed by MTT absorbance. (G) NSG mice (N = 5 per group) were engrafted with 1 × 10⁶ NOMO-1 cells expressing either Scr, shNCAM_2, or shNCAM_3 shRNA clones. All mice received Dox starting on day 15, which was discontinued in living animals on day 55. NOMO-1_Scr and shNCAM3 were either treated with vehicle or Ara-C (10 mg/kg) once daily subcutaneous between day 20 and 30. Survival of mice is shown as Kaplan-Meier curves.