Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1.

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Biodegradable lipophilic polymeric mRNA nanoparticles for ligand-free targeting of splenic dendritic cells for cancer vaccination.

Nanoparticle (NP)-based mRNA cancer vaccines hold great promise to realize personalized cancer treatments. To advance this technology requires delivery formulations for efficient intracellular delivery to antigen-presenting cells. We developed a class of bioreducible lipophilic poly(beta-amino ester) nanocarriers with quadpolymer architecture. The platform is agnostic to the mRNA sequence, with one-step self-assembly allowing for delivery of multiple antigen-encoding mRNAs as well as codelivery of nucleic acid-based adjuvants. We examined structure-function relationships for NP-mediated mRNA delivery to dendritic cells (DCs) and identified that a lipid subunit of the polymer structure was critical. Following intravenous administration, the engineered NP design facilitated targeted delivery to the spleen and preferential transfection of DCs without the need for surface functionalization with targeting ligands. Treatment with engineered NPs codelivering antigen-encoding mRNA and toll-like receptor agonist adjuvants led to robust antigen-specific CD8+ T cell responses, resulting in efficient antitumor therapy in in vivo models of murine melanoma and colon adenocarcinoma.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor.

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Fe/Thiol Cooperative Hydrogen Atom Transfer Olefin Hydrogenation: Mechanistic Insights That Inform Enantioselective Catalysis.

Asymmetric hydrogenation of activated olefins using transition metal catalysis is a powerful tool for the synthesis of complex molecules, but traditional metal catalysts have difficulty with enantioselective reduction of electron-neutral, electron-rich, and minimally functionalized olefins. Hydrogenation based on radical, metal-catalyzed hydrogen atom transfer (mHAT) mechanisms offers an outstanding opportunity to overcome these difficulties, enabling the mild reduction of these challenging olefins with selectivity that is complementary to traditional hydrogenations with H2. Further, mHAT presents an opportunity for asymmetric induction through cooperative hydrogen atom transfer (cHAT) using chiral thiols. Here, we report insights from a mechanistic study of an iron-catalyzed achiral cHAT reaction and leverage these insights to deliver stereocontrol from chiral thiols. Kinetic analysis and variation of silane structure point to the transfer of hydride from silane to iron as the likely rate-limiting step. The data indicate that the selectivity-determining step is quenching of the alkyl radical by thiol, which becomes a more potent H atom donor when coordinated to iron(II). The resulting iron(III)-thiolate complex is in equilibrium with other iron species, including FeII(acac)2, which is shown to be the predominant off-cycle species. The enantiodetermining nature of the thiol trapping step enables enantioselective net hydrogenation of olefins through cHAT using a commercially available glucose-derived thiol catalyst with up to 80:20 enantiomeric ratio. To the best of our knowledge, this is the first demonstration of asymmetric hydrogenation via iron-catalyzed mHAT. These findings advance our understanding of cooperative radical catalysis and act as a proof of principle for the development of enantioselective iron-catalyzed mHAT reactions.

Phosphorylation of cardiac sodium channel at Ser571 anticipates manifestations of the aging myopathy.

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction.NEW & NOTEWORTHY We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy.

Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Immunology beats cancer: a blueprint for successful translation.

Immunology offers an unprecedented opportunity for the science-driven development of therapeutics. The successes of antibodies to the immunomodulatory receptor CTLA-4 and blockade of the immunoinhibitory receptor PD-1 in cancer immunotherapy, from gene discovery to patient benefit, have created a paradigm for driving such endeavors.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

Distinct mechanisms of tumor resistance to NK killing: of mice and men.

Tumors cells can release natural killer (NK) cell ligands for activating receptor NKG2D that are thought to inhibit NK cell function. In a recent issue of Science, Deng et al. (2015) show that, unexpectedly, a soluble NKG2D ligand can enhance anti-tumor NK cell activity.

Time to Wake Up! The Ongoing Search for General Anesthetic Reversal Agents.

