Search

Lung ultrasound has become a part of the daily examination of physicians working in intensive, sub-intensive, and general medical wards. The easy access to hand-held ultrasound machines in wards where they were not available in the past facilitated the widespread use of ultrasound, both for clinical examination and as a guide to procedures; among point-of-care ultrasound techniques, the lung ultrasound saw the greatest spread in the last decade. The COVID-19 pandemic has given a boost to the use of ultrasound since it allows to obtain a wide range of clinical information with a bedside, not harmful, repeatable examination that is reliable. This led to the remarkable growth of publications on lung ultrasounds. The first part of this narrative review aims to discuss basic aspects of lung ultrasounds, from the machine setting, probe choice, and standard examination to signs and semiotics for qualitative and quantitative lung ultrasound interpretation. The second part focuses on how to use lung ultrasound to answer specific clinical questions in critical care units and in emergency departments.

We prove the large deviation principle (LDP) for posterior distributions arising from subfamilies of full exponential families, allowing misspecification of the model. Moreover, motivated by the so-called inverse Sanov Theorem (see e.g. Ganesh and O'Connell 1999 and 2000), we prove the LDP for the corresponding maximum likelihood estimator, and we study the relationship between rate functions. In our setting, even in the non misspecified case, it is not true in general that the rate functions for posterior distributions and for maximum likelihood estimators are Kullback-Leibler divergences with exchanged arguments.

The human gastrointestinal tract hosts a complex and dynamic population of commensal bacterial species, which have coevolved with the host, generating a symbiotic relationship. Some compounds present in foods, such as polyols, prebiotic fibers, or phenolic compounds, are poorly metabolized and absorbed by the host before the transformation guided by the colonic microbiota. By influencing gut microbiota, diet plays a fundamental role in understanding the beneficial effects of the gut microbiota on the host, including its long-term metabolism. The idea that probiotics can act not only by influencing the colonizing microbiota opens the door to a wider range of probiotic possibilities, encouraging innovation in the field. Furthermore, it has been shown both that some probiotics increase phagocytosis or the activity of natural killer cells. Current prebiotics are mainly based on carbohydrates, but other substances, such as polyphenols and polyunsaturated fatty acids, could exert prebiotic effects. A prebiotic substance has been defined as ‘a substrate that is selectively used by host microorganisms that confer a health benefit’, and so can interact with the gut microbiota through competition for nutrients, antagonism, cross-feeding, and support for microbiota stability. Influencing its composition in terms of richness and diversity, food components have a key impact on the intestinal microbiota. Eating habits can strongly influence the composition of the intestinal microbiota. A healthy intestinal microbiota is essential for maintaining general health, and diet is one of the major modulators of this fascinating world of microorganisms. This must give us one more reason to adopt a healthy lifestyle.

Objectives. During coronavirus disease (COVID-19) pandemic, preoperative screening before thoracic surgery is paramount in order to protect patients and staff from undetected infections. This study aimed to determine which preoperative COVID-19 screening tool was the most effective strategy before thoracic surgery. Methods. This retrospective cohort multicenter study was performed at 3 Italian thoracic surgery centers. All adult patients scheduled for thoracic surgery procedures from 4th March until 24th April, 2020, and submitted to COVID-19 preoperative screenings were included. The primary outcome was the yield of screening of the different strategies. Results. A total of 430 screenings were performed on 275 patients; 275 anamnestic questionnaires were administered. 77 patients were screened by an anamnestic questionnaire and reverse transcriptase polymerase chain reaction (RT-PCR). 78 patients were selected to combine screening with anamnestic questionnaire and chest computed tomography (CT). The positive yield of screening using a combination of anamnestic questionnaire and RT-PCR was 7.8% (95% CI: 2.6–14.3), while using a combination of anamnestic questionnaire and chest CT was 3.8% (95% CI: 0–9). Individual yields were 1.1% (95% CI: 0–2.5) for anamnestic questionnaire, 5.2% (95% CI: 1.3–11.7) for RT-PCR, and 3.8% (95% CI: 0–9). Conclusions. The association of anamnestic questionnaire and RT-PCR is able to detect around 8 positives in 100 asymptomatic patients. This combined strategy could be a valuable preoperative SARS-CoV-2 screening tool before thoracic surgery.

