CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer, which is due in part to the paucity of targeted therapies. A systematic analysis of regulatory elements that extend beyond protein-coding genes could uncover avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their noncoding enhancer RNA (eRNA) transcripts. The functions of the top 30 eRNA-producing super-enhancers were systematically probed using high-throughput CRISPR-interference assays coupled to RNA sequencing that enabled unbiased detection of target genes genome-wide. Generation of high-resolution Hi-C chromatin interaction maps enabled annotation of the direct target genes for each super-enhancer, which highlighted their proclivity for genes that portend worse clinical outcomes in patients with TNBC. Illustrating the utility of this dataset, deletion of an identified super-enhancer controlling the nearby PODXL gene or specific degradation of its eRNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared with nonmalignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of TNBC. Significance: Integrative analysis of eRNA-producing super-enhancers defines molecular mechanisms controlling global patterns of gene expression that regulate clinical outcomes in breast cancer, highlighting the potential of enhancers as biomarkers and therapeutic targets.

Racial/ethnic disparities in availability of volunteer unrelated donors for allogeneic transplantation.

ABSTRACT: Despite the global unrelated donor (URD) registry size, the degree to which URD availability is a transplant barrier is not established. We evaluated the availability of 3,843 URDs requested for 455 diverse adult patients (predominantly with acute leukemia). URDs for non-Europeans were more likely to be domestic and had markedly lower Donor Readiness scores. Of URDs requested for confirmatory HLA-typing (CT) alone (ie, without simultaneous workup), 1,894 of 3,529 (54%) were available. Availability of domestic URDs was 45%. Donor Readiness score was highly predictive of CT availability. More non-European patients (n = 120) than Europeans (n = 335) had >10 URDs requested and <5 available. Of workup requests (after CT or CT-workup), <70% (604/889 [68%]) were available. More non-Europeans had <2 URDs available. URD availability for CT was markedly worse for non-Europeans, with availabilities for African, non-Black Hispanic, and Asian patients being 150/458 (33%), 120/258 (47%), and 119/270 (44%), respectively, with further decrements in URD workup availability. Our data suggest the functional size of the URD pool is much smaller than appreciated, mandating major operational changes for transplant centers and donor registries. Likelihood of donor availability should have a high priority in donor selection. Considering patient ancestry and URD Donor Readiness scores, centers should pursue, and registries permit, simultaneous pursuit of many URDs and abandon futile searches. Patients should be informed about their likelihood of donor availability and alternative options. Finally, although registries should address high URD attrition and speed procurement, use of all HLA-disparate graft types is needed to facilitate timely transplant for all.

Anatomical Substrates of Rapid Eye Movement Sleep Rebound in a Rodent Model of Post-sevoflurane Sleep Disruption.

BACKGROUND: Previous research suggests that sevoflurane anesthesia may prevent the brain from accessing rapid eye movement (REM) sleep. If true, then patterns of neural activity observed in REM-on and REM-off neuronal populations during recovery from sevoflurane should resemble those seen after REM sleep deprivation. In this study, the authors hypothesized that, relative to controls, animals exposed to sevoflurane present with a distinct expression pattern of c-Fos, a marker of neuronal activation, in a cluster of nuclei classically associated with REM sleep, and that such expression in sevoflurane-exposed and REM sleep-deprived animals is largely similar. METHODS: Adult rats and Targeted Recombination in Active Populations mice were implanted with electroencephalographic electrodes for sleep-wake recording and randomized to sevoflurane, REM deprivation, or control conditions. Conventional c-Fos immunohistochemistry and genetically tagged c-Fos labeling were used to quantify activated neurons in a group of REM-associated nuclei in the midbrain and basal forebrain. RESULTS: REM sleep duration increased during recovery from sevoflurane anesthesia relative to controls (157.0 ± 24.8 min vs. 124.2 ± 27.8 min; P = 0.003) and temporally correlated with increased c-Fos expression in the sublaterodorsal nucleus, a region active during REM sleep (176.0 ± 36.6 cells vs. 58.8 ± 8.7; P = 0.014), and decreased c-Fos expression in the ventrolateral periaqueductal gray, a region that is inactive during REM sleep (34.8 ± 5.3 cells vs. 136.2 ± 19.6; P = 0.001). Fos changes similar to those seen in sevoflurane-exposed mice were observed in REM-deprived animals relative to controls (sublaterodorsal nucleus: 85.0 ± 15.5 cells vs. 23.0 ± 1.2, P = 0.004; ventrolateral periaqueductal gray: 652.8 ± 71.7 cells vs. 889.3 ± 66.8, P = 0.042). CONCLUSIONS: In rodents recovering from sevoflurane, REM-on and REM-off neuronal activity maps closely resemble those of REM sleep-deprived animals. These findings provide new evidence in support of the idea that sevoflurane does not substitute for endogenous REM sleep.

