Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells.

Glutamine is a critical metabolite for rapidly proliferating cells as it is used for the synthesis of key metabolites necessary for cell growth and proliferation. Glutamine metabolism has been proposed as a therapeutic target in cancer and several chemical inhibitors are in development or in clinical trials. How cells subsist when glutamine is limiting is poorly understood. Here, using an unbiased screen, we identify ALDH18A1, which encodes P5CS, the rate-limiting enzyme in the proline biosynthetic pathway, as a gene that cells can downregulate in response to glutamine starvation. Notably, P5CS downregulation promotes de novo glutamine synthesis, highlighting a previously unrecognized metabolic plasticity of cancer cells. The glutamate conserved from reducing proline synthesis allows cells to produce the key metabolites necessary for cell survival and proliferation under glutamine-restricted conditions. Our findings reveal an adaptive pathway that cancer cells acquire under nutrient stress, identifying proline biosynthesis as a previously unrecognized major consumer of glutamate, a pathway that could be exploited for developing effective metabolism-driven anticancer therapies.

Down syndrome is associated with altered frequency and functioning of tracheal multiciliated cells, and response to influenza virus infection.

Individuals with Down syndrome (DS) clinically manifest severe respiratory illnesses; however, there is a paucity of data on how DS influences homeostatic physiology of lung airway, and its reactive responses to pulmonary pathogens. We generated well-differentiated ciliated airway epithelia using tracheas from wild-type and Dp(16)1/Yey mice in vitro, and discovered that Dp(16)1/Yey epithelia have significantly lower abundance of ciliated cells, an altered ciliary beating profile, and reduced mucociliary transport. Interestingly, both sets of differentiated epithelia released similar quantities of viral particles after infection with influenza A virus (IAV). However, RNA-sequencing and proteomic analyses revealed an immune hyperreactive phenotype particularly for monocyte-recruiting chemokines in Dp(16)1/Yey epithelia. Importantly, when we challenged mice in vivo with IAV, we observed immune hyper-responsiveness in Dp(16)1/Yey mice, evidenced by higher quantities of lung airway infiltrated monocytes, and elevated levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Our findings illuminate mechanisms underlying DS-mediated pathophysiological changes in airway epithelium.

Triplication of the interferon receptor locus contributes to hallmarks of Down syndrome in a mouse model.

Down syndrome (DS), the genetic condition caused by trisomy 21, is characterized by variable cognitive impairment, immune dysregulation, dysmorphogenesis and increased prevalence of diverse co-occurring conditions. The mechanisms by which trisomy 21 causes these effects remain largely unknown. We demonstrate that triplication of the interferon receptor (IFNR) gene cluster on chromosome 21 is necessary for multiple phenotypes in a mouse model of DS. Whole-blood transcriptome analysis demonstrated that IFNR overexpression associates with chronic interferon hyperactivity and inflammation in people with DS. To define the contribution of this locus to DS phenotypes, we used genome editing to correct its copy number in a mouse model of DS, which normalized antiviral responses, prevented heart malformations, ameliorated developmental delays, improved cognition and attenuated craniofacial anomalies. Triplication of the Ifnr locus modulates hallmarks of DS in mice, suggesting that trisomy 21 elicits an interferonopathy potentially amenable to therapeutic intervention.

Multidimensional definition of the interferonopathy of Down syndrome and its response to JAK inhibition.

Individuals with Down syndrome (DS) display chronic hyperactivation of interferon signaling. However, the clinical impacts of interferon hyperactivity in DS are ill-defined. Here, we describe a multiomics investigation of interferon signaling in hundreds of individuals with DS. Using interferon scores derived from the whole blood transcriptome, we defined the proteomic, immune, metabolic, and clinical features associated with interferon hyperactivity in DS. Interferon hyperactivity associates with a distinct proinflammatory phenotype and dysregulation of major growth signaling and morphogenic pathways. Individuals with the highest interferon activity display the strongest remodeling of the peripheral immune system, including increased cytotoxic T cells, B cell depletion, and monocyte activation. Interferon hyperactivity accompanies key metabolic changes, most prominently dysregulated tryptophan catabolism. High interferon signaling stratifies a subpopulation with elevated rates of congenital heart disease and autoimmunity. Last, a longitudinal case study demonstrated that JAK inhibition normalizes interferon signatures with therapeutic benefit in DS. Together, these results justify the testing of immune-modulatory therapies in DS.