PURPOSE Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship with NF2 loss. Given the predominance of NF2 mutations in meningiomas, we evaluated the efficacy of GSK2256098, a FAK inhibitor, as part of the first genomically driven phase II study in recurrent or progressive grade 1-3 meningiomas. PATIENTS AND METHODS Eligible patients whose tumors screened positively for NF2 mutations were treated with GSK2256098, 750 mg orally twice daily, until progressive disease. Efficacy was evaluated using two coprimary end points: progression-free survival at 6 months (PFS6) and response rate by Macdonald criteria, where PFS6 was evaluated separately within grade-based subgroups: grade 1 versus 2/3 meningiomas. Per study design, the FAK inhibitor would be considered promising in this patient population if either end point met the corresponding decision criteria for efficacy. RESULTS Of 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients with NF2 mutations were enrolled and treated: 12 grade 1 and 24 grade 2/3 patients. Across all grades, one patient had a partial response and 24 had stable disease as their best response to treatment. In grade 1 patients, the observed PFS6 rate was 83% (10/12 patients; 95% CI, 52 to 98). In grade 2/3 patients, the observed PFS6 rate was 33% (8/24 patients; 95% CI, 16 to 55). The study met the PFS6 efficacy end point both for the grade 1 and the grade 2/3 cohorts. Treatment was well tolerated; seven patients had a maximum grade 3 adverse event that was at least possibly related to treatment with no grade 4 or 5 events. CONCLUSION GSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive NF2-mutated meningiomas, compared with historical controls. The criteria for promising activity were met, and FAK inhibition warrants further evaluation for this patient population.

Dunbar syndrome is caused by the compression of the celiac artery by the median arcuate ligament (MAL) and the diaphragmatic crura, presenting with abdominal pain. The treatment is surgical, with the aim of dissecting and separating the diaphragmatic crura from the celiac axis, restoring blood flow in the celiac trunk. We propose the use of transesophageal echocardiography to measure the peak systolic velocity in the celiac trunk and to evaluate, in real time, the efficacy of the surgery during MAL resection. Two clinical cases with practical implications are reported. In one of these, the ultrasound exam was useful in providing an intraoperative confirmation of the outcome of the resection.

Despite its decreasing incidence, gastric cancer remains an important global healthcare problem due to its overall high prevalence and high mortality rate. Since the MAGIC and FNLCC/FFCD trials, the neoadjuvant chemotherapy has been recommended throughout Europe in gastric cancer. Potential benefits of preoperative treatments include a higher rate of R0 resection achieved by downstaging the primary tumor, a likely effect on micrometastases and isolated tumor cells in the lymph nodes, and, as a result, improved cancer-related survival. Nevertheless, distortion of anatomical planes of dissection, interstitial fibrosis, and sclerotic tissue changes may increase surgical difficulty. The collection of at least twenty-five lymph nodes after neoadjuvant therapy would seem to ensure removal of undetectable node metastasis and reduce the likelihood of locoregional recurrence. It is not what you take but what you leave behind that defines survival. Therefore, para-aortic lymph node dissection is safe and effective after neoadjuvant chemotherapy, in both therapeutic and prophylactic settings. In this review, the efficacy of adequate lymph node dissection, also in a neoadjuvant setting, has been investigated in the key studies conducted to date on the topic.

Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies. Significance: Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. Significance: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.

Waterhouse-Friderichsen syndrome is a rare disease characterized by bleeding into the adrenal glands, and its symptoms may vary from non-specific abdominal pain to multi-organ failure. Commonly related to a variety of infectious diseases among which sepsis from Neisseria meningitidis is the most frequent, many other etiologies have been described, and in some rare cases, even no apparent cause can be identified. We report a rare case of an idiopathic bilateral adrenal hemorrhage in a 70-year-old male who presented to our emergency department with non-specific abdominal pain. Ultrasonography Scan (US) and Computed Tomography (CT) findings were crucial for making an early diagnosis, which resulted in a short hospitalization time and, eventually, a complete recovery of the adrenal function. After discharge, Magnetic Resonance Imaging (MRI) was also performed, providing a more complete ensemble of multimodality imaging findings that can be associated with this rare syndrome.

