p90RSK Blockade Inhibits Dual BRAF and MEK Inhibitor-Resistant Melanoma by Targeting Protein Synthesis

Abstract

Despite improvements in survival in metastatic melanoma with combined BRAF and mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor treatment, the overwhelming majority of patients eventually acquire resistance to both agents. Consequently, new targets for therapy in resistant tumors are currently being evaluated. Previous studies have identified p90 subfamily of ribosomal S6 kinase (p90RSK) family kinases as key factors for growth and proliferation, as well as protein synthesis via assembly of the 7-methyl-guanosine triphosphate cap-dependent translation complex. We sought to evaluate inhibitors of p90RSK family members: BI-D1870 and BRD7389, for their ability to inhibit both proliferation and protein synthesis in patient-derived melanoma cell lines with acquired resistance to combined treatment with the BRAF inhibitor vemurafenib and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor selumetinib. We found that the RSK inhibitors blocked cell proliferation and protein synthesis in multiple dual-resistant melanoma lines. In addition, single agent RSK inhibitor treatment was effective in drug-naïve lines, two of which are innately vemurafenib resistant. We also used Reverse Phase Protein Array screening to identify differential protein expression that correlates with BI-D1870 sensitivity, and identified prognostic biomarkers for survival in human melanoma patients. These findings establish p90RSK inhibition as a therapeutic strategy in treatment-resistant melanoma and provide insight into the mechanism of action.

Figure 1.. Dual vemurafenib and selumetinib treatment is insufficient to restrict protein translation in lines with dual BRAF and MEK inhibitor resistance.

Western blot of parental YUMAC and YURIF, as well as dual-resistant YUMACdr and YURIFdr, shows significant downregulation of new protein translation at low concentrations in YUMAC and YURIF but only at the highest concentrations in YUMACdr and YURIFdr. Erk, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; p90RSK, p90 subfamily of ribosomal S6 kinase; RPS6, ribosomal protein S6.

Figure 2.. p90RSK inhibition is an effective therapeutic strategy in dual-resistant melanoma lines.

RSK inhibitor treatment with (a) BI-D1870 or (b) BRD 3789 results in significant growth inhibition in both drug-naïve and acquired BRAF/MEK-inhibitor-resistant lines over 72 hours alone or in combination with vemurafenib and selumetinib. Growth inhibition with vemurafenib and selumetinib alone (no RSK inhibitor) plotted for comparison in green with error bars at 0 μM RSK inhibitor concentration. Mean ± SEM, N = 3. (c) Western blotting for signaling nodes involved in translation with 24-hour 5 μM BI-D1870, 5 μM vemurafenib, and 150 nM selumetinib triple therapy shows significant downregulation over dual Vem + Sel therapy alone. (d). shRNA-mediated knockdown of RSK1 and RSK2 leads to significant growth inhibition of YUMACdr treated with 5 μM vemurafenib and 150 nM selumetinib over 72 hours, P < 0.0001, mean ± SEM, N = 3. MEK, MAPK/ERK kinase; p90RSK, p90 subfamily of ribosomal S6 kinase; RPS6, ribosomal protein S6; SEM, standard error of the mean.

Figure 3.. Long-term growth of dual resistant melanoma is greatly inhibited by triple therapy with BI-D1870 and BRD7389.

2D colony formation assays were performed with initial seeding of 5,000 cells per well. Cells were allowed to settle for 48 hours before being treated with 5 μM BI-D1870 or 5 μM BRD7389 ± 5 μM vemurafenib and 150 nM selumetinib for 14 days.Media and drug were replaced every 72 hours after commencement of drug treatment, N = 3.

Figure 4.. p90RSK inhibition reduces proliferation in dual resistant melanoma through a mixture of apoptosis induction and growth arrest.

(a, b) Propidium iodide+BrdU cell cycle flow cytometry showed a significantly greater fold reduction in cells in the S phase after a 30-minute pulse followed by a 2-hour chase with 5 μM BI-D1870 triple therapy over dual 5 μM Vem + 150 nM Sel alone. *P < 0.05, **P < 0.01, mean ± SEM, N = 3. (c) Dual annexin V and propidium iodide flow cytometry showed a significantly larger increase in apoptosis induction by 5 μM BI-D1870 in 72-hour triple therapy over dual Vem + Sel alone. *P < 0.05, **P < 0.01, mean ± SEM, N = 3. APC, antigen-presenting cell; p90RSK, p90 subfamily of ribosomal S6 kinase; SEM, standard error of the mean.

Figure 5.. Growth inhibition with p90RSK inhibitors reduces protein synthesis.

(a) Seventy-hour YUMACdr treatment with 5 μM BI-D1870, 20 μM SL0101, or 10 μM FMK alone or with 5 μM vemurafenib + 150 nM selumetinib inhibits protein synthesis as assessed by 1-hour ³⁵S-labeled methionine/cysteine pulse. (b) m⁷-GTP-sepharose bead cap-dependent translation complex precipitation from YUMACdr lysate after 72 hours of BI-D1870 mono- or triple therapy. Normalized to cell number. (c) After 72 hours of BI-D1870+Vem+Sel triple therapy, approximately 50% reduction in protein content per cell was observed using the Bradford assay normalized to cell number. *P < 0.05, mean ± SEM, N = 3. (d) Screening of a panel of drugs on melanoma lines showed inhibition of protein synthesis with BID1870 equal to or greater than vemurafenib, selumetinib, PI3K/Akt/ mTOR, or MAPK inhibitors. eIF, elongation initiation factor; m⁷-GTP, 7-methyl-guanosine triphosphate; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; p90RSK, p90 subfamily of ribosomal S6 kinase; SEM, standard error of the mean.

Figure 6.. p90RSK is a potential clinical therapeutic target in BRAF-mutant melanoma.

(a) Patients indexed in The Cancer Genome Atlas were stratified into high and low RSK1 expressers, P = 3.9 × 10⁻⁷ patients with high p90RSK levels (n = 36) showed 26.5-month poorer disease-free survival versus patients who were low for both (n = 272), Kaplan-Meier estimate, P = 0.04. (b) RPPA screening of levels of 217 proteins in lysates collected from seven BRAF-mutant drug-naïve melanoma lines showed significant, P < 0.05, positive, and negative interaction between 20 proteins and BI-D1870 GI₅₀. The top 10 genes with the strongest negative correlation of protein levels to drug potency are shown in red, whereas those with the strongest positive correlation between protein levels and drug potency are shown in blue. MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; p90RSK, p90 subfamily of ribosomal S6 kinase; RPPA, reverse phase protein array; RPS6, ribosomal protein S6.