How general anesthetics work remains a topic of ongoing study. A parallel field of research has sought to identify methods to reverse general anesthesia. Reversal agents could shorten patients' recovery time and potentially reduce the risk of postoperative complications. An incomplete understanding of the mechanisms of general anesthesia has hampered the pursuit for reversal agents. Nevertheless, the search for reversal agents has furthered understanding of the mechanisms underlying general anesthesia. The study of potential reversal agents has highlighted the importance of rigorous criteria to assess recovery from general anesthesia in animal models, and has helped identify key arousal systems (e.g., cholinergic, dopaminergic, and orexinergic systems) relevant to emergence from general anesthesia. Furthermore, the effects of reversal agents have been found to be inconsistent across different general anesthetics, revealing differences in mechanisms among these drugs. The presynapse and glia probably also contribute to general anesthesia recovery alongside postsynaptic receptors. The next stage in the search for reversal agents will have to consider alternate mechanisms encompassing the tripartite synapse.

Integrating nutrition and genetic counseling: A case study approach to interprofessional learning.

Interprofessional collaboration is an increasingly important skillset for practicing healthcare professionals including genetic counselors and registered dietitian nutritionists. A multi-part interactive case study activity was created to develop interprofessional skills for graduate students within genetic counseling and medical nutrition training programs at an academic medical center. Feedback from learners who participated in this activity highlights its effect on their post-graduation clinical practice. Additionally, since the implementation of this activity, collaboration between students and faculty members of each program has occurred in other scholarly pursuits, fostering longstanding interprofessional relationships. Similar approaches to interprofessional education could be considered with other healthcare professions or at other institutions as another tool to strengthen practice upon graduation.

Targeting loss of heterozygosity for cancer-specific immunotherapy.

Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Aggression by omission: Redefining and measuring an understudied construct.

Researchers of aggression have classically focused on what has been previously called active aggression-the deliberate infliction of harm through the direct application of deleterious consequences. However, the counterpart to this, what was originally called passive aggression, has gone understudied, and its definition has mutated beyond its original conceptualization. The present two studies (N's 196 and 220, respectively) attempted to examine passive aggression as originally defined-the deliberate withholding of behavior to ensure that a target is harmed-and renaming it aggression by omission (ABO), in contrast to aggression by commission (ABC). These studies found that both fit within a similar nomological network of antagonism, Sadism, and trait aggression. Study 2 additionally found that both were equally affected by provocation and were considered equally harmful. These findings encourage further research into ABO to capture this construct concretely, especially in the context of common paradigms (e.g., the Taylor Aggression Paradigm, Hot Sauce, Point-Subtraction Aggression Paradigm), and trait aggression scales, which typically measure ABC.

Cationic Cobalt(II) Bisphosphine Hydroformylation Catalysis: In Situ Spectroscopic and Reaction Studies.

[HCo(CO)x(bisphosphine)](BF4), x = 1-3, is a highly active hydroformylation catalyst system, especially for internal branched alkenes. In situ infrared spectroscopy (IR), electron paramagnetic resonance (EPR), and nuclear magnetic resonance studies support the proposed catalyst formulation. IR studies reveal the formation of a dicationic Co(I) paramagnetic CO-bridged dimer, [Co2(µ-CO)2(CO)(bisphosphine)2]2+, at lower temperatures formed from the reaction of two catalyst complexes via the elimination of H2. DFT studies indicate a dimer structure with square-pyramidal and tetrahedral cobalt centers. This monomer-dimer equilibrium is analogous to that seen for HCo(CO)4, reacting to eliminate H2 and form Co2(CO)8. EPR studies on the catalyst show a high-spin (S = 3/2) Co(II) complex. Reaction studies are presented that support the cationic Co(II) bisphosphine catalyst as the catalyst species present in this system and minimize the possible role of neutral Co(I) species, HCo(CO)4 or HCo(CO)3(phosphine), as catalysts.