In vivo CRISPR screens identify a dual function of MEN1 in regulating tumor-microenvironment interactions.

Functional genomic screens in two-dimensional cell culture models are limited in identifying therapeutic targets that influence the tumor microenvironment. By comparing targeted CRISPR-Cas9 screens in a two-dimensional culture with xenografts derived from the same cell line, we identified MEN1 as the top hit that confers differential dropout effects in vitro and in vivo. MEN1 knockout in multiple solid cancer types does not impact cell proliferation in vitro but significantly promotes or inhibits tumor growth in immunodeficient or immunocompetent mice, respectively. Mechanistically, MEN1 knockout redistributes MLL1 chromatin occupancy, increasing H3K4me3 at repetitive genomic regions, activating double-stranded RNA expression and increasing neutrophil and CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Pharmacological inhibition of the menin-MLL interaction reduces tumor growth in a CD8+ T cell-dependent manner. These findings reveal tumor microenvironment-dependent oncogenic and tumor-suppressive functions of MEN1 and provide a rationale for targeting MEN1 in solid cancers.

GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma.

Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.

Excessive sucrose consumption reduces synaptic density and increases cannabinoid receptors in Göttingen minipigs.

Diets high in sucrose and fat are becoming more prevalent the world over, accompanied by a raised prevalence of cardiovascular diseases, cancers, diabetes, obesity, and metabolic syndrome. Clinical studies link unhealthy diets with the development of mental health disorders, particularly depression. Here, we investigate the effects of 12 days of sucrose consumption administered as 2 L of 25% sucrose solution daily for 12 days in Göttingen minipigs on the function of brain receptors involved in reward and motivation, regulating feeding, and pre- and post-synaptic mechanisms. Through quantitative autoradiography of cryostat sections containing limbic brain regions, we investigated the effects of sucrose restricted to a 1-h period each morning, on the specific binding of [3H]raclopride on dopamine D2/3 receptors, [3H]UCB-J at synaptic vesicle glycoprotein 2A (SV2A), [3H]MPEPγ at metabotropic glutamate receptor subtype 5 (mGluR5) and [3H]SR141716A at the cannabinoid receptor 1 (CB1). Compared to control diet animals, the sucrose group showed significantly lower [3H]UCB-J and [3H]MPEPγ binding in the prefrontal cortex. The sucrose-consuming minipigs showed higher hippocampal CB1 binding, but unaltered dopamine D2/3 binding compared to the control group. We found that the sucrose diet reduced the synaptic density marker while increasing CB1 binding in limbic brain structures, which may subserve maladaptive changes in appetite regulation and feeding. Further studies of the effects of diets and lifestyle habits on brain neuroreceptor and synaptic density markers are warranted.

Regulation and impact of tumor-specific CD4+ T cells in cancer and immunotherapy.

CD4+ T cells are crucial in generating and sustaining immune responses. They orchestrate and fine-tune mammalian innate and adaptive immunity through cell-based interactions and the release of cytokines. The role of these cells in contributing to the efficacy of antitumor immunity and immunotherapy has just started to be uncovered. Yet, many aspects of the CD4+ T cell response are still unclear, including the differentiation pathways controlling such cells during cancer progression, the external signals that program them, and how the combination of these factors direct ensuing immune responses or immune-restorative therapies. In this review, we focus on recent advances in understanding CD4+ T cell regulation during cancer progression and the importance of CD4+ T cells in immunotherapies.

IL-2 produced by HBV-specific T cells as a biomarker of viral control and predictor of response to PD-1 therapy across clinical phases of chronic hepatitis B.

BACKGROUND: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure. METHODS: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors. RESULTS: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration. CONCLUSIONS: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.

Molecular, metabolic, and functional CD4 T cell paralysis in the lymph node impedes tumor control.

CD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.

A conserved STRIPAK complex is required for autophagy in muscle tissue.

Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 as conserved mediators of muscle tissue maintenance. We performed affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.

Molecular, metabolic and functional CD4 T cell paralysis impedes tumor control.