Inflammation is a physiological, beneficial, and auto-limiting response of the host to alarming stimuli. Conversely, a chronic systemic low-grade inflammation (CSLGI), known as a long-time persisting condition, causes damage to the organs and host tissues, representing a major risk for chronic diseases. Currently, a high global incidence of chronic inflammatory diseases is observed, often linked to the lifestyle-related changes that occurred in the last decade. The main lifestyle-related factors are proinflammatory diet, psychological stress, tobacco smoking, alcohol abuse, physical inactivity, and indoor living and working with its related consequences such as indoor pollution, artificial light exposure, and low vitamin D production. Recent scientific evidence found that gut microbiota (GM) has a main role in shaping the host’s health, particularly as CSLGI mediator. Based on the lastest discoveries regarding the remarkable GM activity, in this manuscript we focus on the elements of actual lifestyle that influence the composition and function of the intestinal microbial community in order to elicit the CSLGI and its correlated pathologies. In this scenario, we provide a broad review of the interplay between modern lifestyle, GM, and CSLGI with a special focus on the COVID symptoms and emerging long-COVID syndrome.

The purpose of this study is to evaluate the association between SARS-CoV-2 viral load in respiratory secretions of infected children and signs/symptoms of COVID-19.We reported the clinical characteristics of SARS-CoV-2-infected children during the study period. We compared viral load for several clinical variables, performed a predictive linear regression analysis to identify signs and symptoms significantly associated with viral load, and searched for discriminant viral load thresholds for symptomatic versus asymptomatic infections based on receiver-operating characteristics.A total of 570 patients were included. The median age was 4.75 years. Comparison of CT values by dichotomous variable showed higher viral loads in children with fever, respiratory symptoms, and previous exposure to SARS-CoV-2. The linear regression analysis confirmed a significant relationship between the CT value with these variables and with age, other symptoms, and asymptomaticity. In particular, infants with fever and SARS-CoV-2 exposure had higher viral loads. No viral load cut-offs were found to distinguish symptomatic from asymptomatic patients.Our study shows that fever, SARS-CoV-2 exposure, and respiratory symptoms are associated with higher viral load in children, especially infants, while age, presence of nonrespiratory symptoms, or absence of any symptoms are associated with lower viral load.Key message: the clinical variables that best predict viral load in infected children are history of previous exposure to a SARS-CoV-2-infected person and presence of fever and respiratory symptoms (higher viral load). Added value to the current literature: this is the first article to prove this point.SARS-CoV-2 viral load should not be used as a measure of clinical severity of COVID-19 in the pediatric population; however, lower viral load appears to be associated with asymptomatic COVID-19 in older children.

We address the questions of identifying pairs of interacting neurons from the observation of their spiking activity. The neuronal network is modeled by a system of interacting point processes with memory of variable length. The influence of a neuron on another can be either excitatory or inhibitory. To identify the existence and the nature of an interaction we propose an algorithm based only on the observation of joint activity of the two neurons in successive time slots. This reduces the amount of computation and storage required to run the algorithm, thereby making the algorithm suitable for the analysis of real neuronal data sets. We obtain computable upper bounds for the probabilities of false positive and false negative detection. As a corollary we prove the consistency of the identification algorithm.

Background:The role of gut microbiota in human disease is fascinating for hundreds of researchers worldwide. Many works have highlighted that gut microbiota modulates the immune system and that its disruption can trigger autoimmune and inflammatory immune-mediated diseases. Probiotics are able to positively modify microbiota composition.Objective:The aim of this review is to report the most important findings regarding the effects of probiotics administration in the most common autoimmune disease and inflammatory immune-mediated diseases.Method:Literature research was performed in PubMed, Google Scholar, and Medline, as well as in specific journal websites using the keywords: “autoimmunity”, “microbiota”, and “probiotics”. The article selection has been made independently by three authors, and controversies have been solved by a fourth researcher. Only English-language articles were included and preference was given to clinical trials, meta-analysis, and case series. After the review process, 68 articles have been considered.Results:Relying on this evidence, many studies have investigated the potential of probiotics in restoring gut eubiosis, thus affecting pathogenesis, clinical manifestations, and course of these pathologies. Even in the light of few and sometimes contradictory studies, physicians should start to consider these preliminary findings when approaching patients suffering from autoimmune disease. After an accurate case-by-case evaluation of potential candidates, probiotics might be introduced besides the standard therapeutic plan as supportive measures.