Excellent leukemia control after second hematopoietic cell transplants with unrelated cord blood grafts for post-transplant relapse in pediatric patients.

Background: Patients with leukemia relapse after allogeneic hematopoietic cell transplant (HCT) have poor survival due to toxicity and disease progression. A second HCT often offers the only curative treatment. Methods: We retrospectively reviewed our bi-institutional experience (MSKCC-USA; Utrecht-NL) with unrelated cord blood transplantation (CBT) for treatment of post-transplant relapse. Overall survival (OS) and event-free survival (EFS) were evaluated using the Kaplan-Meier method, treatment-related mortality (TRM) and relapse were evaluated using the competing risk method by Fine-Gray. Results: Twenty-six patients age < 21 years received a second (n=24) or third (n=2) HCT with CB grafts during the period 2009-2021. Median age at first HCT (HCT1) was 11.5 (range: 0.9-17.7) years and all patients received myeloablative cytoreduction. Median time from HCT1 to relapse was 12.8 (range 5.5-189) months. At CBT, median patient age was 13.5 (range 1.4-19.1) years. Diagnoses were AML: 13; ALL: 4, MDS: 5, JMML: 2; CML: 1; mixed phenotype acute leukemia: 1. Sixteen patients (62%) were in advanced stage, either CR>2 or with active disease. Median time from HCT1 to CBT was 22.2 (range 7-63.2) months. All patients engrafted after CBT. Thirteen patients developed acute GvHD; 7 had grade III or IV. With a median survivor follow-up of 46.6 (range 17.4-155) months, 3-year OS was 69.2% (95% CI 53.6-89.5%) and 3-year EFS was 64.9% (95% CI 48.8-86.4%). Eight patients died, 3 of AML relapse and 5 due to toxicity (respiratory failure [n=4], GvHD [n=1]) at a median time of 7.7 (range 5.9-14.4) months after CBT. Cumulative incidence of TRM at 3 years was 19.2% (95% CI 4.1-34.4%). Notably, all TRM events occurred in patients transplanted up to 2015; no toxicity-related deaths were seen in the 16 patients who received CBT after 2015. Cumulative incidence of relapse was 15.9% (95% CI 1.6-30.2%) at 3 years, remarkably low for these very high-risk patients. Conclusions: Survival was very encouraging following CB transplants in pediatric patients with recurrent leukemia after first HCT, and TRM has been low over the last decade. CBT needs to be strongly considered as a relatively safe salvage therapy option for post-transplant relapse.

Sleep Fragmentation, Electroencephalographic Slowing, and Circadian Disarray in a Mouse Model for Intensive Care Unit Delirium.

