A systems biology understanding of the synergistic effects of arsenic sulfide and Imatinib in BCR/ABL-associated leukemia

Abstract

In this study, we show that combined use of Imatinib (IM) and arsenic sulfide [As(4)S(4) (AS)] exerts more profound therapeutic effects in a BCR/ABL-positive mouse model of chronic myeloid leukemia (CML) than either drug as a single agent. A systematic analysis of dynamic changes of the proteome, phosphoproteome, and transcriptome in K562 cells after AS and/or IM treatment was performed to address the mechanisms underlying this synergy. Our data indicate that AS promotes the activities of the unfolded protein reaction (UPR) and ubiquitination pathway, which could form the biochemical basis for the pharmacological effects of this compound. In this CML model, AS targets BCR/ABL through the ubiquitination of key lysine residues, leading to its proteasomal degradation, whereas IM inhibits the PI3K/AKT/mTOR pathway. Combination of the 2 agents synergistically arrests the cell cycle, decreases activity of BCR/ABL, and leads to activation of intrinsic and extrinsic apoptosis pathways through complex modifications to both transcription and protein levels. Thus, these results suggest potential clinical benefits of IM/AS combination therapy for human CML.

Fig. 1.

Efficacy of AS/IM in a CML mouse model. (A) GFP-positive cells and white blood cell (WBC) counts in peripheral blood (PB) were measured 2 weeks after drug treatment. P values were labeled on the figures. (B) Kaplan–Meier survival analysis for each group.

Fig. 2.

Dynamic changes in the proteome, phosphoproteome, and transcriptome profiles of K562 cells treated with AS and/or IM. (A and B Left) The 2D gel stained by Deep purple or Pro-Q of K562 cells treated by AS and/or IM for 24 or 4 h were shown as representatives, respectively. The protein spots indicated with arrows in the boxed regions are representatives of differentially expressed proteins. (C and D) The SOM outputs of proteome (C), phosphoproteome (B Right), and transcriptome (D) expression data of all treatment series were visually shown. Each presentation illustrates a sample-specific, proteome-wide or transcriptome-wide proteins/genes regulation map. Color-coding index stands for log-transformed (base 2) ratios.

Fig. 3.

Changes in gene/protein expression. (A–C) Functional feature of each protein/gene was determined based on detailed literature reading and GO categories. All of the relevant proteins/genes are grouped by hierarchical clustering based on expression values (log2 ratios) across all of the samples. Log2 ratios are color coded as indicated. (D and E) Western blot analysis was performed with different antibodies (see Materials and Methods). (F) BM cells of CML mice treated with IM, AS, and IM plus AS for 2 weeks were harvested and lysed. Western blot analysis was performed with antibodies to PDIA3, ATF6, and USP5.

Fig. 4.

Schematic representing synergic/additive effects of AS and IM. Genes/proteins modulated in different treatment groups are marked as indicated above the figure. Intracellular compartments in which molecular events occur are also indicated.

Fig. 5.

Effects of IM and/or AS on BCR/ABL. (A) PTK activity was analyzed in K562 cells after treatment with AS and/or IM for 4, 12, 24, and 48 h (n = 3). One-sided paired t test is used for statistical analysis (★, P < 0.05 versus control; ☆, P < 0.01 versus control; ▲, P < 0.05 versus IM, and AS groups.). (B) (Upper) Western blot analysis using an anti-ubiquitin antibody. (Lower) K562 cells were treated as described in Materials and Methods. Then, cell lysates were immunoprecipitated against BCR and Western blot against polyubiquitin (Upper) and c-ABL (Lower). (C) The product ions spectrum of the precursor ion 1427.8 Da from the digestion product of immunoprecipitation. (D and E) The protein crystallographic structures of BCR/ABL from 1 to 67 and 925 to 2,031 aa indicate the K39 and K1990 of BCR/ABL exposed outside.