Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody

Abstract

Background: Alterations in the PI3K/Akt pathway are found in a wide range of cancers and the development of PI3K inhibitors represents a promising approach to cancer therapy. Constitutive PI3K activation, reflecting an intrinsic oncogenic deregulation of primary blast cells, is detected in 50% of patients with acute myeloid leukemia. However, the mechanisms leading to this activation are currently unknown. As we previously reported IGF-1 autocriny in acute myeloid leukemia cells, we investigated whether IGF-1 signaling was involved in the constitutive activation of PI3K.

Design and methods: We analyzed the IGF-1/IGF-1R signaling pathway and PI3K activity in 40 acute myeloid leukemia bone marrow samples. Specific inhibition of IGF-1/IGF-1R signaling was investigated using neutralizing anti-IGF-1R, anti-IGF-1 antibodies or IGF-1 short interfering RNA. The anti-leukemic activity of the neutralizing anti-IGF-1R was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation and survival.

Results: In all samples tested, we found that functional IGF-1R was constantly expressed in leukemic cells. In the acute myeloid leukemia samples with PI3K activation, we found that the IGF-1R was constitutively phosphorylated, although no IGF-1R activating mutation was detected. Specific inhibition of IGF-1R signaling with neutralizing anti-IGF-1R strongly inhibited the constitutive phosphorylation of both IGF-1R and Akt in 70% of the PI3K activated samples. Moreover, both incubation with anti-IGF-1 antibody and IGF-1 short interfering RNA inhibited Akt phosphorylation in leukemic cells. Finally, neutralizing anti-IGF-1R treatment decreased the clonogenicity of leukemic progenitors and the proliferation of PI3K activated acute myeloid leukemia cells.

Conclusions: Our current data indicate a critical role for IGF-1 autocriny in constitutive PI3K/Akt activation in primary acute myeloid leukemia cells and provide a strong rationale for targeting IGF-1R as a potential new therapy for this disease.

Figure 1.

The IGF-1/IGF-1R pathway is functional and constitutively activated in PI3K⁺ AML blast cells. (A) Expression of IGF-1R in total cell lysates from six AML samples was compared with that in normal CD34⁺ hematopoietic progenitors. Protein extracts from 10⁶ cells were analyzed by western blotting. (B) After purification, PI3K⁺ or (C) PI3K⁻ AML cells were starved for 4 h in cytokine and serum-free medium and then stimulated or not with 50 ng/mL IGF-1 for 10 min. Protein extracts from 10⁶ cells were analyzed by western blotting.

Figure 2.

Specific inhibition of IGF-1R signaling with αIR3. (A) PI3K-AML blast cells were cultured for 4 h in cytokine- and serum-free medium with or without αIR3 (5 μg/mL), mouse IgG1 (5 μg/mL) for isotype negative control or NVP-AEW541 (1 μM) during the last hour. AML blast cells were then stimulated for 10 min with either IGF-1 (50 ng/mL), FLT3-L (50 ng/mL) or SCF (30 ng/mL). Protein extracts from 10⁶ cells were then analyzed by western blotting. (B) Phospho-Akt S⁴⁷³ signal intensity was quantified and normalized to Akt signal intensity in three PI3K⁻ AML samples. Results are expressed as a ratio to the control incubation without αIR3 or NVP-AEW541. Vertical bars indicate standard deviations. The statistical significance was calculated by Student’s t test. **P<0.01, ***P<0.001, ns: not significant.

Figure 3.

PI3K constitutive activation is due to IGF-1/IGF-1R interaction in 70% of AML. After purification, AML blast cells were cultured for 4 h in cytokine- and serum-free medium. (A) During the last hour of starvation, cells were treated with or without αIR3 (5μg/mL) and (C) stimulated or not with IGF-1 (50 ng/mL). Protein extracts from 10⁶ cells were analyzed by western blotting. (B) Phospho-Akt S473 and phospho-IGF-1R (Y1135/1136) were quantified and normalized to Akt and IGF-1R signal intensity in 14 and 6 PI3K⁺ samples, respectively. Results are expressed as a ratio to the control incubation without αIR3. Vertical bars indicate standard deviations. ***P<0.001.

Figure 4.

Autocrine IGF-1 production is responsible for PI3K constitutive activation in AML samples. (A) During the last hour of starvation, blast cells from three AML samples were treated with or without an anti-human IGF-1 antibody (50 μg/mL). Protein extracts from 10⁶ cells were analyzed by western blotting and phospho-Akt S⁴⁷³ signal intensity was quantified as described in Figure 3B. ***P<0.001. (B) AML blast cells from five patients were transfected with IGF-1 siRNA or control siRNA, then incubated for 24 h in 10% FCS MEM. IGF-1 mRNA expression was assessed by quantitative RT-PCR and results are expressed as a ratio to the incubation with control siRNA. Protein extracts from 10⁶ cells were analyzed by western blotting and phospho-Akt S⁴⁷³ signal intensity was quantified as described in Figure 3B. **P<0.01; ***P<0.001.

Figure 5.

Inhibition of IGF-1 signaling with αIR3 impairs the clonogenic growth of leukemic progenitors and the cell cycle progression of PI3K⁺ AML blast cells. (A) Blast cells from three PI3K⁺ AML samples were cultured at 10⁵ cells/mL in H4230 methyl cellulose medium, supplemented with 10% 5637 conditioned medium with or without the different inhibitors: αIR3 (5 μg/mL), LY2942002 (25 μM). Cells were then plated in 35-mm Petri dishes in duplicate, and incubated for 7 days. At day 7, CFU-L were scored under an inverted microscope. Upper panel: the picture illustrates the results obtained for sample #33. Lower panel: results are expressed as a percentage of the control for each condition. Vertical bars indicate standard deviations. **P<0.01; ***P<0.001. (B) For proliferation assays, AML blast cells from three PI3K⁺ and three PI3K⁻ samples were cultured for 48 h at 10⁵ cells/mL, in triplicate, in 5% FCS, without or with αIR3 (5 μg/mL) or LY2942002 (25 μM), and then pulsed for 6 h with 1 μCi (37 kBq) [³H]thymidine. The amounts of radioactivity were determined after trichloracetic acid precipitation. Results are expressed as a ratio to the control, for each condition. Vertical bars indicate standard deviations. ***P<0.001; ns: not significant (C) AML cells from two PI3K⁺ AML samples were incubated at 10⁶/mL in FCS 10%, without or with αIR3 for 24 h. Protein extracts from 10⁶ cells were analyzed by western blotting. (D) AML blast cells from seven PI3K⁺ and four PI3K⁻ samples were incubated at 5×10⁵ cells/mL for 48 h in 10% FCS, without or with αIR3 (5 μg/mL) and then stained with annexin V-phycoerythrin. Results are expressed as a percentage of annexin-V stained cells. Vertical bars indicate standard deviations and “ns” means that the difference is not significant.