BACKGROUND: We aimed to further validate our previously published animal model for delirium by testing the hypothesis that in aged mice, Anesthesia, Surgery and simulated ICU conditions (ASI) induce sleep fragmentation, electroencephalographic (EEG) slowing, and circadian disarray consistent with intensive care unit (ICU) patients with delirium. METHODS: A total of 41 mice were used. Mice were implanted with EEG electrodes and randomized to ASI or control groups. ASI mice received laparotomy, anesthesia, and simulated ICU conditions. Controls did not receive ASI. Sleep was recorded at the end of ICU conditions, and hippocampal tissue was collected on EEG recording. Arousals, EEG dynamics, and circadian gene expression were compared with t tests. Two-way repeated measures analysis of variance (RM ANOVA) was used to assess sleep according to light. RESULTS: ASI mice experienced frequent arousals (36.6 ± 3.2 vs 26.5 ± 3.4; P = .044; 95% confidence interval [CI], 0.29-19.79; difference in mean ± SEM, 10.04 ± 4.62) and EEG slowing (frontal theta ratio, 0.223 ± 0.010 vs 0.272 ± 0.019; P = .026; 95% CI, -0.091 to -0.007; difference in mean ± SEM, -0.05 ± 0.02) relative to controls. In ASI mice with low theta ratio, EEG slowing was associated with a higher percentage of quiet wakefulness (38.2 ± 3.6 vs 13.4 ± 3.8; P = .0002; 95% CI, -35.87 to -13.84; difference in mean ± SEM, -24.86 ± 5.19). ASI mice slept longer during the dark phases of the circadian cycle (nonrapid eye movement [NREM], dark phase 1 [D1]: 138.9 ± 8.1 minutes vs 79.6 ± 9.6 minutes, P = .0003, 95% CI, -95.87 to -22.69, predicted mean difference ± SE: -59.28 ± 13.89; NREM, dark phase 2 (D2): 159.3 ± 7.3 minutes vs 112.6 ± 15.5 minutes, P = .006, 95% CI, -83.25 to -10.07, mean difference ± SE, -46.66 ± 13.89; rapid eye movement (REM), D1: 20.5 ± 2.1 minutes vs 5.8 ± 0.8 minutes, P = .001, 95% CI, -24.60 to -4.71, mean difference ± SE, -14. 65 ± 3.77; REM, D2: 21.0 ± 2.2 minutes vs 10.3 ± 1.4 minutes, P = .029, 95% CI, -20.64 to -0.76, mean difference ± SE, -10.70 ± 3.77). The expression of essential circadian genes was also lower in ASI mice (basic helix-loop-helix ARNT like [BMAL1] : -1.3 fold change; circadian locomotor output cycles protein kaput [CLOCK] : -1.2). CONCLUSIONS: ASI mice experienced EEG and circadian changes mimicking those of delirious ICU patients. These findings support further exploration of this mouse approach to characterize the neurobiology of delirium.

An Optimized Search Prognosis Tool to Predict 8/8 HLA Allele-Matched Unrelated Donor Procurement.

For patients in need of allogeneic transplantation who lack an HLA-identical sibling, an 8/8 HLA allele-matched unrelated donor (URD) is a standard alternative. However, delays in URD procurement can adversely impact patient care. Recipient genotype and search assessment (MSKv1.0)-based tools can predict search prognosis for many, but both tools have lower performance in non-European ancestry patients. Using the MSKv1.0 tool, we analyzed searches from 1530 potential allograft recipients (including 863 who underwent transplantation) with the aim of creating an optimized MSKv2.0 search prognosis tool that can classify a URD search as either Good or Poor with a high level of accuracy while also limiting an ambiguous Fair search prognosis regardless of patient ancestry. By MSKv2.0, the 8/8 URD search prognosis distribution was 57% Good, 21% Fair, and 22% Poor in Europeans and 15% Good, 21% Fair, and 63% Poor in non-Europeans. Importantly, compared to MSKv1.0, the likelihood of Fair categorization was reduced to <25% with comparable Fair rates (P = .847) in both European and non-European groups. Moreover, all patients with an MSKv2.0 Good prognosis had an 8/8 URD identified, and almost all of those who underwent transplantation had an 8/8 URD (Europeans, 99%; non-Europeans, 98%; P = .504). The MSKv2.0 tool also was highly accurate at classifying Poor searches, with <10% identifying an 8/8 URD, and almost all patients who underwent transplantation (Europeans, 95%; non-Europeans, 96%) receiving an alternative donor. Using preliminary search results, MSKv2.0 accurately classifies patients by likelihood of 8/8 URD procurement, greatly facilitating triage to 8/8 URD (Good prognosis) or alternative donor (Poor prognosis) transplantations.

International Consensus Statement on Obstructive Sleep Apnea.

