Supplementary Figures s3

Supplementary Figures.

Figure 1S: A) Nampt locus (human chromosome 7q22.3) copy number heatmap for lines included in the Cancer Cell Line Encyclopedia (http://www.broadinstitute.org/ccle) Color indicates degree of copy number loss (blue) or gain (red). B) mRNA Nampt expression plotted vs. DNA copy number (log2 ratio). The Pearson correlation coefficient (R) value, calculated using GraphPad Prism analysis software, is indicated.

Figure 2S: 2,5 x 104 Mec.1 cells were incubated in 96-well plates in the presence or absence of 3 nM APO866 and FK506 (ranging from 1 to 0,1 μM) or their combination. Cell death was assessed 96 h later by PI staining and flow cytometry. Results are means of triplicates ± SD.

Figure 3S: APO866 determination in primary B-CLL cell extract. Extracted ion chromatogram

(panel a) of the precursor ion (m/z 391.5). The corresponding MS spectrum is reported in panel b. The analyses were performed in the positive ion mode. The MS/MS fragmentation pattern in the spectra relative to the precursor ion at m/z 392.5 [M+H]+ obtained for the sample (panel c) and for the standard molecule (panel d) present the same ions and ratios.

Figure 4S: Primary B-CLL and AML cells were pleated in 96-well plates and incubated with 10 nM APO866, Pg-p inhibitors (CsA, Verapamil and Nilotinib), or their combination at the indicated concentrations. Cell death was assessed 96 h later by PI staining and flow cytometry.

Results are means of triplicates ± SD. CI values refer to the highest drug concentrations used.

Figure 5S: A-B) Healthy PBPCs (A) and PBMCs (B) were incubated in 96-well plates and treated with 3 nM APO866 and 1 μM CsA or 6 μM Verapamil alone or in combination where indicated. Viability was assessed 96 h later. One representative experiment out of three is shown.

The results are means ± SD of three separate experiments with three different donors. Figure 6S: A-B) 1 x 105 OCI/AML3 cells/wells were plated in 6-well plates and incubated with or without 3 nM APO866 and Pgp modulators (10 μM Verapamil, 1 μM CsA). 24 h (A) or 48 h

(B) later, cells were harvested and intracellular NAD+(H) and ATP levels were evaluated in comparison with control cells. c-e means with standard deviation of at least three independent experiments are shown. (*** 0.004

Figure 7S: relative expression of BiP, IRE1α and CHOP (WBs showed in Figure 6B) calculated by taking the ratio of densitometry signal for each protein to tubulin by using Image J software

(1.37v; National Institutes of Health, http://rsb.info.nih.gov/ij/).

Figure 8S. A) 5 x106 Mec.1 cells were plated in 24-well plates and treated with or without

APO866 and CsA in the presence or absence of 10 µM NA. Cell death was assessed 96 h later by PI staining and flow cytometry. Results are means of triplicates ± SD. B) Mec.1 cells were treated as in (A). Twenty-four hours later, cells were harvested and NAD+ levels were determined in cell extracts. Results were normalized to the values detected in untreated cells. C)

Schematic representation of a hypothetic mechanism for the synergism between APO866 and

CsA. Pgp blockade leads to preferential accumulation within leukemia cells of APO866 without affecting its ability to inhibit NAMPT. This event leads to reduced the intracellular NAD+ levels and ultimately to cell death by promoting both mitochondrial dysfunction and ER stress.