Abstract
Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.
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Acknowledgements
This work was supported in part by an IDEAS grant from Princess Margaret Hospital, the Head and Neck Translational Research Program (I.W., B.A.C., P.C.B. and J.D.M.), the Ontario Institute for Cancer Research and the government of Ontario (P.C.B. and J.D.M.) and by a Terry Fox Research Institute New Investigator Award (P.C.B.). C.H. and F.-F.L. acknowledge support from the Wharton family, Joe's Team, Gordon Tozer, the Campbell Family Institute for Cancer Research and the Ministry of Health and Long-Term Planning, Canada.
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I.W., L.N., J.D.M., B.A.C., B.W., P.C.B. and J.S.R.-F. conceived the study and supervised the work. I.W., L.D.R.T., S.I.C., R.R.S., A.S., E.B.S., I.F., A.A., C.H., J.W., R.d.B., M.C.-S.-Y., C.J.H., A.C.N., Y.H.W., N.K. and B.P.R. provided samples. I.W., B.P.-O., Y.H.W. and N.K. reviewed the cases. Sample processing, Sanger sequencing and amplicon sequencing were performed by S.P., N.F., A.d.C.P., C.H. and F.-F.L. Massively parallel RNA sequencing and whole-exome sequencing were carried out by A.V., J.D.M. and P.C.B. RNA sequencing analysis was performed by C.K.Y.N. and R.L. Whole-exome sequencing analysis was performed by D.W., C.K.Y.N., N.J.H., R.L., N.B. and L.M. J.D.M., P.C.B. and J.S.R.-F. coordinated the bioinformatics analyses. In silico protein structure analysis was performed by S.P., L.G.M., J.A., T.K., B.G.W. and P.C.B. Functional in vitro experiments were carried out by L.G.M. and B.W. R.A.S. carried out immunohistochemical analysis. J.S.R.-F. and C.M. interpreted the immunohistochemical results. C.K.Y.N., K.C.C. and P.C.B. performed statistical analyses. B.W. and J.S.R.-F. wrote the first draft of the manuscript, which was initially reviewed by I.W., S.P., L.G.M., D.W., L.N., B.P.R. and P.C.B. All authors edited and approved the final draft.
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Integrated supplementary information
Supplementary Figure 1 Validation of PRKD1 p.Glu710Asp mutations in PLGAs by Sanger sequencing.
Representative sequence electropherograms of the six PLGAs of the discovery cohort, of which five were found to have either a c.2130A>T or c.2130A>C PRKD1 p.Glu710Asp mutation.
Supplementary Figure 2 Expression of wild-type and mutant PRKD1 mRNA and protein in PLGAs.
(a) Integrative Genomics Viewer (IGV) pileups of the expression of PRKD1 mRNA as defined by RNA sequencing of PLGAs from the discovery cohort with p.Glu710Asp-mutant (n = 2) and wild-type (n = 1) PRKD1. (b) Representative micrographs of the immunohistochemical analysis of PRKD1 expression in frozen samples of PLGA from the MSKCC discovery cohort with p.Glu710Asp-mutant and wild-type PRKD1. Scale bar, 500 μm. (c) Results of the immunohistochemical analysis of formalin-fixed, paraffin-embedded PLGAs from the validation cohort (n = 18). Cytoplasmic PRKD1 expression was assessed using the H-score system. Error bars, s.d. of the mean. Nuclear expression of PRKD1 was defined by an Allred score of >2.
Supplementary Figure 3 Validation of PRKD1 p.Glu710Asp somatic mutation status in PLGAs from the validation series by Sanger sequencing.
Representative sequence electropherograms of DNA from 15 PLGAs and matched germline samples. PRKD1 c.2130A>T and c.2130A>C (p.Glu710Asp) mutations were found to be somatic in all cases harboring one of these mutations.
Supplementary Figure 4 Model quality assessment of wild-type and p.Glu710Asp-mutant PRKD1.
