Establishment of novel cell lines recapitulating the genetic landscape of uveal melanoma and preclinical validation of mTOR as a therapeutic target

Abstract

Uveal melanoma (UM) is the most common primary tumor of the eye in adults. There is no standard adjuvant treatment to prevent metastasis and no effective therapy in the metastatic setting. We have established a unique panel of 7 UM cell lines from either patient's tumors or patient-derived tumor xenografts (PDXs). This panel recapitulates the molecular landscape of the disease in terms of genetic alterations and mutations. All the cell lines display GNAQ or GNA11 activating mutations, and importantly four of them display BAP1 (BRCA1 associated protein-1) deficiency, a hallmark of aggressive disease. The mTOR pathway was shown to be activated in most of the cell lines independent of AKT signaling. mTOR inhibitor Everolimus reduced the viability of UM cell lines and significantly delayed tumor growth in 4 PDXs. Our data suggest that mTOR inhibition with Everolimus, possibly in combination with other agents, may be considered as a therapeutic option for the management of uveal melanoma.

Keywords: BAP1; Cell lines; Everolimus; Patients-derived tumor xenografts; Uveal melanoma; mTOR.

Figure 1

Morphological analysis of established uveal melanoma cell lines. Light microscopy image of UM cell lines showing predominant epithelioid (MP41) spindle (MP38; MP65) or mixed morphology (MM28; MM33; MP46; MM66). MM28 (A), MM33 (B), MP38 (C), MP41 (D), MP46 (E), MP65 (F), MM66 (G).

Figure 2

Western blot analysis of BAP1 protein expression in UM cell lines. Immunostaining on cell lines MM33, MP41 and MM66 reveals presence of the protein BAP1 while MP28, MP38, MP46 and MP65 show loss of BAP1 protein expression.

Figure 3

Analysis of mTOR and AKT signaling pathway in UM cell. UM cell lines were cultured for 24 h at different serum concentrations. P(Ser473)‐AKT, P(Thr308)‐AKT, AKT, P(Ser235/236)‐S6, S6 and B‐Actin were evaluated on cellular lysates by Western blot analysis.

Figure 4

Sensitivity of a representative panel of uveal melanoma cell lines to mTOR inhibitor Everolimus and effect of Everolimus on UM cell lines viability. A. UM cell lines were treated for 24 h with different concentrations of Everolimus and P(Ser235/236)‐S6, S6 and B‐Actin assessed by Western blot analysis. B. MM28 (GNAQ 11 mutated, BAP1 deficient) MP38 (GNAQ mutated, BAP1 deficient), MP41 (GNA11 mutated), MP46 (GNAQ mutated, BAP1 deficient) MP65 (GNA11 mutated, BAP1 deficient), MM66 (GNA11 mutated), 92.1 (GNAQ mutated, EIF1AX mutated), Mel202 (GNAQ mutated, SF3B1 mutated), OMM1 (GNA11 mutated), OMM2.5 (GNAQ mutated) were seeded at adequate concentration and incubated with the drugs for 5 days. Cell viability was quantified with the MTT assay. Results are expressed as the mean of at least 3 separate experiments. Error bars represent standard errors of the mean.

Figure 5

Effects of mTOR inhibitor Everolimus in the growth of four UM PDXs in vivo. Growth curves of four human uveal melanoma xenografts: MP46 (A), MP55 (B), MP34(C), and MP41(D), treated with Everolimus (Δ) per os at 2 mg/kg 3 times a week, or receiving vehicle (■) with the same schedule as the treated animals for 4 (MP46, MP55, MP34) to 6 (MP41) weeks. Tumor volume and RTV were calculated as described in Materials and Methods. Growth curves were obtained by plotting mean RTV against time. Bars, SD. For the treated groups n = 6–8 mice; for the control groups n = 8–10 mice. P values calculated at the end of the treatment were <0.05 for the four models.

Figure 6

Effect of the combination of MEK inhibitor Trametinib and mTOR inhibitor Everolimus on the viability of a panel of 10 UM cell lines. Cell lines were treated at the indicated doses of inhibitors for 5 days and cell viability was determined by MTT as described in Material and Methods. Drug concentration is expressed as Molarity; Drug concentration in (C) is expressed as sum of the concentration of each drug. A and B: single drug curves for Everolimus and Trametinib, C: combination. Drug concentrations for the combination had been selected maintaining a constant ratio between the two drugs in order to facilitate synergy evaluation.