p62-Dependent Phase Separation of Patient-Derived KEAP1 Mutations and NRF2.

Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.

WNT7B mediates autocrine Wnt/ß-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/ß-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/ß-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/ß-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/ß-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/ß-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/ß-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/ß-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/ß-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/ß-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/ß-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/ß-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.