(a) Homology model quality was assessed by the QMEAN server. The results are summarized in panels for wild-type PRKD1 (left) and p.Glu710Asp-mutant PRKD1 (right). A Q-MEAN score was calculated for each homology model (denoted by red ’X’ characters) as well as for reference Protein Data Bank structures (Online Methods). A variety of statistics regarding the homology model, including the Q-MEAN z score, are also shown. The structure is colored according to predicted local (per-residue) model error. Residues within the active site are well modeled (green). The bulk of the predicted error associated with the model comes from residues distant from the active site (colored red). Protein structures were visualized using the Visual Molecular Dynamics software suite (J. Mol. Graph. 14, 33–38, 1996). (b) The prevalence of glutamic acid (E) and aspartic acid (D) residues at the homologous site of the p.Glu710Asp mutation across the eukaryotic protein kinases (ePKs) is shown. Residues at the homologous column of the alignment provided in Manning et al. (Science 298, 1912–1934, 2002) were examined. The names of those kinases with glutamic acid at the homologous site are colored green, those with aspartic acid at the homologous site are colored red, and those with any other residue are colored black. The glutamic acid wild-type residue is ubiquitously present within the Ca2+/calmodulin-dependent kinase (CAMK) and AGC families, and its presence is not consistently found in other kinase groups. Related kinases within the AGC group having an aspartic acid at this position include a subtree containing PKCs and PKNs, a group containing the ROCKs, DMPKs, MASTs, NDRs and LATSs, and, finally, a group containing the YANKs.
Supplementary Figure 5 Kinase assay and kinase activity in subcellular compartments of wild-type and p.Glu710Asp-mutant PRKD1.
(a) Western blot analysis of 5% of immunoprecipitation (IP) input (top) and an example of immunoprecipitated and eluted proteins from beads of the kinase assay (bottom). Total wild-type/p.Glu710Asp-mutant and normalized phosphorylated wild-type/p.Glu710Asp-mutant (Ser738/Ser742) signal ratios are shown in parentheses. For eluted proteins (bottom), the ratios shown are averages of triplicates. (b) Subcellular localization of wild-type PRKD1 and p.Glu710Asp-mutant PRKD1 in MCF10A (left), MCF12A (middle) and HEK293T (right) cells expressing pCMV6:PRKD1-tGFP or pCMV6:PRKD1(p.Glu710Asp)-tGFP. Scale bar, 50 μm. (c) Cellular fractionation and western blot analysis exemplifying the subcellular distribution of wild-type and p.Glu710Asp-mutant PRKD1 expression in non-malignant breast epithelial MCF12A cells. Phosphorylation status of the activation loop, Ser738/Ser742, and the autocatalytic site Ser910, of PRKD1 in the distinct subcellular compartments in MCF12A cells is shown. The efficiency and purity of separation was evaluated using the expression of MEK1/2 (cytoplasmic localization), AIF (organellular/membranous localization), vimentin (cytoskeletal and nuclear localization) and histone H3 (nuclear localization) according to the manufacturer’s guidelines. Quantification of the results is presented in Figure 2c. Cyto, cytoplasm; mem, membrane; nuc, nuclear; wt, wild type.
Supplementary Figure 6 Impact of the PRKD1 p.Glu710Asp mutation on cell migration and cell growth.
(a) Using Transwell assays, MCF12A non-malignant breast epithelial cells and NIH3T3 mouse fibroblasts show reduced migration upon expression of p.Glu710Asp-mutant PRKD1 in comparison to cells transfected with empty vector or wild-type PRKD1. (b) Quantification of the number of migratory MCF12A cells expressing empty vector or wild-type or p.Glu710Asp-mutant PRKD1. *P < 0.05, ***P < 0.001, n = 3, two-tailed unpaired t test; n.s, not significant. Error bars, s.d. of the mean. (c) Using scratch wound healing assays, MCF12A non-malignant breast epithelial cells and NIH3T3 mouse fibroblasts show reduced migration and gap closure upon expression of p.Glu710Asp-mutant PRKD1 in comparison to cells transfected with empty vector or wild-type PRKD1. In each micrograph, the solid line indicates the center of the scratch and the dashed lines represent the invasive front. Scale bar, 500 μm. (d) Expression of wild-type or p.Glu710Asp-mutant PRKD1 in MCF10A and MCF12A non-malignant breast epithelial cells leads to a significant increase in cell viability in comparison to cells transfected with empty vector control. ****, Holm-Šídák-corrected P < 0.0001 (n = 3), multiple t test. Error bars, s.d. of the mean.
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Weinreb, I., Piscuoglio, S., Martelotto, L. et al. Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands. Nat Genet 46, 1166–1169 (2014). https://doi.org/10.1038/ng.3096
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DOI: https://doi.org/10.1038/ng.3096
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