Proteasome Inhibition Sensitizes Liposarcoma to MDM2 Inhibition with Nutlin-3 by Activating the ATF4/CHOP Stress Response Pathway.

Liposarcoma is the most commonly occurring soft-tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes MDM2 and CDK4. This unique genetic profile makes liposarcoma an attractive candidate for targeted therapeutics. While CDK4/6 inhibitors are currently employed for treatment of several cancers, MDM2 inhibitors have yet to attain clinical approval. Here, we report the molecular characterization of the response of liposarcoma to the MDM2 inhibitor nutlin-3. Treatment with nutlin-3 led to upregulation of two nodes of the proteostasis network: the ribosome and the proteasome. CRISPR/Cas9 was used to perform a genome-wide loss of function screen that identified PSMD9, which encodes a proteasome subunit, as a regulator of response to nutlin-3. Accordingly, pharmacologic studies with a panel of proteasome inhibitors revealed strong combinatorial induction of apoptosis with nutlin-3. Mechanistic studies identified activation of the ATF4/CHOP stress response axis as a potential node of interaction between nutlin-3 and the proteasome inhibitor carfilzomib. CRISPR/Cas9 gene editing experiments confirmed that ATF4, CHOP, and the BH3-only protein, NOXA, are all required for nutlin-3 and carfilzomib-induced apoptosis. Furthermore, activation of the unfolded protein response using tunicamycin and thapsigargin was sufficient to activate the ATF4/CHOP stress response axis and sensitize to nutlin-3. Finally, cell line and patient-derived xenograft models demonstrated combinatorial effects of treatment with idasanutlin and carfilzomib on liposarcoma growth in vivo. Together, these data indicate that targeting of the proteasome could improve the efficacy of MDM2 inhibitors in liposarcoma. SIGNIFICANCE: Targeting the proteasome in combination with MDM2 inhibition activates the ATF4/CHOP stress response axis to induce apoptosis in liposarcoma, providing a potential therapeutic approach for the most common soft-tissue sarcoma.

FAM193A is a positive regulator of p53 activity.

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.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia ­ structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.

IGF1 deficiency integrates stunted growth and neurodegeneration in Down syndrome.

Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), is characterized by stunted growth, cognitive impairment, and increased risk of diverse neurological conditions. Although signs of lifelong neurodegeneration are well documented in DS, the mechanisms underlying this phenotype await elucidation. Here we report a multi-omics analysis of neurodegeneration and neuroinflammation biomarkers, plasma proteomics, and immune profiling in a diverse cohort of more than 400 research participants. We identified depletion of insulin growth factor 1 (IGF1), a master regulator of growth and brain development, as the top biosignature associated with neurodegeneration in DS. Individuals with T21 display chronic IGF1 deficiency downstream of growth hormone production, associated with a specific inflammatory profile involving elevated tumor necrosis factor alpha (TNF-α). Shorter children with DS show stronger IGF1 deficiency, elevated biomarkers of neurodegeneration, and increased prevalence of autism and other conditions. These results point to disruption of IGF1 signaling as a potential contributor to stunted growth and neurodegeneration in DS.

PPM1D suppresses p53-dependent transactivation and cell death by inhibiting the Integrated Stress Response.

The p53 transcription factor is a master regulator of cellular stress responses inhibited by repressors such as MDM2 and the phosphatase PPM1D. Activation of p53 with pharmacological inhibitors of its repressors is being tested in clinical trials for cancer therapy, but efficacy has been limited by poor induction of tumor cell death. We demonstrate that dual inhibition of MDM2 and PPM1D induces apoptosis in multiple cancer cell types via amplification of the p53 transcriptional program through the eIF2α-ATF4 pathway. PPM1D inhibition induces phosphorylation of eIF2α, ATF4 accumulation, and ATF4-dependent enhancement of p53-dependent transactivation upon MDM2 inhibition. Dual inhibition of p53 repressors depletes heme and induces HRI-dependent eIF2α phosphorylation. Pharmacological induction of eIF2α phosphorylation synergizes with MDM2 inhibition to induce cell death and halt tumor growth in mice. These results demonstrate that PPM1D inhibits both the p53 network and the integrated stress response controlled by eIF2α-ATF4, with clear therapeutic implications.

