Response

We thank Cea and coauthors for sharing their results on APO866 activity in hematologic malignancies. We reported that primary cells and cell lines from patients with hematologic malignancies are highly sensitive toward APO866.1  Results were obtained from MTT (thiazolyl blue tetrazolium bromide), trypan blue dye exclusion, annexin V/7AAD stainings, clonogenic assays, and xenochimeric transplantation models. We are aware that NAD (nicotinamide adenine dinucleotide) synthesis inhibitors including APO866 potentially skew MTT assay results, the reason for which we confirmed results on cell lines by determining annexin V-7AAD expressions (Nahimana et al,1  Table 1) and in vivo cell viability assays (Nahimana et al,1  Figure 6). In addition, Cea and coworkers found similar results to ours on all cell lines except for Molt-4. Discrepancy concerning Molt-4 cell sensitivity to APO866 may be due to the time that cells are maintained in culture before testing. Indeed, we found that some cell lines (ML-2, Namalwa, and Jurkat) cultured for long period (ie, over 12 months [> 60 splittings]) developed resistance to APO866, possibly due to secondary genetic events (Figure 1A). We are currently dissecting the mechanisms of resistance of such cell lines.

Figure 1

Cytotoxic effect of APO866 in hematologic malignant cells. (A) ML-2, Namalwa, and Jurkat cells were cultured (in RPMI+10% FCS+1% P/S) after various splittings without or with 10 nM APO866. Cell death was assessed after 96 hours by flow cytometry using annexin V and 7AAD double staining. The percentage of early apoptotic cells (annexin V+ 7AAD) are shown as ▬ and that of late apoptotic cells (annexin V+ 7AAD+) are shown as ▭. Data are presented as means of triplicate + SD. (B) Primary cells from patients with various hematologic malignancies (Nahimana et al,1  Table 2, patients 1-32): Acute myeloid leukemia (AML; n = 10); acute lymphoblastic leukemia (ALL; n = 3); chronic lymphocytic leukemia (CLL; n = 12); T-large granular lymphocyte leukemia (T-LGL; n = 1); T-lymphoma (TL; n = 1); marginal zone lymphoma (ML; n = 3); mantle cell lymphoma (MCL; n = 1); and follicular lymphoma (FL; n = 1) were cultured and cell death monitored as in panel A. Specific cell death (%) induced by drug was calculated according to the formula scd = [(S-C) / (100-C)] × 100; where S = treated sample cell death and C = untreated sample cell death. (C) 0.5-1 × 106 Jurkat cells were cultured in various media as indicated on x-axis in the presence or absence of 10 nM APO866. Specific cell death calculated as mentioned above. #P indicates number of cell splittings.

Figure 1

Cytotoxic effect of APO866 in hematologic malignant cells. (A) ML-2, Namalwa, and Jurkat cells were cultured (in RPMI+10% FCS+1% P/S) after various splittings without or with 10 nM APO866. Cell death was assessed after 96 hours by flow cytometry using annexin V and 7AAD double staining. The percentage of early apoptotic cells (annexin V+ 7AAD) are shown as ▬ and that of late apoptotic cells (annexin V+ 7AAD+) are shown as ▭. Data are presented as means of triplicate + SD. (B) Primary cells from patients with various hematologic malignancies (Nahimana et al,1  Table 2, patients 1-32): Acute myeloid leukemia (AML; n = 10); acute lymphoblastic leukemia (ALL; n = 3); chronic lymphocytic leukemia (CLL; n = 12); T-large granular lymphocyte leukemia (T-LGL; n = 1); T-lymphoma (TL; n = 1); marginal zone lymphoma (ML; n = 3); mantle cell lymphoma (MCL; n = 1); and follicular lymphoma (FL; n = 1) were cultured and cell death monitored as in panel A. Specific cell death (%) induced by drug was calculated according to the formula scd = [(S-C) / (100-C)] × 100; where S = treated sample cell death and C = untreated sample cell death. (C) 0.5-1 × 106 Jurkat cells were cultured in various media as indicated on x-axis in the presence or absence of 10 nM APO866. Specific cell death calculated as mentioned above. #P indicates number of cell splittings.

Cea and colleagues found lower specific cell death on primary cells than what was published in our article (Nahimana et al,1  Table 2) and wondered whether the difference could be due to cell death method calculation. As a note, we determined specific cell killing by APO866 to avoid any bias in apoptosis and autophagy inhibition experiments (Nahimana et al,1  Figure 4 and Figure S1), and we additionally presented raw data of cell death–associated parameters for primary cells from several hematologic cancers highlighting the broad efficacy of APO866 (Nahimana et al,1 Figures 1,2). We have now expanded such determination on all primary hematologic cancer cells originally reported. Results are quite similar to fdc (fraction of dead cells; Figure 1B). Several explanations may account for the difference observed between our data and those from Cea et al. First, our patient samples used to investigate the killing effect of APO866 had cell viability of more than 85% after thawing. High spontaneous cell death may skew specific cell death rate determination. Second, we determined annexin V-7AAD expressions on gated tumoral cells using cell lineage specific labeled antibodies, information we do not know from Cea and colleagues who used samples containing up to 30% nontumoral cells. Third, we would be interested to know in which culture conditions Cea and colleagues determined APO866 killing activity, because they can influence killing efficacy of antimetabolite cytotoxic agents such as APO866. As shown in Figure 1C, we observed variability in APO866 cell killing efficacy according to cell culture. This latter point also stresses the need to validate in vitro results with in vivo data, where we found that APO866 exerts potent antitumor activities in vivo xenochimeric models of human acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and lymphoblastic lymphomas without significant toxicities to the animals.

In summary, the cell-killing effect of APO866 on primary hematologic cancer cells is not dependent on the calculation methods of cell death. The confirmation of the MTT data by other cell cytoxicity readouts demonstrates that APO866 is a potent anticancer agent in numerous hematologic malignancies.

Authorship

Contribution: A.N. designed, executed, analyzed experiments and wrote the letter; D.A. and S.B. analyzed results and wrote the paper; and M.A.D. designed and analyzed experiments and wrote the paper.

Conflict-of-interest disclosure: S.B. is employed by TopoTarget, Denmark SA. TopoTarget has licensed the worldwide development and marketing rights to APO866 from Astellas. The remaining authors declare no competing financial interests.

Correspondence: Michel A. Duchosal, MD, Service of Hematology, University Hospital of Lausanne (CHUV), 46 rue du Bugnon, 1011 Lausanne, Switzerland; e-mail: michel.duchosal@chuv.ch.

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