IGF-1R Inhibition Activates a YES/SFK Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase in Rhabdomyosarcoma

Abstract

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition-induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo. Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo. This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

Figure 1

(A and B) cDNA expression profile shows expression levels of IGF-1, IGF-2, IGF-1R, and SFKs in human RMS (ERMS, embryonal RMS, ARMS, and alveolar RMS) cell lines [C], xenograft tumors of human cell lines [X], and human tumor tissue [T]. For comparison, non-RMS tumors and normal human skeletal muscle are shown. The Affy ID refers to specific probes used in U133 plus 2.0 Arrays and are designed to provide multiple probes for the same gene. The scale is median centered (in log₂) with a saturation threshold of + 2 (red; four-fold higher) to − 2 (green; four-fold lower). (C) By quantitative PCR, YES1 RNA expression in RMS cell lines was higher compared to expression in normal human skeletal muscle (NSM). YES and GADPH SQ values were generated with Bio-RAD CFX software and normalized to GADPH. (D) YES expression and SFK phosphorylation were analyzed by Western blot analysis in RMS cell lines.

Figure 2

IGF-1R inhibition is associated with a compensatory increase in YES/SFK activation. (A and B) Cells were treated with single-agent R1507, BMS-754807, or dasatinib or in combination using dasatinib with R1507/BMS-754807 at 24 (A) or 4 hours (B). Both single-agent R1507 and BMS-754807 induced an increase of SFK phosphorylation at 24-hour treatment (A) and 4-hour treatment (B) by Western blot analysis. (C and D) IGF-1R inhibition–induced up-regulation of SFK activation is specifically associated with YES. Protein lysates from Rh30 and RD cells with or without R1507 treatment at indicated times were immunoprecipitated with normal IgG, SRC, and YES antibodies overnight. These blots were probed with mouse anti-phosphotyrosine, SRC, or YES antibodies (C). (D) The phosphotyrosine intensities from C were scanned and analyzed by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). (E) The levels of cleaved caspase-3 and PARP were significantly increased with combination IGF-1R and SFK inhibition. Apoptosis was determined by cleaved caspase-3 and PARP. Western blot analysis demonstrates a significant increase in cleaved caspase-3 and PARP at 24 hours of combination treatment compared to single-agent treatment.

Figure 3

Effects of dual inhibition of IGF-1R and SFK on RMS cell growth in vitro. (A and B) Both RD and Rh30 cells were plated onto a 96-well plate (5000 cells per well) in regular growth medium. After 16 hours of incubation, cells were treated with single agent or combined agents as indicated in the figures for 48 hours (*P < .0001; 10 μM AZD0530 + R1507 vs R1507 or 10 μM AZD0530 alone in both Rh30 and RD cell lines) (A) or 72 hours (*P < .0001; 10 μM AZD0530 + h7C10/R1507 vs 10 μM AZD0530 or R1507 alone in Rh30 cell line; **P < .0005; 1 μM AZD0530 + h7C10/R1507 vs 1 μM AZD0530 or R1507 alone in Rh30 cell line; ***P < .05-.001; 1 μM AZD0530/10 μM AZD0530 + h7C10/R1507 vs 1 μM AZD0530/10 μM AZD0530 alone in RD cell line) (B). (C) Cells were treated with dasatinib alone at a range of concentrations from 0.01 to 10 μM or in combination with R1507 at a fixed concentration (100 nM) for 72 hours. Cell growth was measured by MTT (*P < .0002-.0001; 10 nM-10 μM dasatinib + R1507 vs 10 nM-10 μM dasatinib alone in both Rh30 and RD cell lines. (D) The combination of dasatinib and BMS-754807, a small-molecule inhibitor of IGF-1R/IR, results in more potent inhibition of RMS cell growth than does either agent alone. Cells were treated with single agent alone or combined agents for 72 hours as indicated. Images were taken 4 hours after incubation with MTT.

Figure 4

Effects of dual inhibition of IGF-1R and SFK on cell growth were examined in additional RMS cell in vitro. Rh5, Rh28, and TTC442 cells were plated onto a 96-well plate and treated with single-agent R1507 (100 nM), BMS-754807 (1 μM), or dasatinib (100 nM) or in combination using dasatinib (100 nM) with R1507 (100 nM)/BMS-754807 (1 μM). These cells were monitored and recorded with the IncuCyte System. Arrow indicates beginning of treatment.

Figure 5

Dual IGF-1R and SFK blockade inhibit RMS xenograft growth. (A and D) Mice were injected with 3 × 10⁶ RD cells per mouse into the gastrocnemius muscle of the left hind leg. Treatment with agents began when tumor was palpable (day 14) in Rh30 tumor–bearing mice (A) and 11 days following injection of cells in RD tumor–bearing mice (B). Dasatinib was given by oral gavage at 100 mg/kg daily × 5. R1507 was given IP at 6 mg/kg twice a week. Tumor growth was measured twice per week, and tumor volume was determined using the formula (D × d²/6 × 3.14). (B and C) Measured tumor volume of each of the individual mice bearing Rh30 xenografts shown on day 45 (B) and day 69 (C) post-injection. (E and F) Measured tumor volume of each of the individual mice bearing RD xenografts shown on day 71 (B) and day 90 (C) post-injection. (G and H) SFK phosphorylation was analyzed by Western blot in RD (G) and Rh30 (H) xenograft tumor samples.