Despite the multiple evidence of the diffuse presence of methane in Jupiter’s auroral regions, the mechanisms leading to the CH4 brightening observed both from ground- and space-based platforms are not yet fully understood. During the first NASA/Juno’s orbit, the on-board imager/spectrometer JIRAM (Jovian Infrared Auroral Mapper) detected the 3.3-µm methane emission on both Jupiter’s poles. The signal was found to be mostly confined within the main auroral ovals, although the lack of spectral coverage over 80°S prevented a deep investigation of the southern methane spot. The CH4 polar emissions at 3.3 µm are likely originated by non-thermal excitation mechanisms occurring above the 1 µbar level, such as auroral particle precipitation and/or Joule heating. However, aurorally driven upwelling of methane inside the main oval might also explain the enhanced concentrations of CH4 observed at the jovian poles. To address this controversy, we derive the effective temperature of methane in Jupiter’s auroral regions, which is a key information to understand the origin of the detected fluorescence. The goal is achieved by exploring three Juno’s orbits and focusing on the spectra with the highest methane emissions and the smallest contribution from other auroral features due to H3+. JIRAM measurements from the first perijove are used to investigate the northern methane brightening, while observations from perijoves 7 and 8 are examined for its southern counterpart. The analysis reveals similar temperatures in the north- and south-polar spots, mainly ranging between 400 K and 670 K.

Urban lakes provide essential ecosystem services (hotspot of biodiversity, stormwater management, reduction of pollutant loadings…).During late summer and early autumn 2021, two whole-lake anoxia events occurred in Lake Champs-sur-Marne (Great Paris, France). This sandpit lake, principally fed by groundwater from the nearby Marne River, is continuously monitored by an autonomous station equipped with underwater sensors (water temperature, oxygen, Chlorophyll-a, CDOM, Nitrate). At the same point, a meteorological station is installed on a buoy, above the lake surface.During the two anoxia events (end of August and mid-September), oxygen concentration dropped from supersaturation level corresponding to a high peak of phytoplankton biomass, to 0% within a few days. During this summer period, successive heavy rainfall events occurred, causing a flood of the Marne River and rising the watertable level to unusual values in Summer. This resulted in high water and nutrient fluxes from the river towards the lake. The observed whole-lake anoxia can be explained according to the following assumptions: (1) the groundwater nutrient loading, favored by the high level of the Marne River, caused a huge phytoplankton production; (2) then, the phytoplankton decline was associated to an intense mineralization of the biomass organic carbon; (3) the lake oxygen was completely exhausted, leading to a massive fish kill. These results highlight the severe impact of a non-extreme but high and long hydro-meteorological event on a lake ecosystem. In Lake Champs-sur-Marne, the nutrient limitation of phytoplankton production generally occurs during late summer. In 2021, the limitation was removed by the Summer exceptional nutrient loading. In temperate regions, summer algal blooms are not limited by water temperature but by nutrient availability. Climate change is expected to increase the frequency of extreme hydro-meteorological events. Higher frequency of summer heavy rainfall may trigger repeated phytoplankton blooms, deteriorating the ecological status of lake ecosystems.