Specialized interferon action in COVID-19.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy.

BACKGROUND: Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. METHODS: Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. RESULTS: PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. CONCLUSIONS: These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in MYC-driven medulloblastoma.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

Global Analyses to Identify Direct Transcriptional Targets of p53.

The transcription factor p53 controls a gene expression program with pleiotropic effects on cell biology including cell cycle arrest and apoptosis. Identifying direct p53 target genes within this network and determining how they influence cell fate decisions downstream of p53 activation is a prerequisite for designing therapeutic approaches that target p53 to effectively kill cancer cells. Here we describe a comprehensive multi-omics approach for identifying genes that are direct transcriptional targets of p53. We provide detailed procedures for measuring global RNA polymerase activity, defining p53 binding sites across the genome, and quantifying changes in steady-state mRNA in response to p53 activation.

Seroconversion stages COVID19 into distinct pathophysiological states.

COVID19 is a heterogeneous medical condition involving diverse underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Low antibody titers associate with hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, and depletion of lymphocytes, neutrophils, and platelets. Upon seroconversion, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased D-dimer, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.

The COVIDome Explorer Researcher Portal.

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. In order to expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients including matched analysis of the whole blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate here the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.

Seroconversion stages COVID19 into distinct pathophysiological states.

COVID19 is a heterogeneous medical condition involving a suite of underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Seronegative COVID19 patients harbor hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, neutropenia, lymphopenia and thrombocytopenia. In seropositive patients, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased markers of platelet activation, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.

JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome.

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.

Nutlin-Induced Apoptosis Is Specified by a Translation Program Regulated by PCBP2 and DHX30.

Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits. In this study, we report a translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to associate with the enhanced translation of mRNAs carrying multiple copies of an identified 3' UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identify PCBP2 and DHX30 as CGPD-motif interactors. We find that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs increases, and the response to Nutlin shifts toward apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells leads to decreased translation of CGPD-motif mRNAs.

Trisomy 21 dysregulates T cell lineages toward an autoimmunity-prone state associated with interferon hyperactivity.

Trisomy 21 (T21) causes Down syndrome (DS), a condition characterized by high prevalence of autoimmune disorders. However, the molecular and cellular mechanisms driving this phenotype remain unclear. Building upon our previous finding that T cells from people with DS show increased expression of interferon (IFN)-stimulated genes, we have completed a comprehensive characterization of the peripheral T cell compartment in adults with DS with and without autoimmune conditions. CD8+ T cells from adults with DS are depleted of naïve subsets and enriched for differentiated subsets, express higher levels of markers of activation and senescence (e.g., IFN-γ, Granzyme B, PD-1, KLRG1), and overproduce cytokines tied to autoimmunity (e.g., TNF-α). Conventional CD4+ T cells display increased differentiation, polarization toward the Th1 and Th1/17 states, and overproduction of the autoimmunity-related cytokines IL-17A and IL-22. Plasma cytokine analysis confirms elevation of multiple autoimmunity-related cytokines (e.g., TNF-α, IL17A-D, IL-22) in people with DS, independent of diagnosis of autoimmunity. Although Tregs are more abundant in DS, functional assays show that CD8+ and CD4+ effector T cells with T21 are resistant to Treg-mediated suppression, regardless of Treg karyotype. Transcriptome analysis of white blood cells and T cells reveals strong signatures of T cell differentiation and activation that correlate positively with IFN hyperactivity. Finally, mass cytometry analysis of 8 IFN-inducible phosphoepitopes demonstrates that T cell subsets with T21 show elevated levels of basal IFN signaling and hypersensitivity to IFN-α stimulation. Therefore, these results point to T cell dysregulation associated with IFN hyperactivity as a contributor to autoimmunity in DS.