Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment.

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates. Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease. Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment.

PURPOSE: Cancer drug development is currently limited by a paradigm of preclinical evaluation that does not adequately recapitulate the complexity of the intact human tumor microenvironment (TME). To overcome this, we combined trackable intratumor microdosing (CIVO) with spatial biology readouts to directly assess drug effects in patient tumors in situ. EXPERIMENTAL DESIGN: In a first-of-its-kind phase 0 clinical trial, we explored the effects of an investigational stage SUMOylation-activating enzyme (SAE) inhibitor, subasumstat (TAK-981) in 12 patients with head and neck carcinoma (HNC). Patients scheduled for tumor resection received percutaneous intratumor injections of subasumstat and vehicle control 1 to 4 days before surgery, resulting in spatially localized and graded regions of drug exposure (∼1,000-2,000 µm in diameter). Drug-exposed (n = 214) and unexposed regions (n = 140) were compared by GeoMx Digital Spatial Profiler, with evaluation at single-cell resolution in a subset of these by CosMx Spatial Molecular Imager. RESULTS: Localized regions of subasumstat exposure revealed SUMO pathway inhibition, elevation of type I IFN response, and inhibition of cell cycle across all tumor samples. Single-cell analysis by CosMx demonstrated cell-cycle inhibition specific to the tumor epithelium, and IFN pathway induction commensurate with a TME shift from immune-suppressive to immune-permissive. CONCLUSIONS: Pairing CIVO with spatial profiling enabled detailed investigation of response to subasumstat across a diverse sampling of native and intact TME. We demonstrate that drug mechanism of action can be directly evaluated in a spatially precise manner in the most translationally relevant setting: an in situ human tumor.

Genotype imputation accuracy and the quality metrics of the minor ancestry in multi-ancestry reference panels.

Large-scale imputation reference panels are currently available and have contributed to efficient genome-wide association studies through genotype imputation. However, whether large-size multi-ancestry or small-size population-specific reference panels are the optimal choices for under-represented populations continues to be debated. We imputed genotypes of East Asian (180k Japanese) subjects using the Trans-Omics for Precision Medicine reference panel and found that the standard imputation quality metric (Rsq) overestimated dosage r2 (squared correlation between imputed dosage and true genotype) particularly in marginal-quality bins. Variance component analysis of Rsq revealed that the increased imputed-genotype certainty (dosages closer to 0, 1 or 2) caused upward bias, indicating some systemic bias in the imputation. Through systematic simulations using different template switching rates (θ value) in the hidden Markov model, we revealed that the lower θ value increased the imputed-genotype certainty and Rsq; however, dosage r2 was insensitive to the θ value, thereby causing a deviation. In simulated reference panels with different sizes and ancestral diversities, the θ value estimates from Minimac decreased with the size of a single ancestry and increased with the ancestral diversity. Thus, Rsq could be deviated from dosage r2 for a subpopulation in the multi-ancestry panel, and the deviation represents different imputed-dosage distributions. Finally, despite the impact of the θ value, distant ancestries in the reference panel contributed only a few additional variants passing a predefined Rsq threshold. We conclude that the θ value substantially impacts the imputed dosage and the imputation quality metric value.

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.

Persistence of Adverse Drug Reaction-Related Hospitalization Risk Following Discharge.

This retrospective cohort study analyzed the administrative hospital records of 91,500 patients with the aim of assessing adverse drug reaction (ADR)-related hospital admission risk after discharge from ADR and non-ADR-related admission. Patients aged ≥18 years with an acute admission to public hospitals in Tasmania, Australia between 2011 and 2015 were followed until May 2017. The index admissions (n = 91,550) were stratified based on whether they were ADR-related (n = 2843, 3.1%) or non-ADR-related (n = 88,707, 96.9%). Survival analysis assessed the post-index ADR-related admission risk using (1) the full dataset, and (2) a matched subset of patients using a propensity score analysis. Logistic regression was used to identify the risk factors for ADR-related admissions within 90 days of post-index discharge. The patients with an ADR-related index admission were almost five times more likely to experience another ADR-related admission within 90 days (p < 0.001). An increased risk persisted for at least 5 years (p < 0.001), which was substantially longer than previously reported. From the matched subset of patients, the risk of ADR-related admission within 90 and 365 days more than doubled in the patients with an ADR-related index admission (p < 0.0001). These admissions were often attributed to the same drug class as the patients' index ADR-related admission. Cancer was a major risk factor for ADR-related re-hospitalization within 90 days; other factors included heart failure and increasing age.