The most direct evidence that the icy Galilean satellite Callisto is able to sustain a significant neutral exosphere dates back to the detection of the CO2 non-LTE emission at 4.3 μm wavelength, measured by the NIMS instrument onboard the NASA Galileo spacecraft [1]. Other exospheric emissions have been observed in the UV spectral range, basically tracing the ionised exospheric component ([2], [3]). The analysis of such emissions in the framework of exospheric models (see e.g. [4], [5]) allowed to establish an overall composition dominated by O2 and H2O, with minor contributions by CO2 and CO. However, direct observations of neutral species other than CO2 are still missing, and their actual abundances, as well as spatial and temporal variability, are poorly constrained.The MAJIS (Moon And Jupiter Imaging Spectrometer, [6]) instrument, on board the ESA JUICE spacecraft, is expected to contribute in this field, by searching for non-LTE emissions falling in its spectral range, from 0.50 to 5.54 μm. In particular, we evaluate the chance of detection of signals at the satellite’s limb emitted by the CO2 complexes at 4.3 μm and 2.3 μm, by the H2O complex at 2.3 μm, by O2 at 1.27 μm, and by the CO bands at 4.7 μm and 2.3 μm. We calculate the populations of molecular levels by using the GRANADA algorithm [7], then the emissions intensities, for reference abundances of the molecular species and for limb-viewing geometry, by taking advantage of the KOPRA algorithm [8].Detection limits for all the abovementioned species are obtained in the approximation of horizontal uniformity of exospheric layers and adopting a vertical scaling compatible with the scale height in [1]. Surface density detection limits around 6.2 .106 cm-3, 6.6 .106 cm-3, 3.4 .109 cm-3, 3.4 .107 cm-3 are found for CO2, H2O, O2, and CO respectively. For both CO2 and H2O, these results indicate a high detection probability during the Callisto flybys planned in the current JUICE trajectory version (crema 5.0, [9]). Detection of O2 could also be possible if appropriate observing strategies are adopted. Detection of CO is instead very challenging, being its expected abundance well below the detection limit. AcknowledgementsThis work is supported by the Italian Space Agency (ASI-INAF grant 2018-25-HH.0). IAA researchers acknowledge financial support from the State Agency for Research of the Spanish MCIU through the Center of Excellence Severo Ochoa" award to the Instituto de Astrofísica de Andalucía (CEX2021-001131-S/fund by MICIN/AEI/10.13039/501100011033).References[1] Carlson,R.W.,1999, Science 283 (5403): 820–21. [2] Kliore,A.J., 2002, Journ.Geophys.Res. doi: 10.1029/2002ja009365. [3] Cunningham,N.J. et al., 2015, Icarus. doi:10.1016/j.icarus.2015.03.021. [4] Vorburger,A., et al., 2015, Icarus. doi:10.1016/j.icarus.2015.07.035. [5] Liang, M., 2005, Journ.Geophys.Res. doi:10.1029/2004je002322. [6] Guerri I., et al., 2018, Proc.of SPIE Vol.10690 106901L-1. doi: 10.1117/12.2312013. [7] Funke,B., et al., 2012, Journ.Quant.Sp.Rad.Tran. doi:10.1016/j.jqsrt.2012.05.001. [8] Stiller,G.P., et al., 2002. Journ.Quant.Sp.Rad.Tran. doi:10.1016/s0022-4073(01)00123-6. [9] ESA SPICE Service, JUICE Operational SPICE Kernel Dataset, doi:10.5270/esa-ybmj68p.

The Jupiter InfraRed Auroral Mapper (JIRAM) instrument on board the Juno spacecraft performed multiple observations of the Jupiter North Equatorial Belt (NEB) around the time of 12th Juno pericenter passage on April 1st 2018. The data consist in thermal infrared images (the JIRAM filter has a band pass centred around 4.8 μm) and show, among other atmospheric features, two bright hot-spots.Images of the same areas at different emission angles were used to constraint the trend of the limb darkening function.Comparison against simulated observations computed for different emission angles, total opacities, single scattering albedo ω0 and asymmetry parameter g suggest that ω0 ~ 0.9 and g ~ 0.32 provide best match with data, with the latter parameter only weakly constrained by JIRAM observations. Then, we computed the ω0 and g resulting from different size distributions (exploring the effective radius reff and variance v space), taking into account the complex refractive indices of ammonium hydrosulphide by [1] and [2].Our analysis suggests that neither sets of refractive indices are consistent with JIRAM observations. A more reasonable agreement is found once tholines are adopted, with an effective radius of 0.6 μm. This value is broadly consistent with the mean radius of Hot Spot’s particles estimated by [3] on the basis of Galileo Entry Probe data. While a composition of pure tholine is not realistic for Jupiter conditions, our results indicate that scattering properties of clouds are largely dominated by optical properties of contaminants, as already suggested in [4]. Indeed, a thin (0.01 of total radius) coating of such compound over a NH4SH particle can effectively mask the optical properties of the latter. An effective radius of 0.4 μm for these coated particles produces the ω0 and g derived from JIRAM data. [1] Howett C. J. A. et al., (2007) J. Opt. Soc. Am. B 24, 126-136.[2] Ferraro J. R et al. (1980) Applied Spectroscopy, 34 (5), 525-533.[3] Ragent B. et al, (1998), J. Geophys. Res., 103 (E10), 22891– 22909.[4] Grassi D. et al. (2021) MNRAS, 503(4), 4892-4907. This work was supported by the Italian Space Agency through ASI-INAF contract 2016-23-H.1-2018.

