IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide

Abstract

Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage.

Keywords: IGF-1R; apoptosis; chemo-sensitization; double strand break; temozolomide.

Figure 1. IGF axis association with TMZ resistance and p53 status

A. Whole cell extracts analyzed by western blotting. Similar results were obtained in a second set of independent lysates. B. Cells were treated with TMZ or vehicle, after 5 days CellTiter Glo viability assays were performed and data were expressed as % viability in TMZ-untreated cultures. Graphs: pooled results from 2 independent assays (6 data points) expressed as mean ± SEM % viability. C. Correlation between TMZ GI₅₀ and upper: MGMT; lower: phospho-IGF-1R, quantified from western blots in A, corrected for actin loading. D. Serum-starved A375M cells treated with: upper, OSI-906 for 1 hr; lower: 1 μM OSI-906 for 1–72 hr, and in the final 15 min with 50 nM IGF-1. E. Cells were treated with OSI-906 or vehicle, and after 5 days viability assayed as B). F. B231 cells treated with solvent or 2 μg/ml doxycycline for 24 hr, and lysed for western blot (inset) or treated with solvent or OSI-906 as E) and viability assayed after 5 days. Graph: data from n = 3 assays; bars, SEM. GI₅₀ values were > 1 μM in p53 null cells, 116 nM in p53-positive cells.

Figure 2. IGF-1R inhibition induces MGMT-independent sensitization of BRAF WT and mutant melanoma cells to TMZ

A. Serum-starved CHL1 cells treated with: upper, AZ3801 for 1 hr; lower, 30 nM AZ12253801 for 1–72 hr, and with IGF-1 as Figure 1D. B. CHL1 and C. A375M cells treated with TMZ alone or with AZ3801 or OSI-906. Graphs: mean ± SEM % cell survival from n = 3 independent assays in each cell line. IGF-1Ri-treated cells were sensitized to TMZ (*p < 0.05, **p < 0.01, ***p < 0.001). D. Graphs: mean ± SEM caspase activity (% signal in solvent-treated controls) from n = 2 independent assays in CHL1 cells, each with triplicate samples. AZ3801 enhanced apoptosis after 24 hr (upper) and 48 hr (lower; *p < 0.05, **p < 0.01, ***p < 0.001). E. SKmel23 cells treated with solvent or 50 μM TMZ alone or with 75 nM AZ3801. Graph: mean ± SEM % survival from n = 3 assays (*p < 0.05). The sensitivity of SKmel23 to IGF-1Ri precluded testing of AZ3801 > 75 nM. F. CHL1 cells pre-treated for 2 hr with 10 μM O6BG prior to TMZ alone or with AZ3801. Graph: pooled data from n = 2 survival assays from which were derived TMZ SF₅₀ values, shown in legend. In cultures treated with 300 μM TMZ, O6BG suppressed survival of AZ3801-untreated controls from 59 ± 3% to 24 ± 4% (p < 0.001), with 50 nM AZ3801 from 34 ± 4% to 13 ± 2% ( p < 0.001), and 100 nM AZ3801 from 16 ± 4% to 2 ± 0.5% ( p < 0.05).

Figure 3. IGF-1R inhibition induces schedule-dependent sensitization to TMZ

A. A375M, B. CHL1 cells were treated with solvent, OSI-906 or TMZ. Graphs: mean ± SEM % survival from n = 2 assays, each with triplicate dishes (6 data points). Cell survival was inhibited in both cell lines by 250 μM TMZ and by OSI-906 at 300–1000 nM in A375M, and 30–1000 nM in CHL1 (*p < 0.05, ***p < 0.001). C. Treatment schedules for chemo-sensitivity testing. D. A375M cells treated simultaneously or sequentially with solvent, 30–300 nM OSI-906 and/or TMZ. Graphs: mean ± SEM % survival from n = 3 assays, showing significance of differences from control (OSI-906-untreated) cultures (*p < 0.05, **p < 0.01, ***p < 0.001). Legends: TMZ SF₅₀ (μM) at each OSI-906 concentration.

Figure 4. IGF-1R inhibition pre-TMZ induces G1 arrest and reduces DSB induction

A. Cells were treated with 300 nM OSI-906 and IGF-1 as Figure 1D. B. A375M cells treated with solvent, 300 nM OSI-906 or 300 μM TMZ. After 72 hr, cell cycle profiles were determined by analysis of BrdU incorporation vs DNA content (PI staining). C. A375M, D. CHL1 cells treated with solvent, 300 nM OSI-906, 300 μM TMZ alone or with 300 nM OSI-906 applied 24 hr before, simultaneously or 24 hr post-TMZ, and collected 72 hr post-TMZ. Graphs: mean ± SEM data from n = 4 analyses in each cell line. Compared with TMZ alone, TMZ-treated/IGF-1R-inhibited cells showed significant differences in pre-G1, G1 and non-cycling S populations of A375M, and only in the pre-G1 population of CHL1 (*p < 0.05, **p < 0.01, ***p < 0.001). E. Cultures treated as C, D were lysed 72 hr post-TMZ for western blotting. F. CHL1 cells were treated with solvent or 300 μM TMZ and after 72 hr stained for γH2AX. G. Cells were treated as C), D) and after 120 hr, 50–2300 cells per condition were analysed for γH2AX foci. Graphs: mean ± SEM % cells with > 3 foci. Pre- or co- inhibition of IGF-1R significantly reduced TMZ-induced foci in A375M (*p < 0.05, **p < 0.01 compared with TMZ alone).

Figure 5. IGF-1R inhibition sensitizes melanoma to TMZ in vivo

A. Mice bearing A375M xenografts were treated with Ora-Plus (OP, Group A, 2 mice) or 50 mg/kg TMZ in Ora-Plus by gavage days 1–5 (Group B, 8 mice). On day 6, group B was randomly divided into 4 groups of 2 for gavage on days 6–8 with 50 mg/kg OSI-906 in 25 mM tartaric acid (TA, B1), Ora-Plus (OP, B2), corn oil (CO, B3), or vehicle (Ora-Plus) alone (B4). Graph: mean ± range tumor volume (% baseline). B. Tumors harvested 4 hr after final OSI-906 dosing were stained for Ki67 and activated caspase 3. Scale bar 50 μm. C. Quantification of Ki67 (white bars) and activated caspase 3 (black bars). Ki67 counts were lower in group B4 (TMZ alone; *p < 0.05, **p < 0.01). Activated caspase 3 was detected only in tumors treated with TMZ followed by OSI-906. D. OSI-906 levels in plasma and tumors. Dotted line: steady state OSI-906 levels achieved at recommended Phase 2 dose [18]. OSI-906 levels in groups A (control) and B4 (TMZ alone) were below limits of quantitation: ~20 ng/ml (0.05 μM) for plasma, 200 ng/g (0.5 μM) for tumor. E. Mice bearing A375M xenografts were treated with Ora-Plus (control), 50 mg/kg TMZ (T), 50 mg/kg OSI-906 (O) or combination treatment, scheduling OSI-906 to avoid administration immediately before/with TMZ. Graph: mean ± SEM tumor volumes, n = 6. F. Graph: linear regression analysis of slopes of tumor growth, showing individual data points and mean ± SEM slope as % control. There were no differences in growth rates of control, TMZ or OSI-906 –treated tumors, and significant reduction in the combination group compared with each of the other groups (*p < 0.05, **p < 0.01).