Loss of BTK potentiates ASNase-induced apoptosis by repression of GCN2 activity mediated by c-Myc. (A) Upstream regulators of genes differentially expressed in Nalm6 and Sem treated with ASNase alone vs treatment with ASNase and ibrutinib, determined by Ingenuity software. (B) Immunoblot analysis of c-Myc protein expression. Nalm6 and Sem cells were treated for 72 hours (Sem) or 96 hours (Nalm6) with indicated doses of ASNase and ibrutinib. (C) Cell death determined by quantification of cells positive for amine-reactive dyes using flow cytometry. Nalm6 and Sem cells were treated with indicated doses of JQ1 and/or ASNase for 3 days (Sem) or 6 days (Nalm6). Each bar represents a mean of 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test). (D) Immunoblot analysis of PARP, ATF4, ASNS, and actin. Nalm6 and Sem cells were treated for 72 hours with indicated doses of ASNase and JQ1. (E) Working model explaining the synergistic interaction of BTK inhibition and ASNase treatment. In response to ASNase treatment, cells upregulate the AAR pathway via GCN2 to adapt to nutrient stress. BTK inhibition renders cells incapable of activating c-Myc, thus preventing activation of the GCN2-ATF4 axis. As a consequence, cells cannot mount an appropriate amino acid stress response and eventually die.