The electromagnetic coupling between the Galilean satellites at Jupiter and the planetary ionosphere generates an auroral footprint, whose ultimate source is the relative velocity between the moons and the corotating magnetospheric plasma. The footprint can be detected in the infrared L band (3.3-3.6 microns) by the Jovian InfraRed Auroral Mapper (JIRAM) onboard the Juno spacecraft, which can observe the footprint position with high precision. Here, we report the JIRAM data acquired since August 27th 2016 until May 23rd 2022, corresponding to the first 42 orbits of Juno. The dataset is used to compute the average position of the footprint tracks of Io, Europa and Ganymede. The result of the present analysis can help to test the reliability of magnetic field models, to calibrate ground-based observations and to highlight episodes of variability in the footprint positions, which in turn can point out specific conditions of the Jovian magnetospheric environment.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.Significance:We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells.See related commentary Leli and Koumenis, p. 535.This article is highlighted in the In This Issue feature, p. 517

Supplementary Figure S1 shows the strategy and result of co-essentiality module identificationfrom the DepMap CRISPR screen dataset and further characterization of the BIRC6 module. Supplementary Figure S2 shows the viability effect of BIRC6 depletion in BIRC6-dependent and -nondependent cancer cells lines as well asin nontransformed cells. Supplementary Figure S3 shows in vitro confirmations of the inducible BIRC6 depletion (by RNAi and CRISPR) systems as well as the effect of BIRC6 depletion on metastasis in xenograft models. Supplementary Figure S4 shows detailed results for the allele competition assay to evaluate the role of the BIR and UBC domains of BIRC6 and further evidence for the physical assembly of BIRC6 ubiquitin ligase complex. Supplementary Figure S5 shows evidence for the selective activation of the p-eIF2alpha/ATF4 arm of UPR (or ISR) upon BIRC6 depletion. Supplementary Figure S6 shows evidence supporting the importance of HRI-mediated ISR activation as a mechanism underlying the loss of viability caused by BIRC6 complex suppression. Supplementary Figure S7 shows evidence for the direct regulation of HRI ubiquitination and degradation by the BIRC6 complex Supplementary Figure S8 shows elevated expression of HRI mRNA in the tumor samples compared to the normal samples in large-scale datasets (TCGA, TARGET and GTEx). Supplementary Figure S9 shows the inefficiency of the gene expression and copy number of the functionally related genes in predicting BIRC6 dependency. Supplementary Table S1 lists the top 50 co-essentiality modules identified in the analysis explained in Supplementary Figure S1A. Supplementary Table S2 lists the key DNA and RNA sequences used in this study.

Supplementary Table from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Supplementary Figure from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.Significance:Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.See related commentary by Beytagh and Weiss, p. 2730.See related article by Mo et al., p. 2906.This article is highlighted in the In This Issue feature, p. 2711

Despite the increased use of molecular diagnostics, the extent to which patients who have these tests harbor potentially actionable aberrations is unclear. We retrospectively reviewed 439 patients with diverse cancers, for whom next-generation sequencing (mostly 236-gene panel) had been performed. Data pertaining to the molecular alterations identified, as well as associated treatment suggestions (on- or off-label, or experimental), were extracted from molecular diagnostic reports. Most patients (420/439; 96%) had at least one molecular alteration: 1,813 alterations (in 207 distinct genes) were identified [the majority being mutations (62%) or amplifications (29%)]. The three most common gene abnormalities were TP53 (44%), KRAS (16%), and PIK3CA (12%). The median number of alterations per patient was 3 (range, 0–16). Nineteen patients (4%) had no alterations; 48 patients (11%) had only one alteration; and 372 patients had two or more abnormalities (85%). The median number of potentially actionable anomalies per patient was 2 (range, 0–8). Most patients (393/439; 90%) had at least one potentially actionable alteration, and in all these cases the aberration could at least be targeted by an experimental drug in a clinical trial. A total of 307 patients (70%) had an alteration that was actionable with an approved drug, but in only 89 patients (20%) was the drug approved for their disease (on-label). Next-generation sequencing identified theoretically actionable aberrations in 90% of our patients. Many of the drugs are, however, experimental or would require off-label use. Strategies to address drug access for patients harboring potentially actionable mutations are needed. Mol Cancer Ther; 14(6); 1488–94. ©2015 AACR.

