Synergistic action of dual IGF1/R and MEK inhibition sensitizes childhood acute lymphoblastic leukemia (ALL) cells to cytotoxic agents and involves downregulation of STAT6 and PDAP1

Abstract

Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024 + U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024 + U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024 + U0126 in samples with variable basal levels of phosphorylated IGF1/Rβ and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 "synergy genes" differentially targeted by IGF1/R + MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analysesrevealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key "synergy" genes, supporting their predicted role in an STAT6-ERK-nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.

Figure1

The combined inhibition of IGF1/R + MEK pathways by AG1024 + U0126 sensitizes ALL cells to cytotoxic agents. (A) Representative scatter plots of Annexin/PI staining in ALL-141 are shown. The average total percentage of apoptotic cells (Ann+/PI+, quadrant 2) induced by each treatment is shown, as well as the average IR-induced percentage of apoptosis (shown in parentheses), which were determined by subtracting the background apoptosis from the IR plots. (B) The combination of AG1024 + U0126 (shaded) consistently sensitizes ALL cells to IR-induced apoptosis regardless of the effect of AG1024 and U0126 as single agents in four cases (ALL-111, ALL-102, ALL-106, and ALL-141) after 24hours of treatment, as determined by Annexin V/PI staining. (The maximum concentration of DMSO reached in this experiment was 0.6% v/v DMSO.) CIs could be determined from individual drug dose curves (Supplementary Figure E3, online only, available at www.exphem.org) for ALL-102 (++, moderate synergism), ALL-106 (+, nearly additive), and ALL-141 (+++, synergism). For ALL-111, the PE of the combination of AG1024 + U0126 was determined using unpaired t test analysis as described (***, strong synergism) (Supplementary Table E2, online only, available at www.exphem.org). (C) AG1024 and U0126 together sensitize ALL (S025117, S032957, S026767, ALL-202, ALL-201R, ALL-211, ALL-203, and ALL-212) to 100nmol/L dexamethasone (top) 100nmol/L vincristine (middle), and 10nmol/L daunorubicin (bottom) after 72hours of treatment. (The maximum DMSO concentration reached was 0.2% v/v DMSO.) The PE of AG1024 + U0126 with each drug was determined using paired t test analysis. **p ≤ 0.005 indicates moderate synergism and *p ≤ 0.05 indicates synergism.

Figure2

The synergistic effect of combined inhibition of IGF1/R + MEK pathways by AG1024 + U0126 induces a distinct “synergy” gene signature. (A) In the absence of cytotoxic agents, primary ALL samples (n = 10) display synergistic sensitivity (PE = ***) to treatment with both AG0124 + U0126 after 72hours. (The maximum DMSO concentration reached was 0.2% v/v DMSO.) (B) Western blot analysis revealing samples displaying synergism to AG1024 + U0126 and variable basal MEK and IGF1/R pathway activity, as indicated by phosphorylation of IGF1/Rβ + IRβ and ERK1/2, suggesting that additional mechanisms underlie the synergistic effect. (C) Heat map showing differential expression of 32 genes (“synergy” signature) induced by 6hours of treatment with AG1024 + U0126 compared with the respective individual inhibitors + IR, IR alone, and untreated cells in ALL-102, ALL-141, ALL-150, and ALL-200. *p ≤ 0.05 indicates synergism; ***p ≤ 0.0005 indicates strong synergism.

Figure3

Predicted interactions of “synergy” genes STAT6 and PDAP1. (A) Heat map showing that STAT6 and PDAP1 are highly differentially downregulated by AG1204 + U0126. (B) Pearson correlation coefficient analyses from 20 microarrays indicating that STAT6 and PDAP1 expression are highly codependent. (C)STAT6 and PDAP1 display common patterns of expression c-dependency with specific “synergy” genes highlighted in bold text. (Pearson coefficient r values: below 0.4 = weak; 0.4–0.6 = modest; above 0.6 = marked codependency). (D) Ingenuity network analysis predicting interaction of PDAP1 and STAT6 in a STAT6–ERK–NF-κB regulatory network seeded with “synergy” genes that might be involved in apoptosis resistance (green = downregulated; red = upregulated). *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005.

Figure4

Cellular consequences of siRNA-mediated knockdown of synergy genes. (A) Western blot showing siRNA mediated knockdown of STAT6 and PDAP1 in HeLa cells. (B) siRNA-mediated knockdown of STAT6 results in a significant loss in cell viability, whereas PDAP1 knockdown has no impact after 1 week. (C) siRNA-mediated knockdown of STAT6 causes a 20% increase in cells in the G₁ phase of the cell cycle, indicating decreased proliferation, whereas PDAP1 knockdown has no impact on the cell cycle in HeLa cells. (D) Effect on loss of cell viability by STAT6 knockdown leads to an additive effect with daunorubicin after 72hours of treatment, whereas PDAP1 knockdown is synergistic with daunorubicin at more than one dose in HeLa cells. *p ≤ 0.05; **p ≤ 0.005.

Supplementary Figure E1

Graphs show minimal effect on survival of cells from 5 representative primary ALL samples following 24h and 72h treatment with doses of DMSO (drug vehicle) reflecting those doses reached within the experimental data (<0.6% DMSO). In contrast, 1% DMSO did cause a cytotoxic effect after 72h.

Supplementary Figure E2

Western blot analysis comparing caspase-dependent apoptosis induced by AG1024+U0126 with the single most potent single inhibitor 8h following treatment in two representative ALL (ALL-111 and ALL-141). In ALL-111, which exhibited equal sensitivity to LY294002 and U0126+AG1024, caspase and PARP cleavage were comparable following 8h treatment. In the completely IR-resistant leukaemia, ALL-141, caspase and PARP1 cleavage was significantly induced by treatment with U0126+AG1024 compared with AG1024 alone, the only single inhibitor with some effect.

Supplementary Figure E3

Graphs show dose-curves for U0126 and for AG1024 for four ALLs which were used for Calcusyn analysis to determine the effect of combined U0126+AG1024 treatment.

Supplementary Figure E4

Graph shows absence of a combined effect of AG1024+U1026 on IR-induced apoptosis in peripheral blood mononuclear cells (PBMCs) from three healthy individuals, measured by Annexin V/PI staining and FACS analysis after 72h.

Supplementary Figure E5

Graph shows no synergism between AG1024 and U0126 at a dose of 1µM following 72h treatment in 10 primary ALL.

Supplementary Figure E6

Identification of genes contributing to AG1024+U0126 synergism. Venn analysis identifying ‘synergy’ genes shows 26 genes are differentially down-regulated (left) and 6 genes are differentially upregulated (right) by treatment of ALL cells with AG1024+U0126+IR compared with AG1024+IR and U0126+IR in samples ALL-102, ALL-141, ALL-150 and ALL-200.

Supplementary Figure E7

Independent qRT-PCR validation of the most differentially altered ‘synergy’ genes, STAT6 and PDAP1, in ALL employed in the microarray analysis as well as others treated under the same conditions.

Supplementary Figure E8

Sensitization of HeLa cells by siRNA silencing of ‘synergy’ candidate genes, STAT6 and PDAP1. Graphs show the decrease in cell proliferation caused by STAT6 knockdown protects HeLa cells from vincristine-induce killing whereas PDAP1 knockdown has no effect compared with scrambled siRNA, following 72h treatment in vitro.