Multipurposing CARs: Same engine, different vehicles.

T cells genetically engineered to recognize and eliminate tumor cells through synthetic chimeric antigen receptors (CARs) have demonstrated remarkable clinical efficacy against B cell leukemia over the past decade. This therapy is a form of highly personalized medicine that involves genetically modifying a patient's T cells to recognize and kill cancer cells. With the FDA approval of 5 CAR T cell products, this approach has been validated as a powerful new drug in the therapeutic armamentarium against cancer. Researchers are now studying how to expand this technology beyond its use in conventional polyclonal αß T cells to address limitations to the current therapy in cancer and applications beyond it. Considering the specific characteristics of immune cell from diverse lineages, several preclinical and clinical studies are under way to assess the advantages of CAR-redirected function in these cells and apply the lessons learned from CAR T cell therapy in cancer to other diseases.

Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.

Rapid Arterial Occlusion Evaluation Scale Agreement between Emergency Medical Services Technicians and Neurologists.

BACKGROUND: Rapid arterial occlusion evaluation (RACE) scale is a valid prehospital tool used to predict large vessel occlusion of major cerebral arteries in patients with suspected acute stroke. RACE scale administered by Emergency medicine services (EMS) technicians in the prehospital setting correlates well with NIH Stroke Scale score after patient arrival at a hospital. Despite this, the RACE scale is often characterized as too difficult for EMS technicians to accurately utilize. There are no data examining RACE scale accuracy in the prehospital setting comparing EMS technicians with neurologists. We sought to examine agreement between RACE scores calculated by EMS technicians and stroke neurologists in the prehospital setting during telestroke consultation. METHODS: Data for this observational cohort study were prospectively collected and retrospectively analyzed. EMS technicians in person and stroke specialized neurologists via televideo connection independently assessed suspected stroke patients and calculated RACE scores in the prehospital setting. We used a linearly weighted Cohen's kappa (kw) to estimate the extent of agreement for RACE score between EMS technicians and stroke neurologists. RESULTS: Thirty-one patients with stroke symptoms were independently examined and assessed with the RACE scale by EMS technicians and stroke neurologists in the prehospital setting. Exact agreement on the RACE score was found in 24 of 31 (77%) patients. We found very good agreement between EMS technicians and stroke neurologists, kw = .818 (95% CI, .677-.960), P< .001. CONCLUSIONS: EMS technicians provide reliable RACE assessments in patients with suspected stroke, with agreement similar to stroke specialized neurologists in the prehospital setting.

What every dentist needs to know about liquid biopsies.

Integral components of the examination of the oral and maxillofacial region include the early detection of benign and malignant lesions and recognition of manifestations of systemic disorders. The gold standard for establishing a diagnosis has entailed excision of tissue and submission to a laboratory for histopathologic review or, to a lesser extent, microcollection of cells via a fine-needle aspiration biopsy. A revolutionary technology in the medical and dental fields, referred to as a liquid biopsy, involves the harvesting of fragments of DNA or RNA for surveillance of an array of pathological processes from various body fluids, such as blood, plasma, and saliva. This article provides an overview of this developing diagnostic field.

Pleiotropic role for MYCN in medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Large-Pore Functionalized Mesoporous Silica Nanoparticles as Drug Delivery Vector for a Highly Cytotoxic Hybrid Platinum-Acridine Anticancer Agent.

Large-pore mesoporous silica nanoparticles (MSN) were prepared and functionalized to serve as a highly robust and biocompatible delivery platform for platinum-acridine (PA) anticancer agents. The material showed a high loading capacity for the dicationic, hydrophilic hybrid agent [PtCl(en)(N-[acridin-9-ylaminoethyl]-N-methylpropionamidine)] dinitrate salt (P1A1) and virtually complete retention of payload at neutral pH in a high-chloride buffer. In acidic media mimicking the pH inside the cell lysosomes, rapid, burst-like release of P1A1 from the nanoparticles is observed. Coating of the materials in phospholipid bilayers resulted in nanoparticles with greatly improved colloidal stability. The lipid and carboxylate-modified nanoparticles containing 40 wt % drug caused S-phase arrest and inhibited cell proliferation in pancreatic cancer cells at submicromolar concentrations similar to carrier-free P1A1. The most striking feature of nanoparticle-delivered P1A1 was that the payload did not escape from the acidified lysosomal vesicles into the cytoplasm, but was shuttled to the nuclear membrane and released into the nucleus.

CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit.

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

Spatiotemporal characteristics of the neural representation of event concepts.

Events are a fundamentally important part of our understanding of the world. How lexical concepts denoting events are represented in the brain remains controversial. We conducted two experiments using event and object nouns matched on a range of psycholinguistic variables, including concreteness, to examine spatial and temporal characteristics of event concepts. Both experiments used magnitude and valence tasks on event and object nouns. The fMRI experiment revealed a distributed set of regions for events, including the angular gyrus, anterior temporal lobe, and posterior cingulate across tasks. In the EEG experiment, events and objects differed in amplitude within the 300-500 ms window. Together these results shed light into the spatiotemporal characteristics of event concept representation and show that event concepts are represented in the putative hubs of the semantic system. While these hubs are typically associated with object semantics, they also represent events, and have a likely role in temporal integration.

Outcomes of endovascular thrombectomy in patients selected by computed tomography perfusion imaging - a matched cohort study comparing nonagenarians to younger patients.

BACKGROUND: Endovascular thrombectomy (EVT) is efficacious for appropriately selected patients with large vessel occlusions (LVO) up to 24 hours from symptom onset. There is limited information on outcomes of nonagenarians, selected with computed tomography perfusion (CTP) imaging. METHODS: We retrospectively analyzed data from a large academic hospital between December 2017 and October 2019. Patients receiving EVT for anterior circulation LVO were stratified into nonagenarian (≥90 years) and younger (<90 years) groups. We performed propensity score matching on 18 covariates. In the matched cohort we compared: primary outcome of inpatient mortality and secondary outcomes of successful reperfusion (TICI ≥2B), symptomatic intracranial hemorrhage (sICH), and functional independence. Subgroup analysis compared CTP predicted core volumes in nonagenarians with outcomes. RESULTS: Overall, 214 consecutive patients (26 nonagenarians, 188 younger) underwent EVT. Nonagenarians were aged 92.8±2.9 years and younger patients were 74.5±13.5 years. Mortality rate was significantly greater in nonagenarians compared with younger patients (43.5% vs 10.4%, OR 9.33, 95% CI 2.88 to 47.97, P<0.0001) and a greater proportion of nonagenarians developed sICH (13.0% vs 3.0%, OR 6.00, 95% CI 1.34 to 55.20, P=0.02). There were no significant differences for successful reperfusion (P=1.00) or functional independence (P=0.75). Nonagenarians selected with smaller ischemic core volumes had decreased mortality rates (P=0.045). CONCLUSIONS: Nonagenarians were noted to have greater mortality and sICH rates following EVT compared with matched younger patients, which may be ameliorated by selecting patients with smaller CTP core volumes. Nonagenarians undergoing EVT had similar rates of successful reperfusion and functional independence compared with the younger cohort.

Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis.

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty-mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/ß-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non-small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression. SIGNIFICANCE: Loss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

Evaluation of a Platinum-Acridine Anticancer Agent and Its Liposomal Formulation in an in vivo Model of Lung Adenocarcinoma.

Liposomal formulations have been developed for a highly cytotoxic platinum-acridine agent, [PtCl(pn)(C18 H21 N4 )](NO3 )2 (PA, pn=propane-1,3-diamine), and fully characterized. Nanoliposomes consisting of hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and polyethylene glycol-2000-distearoylphosphatidylethanolamine (DSPE-mPEG2k ) were able to stably encapsulate PA at payload-to-lipid ratios of 2-20 %. The fusogenic properties of the liposomes promote efficient cellular uptake of PA across the plasma membrane, which results in vesicular transport of payload to the nucleus in cultured lung cancer cells. Unencapsulated PA and one of the newly designed liposomal formulations show promising tumor growth inhibition in tumor xenografts derived from A549 lung adenocarcinoma cells of 76 % and 72 %, respectively. Cisplatin showed no significant efficacy at a 10-fold higher dose. These findings underscore the utility of platinum-acridine agents for treating aggressive, chemoresistant forms of cancer and validate nanoliposomes as a biocompatible, expandable platform for their intravenous delivery and other potential routes of administration.

Engineering strategies to overcome the current roadblocks in CAR T cell therapy.