Supplementary Tables from An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

ORF overexpression screen data

Supplementary detailed methods

IC50, pYome results in MV4-11 and MOLM14, ORF-Screen Data in MV4-11 and MOLM14 and merged hits, SNV indels and copy number alteration of MV4-11 naive and resistant cells

Information on FANCJ mutation library, including source of mutation

Sensitivity data from validated FANCJ LOF mutations

Spleen tyrosine kinase (SYK) is a nonmutated therapeutic target in acute myeloid leukemia (AML). Attempts to exploit SYK therapeutically in AML have shown promising results in combination with chemotherapy, likely reflecting induced mechanisms of resistance to single-agent treatment in vivo. We conducted a genome-scale open reading frame (ORF) resistance screen and identified activation of the RAS–MAPK–ERK pathway as one major mechanism of resistance to SYK inhibitors. This finding was validated in AML cell lines with innate and acquired resistance to SYK inhibitors. Furthermore, patients with AML with select mutations activating these pathways displayed early resistance to SYK inhibition. To circumvent SYK inhibitor therapy resistance in AML, we demonstrate that a MEK and SYK inhibitor combination is synergistic in vitro and in vivo. Our data provide justification for use of ORF screening to identify resistance mechanisms to kinase inhibitor therapy in AML lacking distinct mutations and to direct novel combination-based strategies to abrogate these.Significance:The integration of functional genomic screening with the study of mechanisms of intrinsic and acquired resistance in model systems and human patients identified resistance to SYK inhibitors through MAPK signaling in AML. The dual targeting of SYK and the MAPK pathway offers a combinatorial strategy to overcome this resistance.This article is highlighted in the In This Issue feature, p. 161

By profiling their patients' tumors, oncologists now have the option to use molecular results to match patients with drug(s) based on specific biomarkers. In this observational study, 347 patients with solid advanced cancers and next-generation sequencing (NGS) results were evaluated. Outcomes for patients who received a “matched” versus “unmatched” therapy following their NGS results were compared. Eighty-seven patients (25%) were treated with a “matched” therapy, 93 (26.8%) with an “unmatched” therapy. More patients in the matched group achieved stable disease (SD) ≥ 6 months/partial response (PR)/complete response (CR), 34.5% vs. 16.1%, (P ≤ 0.020 multivariable or propensity score methods). Matched patients had a longer median progression-free survival (PFS; 4.0 vs. 3.0 months, P = 0.039 in the Cox regression model). In analysis using PFS1 (PFS on the prior line of therapy) as a comparator to PFS after NGS, as expected, the unmatched group demonstrated a PFS2 significantly shorter than PFS1 (P = 0.009); however, this shortening was not observed in the matched patients (P = 0.595). Furthermore, 45.3% of the matched patients (24/53) had a PFS2/PFS1 ratio ≥1.3 compared with 19.3% of patients (11/57) in the unmatched group (P = 0.004 univariable and P ≥ 0.057 in multivariable/propensity score analysis). Patients with a “matching-score” (the number of matched drugs divided by the number of aberrations; unmatched patients had a score of zero) > 0.2 had a median overall survival (OS) of 15.7 months compared with 10.6 months when their matching-score was ≤ 0.2, (P = 0.040 in the Cox regression model). Matched versus unmatched patients had higher rates of SD ≥ 6 months/PR/CR and longer PFS, and improvement in OS correlated with a higher matching score in multivariable analysis. Mol Cancer Ther; 15(4); 743–52. ©2016 AACR.

Secondary mini-pool overexpression screen data

Supplemental Methods 1) Sequencing: (182 and 236 gene lists) 2)Therapy 3) Matching Score 4) Statistical Analysis (patient's characteristics, study endpoints and definitions, analyses performed);Supplemental Table 1. Patient characteristics; Supplemental Table 2. List of alterations and matched drugs; Supplemental Figure 1. Overall survival analysis

Controls of mutation screen

FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient–derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information.Implications:We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients.

Supplementary Table 1 and 2 describe pharmacologic properties of BGJ398 and FIIN-3 and Supplementary Table 4 describes the characteristics of cellular models employed in the colony formation assays.

ORF constructs in secondary mini-pool overexpression screen

Supplement Methods

Pool composition of in vivo screen.

Figure S2 shows ORF screen for resistance to FGFR inhibition and Figure S3 shows candidate mediators of resistance to FGFR inhibition based on ORF screen.

Figure S4 shows immunoblots of cells expressing a subset of resistance ORFs

Genes and alleles selected for the project.

Annotation of 1163 ORFs.

CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.Significance:There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275

Figure Legends