Hospital Readmission Due to Chronic Obstructive Pulmonary Disease: A Longitudinal Study.

BACKGROUND: This study aimed to investigate the prevalence of hospital readmission for chronic obstructive pulmonary disease (COPD) at 30, 90 and 365 days, and to determine demographic and socioeconomic risk factors for 30-day and 90-day readmission and time to COPD-related readmission within 365 days in Tasmania. METHODS: Patients ≥40 years admitted for COPD between 2011 and 2015 were identified using administrative data from all major public hospitals in Tasmania, Australia. Factors associated with readmission and time to readmission were identified using logistic and Cox regression, respectively. RESULTS: The rates of COPD-related readmission were 6.7% within 30 days, 12.2% within 90 days and 23.7% within 365 days. Being male (odds ratio [OR]: 1.49, CI: 1.06-2.09), Indigenous (OR: 2.47, CI: 1.31-4.66) and living in the lower socioeconomic North-West region of Tasmania (OR: 1.80, CI: 1.20-2.69) were risk factors for 30-day readmission. Increased COPD-related (OR: 1.48, CI: 1.22-1.80; OR: 1.52, CI: 1.29-1.78) and non-COPD-related (OR: 1.12, CI: 1.03- 1.23; OR: 1.11, CI: 1.03-1.21) emergency department (ED) visits in the preceding six months were risk factors for both 30-day and 90-day readmissions. Being Indigenous (hazard ratio [HR]: 1.61, CI: 1.10-2.37) and previous COPD-related ED visits (HR: 1.30, CI: 1.21-1.39) decreased, while a higher Charlson Comorbidity Index (CCI) (OR: 0.91, CI: 0.83- 0.99) increased the time to readmission within 365 days. CONCLUSION: Being male, Indigenous, living in the North-West region and previous ED visits were associated with increased risk of COPD readmission in Tasmania. Interventions to improve access to primary healthcare for these groups may reduce COPD-related readmissions.

Profile of the Ovation ALTO abdominal stent graft for the treatment of abdominal aortic aneurysms: overview of its safety and efficacy.

INTRODUCTION: The Ovation ALTO is the next generation aortic stent graft from Endologix for the treatment of infra-renal abdominal aortic aneurysms. The device uses polymer-injected rings to create a proximal seal at the aneurysm neck. AREAS COVERED: Results from the first clinical study of the ALTO graft are analyzed and the potential benefits of the graft in minimizing post-treatment aneurysmal neck dilation discussed. The implications of the ALTO's Instructions-For-Use (IFU) and low-profile delivery system are also reviewed. EXPERT OPINION: The re-positioning of the sealing rings higher on the graft and an integrated compliant balloon are the most significant improvements on the Ovation iX, facilitating accurate placement of the proximal sealing ring and prompt balloon dilation of the polymer rings. The expansion the IFU to include neck lengths of ≥7 mm will mean more patients are eligible for infra-renal EVAR within IFU with the ALTO device. The published data on the device to date is limited. With over 1000 implants worldwide we would hope for more published data to become available. If this demonstrates similar mid-term results to that seen with the Ovation iX in arguably more hostile neck anatomy, then the Ovation ALTO is likely to be more widely used.

Balloon assisted, ultrasound guided percutaneous thrombin injection of a large radial artery pseudoaneurysm using a trans-venous approach via an Ipsilateral Arteriovenous Fistula.