CD4 T cells are important effectors of anti-tumor immunity, yet the regulation of CD4 tumor-specific T (T TS ) cells during cancer development is still unclear. We demonstrate that CD4 T TS cells are initially primed in the tumor draining lymph node and begin to divide following tumor initiation. Distinct from CD8 T TS cells and previously defined exhaustion programs, CD4 T TS cell proliferation is rapidly frozen in place and differentiation stunted by a functional interplay of T regulatory cells and both intrinsic and extrinsic CTLA4 signaling. Together these mechanisms paralyze CD4 T TS cell differentiation, redirecting metabolic and cytokine production circuits, and reducing CD4 T TS cell accumulation in the tumor. Paralysis is actively maintained throughout cancer progression and CD4 T TS cells rapidly resume proliferation and functional differentiation when both suppressive reactions are alleviated. Strikingly, Treg depletion alone reciprocally induced CD4 T TS cells to themselves become tumor-specific Tregs, whereas CTLA4 blockade alone failed to promote T helper differentiation. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 T TS cells to favor tumor progression.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Biomarker opportunities in the treatment of cancer-related depression.

Depression comorbid with cancer is common and associated with a host of negative health outcomes. The inflammatory basis of depression is a growing area of research in cancer, focused on how stressors transduce into inflammation and contribute to the emergence of depression. In this review, we synthesize inflammatory biomarker associations with both depression and the currently available pharmacotherapies and psychotherapies in cancer, underscoring the need for expanding research on anti-inflammatory agents with antidepressant effects. Modulation of inflammatory neuroimmune pathways can slow tumor progression and reduce metastases. Biomarkers associated with depression in cancer may help with diagnosis and treatment monitoring, as well as inform research on novel drug targets to potentially improve cancer survival.

Mass cytometry immunostaining protocol for multiplexing clinical samples.

This is a cytometry by time-of-flight (CyTOF) staining protocol for hematopoietic-derived cells, that leverages live-cell barcoding using receptor-type tyrosine-protein phosphatase C (CD45) antibodies conjugated to metal isotopes in combination with DNA-based palladium barcoding to multiplex up to 40 samples. In this protocol, DNA-based barcoding is performed before surface and intracellular immunostaining, which reduces the batch effects that result from day-to-day variations in staining and instrument sensitivity. This protocol also reduces antibody consumption and eliminates the need for repeated instrument adjustment.

Hip fracture specialists facilitate low-dose spinal anaesthesia in fractured neck of femur surgery.

Fractured neck of femur is a common but potentially devastating complication of frailty. In other surgical specialities, there is an inverse relationship between surgical experience and duration of surgery; however, this has not been quantified in hip trauma. In perioperative hip fracture care, prolonged surgery may be associated with increased morbidity and significantly impacts on the conduct of anaesthesia. Specifically, low-dose spinal anaesthesia, which is associated with improved haemodynamic stability, cannot be used if surgery is likely to be prolonged. We studied the duration of hip fracture surgery undertaken in our institution and compared this to surgical expertise. We retrospectively explored our theatre database to identify patients who underwent hip fracture surgery in our hospital over a 62-month period, recording duration of surgery and primary operating surgeon. Surgeons were classified into one of 3 groups: Consultant hip surgeon (specialist interest in hip surgery), Consultant orthopaedic surgeon but non-hip specialist, or Non-consultant (trainee or non-training grade). We identified 1426 hip fracture procedures. Consultant hip surgeons performed all types of hip fracture surgery faster, and with reduced variation in surgical duration, than did either non-hip specialist consultants or non-consultant grades. Consultant hip surgeons consistently performed hip fracture surgery in under 60 minutes. Specialist consultant hip surgeons make low-dose spinal anaesthesia (with shorter block duration but increased haemodynamic stability) feasible. Our data supports the development of dedicated hip fracture trauma lists where patients should be operated on by specialist hip surgeons or trainees directly under their supervision.

Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

Translational randomized phase II trial of cabozantinib in combination with nivolumab in advanced, recurrent, or metastatic endometrial cancer.

