BTK signaling intersects with the amino acid stress response pathway. (A) Fold induction of fragments per kilobase million (FPKM) values of RNA expression of genes upregulated (upper panel) or downregulated (lower panel) in response to ASNase treatment while suppressed (upper panel) or induced (lower panel) when combined with ibrutinib. RNA expression in Nalm6 and Sem cells treated with ASNase, ibrutinib, or a combination of both was determined by RNA sequencing. (B) Upstream regulators of gene products identified in panel (A) determined by Ingenuity software. (C) Protein expression in Nalm6 and Sem cells treated with ASNase, ibrutinib, or a combination of both as determined by RPPA. Quantified protein expression levels from treated samples were normalized relative to untreated samples and subjected to principal component analysis. The top 50 proteins that contributed most to the difference between the treatments (PC1) were selected, and unsupervised hierarchical clustering with Ward’s linkage was applied. (D) Immunoblot analysis of protein expression. Nalm6 and Sem cells were treated for 72 hours (Sem) or 96 hours (Nalm6) with indicated doses of ASNase and ibrutinib. Representative of 3 independent experiments is shown. (E) Cell death determined by quantification of cells positive for amine-reactive dyes using flow cytometry. Nalm6 BTK KO control cells or cells made to express an ASNS transgene were treated with indicated doses of ASNase. Each bar represents a mean of 3 independent experiments. ***P < .001; **P < .01; ***P < .05 (2-tailed, unpaired Student t test). (F) Apoptosis induction, measured by immunoblot analysis of PARP in Nalm6 BTK KO control cells or cells with ASNS overexpression. Cells were treated with indicated doses of ASNase. A representative of 3 independent experiments is shown.