BACKGROUND: Evaluation and interpretation of the literature on obstructive sleep apnea (OSA) allows for consolidation and determination of the key factors important for clinical management of the adult OSA patient. Toward this goal, an international collaborative of multidisciplinary experts in sleep apnea evaluation and treatment have produced the International Consensus statement on Obstructive Sleep Apnea (ICS:OSA). METHODS: Using previously defined methodology, focal topics in OSA were assigned as literature review (LR), evidence-based review (EBR), or evidence-based review with recommendations (EBR-R) formats. Each topic incorporated the available and relevant evidence which was summarized and graded on study quality. Each topic and section underwent iterative review and the ICS:OSA was created and reviewed by all authors for consensus. RESULTS: The ICS:OSA addresses OSA syndrome definitions, pathophysiology, epidemiology, risk factors for disease, screening methods, diagnostic testing types, multiple treatment modalities, and effects of OSA treatment on multiple OSA-associated comorbidities. Specific focus on outcomes with positive airway pressure (PAP) and surgical treatments were evaluated. CONCLUSION: This review of the literature consolidates the available knowledge and identifies the limitations of the current evidence on OSA. This effort aims to create a resource for OSA evidence-based practice and identify future research needs. Knowledge gaps and research opportunities include improving the metrics of OSA disease, determining the optimal OSA screening paradigms, developing strategies for PAP adherence and longitudinal care, enhancing selection of PAP alternatives and surgery, understanding health risk outcomes, and translating evidence into individualized approaches to therapy.

Analysis of disparities in time to allogeneic transplantation in adults with acute myelogenous leukemia.

Although alternative donors extend transplant access, whether recipient ancestry affects the time to allogeneic transplant is not established. We analyzed the likelihood of clinically significant delays to allograft by patient ancestry in 313 adult patients with acute myelogenous leukemia (AML) who underwent transplantation. Non-European ancestry patients (n = 99) were more likely than Europeans (n = 214) to receive HLA-mismatched donor allografts (45% vs 24%). Overall, the median time from transplant indication to allograft was 127 days (range, 57-1683). In multivariable analysis, non-Europeans had an increased risk of prolonged indication to transplant time >180 days owing to significant delays in indication to consult >90 days and consult to transplant >120 days. Compared with recipients of HLA-matched unrelated donors (URDs), HLA-mismatched adult donor recipients were at an increased risk of delayed indication to transplant, whereas HLA-identical sibling and cord blood recipients were at a lower risk. Subanalysis showed more indication to transplant delays >180 days in non-European (44%) vs European (19%) 8/8 URD recipients. Finally, the pandemic further exacerbated delays for non-Europeans. In summary, although non-European patients with AML are less likely to receive 8/8 URDs as expected, if they do, their transplants are delayed. HLA-identical siblings and cord blood facilitate the fastest transplants regardless of patient ancestry, whereas other adult donor transplants are delayed. Strategies to mitigate referral barriers, hasten donor evaluation, and use all alternative donor sources are critical to ensure timely transplantation for patients with AML.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer.

The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we utilize an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We systematically probe the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we construct chromatin interaction maps that enable the annotation of direct target genes for these super-enhancers and confirm their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach examines a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.

Chronic E-Cigarette Exposure Alters Human Alveolar Macrophage Morphology and Gene Expression.

INTRODUCTION: Alveolar macrophages (AMs) are lung-resident immune cells that phagocytose inhaled particles and pathogens, and help coordinate the lung's immune response to infection. Little is known about the impact of chronic e-cigarette use (ie, vaping) on this important pulmonary cell type. Thus, we determined the effect of vaping on AM phenotype and gene expression. AIMS AND METHODS: We recruited never-smokers, smokers, and e-cigarette users (vapers) and performed research bronchoscopies to isolate AMs from bronchoalveolar lavage fluid samples and epithelial cells from bronchial brushings. We then performed morphological analyses and used the Nanostring platform to look for changes in gene expression. RESULTS: AMs obtained from smokers and vapers were phenotypically distinct from those obtained from nonsmokers, and from each other. Immunocytochemistry revealed that vapers AMs had significantly elevated inducible nitric oxide synthase (M1) expression and significantly reduced CD301a (M2) expression compared with nonsmokers or smokers. Vapers' AMs and bronchial epithelia exhibited unique changes in gene expression compared with nonsmokers or smokers. Moreover, vapers' AMs were the most affected of all groups and had 124 genes uniquely downregulated. Gene ontology analysis revealed that vapers and smokers had opposing changes in biological processes. CONCLUSIONS: These data indicate that vaping causes unique changes to AMs and bronchial epithelia compared with nonsmokers and smokers which may impact pulmonary host defense. IMPLICATIONS: These data indicate that normal "healthy" vapers have altered AMs and may be at risk of developing abnormal immune responses to inflammatory stimuli.

