Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.

The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

The ubiquitin ligase FBXW7 modulates leukemia-initiating cell activity by regulating MYC stability.

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Yap/Taz promote the scavenging of extracellular nutrients through macropinocytosis.

The uptake of macromolecules and cellular debris through macropinocytosis has emerged as an important nutrient acquisition strategy of cancer cells. Genetic alterations commonly found in human cancers (e.g. mutations in KRAS or loss of PTEN) have been shown to increase macropinocytosis. To identify additional effectors that enable cell growth dependent on the uptake of extracellular proteins, pancreatic ductal adenocarcinoma (PDA) cells were selected for growth in medium where extracellular albumin was the obligate source of the essential amino acid leucine. Analysis of global changes in chromatin availability and gene expression revealed that PDA cells selected under these conditions exhibited elevated activity of the transcriptional activators Yap/Taz. Knockout of Yap/Taz prevented growth of PDA cells in leucine-deficient medium, but not in complete medium. Furthermore, constitutively active forms of Yap or Taz were sufficient to stimulate macropinocytosis of extracellular protein. In addition to promoting the uptake of plasma proteins, Yap/Taz also promoted the scavenging of apoptotic cell bodies and necrotic debris by PDA cells. The Yap/Taz transcriptional target Axl was found to be essential for cell growth dependent on the uptake of dead cells and cell debris. Together, these studies suggest that the Hippo pathway effectors Yap and Taz are important transcriptional regulators of endocytic nutrient uptake.

TET1 is a tumor suppressor of hematopoietic malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

Intranasal Oxytocin in Children and Adolescents with Autism Spectrum Disorder.

BACKGROUND: Experimental studies and small clinical trials have suggested that treatment with intranasal oxytocin may reduce social impairment in persons with autism spectrum disorder. Oxytocin has been administered in clinical practice to many children with autism spectrum disorder. METHODS: We conducted a 24-week, placebo-controlled phase 2 trial of intranasal oxytocin therapy in children and adolescents 3 to 17 years of age with autism spectrum disorder. Participants were randomly assigned in a 1:1 ratio, with stratification according to age and verbal fluency, to receive oxytocin or placebo, administered intranasally, with a total target dose of 48 international units daily. The primary outcome was the least-squares mean change from baseline on the Aberrant Behavior Checklist modified Social Withdrawal subscale (ABC-mSW), which includes 13 items (scores range from 0 to 39, with higher scores indicating less social interaction). Secondary outcomes included two additional measures of social function and an abbreviated measure of IQ. RESULTS: Of the 355 children and adolescents who underwent screening, 290 were enrolled. A total of 146 participants were assigned to the oxytocin group and 144 to the placebo group; 139 and 138 participants, respectively, completed both the baseline and at least one postbaseline ABC-mSW assessments and were included in the modified intention-to-treat analyses. The least-squares mean change from baseline in the ABC-mSW score (primary outcome) was -3.7 in the oxytocin group and -3.5 in the placebo group (least-squares mean difference, -0.2; 95% confidence interval, -1.5 to 1.0; P = 0.61). Secondary outcomes generally did not differ between the trial groups. The incidence and severity of adverse events were similar in the two groups. CONCLUSIONS: This placebo-controlled trial of intranasal oxytocin therapy in children and adolescents with autism spectrum disorder showed no significant between-group differences in the least-squares mean change from baseline on measures of social or cognitive functioning over a period of 24 weeks. (Funded by the National Institute of Child Health and Human Development; SOARS-B ClinicalTrials.gov number, NCT01944046.).

Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Autism Spectrum Disorder: A Review.

Importance: Autism spectrum disorder (ASD), characterized by deficits in social communication and the presence of restricted, repetitive behaviors or interests, is a neurodevelopmental disorder affecting approximately 2.3% children aged 8 years in the US and approximately 2.2% of adults. This review summarizes evidence on the diagnosis and treatment of ASD. Observations: The estimated prevalence of ASD has been increasing in the US, from 1.1% in 2008 to 2.3% in 2018, which is likely associated with changes in diagnostic criteria, improved performance of screening and diagnostic tools, and increased public awareness. No biomarkers specific to the diagnosis of ASD have been identified. Common early signs and symptoms of ASD in a child's first 2 years of life include no response to name when called, no or limited use of gestures in communication, and lack of imaginative play. The criterion standard for the diagnosis of ASD is a comprehensive evaluation with a multidisciplinary team of clinicians and is based on semistructured direct observation of the child's behavior and semistructured caregiver interview focused on the individual's development and behaviors using standardized measures, such as the Autism Diagnostic Observation Schedule-Second Edition and the Autism Diagnostic Interview. These diagnostic measures have sensitivity of 91% and 80% and specificity of 76% and 72%, respectively. Compared with people without ASD, individuals with ASD have higher rates of depression (20% vs 7%), anxiety (11% vs 5%), sleep difficulties (13% vs 5%), and epilepsy (21% with co-occurring intellectual disability vs 0.8%). Intensive behavioral interventions, such as the Early Start Denver Model, are beneficial in children 5 years or younger for improvement in language, play, and social communication (small to medium effect size based on standardized mean difference). Pharmacotherapy is indicated for co-occurring psychiatric conditions, such as emotion dysregulation or attention-deficit/hyperactivity disorder. Risperidone and aripiprazole can improve irritability and aggression (standardized mean difference of 1.1, consistent with a large effect size) compared with placebo. Psychostimulants are effective for attention-deficit/hyperactivity disorder (standardized mean difference of 0.6, consistent with a moderate effect size) compared with placebo. These medications are associated with adverse effects including, most commonly, changes in appetite, weight, and sleep. Conclusions and Relevance: ASD affects approximately 2.3% of children aged 8 years and approximately 2.2% of adults in the US. First-line therapy consists of behavioral interventions, while co-occurring psychiatric conditions, such as anxiety or aggression, may be treated with specific behavioral therapy or medication.

GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2.

Mammalian cells possess two amino acid-sensing kinases: general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). Their combined effects orchestrate cellular adaptation to amino acid levels, but how their activities are coordinated remains poorly understood. Here, we demonstrate an important link between GCN2 and mTORC1 signaling. Upon deprivation of various amino acids, activated GCN2 up-regulates ATF4 to induce expression of the stress response protein Sestrin2, which is required to sustain repression of mTORC1 by blocking its lysosomal localization. Moreover, Sestrin2 induction is necessary for cell survival during glutamine deprivation, indicating that Sestrin2 is a critical effector of GCN2 signaling that regulates amino acid homeostasis through mTORC1 suppression.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Suppressed expression of oxidoreductin-like protein, Oxidor, increases follicle degeneration and decreases survival during the overwintering diapause of the mosquito Culex pipiens.

Throughout diapause in mosquitoes, stress resistance and subsequent prolonged lifespan are a few important features of diapause that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that oxidoreductin-like protein is involved with these diapause characteristics for overwintering survival. Expression of oxidor was more than two-fold higher in early stage diapausing females compared to their non-diapausing counterparts. Suppression of the gene that encodes oxidoreductin-like protein by RNAi significantly increased the proportion of degenerating follicles in early-stage adult diapausing females. Inhibition of oxidor also significantly reduced the survivability of diapausing females which indicates that this protein plays a key role in protecting multiple tissues during early diapause.

Promoter demethylation of the asparagine synthetase gene is required for ATF4-dependent adaptation to asparagine depletion.

Tumor cells adapt to nutrient-limited environments by inducing gene expression that ensures adequate nutrients to sustain metabolic demands. For example, during amino acid limitations, ATF4 in the amino acid response induces expression of asparagine synthetase (ASNS), which provides for asparagine biosynthesis. Acute lymphoblastic leukemia (ALL) cells are sensitive to asparagine depletion, and administration of the asparagine depletion enzyme l-asparaginase is an important therapy option. ASNS expression can counterbalance l-asparaginase treatment by mitigating nutrient stress. Therefore, understanding the mechanisms regulating ASNS expression is important to define the adaptive processes underlying tumor progression and treatment. Here we show that DNA hypermethylation at the ASNS promoter prevents its transcriptional expression following asparagine depletion. Insufficient expression of ASNS leads to asparagine deficiency, which facilitates ATF4-independent induction of CCAAT-enhancer-binding protein homologous protein (CHOP), which triggers apoptosis. We conclude that chromatin accessibility is critical for ATF4 activity at the ASNS promoter, which can switch ALL cells from an ATF4-dependent adaptive response to ATF4-independent apoptosis during asparagine depletion. This work may also help explain why ALL cells are most sensitive to l-asparaginase treatment compared with other cancers.

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

Critical role for PI3-kinase in regulating the use of proteins as an amino acid source.

Ras-transformed cells can grow in amino acid-poor environments by recovering amino acids through macropinocytosis and lysosomal catabolism of extracellular proteins. However, when studying nontransformed fibroblasts, we found that Ras GTPases are dispensable for growth-factor-stimulated macropinocytosis and lysosomal catabolism of extracellular proteins. Instead, we establish a critical role for phosphatidylinositol 3-kinase (PI3-kinase) signaling in cell proliferation that is supported by protein macropinocytosis. Downstream of PI3-kinase, distinct effectors have opposing roles in regulating uptake and catabolism of extracellular proteins. Rac1 and PLC are required for nutritional use of extracellular proteins. In contrast, Akt suppresses lysosomal catabolism of ingested proteins when free amino acids are abundant. The interplay between these pathways allows cells with oncogenic PIK3CA mutations or PTEN deletion to grow using diverse amino acid sources. Thus, the prevalence of PI3-kinase and PTEN mutations in cancer may result in part because they allow cells to cope with fluctuating nutrient availability.

An exploration of concomitant psychiatric disorders in children with autism spectrum disorder.

