Located on chromosome 9p21.3, p16INK4a seems lost amongst a cluster of neighboring tumor suppressor genes. While best known for inhibiting cyclin dependent kinase (CDK) activity, p16INK4a is not a one trick pony. Long term p16INK4a expression pushes cells to enter senescence, an irreversible cell cycle arrest that prevents the growth of would-be cancer cells, but also contributes to aging. Loss of p16INK4a is one of the most frequent events in human tumors and allows pre-cancerous lesions to bypass senescence. Therefore, precise regulation of p16INK4a is essential to tissue homeostasis, maintaining a tight balance between tumor suppression and aging. Here, we outline the pathways required for proper p16INK4a regulation and highlight the critical functions of p16INK4a in cancer, aging and human physiology that make this gene special.

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 identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of 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. Though 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.

Senescent cells within the tumor microenvironment (TME) adopt a pro-inflammatory, senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression and therapeutic resistance. Here, exposure to Palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA damage-independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure of non-transformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined melanoma cells (i.e. Ras mutant, Braf mutant, and Ras/Braf wild type). When cultured in the presence of CDK4/6i–induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative responses. However, in vivo, PD-0332991-treated fibroblasts enhanced the growth of all melanoma lines tested and promoted the recruitment of Gr-1-positive immune cells. These data indicate that prolonged CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such senescent cells suppress the anti-tumor immune response and promote melanoma growth in immunocompetent, in vivo models. Implications The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences that should be considered in clinical trials aiming to treat melanoma and other cancer types.

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.

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin–positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a KrasG12D-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2–deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic KrasG12D signaling during the initial stages of tumor development.

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 overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.

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