BACKGROUND: Pseudoaneurysm formation is known to complicate arteriovenous haemodialysis access. Ultrasound guided thrombin injection is a recognised treatment option, but is not possible in pseudoaneurysms with no measurable neck. Balloon assisted techniques have been described in such cases, which transiently obstruct flow out of the pseudoaneurysm and thereby prevent non-target embolization during ultrasound guided percutaneous thrombin injection. We describe a balloon assisted technique for the treatment of a radial artery pseudoaneurysm, via retrograde access from the draining cephalic vein of an arteriovenous fistula. METHOD: A 61-year-old male with a radio-cephalic fistula was found on duplex ultrasound to have a large radial artery pseudoaneurysm with no measurable neck, as well as a juxta-anastomotic cephalic vein stenosis. Endovascular treatment was selected over open surgery. Retrograde cephalic venous access was established, which allowed for concurrent treatment of both the venous stenosis and the arterial pseudoaneurysm. After balloon dilation of the juxta-anastomotic stenosis, a percutaneous transluminal angioplasty balloon catheter was advanced across the arteriovenous anastomosis and inflated across the neck of the radial artery pseudoaneurysm, to transiently obstruct blood flow. This allowed for safe injection of thrombin into the pseudoaneurysm by direct ultrasound guided sac puncture; thereby achieving thrombosis. CONCLUSIONS: Balloon assisted ultrasound guided thrombin injection is an endovascular treatment option that can obviate the need for open surgery in cases involving pseudoaneurysms with no measurable neck. The technique described allowed both concurrent treatment of a juxta-anastomotic venous stenosis and treatment of an arterial pseudoaneurysm from a single venous puncture. This technique avoided arterial access and its inherent complications.

Multiple deprotonation paths of the nucleophile 3'-OH in the DNA synthesis reaction.

DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3'-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3'-OH in the reaction catalyzed by human DNA polymerase (Pol) η and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3'-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3'-OH using the 2'-OH of a ribonucleotide or 2'-F has little effect on catalysis. Moreover, combining the S113A mutation with 2'-F replacement, which removes two potential hydrogen acceptors of the 3'-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3' via alternative paths, supporting the hypothesis that binding of the third Mg2+ initiates the reaction by breaking the α-ß phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

A small number of early introductions seeded widespread transmission of SARS-CoV-2 in Quebec, Canada

Using genomic epidemiology, we investigated the arrival of SARS-CoV-2 to Quebec, the Canadian province most impacted by COVID-19, with >280,000 positive cases and >10,000 deaths in a population of 8.5 million as of March 1st, 2021. We report 2,921 high-quality SARS-CoV-2 genomes in the context of >12,000 publicly available genomes sampled globally over the first pandemic wave (up to June 1st, 2020). By combining phylogenetic and phylodynamic analyses with epidemiological data, we quantify the number of introduction events into Quebec, identify their origins, and characterize the spatio-temporal spread of the virus. Conservatively, we estimated at least 500 independent introduction events, the majority of which happened from spring break until two weeks after the Canadian border closed for non-essential travel. Subsequent mass repatriations did not generate large transmission lineages (>50 cases), likely due to mandatory quarantine measures in place at the time. Consistent with common spring break and snowbird destinations, most of the introductions were inferred to have originated from Europe via the Americas. Fewer than 100 viral introductions arrived during spring break, of which 5-10 led to the largest transmission lineages of the first wave (accounting for 36-58% of all sequenced infections). These successful viral transmission lineages dispersed widely across the province, consistent with founder effects and superspreading dynamics. Transmission lineage size was greatly reduced after March 11th, when a quarantine order for returning travelers was enacted. While this suggests the effectiveness of early public health measures, the biggest transmission lineages had already been ignited prior to this order. Combined, our results reinforce how, in the absence of tight travel restrictions or quarantine measures, fewer than 100 viral introductions in a week can ensure the establishment of extended transmission chains.

Frequently overlooked realistic moral bioenhancement interventions.