T cells genetically engineered to express chimeric antigen receptors (CARs) have proven - and impressive - therapeutic activity in patients with certain subtypes of B cell leukaemia or lymphoma, with promising efficacy also demonstrated in patients with multiple myeloma. Nevertheless, various barriers restrict the efficacy and/or prevent the widespread use of CAR T cell therapies in these patients as well as in those with other cancers, particularly solid tumours. Key challenges relating to CAR T cells include severe toxicities, restricted trafficking to, infiltration into and activation within tumours, suboptimal persistence in vivo, antigen escape and heterogeneity, and manufacturing issues. The evolution of CAR designs beyond the conventional structures will be necessary to address these limitations and to expand the use of CAR T cells to a wider range of malignancies. Investigators are addressing the current obstacles with a wide range of engineering strategies in order to improve the safety, efficacy and applicability of this therapeutic modality. In this Review, we discuss the innovative designs of novel CAR T cell products that are being developed to increase and expand the clinical benefits of these treatments in patients with diverse cancers.

Medicine and surgery residents' perspectives on the impact of COVID-19 on graduate medical education.

The COVID-19 crisis has had an unprecedented impact on resident education and well-being: social distancing guidelines have limited patient volumes and forced virtual learning, while personal protective equipment (PPE) shortages, school/daycare closures, and visa restrictions have served as additional stressors. Our study aimed to analyze the effects of COVID-19 crisis-related stressors on residents' professional and personal lives. In April 2020, we administered a survey to residents at a large academic hospital system in order to assess the impact of the pandemic on residency training after >6 weeks of a modified schedule. The primary outcome was to determine which factors or resident characteristics were related to stress during the pandemic. Our secondary goals were to examine which resident characteristics were related to survey responses. Data were analyzed with regression analyses. Ninety-six of 205 residents completed the survey (47% response rate). For our primary outcome, anxiety about PPE (P < 0.001), female gender (P = 0.03), and the interaction between female gender and anxiety about PPE (P = 0.04) were significantly related to increased stress during the COVID-19 pandemic. Secondary analyses suggested that medicine residents were more comfortable than surgical residents using telemedicine (P > 0.001). Additionally, compared to juniors, seniors believed that the pandemic was more disruptive, modified schedules were effective, and virtual meetings were less effective while virtual lectures were more effective (all P ≤ 0.05) Furthermore, the pandemic experience has allowed seniors in particular to feel more confident to lead in future health crises (P ≤ 0.05). Medicine and surgery residency programs should be cognizant of and closely monitor the effects of COVID-19 crisis-related factors on residents' stress and anxiety levels. Transparent communication, telemedicine, online lectures/meetings, procedure simulations, advocacy groups, and wellness resources may help to mitigate some of the challenges posed by the pandemic.

A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma.

We have shown previously that blockade of epidermal growth factor receptor (EGFR) cooperates with a pan-selective inhibitor of phosphoinositide-3-kinase (PI3K) in EGFR-driven glioma. In this communication, we tested EGFR-driven glioma differing in PTEN status, treating with the EGFR inhibitor erlotinib and a novel dual inhibitor of PI3Kalpha and mTOR (PI-103). Erlotinib blocked proliferation only in PTEN(wt) cells expressing EGFR. Although erlotinib monotherapy showed little effect in PTEN(mt) glioma, PI-103 greatly augmented the antiproliferative efficacy of erlotinib in this setting. To address the importance of PI3K blockade, we showed in PTEN(mt) glioma that combining PI-103 and erlotinib was superior to either monotherapy or to therapy combining erlotinib with either rapamycin (an inhibitor of mTOR) or PIK-90 (an inhibitor of PI3Kalpha). These experiments show that a dual inhibitor of PI3Kalpha and mTOR augments the activity of EGFR blockade, offering a mechanistic rationale for targeting EGFR, PI3Kalpha, and mTOR in the treatment of EGFR-driven, PTEN-mutant glioma.

Structure-based prediction of insertion-site preferences of transposons into chromosomes.

Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences, which are critically important for these uses, are poorly understood. We analyzed the insertion sites of several transposons and retroviruses to detect patterns of integration that might be useful for prediction of preferred integration sites. Initially we found that a mathematical description of DNA-deformability, called V(step), could be used to distinguish preferential integration sites for Sleeping Beauty (SB) transposons into a particular 100 bp region of a plasmid [G. Liu, A. M. Geurts, K. Yae, A. R. Srinivassan, S. C. Fahrenkrug, D. A. Largaespada,J. Takeda, K. Horie, W. K. Olson and P. B. Hackett (2005) J. Mol. Biol., 346, 161-173 ]. Based on these findings, we extended our examination of integration of SB transposons into whole plasmids and chromosomal DNA. To accommodate sequences up to 3 Mb for these analyses, we developed an automated method, ProTIS, that can generate profiles of predicted integration events. However, a similar approach did not reveal any structural pattern of DNA that could be used to predict favored integration sites for other transposons as well as retroviruses and lentiviruses due to a limitation of available data sets. Nonetheless, ProTIS has the utility for predicting likely SB transposon integration sites in investigator-selected regions of genomes and our general strategy may be useful for other mobile elements once a sufficiently high density of sites in a single region are obtained. ProTIS analysis can be useful for functional genomic, gene transfer and human gene therapy applications using the SB system.

Cell lines from MYCN transgenic murine tumours reflect the molecular and biological characteristics of human neuroblastoma.

Overexpression of the human MYCN oncogene driven by a tyrosine hydroxylase promoter causes tumours in transgenic mice that recapitulate the childhood cancer neuroblastoma. To establish an in vitro model to study this process, a series of isogenic cell lines were developed from these MYCN-driven murine tumours. Lines were established from tumours arising in homozygous and hemizygous MYCN transgenic mice. Hemizygous tumours gave rise to cell lines growing only in suspension. Homozygous tumours gave rise to similar suspension lines as well as morphologically distinct substrate-adherent lines characteristic of human S-type neuroblastoma cells. FISH analysis demonstrated selective MYCN transgene amplification in cell lines derived from hemizygous mice. Comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) analysis confirmed a range of neuroblastoma-associated genetic changes in the various lines, in particular, gain of regions syntenic with human 17q. These isogenic lines together with the transgenic mice thus represent valuable models for investigating the biological characteristics of aggressive neuroblastoma.

Genetic determinants of malignancy in a mouse model for oligodendroglioma.

Oligodendrogliomas of all grades overexpress epidermal growth factor receptor (EGFR), whereas deletion of ink4a/arf is found only in high-grade tumors. We used the S100 beta promoter to generate transgenic mice expressing v-erbB, a transforming allele of EGFR. These mice developed low-grade oligodendroglioma. Transgenic animals heterozygous for ink4a/arf or p53 developed high-grade tumors. Comparative genomic hybridization revealed loss of distal mouse chromosome 4, a region orthologous with human chromosome 1p, which is commonly lost in oligodendroglioma. Our results demonstrate that overexpression of EGFR, an epigenetic observation of uncertain significance in human oligodendroglioma, can initiate oligodendroglioma in the mouse. Furthermore, p53 pathway mutations can mediate the transition from low to high grade. These models hold promise for studying tumor lineage, identifying contributing genetic alterations and evaluating preclinical therapies in this important neoplasm.

Genome-wide array CGH analysis of murine neuroblastoma reveals distinct genomic aberrations which parallel those in human tumors.

Neuroblastoma, the third most common tumor of childhood, is a complex disease in which few genetic mutations have been identified.Mice expressing a human MYCN oncogene driven by the rat tyrosine hydroxylase promoter (TH-MYCN) represent an animal model for this disorder. We performed microarray-based comparative genomic hybridization analysis on murine tumors, identifying gains on chromosomes 1, 3, 11, 14, 17, and 18 and losses on chromosomes 5, 9, and 16. Fluorescence in situ hybridization analysis confirmed an amplicon on chromosome 18 as the site of TH-MYCN transgene integration. Selected tumors with localized gains of chromosome 11 delineate a 15-Mb region orthologous to human chromosome 17q and help to narrow the minimal region gained in human tumors. We observed clustered loss of chromosomes 5, 9, and 16, orthologous to a similar pattern of combined loss of chromosomes 3p, 4p, and 11q in human tumors. These data demonstrate conservation of many genetic changes in murine and human neuroblastoma and suggest that further delineation of genetic abnormalities in murine tumors may identify genes important in human disease.