GLI inhibitor GANT61 kills melanoma cells and acts in synergy with obatoclax

Abstract

MEK kinase inhibitors (trametinib and selumetinib) or kinase inhibitors directed against mutated BRAF(V600E) (vemurafenib and dabrafenib) have initial encouraging effects in the treatment of melanoma but acquired resistance appears almost invariably after some months. Studies revealed mutually exclusive NRAS and BRAF activating mutations driving the MAPK/ERK pathway among human melanomas. Although combination therapy exerts significantly better antitumor cell efficacy, complete remission is rarely achieved. To employ an alternative approach, we have targeted the Hedgehog/GLI pathway, which is deregulated in melanomas, through the GLI1/2 inhibitor GANT61, alone or accompanied with the treatment by the BCL2 family inhibitor obatoclax in 9 melanoma cell lines. Thus, we targeted melanoma cells irrespective of their NRAS or BRAF mutational status. After GANT61 treatment, the cell viability was drastically diminished via apoptosis, as substantial nuclear DNA fragmentation was detected. In all tested melanoma cell lines, the combined treatment was more efficient than the application of each drug alone at the end of the cell growth with inhibitors. GANT61 was efficient also alone in most cell lines without the addition of obatoclax, which had only a limited effect when used as a single drug. In most cell lines, tumor cells were eradicated after 5-9 days of combined treatment in colony outgrowth assay. To conclude, GANT61 treatment might become a hopeful and effective anti-melanoma targeted therapy, especially when combined with the BCL2 family inhibitor obatoclax.

Widespread Expression of Hedgehog Pathway Components in a Large Panel of Human Tumor Cells and Inhibition of Tumor Growth by GANT61: Implications for Cancer Therapy

“…We have previously tested melanoma cells and found that GANT61 was variably effective in all tumors. The combination with obatoclax (a BCL-2 family inhibitor) revealed a better effect, showing clear synthetic lethality in six of nine melanoma lines [ 29 ] ( Figure S1 ). The most sensitive cell line was SK-MEL-3.…”

“…To gain insight into the mechanism underlying the eradication of cells in proliferation assays, we carried out the TUNEL assay that detects apoptosis. Many previous papers indicate that GANT61 kills the cells through apoptosis [ 29 , 49 , 57 , 61 ]. We have chosen two GANT61-sensitive tumors cell lines, SK-MEL-3 (see Figure 2 ) and SK-MEL-5 (see Figure S1 ).…”

“…It is believed that MITF (microphthalmia-associated trancription factor), a key factor in melanoma transcription circuitry, maintains antiapoptosis in melanomas [ 68 ]. It has been nevertheless demonstrated that low-MITF melanoma cell lines can also proliferate very fast, implicating sufficient antiapoptotic protection [ 29 , 69 ]. HH-GLI signaling has been recognized to keep melanoma stemness and maintain the presence of CSC [ 70 ].…”

“…All cell lines were of human origin. Melanoma cell lines DOR, Beu, and Hbl were previously described [ 29 ]. Other melanoma cell lines (MeWo, SK-MEL-2, SK-MEL-28, SK-MEL-5, SK-MEL-3, Malme 3M, HT144, WM35, WM1552C, and RPMI-7931) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA).…”

“…In many other cancer types, the HH pathway inhibition decreases the oncogenicity and has been beneficial for the patients. Melanomas critically require HH signaling [ 27 , 28 , 29 ], presumably with activated RAS-MAPK and AKT signaling cascades [ 27 ]. HH has been described to promote oncogenesis in leukemias [ 30 , 31 , 32 , 33 , 34 ], bladder cancer [ 35 ], and prostate cancer [ 36 , 37 , 38 , 39 ].…”