Fig. 3: NRAS ASO treatment significantly induces apoptosis and specifically reduces cell and tumor growth in NRAS-mutant melanoma. | Communications Medicine

Fig. 3: NRAS ASO treatment significantly induces apoptosis and specifically reduces cell and tumor growth in NRAS-mutant melanoma.

From: Suppression of NRAS-mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

Fig. 3

a Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the NRAS-mutant melanoma cell lines D04 (p = 0.000002), MM415 (p = 0.00002), WM1366 (p = 0.0005), Sk-Mel-2 (p = 0.00001), VMM39 (p = 0.00004), WM3060 (p = 0.003), NZM40 (p = 0.0006), WM3629 (p = 0.0008), and the primary derived cell line Hs852T (p = 0.000006). b Treatment with NRAS ASO-2 caused significant inhibition of cell growth in the NRAS-mutant melanoma cell lines D04 (p = 0.000004) and MM415 (p = 0.0001). The antiproliferative outcomes are similar when compared to treatment with NRAS ASO-1. c NRAS ASO treatment did not cause significant antiproliferative effects in primary human melanocytes (PHM, p = 0.33), primary human liver cells (Hs775li, p = 0.29), human colon cells (FHC, p = 0.29), and BRAF-mutant melanoma cells (Sk-Mel-28, p = 0.13). d NRAS ASO treatment significantly inhibited colony formation in the D04 (p = 0.0017) and MM415 (p = 0.008) cell lines compared to treatment with non-targeting Control ASOs. Treatment period was 7 days (50 nM final oligonucleotide concentration, n = 3). e Representative images of D04 colonies in 6 cm dishes after ASO treatment. f Dot plot graph of flow cytometric analysis of PI and Annexin V staining after 1 day of ASO-treatment (100 nM) shows increased apoptotic cell death in D04-cells treated with NRAS ASO (15,780 total events) compared to Control ASO treatment (44,285 total events). g Distribution of overall cell populations from panel f) in regards of their apoptotic state. Bars represent the percentage of vital (Q2), early apoptotic (Q3), late apoptotic (Q4) and dead (Q1) cells. h NRAS ASO-mediated induction of apoptosis was confirmed by measurement of significantly increased activity levels of the apoptosis markers Caspase-3 & -7 after 1 day of treatment with either NRAS or Control ASOs (100 nM) in the D04 (p = 0.002) and MM415 (p = 0.0002) cell lines (n = 4). i Treatment with NRAS ASO−1 caused significant inhibition of cell growth in the NRAS-mutant multiple myeloma (MM) cell line H929 (p = 0.0005), and small cell lung cancer (SCLC) cell line SW1271 (p = 0.0001). j Significant tumor growth reduction was observed when comparing treatment groups for subcutaneous systemic treatment with either NRAS ASO (X) or Control ASO (O) in mouse models carrying xenografts of the D04 melanoma cell line (3 × 200 µg ASO/week, n = 6, days of measurement and p-values: −3 –0.38, 1 –0.27, 3 –0.02, 5 –0.04, 8 –0.05, 10 – 0.02, 12 –0.06, 15 –0.02, 17 – 0.02, 19 – 0.03). At the endpoint of the experiment (day 19), the average tumor size in the NRAS ASO treatment group was 48% smaller compared to control. k NRAS-mRNA levels were significantly reduced (0.68-fold, s.e.m = 0.03, p = 0.0003) in tumors of the NRAS ASO treatment group compared to the Control ASO treatment group at the end of study period. Tumors were harvested at end of treatment period; gene expression was normalized to Β-ACTIN expression and NRAS-mRNA expression in NRAS ASO treated tumors was normalized to expression in Control ASO treated tumors (n of each group = 5). l No significant weight changes were observed between the NRAS ASO (X) and Control ASO (O) groups at any time-point (days of measurement and p-values: -3 – 0.3, 1 – 0.36, 3 – 0.33, 5 – 0.46, 8 – 0.43, 10 – 0.5, 12 – 0.47, 15 – 0.49, 17 – 0.48, 19 – 0.49). m Blood of mice that either received a dose of NRAS ASO-1 (200 µg/injection), or ASO-free PBS was drawn 24 hours after injection and analyzed for parameters of liver function (Serum transaminases – ALT, AST, bilirubin - TBIL, direct (conjugated) bilirubin - DBIL, total protein - TP, albumin - ALB, and alkaline phosphatase – ALKP). All growth and weight curves are presented as polynomial trend lines (order: 2). Data in (a–c, i) were normalized to treatment with non-targeting Control ASO, final oligonucleotide concentration was 50 nM, treatment period was 5 days (n = 3). The error bars in a–d), h, i, m) represent s.d., in j−l they represent s.e.m. Significance is shown as p-values calculated by Student’s t-test. * =p < 0.05, ** =p < 0.01, *** =p < 0.001.

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