Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies

Abstract

In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.

Fig. 1. CRO15 exerts a deleterious effect on melanoma cell viability.

A Chemical structure of metformin and CRO15. B–F Cell viability assay using trypan blue exclusion method on B. A375 melanoma cells treated with 5 μM CRO15 at different times. C A375 melanoma cells were treated with the indicated concentrations of CRO15 for 48 h. D Melanoma cell lines of various genotypes treated with 5 μM CRO15 for 48 h. E Human melanoma cells freshly isolated from tumors treated for 48 h with 5 μM CRO15. F Primary human melanocytes, keratinocytes and fibroblasts treated 48 h with 5 μM CRO15. For B to F, results are expressed as percentages of control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2. CRO15 induces a mitochondrial disorder and activates AMPK pathway.

A Measurement of mitochondrial function in A375 melanoma cells with a Seahorse XF Cell Mito stress test after 6 h of treatment with 5 μM CRO15, 10 mM metformin or DMSO used as control. At 20 min, cells are treated with oligomycin, an inhibitor of complex V of respiratory chain; at 50 min, with FCCP, which disrupts the mitochondrial membrane potential; and at 80 min, with Rotenone and antimycin A, inhibitors of complex I and III, respectively. B A375 cells were treated with 5 μM CRO15 (6H), 10 mM metformin (6H) for or 20 μM CCCP (20 min). Measurement of mitochondrial potential membrane was performed by flow cytometry with a TMRE probe. C Lysates from A375 melanoma cells exposed for the indicated durations to CRO15 were analyzed by western blotting using the indicated antibodies after cell fractionation. One representative experiment of three is shown. D Lysates from A375 cells treated with 5 μM CRO15 for the indicated durations were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. E Lysates form A375 cells transduced with dominant negative AMPK subunits α1 and α2 for 24 h and treated with 5 μM CRO15 were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. F Quantification of cell viability from E using trypan blue exclusion method. Results are expressed as percentages of DMSO control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. CRO15 inhibits MELK kinase activity.

A Scatter plot representing the percentage of kinase inhibition and their Z′ factor measured during two independent in vitro kinome assay with 1 μM CRO15. B Immunofluorescence pictures of CT26 cancer cells treated with 5 μM CRO15 for 2 h. MELK was labeled with a specific antibody (in red), CRO15 was coupled to a fluorescent tag (in green) and nuclei were stained with DAPI (in blue). C In vitro test of kinase activity using recombinant MELK protein treated with CRO15 with the MELK inhibitor OTS167. D A375 cells were infected with MELK adenovirus for 24 h. For the pull down assay, cells lysates were incubated overnight with 100 μM Biotin-CRO15 or its inactive analog MTF465. For Biotin precipitation, cells were treated for 2 h with 20 μM Biotin-CRO15 or MTF465 before lysis and western blot analysis with the indicated antibodies. One representative experiment of three is shown. E and F A375 cells were treated with 5 μM CRO15 with the indicated durations, and lysates were analyzed by western blotting using the indicated antibodies. One representative experiment of three is shown. G A375 cells were transfected with siRNA directed against AMPK α1 and AMPK α2 (20 nM each) or 40 nM of control siRNA. Twenty-four hours later, cells were infected with a MELK adenovirus or a control adenovirus before treatment with 5 µM of CRO15. After 24H, viable cells were counted using the trypan blue dye exclusion method (n = 3) (right panel). In parallel, cell lysates were analyzed by western blotting with the indicated antibodies (left panel). One representative experiment of three is shown.

Fig. 4. CRO15 treatment induces p53 pathway activation.

A Indicated melanoma cell lines were treated with 5 μM CRO15 for 48 h before viable cells were counted using the trypan blue dye exclusion method. B A375 cells were treated with 5 μM CRO15 for the indicated duration, and lysates were analyzed by western blotting with indication antibodies. One representative experiment of three is shown. C Luciferase activity in A375 cells transfected with a p53 luciferase reporter upon 5 μM CRO15 treatment or actinomycin used as a positive control for p53 induction. D (left part) A375 cells were transfected with sip53 and then treated with 5 μM CRO15. After 24 h of treatment, viable cells were counted using the trypan blue exclusion method. The results are expressed as percentages of the DMSO control. (Right part) SK-MEL-28 cells were infected with control or p53-encoding adenoviruses and transfected with a p53 luciferase reporter. Cells were treated 24H with 5 μM CRO15 and a luciferase assay was performed. E A375 cells were transfected with control or p53 siRNA and treated with 5 μM CRO15 for the indicated duration. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. For C and D, data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5. CRO15 mediates the activation of autophagy and apoptosis in melanoma cells.

