Identification of a novel subgroup of melanomas with KIT/cyclin-dependent kinase-4 overexpression

Abstract

Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G(1) phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27(KIP). Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy.

Figure 1. Identification of a sub-group of melanoma lines that express high CDK4 levels

A) Microarray analysis, showing increased CDK4 mRNA in a panel of BRAF/NRAS wt melanomas. Expression profile shows a panel of primary human melanocytes (mel), V600E-mutated melanoma cell lines (BRAF V600E), NRAS/BRAF wild-type melanomas and NRAS mutated melanomas (NRAS). B) Increased CDK4 expression in the BRAF/NRAS wt cell lines identified from A). Protein expression of CDK4, phospho-ERK (pERK), total ERK (tERK), cyclin D1 (CycD1) across a panel of human melanocytes (mel), and human melanoma cell lines. Note, the WM8 and WM1382 cell lines exhibit high CDK4/cyclin D1 expression and low levels of phospho-ERK.

Figure 2. The CDK4 overexpressing melanoma cell lines are resistant to the anti-proliferative effects of the BRAF inhibitor SB590885

A) The CDK4 overexpressing melanoma lines are resistant to SB590885 in an MTT assay. Cells were treated with increasing concentrations of SB590885 (1nM - 10 μM) for 72 hrs before being treated with MTT. B) SB590885 preferentially reduces S-phase entry in BRAF V600E mutated melanoma cell lines. The BRAF V600E mutated cell line (1205Lu) and the CDK4 overexpressing cell line (WM1382) were treated with U0126 (10 μM) or SB590885 (1 μM) for 24 hrs, before being fixed, stained with propidium iodide and analyjed using flow cytometry.

Figure 3. CDK4 overexpressing melanomas also show high KIT expression

A) CDK4 overexpressing melanoma lines have higher KIT mRNA expression. Microarray analysis, showing KIT mRNA expression in a panel of primary human melanocytes (mel), V600E-mutated melanoma cell lines (BRAF V600E), NRAS/BRAF wild-type melanomas and NRAS mutated melanomas (NRAS). B) CDK4 overexpressing melanoma cell lines have high KIT expression and phospho-KIT activity (Left panel): Protein expression of total KIT (tKIT) and phospho-KIT (pKIT) receptor across a panel of human melanocytes (mel), and human melanoma cell lines (WM35, WM793, 1205Lu, C8161, WM164, 451Lu, WM983A, WM983B, WM8 and WM1382) (Right panel): Expression of KIT in melanoma lines harboring the BRAF V600E mutation. Western blots showing the expression of total KIT and phospho-c-KIT across a panel of melanoma cell lines (WM39, WM46, WM902B, SK-MEL-28) harboring the BRAF mutation. (C) Human melanoma samples with increased CDK4 expression also have higher KIT expression. Data show quantitative RT-PCR results for KIT expression in 14 melanoma samples without CDK4 amplification (control) or 3 with CDK4 amplification confirmed by array CGH (CDK4). Data shown are -log2 values normalized to KIT expression in normal human melanocytes (defined as zero). Bar shows mean value KIT expression for the Control (non-CDK amplified) and CDK4 amplified groups.

Figure 4. CDK4/KIT overexpressing melanomas are sensitive to imatinib treatment

(A) CDK4/KIT overexpressing cells were sensitive to the growth inhibitory effects of imatinib in an MTT assay. Cells were treated with increasing concentrations of imatinib (10 nM - 10 μM) for 72 hrs before being treated with MTT. Absorbances were read at 570 nm and expressed as a percentage of control absorbance. Data show the mean of three independent experiments +/- S.E. mean. Only the WM8 and WM1382 were sensitive to imatinib treatment (B) Imatinib reduces the viability and survival of melanoma cells grown as 3D collagen-implanted spheroids. Pre-formed spheroids that either overexpressed CDK4 (WM1382) or harbored the BRAF V600E mutation (1205Lu) were embedded into collagen and overlayed with medium. Cells were then treated with Imatininb (3 and 10 μM) for 72 hrs, before being treated with calcein-AM and propidium iodide. Green, viable cells; red, dead cells. Lack of green staining also indicates loss of viability. (C) Imatinib treatment induces regression of established CDK4/KIT melanoma xenografts. CDK4/KIT overexpressing (WM1382) cells were grown as tumor xenografts in SCID mice. After tumor establishment, mice were dosed twice daily with either vehicle (distilled water) or imatinib mesylate (100 mg/kg in distilled water) by oral gavage for 14 days. Tumor volumes were measured twice per week. Left panel: Photographs of representative vehicle and imatinib treated tumors taken after day 14 of treatment. Right panel: Growth curves were normalized to the start volumes. Imatinib treatment led to significant regression of the established xenografts.

Figure 5. KIT inhibition blocks MAP kinase activity and induces cell cycle arrest and apoptosis in CDK4/KIT overexpressing melanomas

(A) Imatinib preferentially reduces S-phase entry and induces apoptosis in a KIT/CDK4 overexpressing melanoma cell line but not BRAF V600E mutated melanoma cell line. The BRAF V600E mutated cell line (1205Lu) and the CDK4 overexpressing cell line (WM1382) were treated with Imatinib (3 and 10 μM) for 24 hrs, before being fixed, stained with propidium iodide and analyzed using flow cytometry. (B) Imatininb treatment preferentially reduces phospho-KIT, and phospho-ERK expression in CDK4 overexpressing cells (WM1382) but not in BRAF V600E mutated cells (1205Lu). Cells were treated with imatinib (1-10 μM) for 24 hrs, protein was then harvested and resolved by Western blotting. Figure shows expression of total KIT (tKIT), phospho-KIT (pKIT), phospho-ERK (pERK), total ERK (tERK), p21 and p27. Equal protein loading was confirmed by stripping the blot once and probing for actin expression. (C) Knockdown of KIT levels using a lentiviral shRNA construct. CDK4 overexpressing cells were infected with a lentivirus encoding for shRNA against KIT and were selected using puromycin. Blot shows knockdown of KIT protein and was scored using a densitometer. (D) Knockdown of KIT reduces the growth of WM1382 cells. CDK4 overexpressing cells were infected with lentiviral shRNA against KIT. A scrambled shRNA sequence was used as the control. Following drug selection, 30,000 cells/ml were plated out and were counted at 24, 48 and 72 hrs.