Many supporters of 'moral bioenhancement' (MBE), the use of biomedical interventions for moral improvement, have been criticised for having unrealistic proposals. The interventions they suggest have often been called infeasible and their implementation plans vague or unethical. I dispute these criticisms by showing that various interventions to implement MBE are practically and ethically feasible enough to warrant serious consideration. Such interventions include transcranial direct current stimulation over the medial and dorsolateral prefrontal cortex, as well as supplementation with lithium and omega-3. Considering their efficacy and feasibility, it is strange that these interventions have rarely been proposed or discussed as MBE. I review evidence that each of those interventions can reduce antisocial behaviour, reduce racial bias, increase executive function or increase prosocial traits like fairness and altruism. I then specify and defend realistic, ethically permissible ways to implement these interventions, especially for violent offenders and public servants-the former as rehabilitation and the latter to meet the high standards of their occupations. These interventions could be given to violent offenders in exchange for a reduced sentence or compulsorily in some cases. Potential intervention methods for non-prisoners include increasing the USDA-recommended dose of omega-3, encouraging food companies to supplement their products with omega-3 or trace lithium, requiring MBE for employment as a police officer or political leader, and insurance companies providing discounts for undergoing MBE. In some reasonably limited form, using these interventions may be a good first step to implement the project of MBE.

Targeting Glutamine Metabolism and Redox State for Leukemia Therapy.

PURPOSE: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. EXPERIMENTAL DESIGN: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. RESULTS: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. CONCLUSIONS: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Female vocalizations predict reproductive output in brown-headed cowbirds (Molothrus ater).

Pair bonds are often maintained through the reciprocal and coordinated exchange of communicative signals. The ability to recognize and appropriately respond to a partner's signals will define a pair's ability to reproduce. Individual variation in responsiveness, by shaping the formation and maintenance of strong pair bonds, will ultimately influence an individual's reproductive output. Throughout the breeding period, female cowbirds (Molothrus ater) respond to male song displays using a vocalization known as the chatter. In this study, we investigated whether variation in chatters remained repeatable across years and predicted reproductive performance. A flock of cowbirds housed in a large aviary complex was observed during the spring of 2011 to 2012. We recorded courtship interactions, including singing behavior for males, and chatters and eggs laid by females. The rate with which females responded to song using chatters remained consistent across years, with some females predictably responding to more songs using chatters than others. During 2012, chattering predicted the number of eggs females laid and her paired status. Paired females were more likely to respond to songs with chatters, and there was a strong positive relationship between the number of eggs laid and the proportion of songs she responded to using chatters. Overall, these findings suggest that variation in female vocal behavior is associated with their reproductive success.

Bypassing a 8,5'-cyclo-2'-deoxyadenosine lesion by human DNA polymerase η at atomic resolution.

Oxidatively induced DNA lesions 8,5'-cyclopurine-2'-deoxynucleosides (cdPus) are prevalent and cytotoxic by impeding DNA replication and transcription. Both the 5'R- and 5'S-diastereomers of cdPu can be removed by nucleotide excision repair; however, the 5'S-cdPu is more resistant to repair than the 5'R counterpart. Here, we report the crystal structures of human polymerase (Pol) η bypassing 5'S-8,5'-cyclo-2'-deoxyadenosine (cdA) in insertion and the following two extension steps. The cdA-containing DNA structures vary in response to the protein environment. Supported by the "molecular splint" of Pol η, the structure of 5'S-cdA at 1.75-Šresolution reveals that the backbone is pinched toward the minor groove and the adenine base is tilted. In the templating position, the cdA takes up the extra space usually reserved for the thymine dimer, and dTTP is efficiently incorporated by Pol η in the presence of Mn2+ Rigid distortions of the DNA duplex by cdA, however, prevent normal base pairing and hinder immediate primer extension by Pol η. Our results provide structural insights into the strong replication blockage effect and the mutagenic property of the cdPu lesions in cells.

Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.

Detection of membrane-bound and soluble antigens by magnetic levitation.

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.