OBJECTIVE: We explored patterns of concomitant psychiatric disorders in a large sample of treatment-seeking children and adolescents with autism spectrum disorder (ASD). METHODS: Participants were 658 children with ASD (age 3-17 years; mean = 7.2 years) in one of six federally-funded multisite randomized clinical trials (RCT) between 1999 and 2014. All children were referred for hyperactivity or irritability. Study designs varied, but all used the Child and Adolescent Symptom Inventory or Early Childhood Inventory to assess Attention Deficit Hyperactivity Disorder (ADHD), Oppositional-Defiant Disorder (ODD), Conduct Disorder (CD), Anxiety Disorders, and Mood Disorders. In addition, several measures in common were used to assess demographic and clinical characteristics. RESULTS: Of the 658 children, 73% were Caucasian and 59% had an IQ >70. The rates of concomitant disorders across studies were: ADHD 81%, ODD 46%, CD 12%, any anxiety disorder 42%, and any mood disorder 8%. Two or more psychiatric disorders were identified in 66% of the sample. Of those who met criteria for ADHD, 50% also met criteria for ODD and 46% for any anxiety disorder. Associations between types of concomitant disorders and a number of demographic and clinical characteristics are presented. CONCLUSION: In this well-characterized sample of treatment-seeking children with ASD, rates of concomitant psychiatric disorders were high and the presence of two or more co-occurring disorders was common. Findings highlight the importance of improving diagnostic practice in ASD and understanding possible mechanisms of comorbidity.

Psychiatric comorbidities in neurodevelopmental disorders.

PURPOSE OF REVIEW: Recent changes in the diagnostic criteria for psychiatric and neurodevelopmental disorders as well as increases in the prevalence of both have elevated the focus on these areas of medicine and their clinical overlap. RECENT FINDINGS: Several recent studies have examined psychiatric comorbidities in neurodevelopmental disorders including autism and specific genetic syndromes. A growing number of reports underscore the genetic overlap between previously distinct clinical disorders. Behavioral and psychiatric features are increasingly identified in association with intellectual developmental disorders. SUMMARY: As there have been advances in our collective understanding of the genetic underpinnings of certain disorders and the downstream physiological consequences of those genetic alterations, challenges to the way boundaries have been drawn around psychiatric disorders, and by extension, the concept of comorbidity, warrant review.

Children with autism spectrum disorder and social skills groups at school: a randomized trial comparing intervention approach and peer composition.

BACKGROUND: Peer relationships improve for children with autism spectrum disorder (ASD) in clinic-based social skills groups but rarely generalize to real world contexts. This study compares child outcomes of two social skills interventions conducted in schools with children in Kindergarten through fifth grade. METHOD: Children with ASD were randomized to one of two interventions that varied on group composition (mixed typical and ASD vs. all ASD or social difficulties) and intervention approach (didactic SKILLS based vs. activity-based ENGAGE groups). Interventions were implemented at school for 8 weeks (16 sessions) with an 8-week follow-up. Innovative measures of peer nomination and playground peer engagement, as well as teacher reports of child behavior problems and teacher-child relationship were analyzed for 137 children with ASD across four sites. RESULTS: On the primary outcome of social network connections from the peer nomination measure, there was no main effect of treatment, but there were moderator effects. Children with low teacher-child closeness or high conflict improved more in their social connections if they received the SKILLS intervention, whereas children with higher teacher-child closeness improved more if they received the ENGAGE intervention. Only two secondary outcome measures yielded significant effects of treatment. Children in the SKILLS groups increased peer engagement and decreased isolation during recess. Child behavior problems and teacher-child closeness moderated peer engagement such that children with higher behavior problems and lower closeness benefitted more from SKILLS groups. CONCLUSIONS: These findings suggest that social skills groups conducted at school can affect both peer engagement during recess as well as peer acceptability. Child characteristics and teacher-child relationship prior to intervention yield important information on who might benefit from a specific social skills intervention.

Dendritic cell-based therapeutic vaccine elicits polyfunctional HIV-specific T-cell immunity associated with control of viral load.

Efforts aimed at restoring robust immune responses limiting human immunodeficiency virus (HIV)-1 replication therapeutically are warranted. We report that vaccination with dendritic cells generated ex vivo and loaded with HIV lipopeptides in patients (n = 19) on antiretroviral therapy was well tolerated and immunogenic. Vaccination increased: (i) the breadth of the immune response from 1 (1-3) to 4 (2-5) peptide-pool responses/patient (p = 0.009); (ii) the frequency of functional T cells (producing at least two cytokines among IFN-γ, TNF-α, and IL-2) from 0.026 to 0.32% (p = 0.002) and from 0.26 to 0.35% (p = 0.005) for CD4(+) and CD8(+) T cells, respectively; and (iii) the breadth of cytokines secreted by PBMCs upon antigen exposure, including IL-2, IFN-γ, IL-21, IL-17, and IL-13. Fifty percent of patients experienced a maximum of viral load (VL) 1 log10 lower than the other half following antiretroviral treatment interruption. An inverse correlation was found between the maximum of VL and the frequency of polyfunctional CD4(+) T cells (p = 0.007), production of IL-2 (p = 0.006), IFN-γ (p = 0.01), IL-21 (p = 0.006), and IL-13 (p = 0.001). These results suggest an association between vaccine responses and a better control of viral replication. These findings will help in the development of strategies for a functional cure for HIV infection.