Targeting RRM2 and Mutant BRAF Is a Novel Combinatorial Strategy for Melanoma

Abstract

The majority of patients with melanoma harbor mutations in the BRAF oncogene, thus making it a clinically relevant target. However, response to mutant BRAF inhibitors (BRAFi) is relatively short-lived with progression-free survival of only 6 to 7 months. Previously, we reported high expression of ribonucleotide reductase M2 (RRM2), which is rate-limiting for de novo dNTP synthesis, as a poor prognostic factor in patients with mutant BRAF melanoma. In this study, the notion that targeting de novo dNTP synthesis through knockdown of RRM2 could prolong the response of melanoma cells to BRAFi was investigated. Knockdown of RRM2 in combination with the mutant BRAFi PLX4720 (an analog of the FDA-approved drug vemurafenib) inhibited melanoma cell proliferation to a greater extent than either treatment alone. This occurred in vitro in multiple mutant BRAF cell lines and in a novel patient-derived xenograft (PDX) model system. Mechanistically, the combination increased DNA damage accumulation, which correlated with a global decrease in DNA damage repair (DDR) gene expression and increased apoptotic markers. After discontinuing PLX4720 treatment, cells showed marked recurrence. However, knockdown of RRM2 attenuated this rebound growth both in vitro and in vivo, which correlated with maintenance of the senescence-associated cell-cycle arrest.

Implications: Inhibition of RRM2 converts the transient response of melanoma cells to BRAFi to a stable response and may be a novel combinatorial strategy to prolong therapeutic response of patients with melanoma. Mol Cancer Res; 14(9); 767-75. ©2016 AACR.

Figure 1. The combination of shRRM2 with PLX4720 inhibits cell proliferation to a greater extent than either treatment alone

A, WM793 cells were stably infected with control or shRRM2 lentivirus and treated with DMSO or 1μM PLX4720. After 7 days in culture, RRM2, cyclin A, and PCNA protein expression was determined by western immunoblotting. GAPDH was used as a loading control. B, Same as (A) but cells were labeled with 10μM BrdU for 30 min. The incorporated BrdU was visualized by immunofluorescence. DAPI was used as a counterstain to visualize cell nuclei. C, Quantification of (B). Mean of 3 independent experiments with SEM. D, Same as (A) but an equal number of cells (1000 cells/well) were seeded in 12-well plates, and after 2 weeks in culture the plates were stained with 0.05% crystal violet in PBS to visualize focus formation. Shown are representative images of 3 independent experiments. E, The intensity of focus formed by the indicated cells was quantified using NIH image J software (n=3). Note the log scale. *p<0.05 compared with control. #p<0.05 compared with shRRM2 or PLX4720 alone.

Figure 2. The combination of shRRM2 with PLX4720 induces DNA damage accumulation and melanoma cell apoptosis

A, WM793 cells were stably infected with control or shRRM2 lentivirus and treated with DMSO or 1μM PLX4720. After 7 days in culture, cells were stained for SA-B-Gal activity. B, Quantification of (A). Mean of 3 independent experiments with SEM *p<0.01 compared with control. C, Same as (A) but γH2AX, cleaved lamin A, cleaved PARP, and cleaved caspase 3 (cas3) protein expression was determined by western immunoblotting. GAPDH was used as a loading control. D, Same as (A) but cell cycle analysis was performed after staining cells with propidium iodide. E, Same as (A) but on day 2 in culture, gene expression of the indicated mRNAs was determined by qRT-PCR. *p<0.05 compared with control. #p<0.01 compared with PLX4720 alone. F, Same as (E) but RRM2 and c-MYC protein expression was determined by immunoblotting. GAPDH was used as a loading control.

Figure 3. Knockdown of RRM2 prolongs PLX4720 treatment response after drug withdrawal

A, WM793 cells were treated with 1μM PLX4720 for 7 days and then withdrawn from drug treatment. RRM2, p-ERK1/2, and total ERK1/2 expression was determined on day 2 (D2) during PLX4720 treatment and day 7 after withdrawal (WD). B, WM793 cells were stably infected with shRRM2 lentivirus and treated with DMSO or 1uM PLX4720. After 7 days in culture, PLX4720 was withdrawn (See Fig. S1A). An equal number of cells (1000 cells/well) were seeded in 12-well plates, and after 2 weeks in culture the plates were stained with 0.05% crystal violet in PBS to visualize focus formation. C, Quantification of (B). Mean of 3 independent experiments with SEM. D, Same as (B) an equal number of cells were seeded in 6-well plates, and cell number was determined at the indicated time points. E, Same as (B) but an equal number of cells (4000 cells/well) were seeded in matrigel in 8-well chamber slides. Representative images after an additional 2 weeks in culture are shown. F–G, Quantification of (E). Acini size (F) and cell number (G) were determined. H, Same as (B) but cells were stained for SA-β-Gal activity. I, Quantification of (G). *p<0.05 compared with control and PLX4720 withdrawal cells.

Figure 4. Knockdown of RRM2 in combination with PLX4720 inhibits patient-derived melanoma tumor growth in vivo

A, ND238 patient-derived melanoma cells were subcutaneously injected into immunocompromised NSG mice. Once tumors reached 200mm³ volume, mice were randomized into the 4 indicated groups (See Fig. S4A). Graphs show tumor growth of each individual mouse in the indicated groups. Slope based on mean tumor volume of each group is indicated along with a trend line for averages from the entire group. B, Same as (A) but graphs indicate % change in tumor volume 21 days later. *p<0.05 vs. control; #p<0.05 vs. PLX4720 alone (calculated using Mann–Whitney test). C, Immunohistochemical staining of RRM2 and Ki67 in tumors from (B). D, Quantification of RRM2 staining in (C). Expression of RRM2 in the indicated groups was quantified using the histological score. *p<0.05 vs. control; #p<0.05 vs. PLX4720 alone.

Figure 5. Knockdown of RRM2 attenuates patient derived tumor growth after withdrawal from PLX4720 in vivo

A, ND238 patient derived tumors were discontinued from PLX4720 treatment after 21 days. Graphs show tumor growth of each individual mouse in the indicated groups. Slope based on mean tumor volume of each group is indicated along with a trend line for averages from the entire group. WD = withdrawal. B, Shown is % change in tumor volume on Day 74. *p=0.0061 PLX4720 WD vs. shRRM2/PLX4720 WD (calculated using linear mixed-effect models). C, RRM2 and cyclin A protein expression was assessed in individual tumors from (B) on day 89. β-actin was used as a loading control. D, Quantification of RRM2 expression in (C). *p<0.05 PLX4720 WD vs. shRRM2/PLX4720 WD. E, SA-β-Gal expression in tumors from (B). F, Quantification of (E) using ImageJ. *p<0.05 vs. control; #p<0.05 vs. PLX4720 WD.