A A375 cells were treated with 5 μM CRO15 for the indicated duration. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. B Immunofluorescence pictures of A375 melanoma cells treated with DMSO, 5 μM CRO15 or 400 nM of the autophagy inducer rapamycin for 6 h. LC3B was labeled with antibody (green), and DNA was visualized with DAPI (blue). C A375 melanoma cells were treated for 24 h with 5 μM CRO15, 5 μM PLX4032 or with 1 μM of the pan-kinase inhibitor staurosporine for 6 h used as positive control of apoptosis induction. Cells were analyzed by flow cytometry using a specific antibody against Caspase 3 active protein conjugated with FITC (upper panel) or Annexin V conjugated with ALEXA and DAPI (lower panel) or against. *p < 0.05; **p < 0.01; ***p < 0.001. D A375 cells were infected with dominant negative adenoviruses against α1 and α2 AMPK subunits for 24 h before transfection with sip53 for another 24 h. Cells was determined by trypan blue staining after a 24 h treatment with 5 μM CRO15 or DMSO control. E A375 cells were infected with dominant negative adenoviruses against α1 and α2 AMPK subunits for 24 h and were transfected with sip53 for another 24 h. Cells were treated with 5 μM CRO15 for the indicated durations, and lysates were analyzed by western blot with the indicated antibodies. One experiment of three is shown. F A375 cells were treated with 5 μM CRO15 for the indicated durations, and lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. G A375 cells were transfected with siREDD1 for 24 h and then treated with 5 μM CRO15 for 24 h before cell viability was determined using the trypan blue exclusion method. H. A375 cells were transfected with siREDD1 for 24 h and then treated with 5 μM CRO15 for the indicated duration. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. For D and G, the results are expressed as percentages of the control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6. CRO15 decreases tumor growth in mouse models.

A Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with 1.0 × 10⁶ A375 cells. After 4 days, mice (n = 6 in each group) were treated with CRO15 (0.7 mg/mouse/day), PLX4032 (0.7 mg/mouse/day) or vehicle (Labrafil). Tumor growth curves were determined by measuring the tumor volume. The bars indicate the mean ± SEM. *p < 0.05; **p < 0.01. B Tumor weight of A375 xenograft after mouse euthanasia. The bars indicate the mean ± SEM *p < 0.05. C. Mice weight over the course of the experiment. D Quantification of hepatic transaminases (AST and ALT) on plasma of nude mice treated with vehicle or CRO15 at end point. E Immunofluorescence staining on xenograft tumor sections. LC3B (green) and active caspase 3 (red) were immunolabeled, and apoptotic cells were visualized with a TUNEL kit. Nuclei were visualized with DAPI (blue). Integrated density was determined for each condition on Fuji. Data are geometric means with 95% CI of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 7. CRO15 induced death in melanoma cells resistant to BRAF inhibitors.

A Indicated melanoma cell lines were treated with 5 μM CRO15 or 5 μM PLX4032. After 48 h, cell viability was determined by Trypan blue exclusion method. The results are expressed as percentages of the corresponding control and data given as the means ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001. B A375-resistant cells were treated with 5 μM CRO15 for the indicated durations. Lysates were analyzed by western blotting with the indicated antibodies. One representative experiment of three is shown. C Immunofluorescence images of A375-resistant melanoma cells treated with 5 μM CRO15 or 400 nM rapamycin for 6 h or DMSO control. LC3B was labeled with antibody (green), and the nuclei were visualized with DAPI (blue). D A375-resistant melanoma cells were treated for 24 h with 5 μM of CRO15 or PLX4032 for 24 h or with 1 μM staurosporine for 6 h. Cells were analyzed in flow cytometry with a specific antibody directed Annexin V (conjugated with ALEXA) or Caspase 3 active protein (conjugated with FITC). *p < 0.05; **p < 0.01; ***p < 0.001. E Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with 1.0 × 10⁶ A375-resistant cells. After 7 days, mice (n = 6 in each group) were treated with CRO15 (0.7 mg/mouse/day), PLX4032 (0.7 mg/mouse/day) or vehicle (Labrafil). Tumor growth curves were determined by measuring the tumor volume. The bars indicate the mean ± SEM. *p < 0.05; **p < 0.01. F Tumor weight of A375-resistant xenograft after mouse euthanasia. The bars indicate the mean ± SEM *p < 0.05; **p < 0.01.