Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands

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.

Figure 1

PRKD1 mutations in PLGAs and other cancer types. (a) Multiple sequence alignment of the catalytic and activation loops within the PRKD1 kinase domain. Conserved and non-conserved amino acids are highlighted in yellow and green, respectively. (b) Frequencies of the PRKD1 c.2130A>T and c.2130A>C (p.Glu710Asp) mutations in the discovery and validation PLGA cohorts. Error bars show 95% confidence intervals. (c) PRKD1 protein expression assessed by immunohistochemistry in PRKD1 mutant and wild-type PLGAs. (d) PRKD1 mutational frequencies in PLGAs and in other cancer types from published datasets available from cBioPortal. (e) Domain structure of the PRKD1 gene and the mutations identified in PLGAs (top) and in other cancer types from published datasets (bottom) available from cBioPortal. (f) PRKD1 mutational frequencies in 12 PLGAs and in 299 salivary gland tumors of other histologic types. (g) Metastasis-free survival of 149 patients with malignant salivary gland tumors according to PRKD1 mutation status.

Figure 2

Functional analysis of the PRKD1 p.Glu710Asp mutation. (a) Surface render of a homology model for wild-type and p.Glu710Asp-mutant PRKD1 onto the structure of CHEK2 places the substitution in the midst of the active site between the ATP-binding pocket and the putative proton acceptor. (b) Cell-free in vitro kinase assay determining transphosphorylation of the serine-threonine substrate CREBtide and the autocatalytic activity of wild-type (grey) and p.Glu710Asp-mutant (orange) PRKD1. ****, Holm-Šídák-adjusted-P<0.0001, multiple t-test. (c) Subcellular distribution of wild-type (grey) and p.Glu710Asp-mutant PRKD1 (orange) in non-malignant breast epithelial MCF10A and MCF12A cells, and Ser738/S742 and Ser910 PRKD1 phosphorylation in distinct subcellular compartments. Cyto, cytoplasm; Mem, membrane; Nuc, nuclear; wt, wild-type. (d) Impact of wild-type and p.Glu710Asp-mutant PRKD1 expression on growth and glandular architecture of MCF10A and MCF12A cells grown in three-dimensional basement membrane cultures.