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The authors describe a functional crosstalk between the p53 network and the integrated stress response through the p53 repressor PPM1D. Inhibition of PPM1D potentiates p53-dependent transactivation and apoptosis via induction of the HRI-eIF2α-ATF4 pathway.

Summary: Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.

The response to hypoxia can significantly impact oncogenic processes. Here, the authors define the early transcriptional response to acute hypoxia and identify HIF1A target genes as part of this acute response, providing a resource for investigating context-dependent roles of HIF1A in the biology of cancer.

Summary: Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.

Summary: The transcriptional repressor ΔNp63α is a potent oncogene widely overexpressed in squamous cell carcinomas (SCCs) of diverse tissue origins, where it promotes malignant cell proliferation and survival. We report here the results of a genome-wide CRISPR screen to identify pathways controlling ΔNp63α-dependent cell proliferation, which revealed that the small GTPase RHOA blocks cell division upon ΔNp63α knockdown. After ΔNp63α depletion, RHOA activity is increased, and cells undergo RHOA-dependent proliferation arrest along with transcriptome changes indicative of increased TGF-β signaling. Mechanistically, ΔNp63α represses transcription of TGFB2, which induces a cell cycle arrest that is partially dependent on RHOA. Ectopic TGFB2 activates RHOA and impairs SCC proliferation, and TGFB2 neutralization restores cell proliferation during ΔNp63α depletion. Genomic data from tumors demonstrate inactivation of RHOA and the TGFBR2 receptor and ΔNp63α overexpression in more than 80% of lung SCCs. These results reveal a signaling pathway controlling SCC proliferation that is potentially amenable to pharmacological intervention. : Abraham et al. employ a genome-wide CRISPR screening strategy to characterize the mechanism of action of the ΔNp63α oncogene in SCC. ΔNp63α suppresses TGFB2 expression and RHOA activity to drive SCC proliferation. TGFB2 is sufficient to impair SCC proliferation and necessary to enforce cell cycle arrest upon depletion of ΔNp63α. Keywords: p63, CRISPR screen, p53, lung cancer, head and neck carcinoma

Summary: Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancerous tissues. Despite its importance in cancer development, our understanding of mechanisms driving this form of metabolic reprogramming is incomplete. We report here an analysis of colorectal cancer cells engineered to carry a single point mutation in the active site of the Mediator-associated kinase CDK8, creating hypomorphic alleles sensitive to bulky ATP analogs. Transcriptome analysis revealed that CDK8 kinase activity is required for the expression of many components of the glycolytic cascade. CDK8 inhibition impairs glucose transporter expression, glucose uptake, glycolytic capacity and reserve, as well as cell proliferation and anchorage-independent growth, both in normoxia and hypoxia. Importantly, CDK8 impairment sensitizes cells to pharmacological glycolysis inhibition, a result reproduced with Senexin A, a dual inhibitor of CDK8/CDK19. Altogether, these results contribute to our understanding of CDK8 as an oncogene, and they justify investigations to target CDK8 in highly glycolytic tumors. : Galbraith et al. use a chemical genetics approach to examine the role of CDK8 kinase activity in cancer cells. CDK8 activity is required for the transcription of multiple genes encoding enzymes required for glucose metabolism. Impaired CDK8 activity reduces glucose uptake and glycolysis and sensitizes cells to the glucose analog 2-deoxy-D-glucose. Keywords: CDK8, CDK19, Mediator, glycolysis, Warburg effect, chemical genetics, HCT116, SW480, A549, H460

Macroautophagy is thought to protect against apoptosis; however, underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells, and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP, after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterward. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient, thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell-fate decisions can be determined by autophagy.

The p53 transcription factor participates in diverse cellular responses to stress, including cell-cycle arrest, apoptosis, senescence, and autophagy. The molecular mechanisms defining the ultimate outcome of p53 activation remain poorly characterized. We performed a genome-wide genetic screen in human cells to identify pathway-specific coregulators of the p53 target gene CDKN1A (p21), an inhibitor of cell-cycle progression, versus BBC3 (PUMA), a key mediator of apoptosis. Our screen identified numerous factors whose depletion creates an imbalance in the p21:PUMA ratio upon p53 activation. The transcription factor TCF3, also known as E2A, drives p21 expression while repressing PUMA across cancer cell types of multiple origins. Accordingly, TCF3/E2A depletion impairs the cell-cycle-arrest response and promotes apoptosis upon p53 activation by chemotherapeutic agents. In contrast, TRIAP1 is a specific repressor of p21 whose depletion slows down cell-cycle progression. Our results reveal strategies for driving cells toward specific p53-dependent responses.

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.