BACKGROUND: Combining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling. PATIENTS AND METHODS: In this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored. RESULTS: Median PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009). CONCLUSIONS: Adding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

Prevalence Estimates of Amyloid Abnormality Across the Alzheimer Disease Clinical Spectrum.

IMPORTANCE: One characteristic histopathological event in Alzheimer disease (AD) is cerebral amyloid aggregation, which can be detected by biomarkers in cerebrospinal fluid (CSF) and on positron emission tomography (PET) scans. Prevalence estimates of amyloid pathology are important for health care planning and clinical trial design. OBJECTIVE: To estimate the prevalence of amyloid abnormality in persons with normal cognition, subjective cognitive decline, mild cognitive impairment, or clinical AD dementia and to examine the potential implications of cutoff methods, biomarker modality (CSF or PET), age, sex, APOE genotype, educational level, geographical region, and dementia severity for these estimates. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional, individual-participant pooled study included participants from 85 Amyloid Biomarker Study cohorts. Data collection was performed from January 1, 2013, to December 31, 2020. Participants had normal cognition, subjective cognitive decline, mild cognitive impairment, or clinical AD dementia. Normal cognition and subjective cognitive decline were defined by normal scores on cognitive tests, with the presence of cognitive complaints defining subjective cognitive decline. Mild cognitive impairment and clinical AD dementia were diagnosed according to published criteria. EXPOSURES: Alzheimer disease biomarkers detected on PET or in CSF. MAIN OUTCOMES AND MEASURES: Amyloid measurements were dichotomized as normal or abnormal using cohort-provided cutoffs for CSF or PET or by visual reading for PET. Adjusted data-driven cutoffs for abnormal amyloid were calculated using gaussian mixture modeling. Prevalence of amyloid abnormality was estimated according to age, sex, cognitive status, biomarker modality, APOE carrier status, educational level, geographical location, and dementia severity using generalized estimating equations. RESULTS: Among the 19 097 participants (mean [SD] age, 69.1 [9.8] years; 10 148 women [53.1%]) included, 10 139 (53.1%) underwent an amyloid PET scan and 8958 (46.9%) had an amyloid CSF measurement. Using cohort-provided cutoffs, amyloid abnormality prevalences were similar to 2015 estimates for individuals without dementia and were similar across PET- and CSF-based estimates (24%; 95% CI, 21%-28%) in participants with normal cognition, 27% (95% CI, 21%-33%) in participants with subjective cognitive decline, and 51% (95% CI, 46%-56%) in participants with mild cognitive impairment, whereas for clinical AD dementia the estimates were higher for PET than CSF (87% vs 79%; mean difference, 8%; 95% CI, 0%-16%; P = .04). Gaussian mixture modeling-based cutoffs for amyloid measures on PET scans were similar to cohort-provided cutoffs and were not adjusted. Adjusted CSF cutoffs resulted in a 10% higher amyloid abnormality prevalence than PET-based estimates in persons with normal cognition (mean difference, 9%; 95% CI, 3%-15%; P = .004), subjective cognitive decline (9%; 95% CI, 3%-15%; P = .005), and mild cognitive impairment (10%; 95% CI, 3%-17%; P = .004), whereas the estimates were comparable in persons with clinical AD dementia (mean difference, 4%; 95% CI, -2% to 9%; P = .18). CONCLUSIONS AND RELEVANCE: This study found that CSF-based estimates using adjusted data-driven cutoffs were up to 10% higher than PET-based estimates in people without dementia, whereas the results were similar among people with dementia. This finding suggests that preclinical and prodromal AD may be more prevalent than previously estimated, which has important implications for clinical trial recruitment strategies and health care planning policies.

Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4-TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

Integration of proteomic and genetic approaches to assess developmental muscle atrophy.

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, we chose to investigate changes in protein profiles during muscle atrophy in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms as these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, we first defined protein signature changes during M. sexta intersegmental muscle (ISM) atrophy and then used genetic approaches to confirm their functional importance in the D. melanogaster dorsal internal oblique muscles (DIOMs). Our data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A-band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. We also uncovered the actin filament regulator ciboulot (Cib) as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may eventually lead to therapeutic targets.