Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling.

Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor.

Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

Identification of a PEAK1/ZEB1 signaling axis during TGFß/fibronectin-induced EMT in breast cancer.

Transforming Growth Factor beta (TGFß) is the archetypal member of the TGFß superfamily of ligands and has pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer. In epithelial cancers TGFß signaling can either suppress tumor growth or promote metastasis via the induction of a well-characterized epithelial-mesenchymal transition (EMT) program. We recently reported that PEAK1 kinase mediates signaling cross talk between TGFß receptors and integrin/Src/MAPK pathways and functions as a critical molecular regulator of TGFß-induced breast cancer cell proliferation, migration, EMT and metastasis. Here, we examined the breast cancer cell contexts in which TGFß induces both EMT and PEAK1, and discovered this event to be unique to oncogene-transformed mammary epithelial cells and triple-negative breast cancer cells. Using the Cancer BioPortal database, we identified PEAK1 co-expressors across multiple malignancies that are also common to the TGFß response gene signature (TBRS). We then used the ScanSite database to identify predicted protein-protein binding partners of PEAK1 and the PEAK1-TBRS co-expressors. Analysis of the Cytoscape interactome and Babelomics-derived gene ontologies for a novel gene set including PEAK1, CRK, ZEB1, IL11 and COL4A1 enabled us to hypothesize that PEAK1 may be regulating TGFß-induced EMT via its interaction with or regulation of these other genes. In this regard, we have demonstrated that PEAK1 is necessary for TGFß to induce ZEB1-mediated EMT in the context of fibronectin/ITGB3 activation. These studies and future mechanistic studies will pave the way toward identifying the context in which TGFß blockade may significantly improve breast cancer patient outcomes.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers.

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop.

ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.

PEAK1 Acts as a Molecular Switch to Regulate Context-Dependent TGFß Responses in Breast Cancer.

Transforming Growth Factor ß (TGFß) has dual functions as both a tumor suppressor and a promoter of cancer progression within the tumor microenvironment, but the molecular mechanisms by which TGFß signaling switches between these outcomes and the contexts in which this switch occurs remain to be fully elucidated. We previously identified PEAK1 as a new non-receptor tyrosine kinase that associates with the cytoskeleton, and facilitates signaling of HER2/Src complexes. We also showed PEAK1 functions downstream of KRas to promote tumor growth, metastasis and therapy resistance using preclinical in vivo models of human tumor progression. In the current study, we analyzed PEAK1 expression in human breast cancer samples and found PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse. At the cellular level, we also observed that PEAK1 expression was highest in mesenchymal breast cancer cells, correlated with migration potential and increased in response to TGFß-induced epithelial-mesenchymal transition (EMT). Thus, we sought to evaluate the role of PEAK1 in the switching of TGFß from a tumor suppressing to tumor promoting factor. Notably, we discovered that high PEAK1 expression causes TGFß to lose its anti-proliferative effects, and potentiates TGFß-induced proliferation, EMT, cell migration and tumor metastasis in a fibronectin-dependent fashion. In the presence of fibronectin, PEAK1 caused a switching of TGFß signaling from its canonical Smad2/3 pathway to non-canonical Src and MAPK signaling. This report is the first to provide evidence that PEAK1 mediates signaling cross talk between TGFß receptors and integrin/Src/MAPK pathways and that PEAK1 is an important molecular regulator of TGFß-induced tumor progression and metastasis in breast cancer. Finally, PEAK1 overexpression/upregulation cooperates with TGFß to reduce breast cancer sensitivity to Src kinase inhibition. These findings provide a rational basis to develop therapeutic agents to target PEAK1 expression/function or upstream/downstream pathways to abrogate breast cancer progression.