p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

Putting p53 in Context.

TP53 is the most frequently mutated gene in human cancer. Functionally, p53 is activated by a host of stress stimuli and, in turn, governs an exquisitely complex anti-proliferative transcriptional program that touches upon a bewildering array of biological responses. Despite the many unveiled facets of the p53 network, a clear appreciation of how and in what contexts p53 exerts its diverse effects remains unclear. How can we interpret p53's disparate activities and the consequences of its dysfunction to understand how cell type, mutation profile, and epigenetic cell state dictate outcomes, and how might we restore its tumor-suppressive activities in cancer?

p53-dependent Nestin regulation links tumor suppression to cellular plasticity in liver cancer.

The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Deletions linked to TP53 loss drive cancer through p53-independent mechanisms.

Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.

DNAJB1-PRKACA fusion kinase interacts with ß-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of ß-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

Pten loss promotes MAPK pathway dependency in HER2/neu breast carcinomas.

Loss of the tumor suppressor gene PTEN is implicated in breast cancer progression and resistance to targeted therapies, and is thought to promote tumorigenesis by activating PI3K signaling. In a transgenic model of breast cancer, Pten suppression using a tetracycline-regulatable short hairpin (sh)RNA cooperates with human epidermal growth factor receptor 2 (HER2/neu), leading to aggressive and metastatic disease with elevated signaling through PI3K and, surprisingly, the mitogen-activated protein kinase (MAPK) pathway. Restoring Pten function is sufficient to down-regulate both PI3K and MAPK signaling and triggers dramatic tumor regression. Pharmacologic inhibition of MAPK signaling produces similar effects to Pten restoration, suggesting that the MAPK pathway contributes to the maintenance of advanced breast cancers harboring Pten loss.

Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates.

Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.

Genomic dissection of the epidermal growth factor receptor (EGFR)/PI3K pathway reveals frequent deletion of the EGFR phosphatase PTPRS in head and neck cancers.

Activation of the PI3K and epidermal growth factor receptor (EGFR) pathway is able to drive oncogenesis in multiple human cancers, including head and neck squamous cell carcinoma. Targeted agents such as cetuximab and erlotinib are currently used in patients with head and neck squamous cell carcinoma, but, in this disease, the genomic alterations that cause pathway activation and determine response to pharmacologic inhibition remain ill-defined. Here, we present a detailed dissection of the EGFR/PI3K pathway, composed of sequencing of the core pathway components, and high-resolution genomic copy number assessment. Mutations were found in PIK3CA (6%), but no point mutations were observed in other pathway genes such as PTEN and EGFR. In contrast, we observed frequent copy number alterations of genes in the pathway, including PIK3CA, EGFR, protein tyrosine phosphatase receptor S (PTPRS), and RICTOR. In total, activating genetic pathway alterations were identified in 74% of head and neck tumors. Importantly, intragenic microdeletions of the EGFR phosphatase PTPRS were frequent (26%), identifying this gene as a target of 19p13 loss. PTPRS loss promoted EGFR/PI3K pathway activation, modulated resistance to EGFR inhibition, and strongly determined survival in lung cancer patients with activating EGFR mutations. These findings have important implications for our understanding of head and neck cancer tumorigenesis and for the use of targeted agents for this malignancy.

A Molecular Switch between Mammalian MLL Complexes Dictates Response to Menin-MLL Inhibition.

Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin-modifying complexes that dictates response to Menin-MLL inhibitors. MLL1-Menin safeguards leukemia survival by impeding the binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the Menin-MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1-Menin and MLL3/4-UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials. SIGNIFICANCE: Menin-MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin-MLL inhibitor-resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses. This article is highlighted in the In This Issue feature, p. 1.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Dicer-regulated microRNAs 222 and 339 promote resistance of cancer cells to cytotoxic T-lymphocytes by down-regulation of ICAM-1.

The RNase III endonuclease Dicer plays a key role in generation of microRNAs (miRs). We hypothesized that Dicer regulates cancer cell susceptibility to immune surveillance through miR processing. Indeed, Dicer disruption up-regulated intercellular cell adhesion molecule (ICAM)-1 and enhanced the susceptibility of tumor cells to antigen-specific lysis by cytotoxic T-lymphocytes (CTLs), while expression of other immunoregulatory proteins examined was not affected. Blockade of ICAM-1 inhibited the specific lysis of CTLs against Dicer-disrupted cells, indicating a pivotal role of ICAM-1 in the interaction between tumor cells and CTL. Both miR-222 and -339 are down-regulated in Dicer-disrupted cells and directly interacted with the 3' untranslated region (UTR) of ICAM-1 mRNA. Modulation of Dicer or these miRs inversely correlated with ICAM-1 protein expression and susceptibility of U87 glioma cells to CTL-mediated cytolysis while ICAM-1 mRNA levels remained stable. Immunohistochemical and in situ hybridization analyses of 30 primary glioblastoma tissues demonstrated that expression of Dicer, miR-222, or miR-339 was inversely associated with ICAM-1 expression. Taken together, Dicer is responsible for the generation of the mature miR-222 and -339, which suppress ICAM-1 expression on tumor cells, thereby down-regulating the susceptibility of tumor cells to CTL-mediated cytolysis. This study suggests development of novel miR-targeted therapy to promote cytolysis of tumor cells.

Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses.

Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.

Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry.

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging.

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples. KHK overexpression in liver cancer cells results in decreased fructose flux through glycolysis. We then developed a strategy to detect this metabolic switch in vivo using hyperpolarized magnetic resonance spectroscopy. Uniformly deuterating [2-13C]-fructose and dissolving in D2O increased its spin-lattice relaxation time (T1) fivefold, enabling detection of F1P and its loss in models of HCC. In summary, we posit that in the liver, fructolysis to F1P is lost in the development of cancer and can be used as a biomarker of tissue function in the clinic using metabolic imaging.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry.

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases as well as coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

Poly-ICLC promotes the infiltration of effector T cells into intracranial gliomas via induction of CXCL10 in IFN-alpha and IFN-gamma dependent manners.

Stimulation of double-stranded (ds)RNA receptors can increase the effectiveness of cancer vaccines, but the underlying mechanisms are not completely elucidated. In this study, we sought to determine critical roles of host IFN-alpha and IFN-gamma pathways in the enhanced therapeutic efficacy mediated by peptide vaccines and polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in the murine central nervous system (CNS) GL261 glioma. C57BL/6-background wild type (WT), IFN-alpha receptor-1 (IFN-alphaR1)(-/-) or IFN-gamma(-/-) mice bearing syngeneic CNS GL261 glioma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes with or without intramuscular (i.m.) injections of poly-ICLC. The combinational treatment induced a robust transcription of CXCL10 in the glioma site. Blockade of CXCL10 with a specific monoclonal antibody (mAb) abrogated the efficient CNS homing of antigen-specific type-1 CTL (Tc1). Both IFN-alphaR(-/-) and IFN-gamma(-/-) hosts failed to up-regulate the CXCL10 mRNA and recruit Tc1 cells to the tumor site, indicating non-redundant roles of type-1 and type-2 IFNs in the effects of poly-ICLC-assisted vaccines. The efficient trafficking of Tc1 also required Tc1-derived IFN-gamma. Our data point to critical roles of the host-IFN-alpha and IFN-gamma pathways in the modulation of CNS glioma microenvironment, and the therapeutic effectiveness of poly-ICLC-assisted glioma vaccines.