Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4

Abstract

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. We performed a mutational analysis of the protein tyrosine kinase (PTK) gene family in cutaneous metastatic melanoma. We identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. We found ERBB4 mutations in 19% of individuals with melanoma and found mutations in two other kinases (FLT1 and PTK2B) in 10% of individuals with melanomas. We examined seven missense mutations in the most commonly altered PTK gene, ERBB4, and found that they resulted in increased kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies could lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.

Figure 1. Distribution of mutations in ERBB4

Red arrows indicate the location of ERBB4 somatic mutations found in this screen. Red stars indicate ERBB4 mutants used in subsequent analysis. Boxes represent functional domains (I, Extracellular Domain Subregion I; II, Extracellular Domain Subregion II; III, Extracellular Domain Subregion III; IV, Extracellular Domain Subregion IV; Kinase, Tyrosine Kinase Domain).

Figure 2. ERBB4 mutants exhibit increased basal activation

A. ERBB4 mutants have increased tyrosine phosphorylation. HEK 293T cells were transiently transfected with the indicated constructs. 24 hrs after transfection, cells were serum starved, lysed and immunoprecipitated with α-ERBB4. After immunoprecipitation of ERBB4, immunoblots were performed using specific antibodies, as indicated. Lysates were immunoblotted with an α-tubulin antibody. B. ERBB4 mutants exhibit increased in vitro kinase activity. HEK 293T cells were transiently transfected as in A. Equivalent amounts of protein from cell lysates were immunoprecipitated and used in a kinase assay to measure receptor autophosphorylation. The same samples that were used in the kinase assay were immunoblotted with ERBB4 antibody and lysates were blotted with α-tubulin. KD: kinase dead. C. Increased basal activation of endogenous mutant ERBB4. Melanoma lines that harbor either WT or mutant ERBB4 were serum starved and then lysed, immunoprecipitated for ERBB4, then immunoblotted withα-PY20 or α-ERBB4. D. Mutant ERBB4 has increased basal activity. Melanoma lines harboring either WT or mutant ERBB4 were serum deprived, lysed, immunoprecipitated for ERBB4, and analyzed by immunoblotting with α-P-ERBB4 (P-Y1162) or α-ERBB4.

Figure 3. Mutant ERBB4 induces cell transformation and anchorage-independent growth in NIH 3T3 and SK-Mel-2 cells

A. NIH 3T3 cells were transfected with the indicated constructs. The photographs show foci stained with Hema3 after 10 days. Ras ^G12V was included as a positive control for cell transformation. B. The graph indicates the average number of foci visualized in A. C. Transformation ability of melanoma SK-Mel-2 cells stably expressing either vector, WT ERBB4 or various ERBB4 missense mutants was assessed by foci formation in tissue culture flasks. The graph indicates the number of foci formed after 14 days.

Figure 4. Expression of mutant ERBB4 provides an essential cell survival signal in melanoma

A. HEK 293 cells were transiently co-transfected with either vector or WT ERBB4 together with either control vector or shRNAs that target ERBB4. Cell lysates were analyzed by immunoblotting using α-ERBB4. For normalization, lysates were analyzed in parallel by α-tubulin immunoblotting. B. Cells transduced with shRNA targeting ERBB4 were lysed and immunopreciptitated using α-ERBB4 beads. Immunoprecipitates were blotted with specific antibodies, as indicated. C–G. shRNA-mediated ERBB4 knockdown in melanoma lines containing ERBB4 mutations results in reduced cell growth. Cells were seeded in 96-well plates and incubated for 13–17 days. Plates were analyzed every other day for cell proliferation where the average cell number at each time point was measured by determining DNA content using SYBR Green I. Melanoma cells harboring ERBB4 mutations stably transduced with shRNA constructs targeting ERBB4, but not those stably transduced with the control vector only, showed decreased growth relative to control. This did not occur in melanoma cells harboring WT ERBB4.

Figure 5. Melanoma lines expressing ERBB4 mutants exhibit increased sensitivity to ERBB inhibition by lapatinib

A. Representative dose response curves showing lapatinib efficacy against ERBB4 mutant lines compared to WT ERBB4 lines. Cells were treated for 72 hours in the presence of increasing concentrations (0.01–30 μM) of lapatinib, and relative cell number was estimated by methylene blue protein staining and plotted as percent survival when compared to vehicle-treated control versus Log (lapatinib) nM (where 1 is 10 nM lapatinib). Fitted lines were generated using 4-parameter nonlinear regression via GraphPad Prism. B. ERBB4 mutant lines have increased sensitivity to lapatinib compared to WT ERBB4 lines. The IC₅₀ values for inhibition of cell growth by 72 hr treatment with lapatinib of a larger panel of lines harboring WT and mutant ERBB4 were analyzed using GraphPad Prism v.5 (n=3). C. Immunoprecipitation and western blot analysis of ERBB4 autophosphorylation in cells treated with lapatinib. Cells were treated for 1 hr with lapatinib or vehicle alone as control. Lysates were immunoprecipitated with α-ERBB4 followed by western blot analysis with α-ERBB4 and α-P-ERBB4 (Y1162). D. Melanoma lines expressing mutant ERBB4 exhibit increased lapatinib sensitivity with respect to ERBB4 as well as AKT phosphorylation. The activity of ERBB4, AKT as well as ERBB2 was determined by immunoblotting with phospho-specific antibodies. Cells were treated for 1 hr with 5 μM lapatinib or vehicle alone. Lysates were immunoprecipitated using α-ERBB2 or α-ERBB4. Lysates and immunoprecipitates were analyzed by western blotting using the indicated antibodies. Shown are representative blots. E. Quantitative assessment of data from 2 lines harboring WT ERBB4 and 3 lines harboring mutant ERBB4 that were performed similarly to D. D. The ratio of band intensities of (P-Y1162)-ERBB4/ERBB4, (P)-S473-AKT/AKT and (P-Y1248)-ERBB2/ERBB2 for each cell line are shown. F. Mutant ERBB4 cells have increased sub-G1 population in the presence of lapatinib compared to WT ERBB4 cells. FACS analysis of 31T (WT) and 12T (E563K) showing cell cycle distribution (PI staining, x-axis) vs cell counts (y-axis). Shown are representative plots. G. Quantitation of FACS-sorted lapatinib-treated cells. The percent apoptotic cells were determined based on the sub G1 population for vehicle treated cells or lapatinib treated cells.