Evidence for triple killing mechanism of obatoclax in primary infant ALL cells from flow cytometry, Western blot analyses, and MTT assays with cell death inhibitors. Molecular cytogenetic subtype and 72-hour obatoclax EC₅₀s determined from surviving fraction plots in Figure 1 for each case are shown. In cases in (A-D), all 3 types of assays were performed; in 2 cases in (E), apoptosis and autophagy proteins were studied by Western blot. In the other MLL-R case in (C), AF4, AF9, and ENL partner genes were excluded.¹⁶ (A-D; left) show contour plots of FSC vs SSC in cells treated for indicated times at respective 72-hour obatoclax EC₇₅ determined from Figure 1. Progressive decrease in FSC signal in all 4 cases indicates apoptosis. (A-D; middle) and (E) are Western blot analyses of apoptosis and autophagy. Note increases in cleaved PARP in all 6 cases in response to obatoclax consistent with apoptosis. Also note increases in LC3-II in all 6 cases in response to obatoclax (A-E); this occurs without increase in p62 in 4 cases in (A, D, E), indicating autophagy with autophagic flux. In MLL-AF4 case in (B), note increase in p62 at 24 hours but decrease by 48 hours after obatoclax treatment, suggesting that autophagy is not blocked. Also note increase in p62 at 48 hours in the other MLL-R case (C, middle panel), indicating either p62 induction or accumulation, the former of which is supported by gene expression profiling data in SEM-K2 (supplemental Table 2). (A-D; right) Surviving fraction plots for MTT assays 72 hours after obatoclax treatment at increasing concentrations by itself or with 50 μM Nec-1, 20 μM zVAD-fmk, or 0.5 mM 3-MA alone or altogether. Inhibitors were used at concentrations determined minimally toxic in cell lines as well as primary cases (supplemental Figure 1), rather than at case-by-case titrated concentrations, and were tested less extensively in obatoclax combinations. Data were normalized to respective primary samples only treated with relevant inhibitors alone or with each other (supplemental Figure 1B). Note inhibition of obatoclax-induced death by Nec-1 in both MLL-AF4 cases (A, B; right) and reduced obatoclax-induced death when all 3 pathways were inhibited (A; right).