Preoperative REM sleep is associated with complication development after colorectal surgery.

BACKGROUND: While total sleep duration and rapid eye movement (REM) sleep duration have been associated with long-term mortality in non-surgical cohorts, the impact of preoperative sleep on postoperative outcomes has not been well studied. METHODS: In this secondary analysis of a prospective observational cohort study, patients who recorded at least 1 sleep episode using a consumer wearable device in the 7 days before elective colorectal surgery were included. 30-day postoperative outcomes among those who did and did not receive at least 6 h of total sleep, as well as those who did and did not receive at least 1 h of rapid eye movement (REM) sleep, were compared. RESULTS: 34 out of 95 (35.8%) patients averaged at least 6 h of sleep per night, while 44 out of 82 (53.7%) averaged 1 h or more of REM sleep. Patients who slept less than 6 h had similar postoperative outcomes compared to those who slept 6 h or more. Patients who averaged less than 1 h of REM sleep, compared to those who achieved 1 h or more of REM sleep, had significantly higher rates of complication development (29.0% vs. 9.1%, P = 0.02), and return to the OR (10.5% vs. 0%, P = 0.04). After adjustment for confounding factors, increased REM sleep duration remained significantly associated with decreased complication development (increase in REM sleep from 50 to 60 min: OR 0.72, P = 0.009; REM sleep ≥ 1 h: OR 0.22, P = 0.03). CONCLUSION: In this cohort of patients undergoing elective colorectal surgery, those who developed a complication within 30 days were less likely to average at least 1 h of REM sleep in the week before surgery than those who did not develop a complication. Preoperative REM sleep duration may represent a risk factor for surgical complications; however additional research is necessary to confirm this relationship.

ATS Core Curriculum 2021. Adult Sleep Medicine: Sleep Apnea.

The American Thoracic Society Sleep Core Curriculum updates clinicians on important sleep topics, presented during the annual meeting, and appearing in summary here. This year's sleep core theme is sleep-disordered breathing and its management. Topics range from pathophysiological mechanisms for the association of obstructive sleep apnea (OSA) and metabolic syndrome, surgical modalities of OSA treatment, comorbid insomnia and OSA, central sleep apnea, and sleep practices during a pandemic. OSA has been associated with metabolic syndrome, independent of the role of obesity, and the pathophysiology suggests a role for sleep fragmentation and intermittent hypoxia in observed metabolic outcomes. In specific patient populations, surgical treatment modalities for OSA have demonstrated large reductions in objective disease severity compared with no treatment and may facilitate adherence to positive airway pressure treatment. Patient-centered approaches to comorbid insomnia and sleep apnea include evaluating for both OSA and insomnia simultaneously and using shared-decision making to determine the order and timing of positive airway pressure therapy and cognitive behavioral therapy for insomnia. The pathophysiology of central sleep apnea is complex and may be due to the loss of drive to breathe or instability in the regulatory pathways that control ventilation. Pandemic-era sleep practices have evolved rapidly to balance safety and sustainability of care for patients with sleep-disordered breathing.

Feasibility and Preliminary Efficacy of an Internet-Delivered Intervention for Insomnia in Individuals with Mild Cognitive Impairment.

BACKGROUND: Approximately 50% of older adults with cognitive impairment suffer from insomnia. When untreated, pre-existing cognitive problems may be exacerbated and potentially contribute to further cognitive decline. One promising approach to maintain cognitive health is to improve sleep quantity and quality. OBJECTIVE: To determine feasibility, acceptability, and preliminary efficacy of Sleep Health Using the Internet for Older Adult Sufferers of Insomnia and Sleeplessness (SHUTi OASIS), an Internet-delivered cognitive behavioral therapy for insomnia (CBT-I) program in older adults with mild cognitive impairment (MCI). METHODS: Older adults with MCI and insomnia were recruited from hospital-based memory and sleep disorders clinics and enrolled in a single-arm pilot study. Participants completed the six cores of SHUTi OASIS, over nine weeks with two-week baseline and post-assessments using self-reported sleep diaries. Feasibility and acceptability were informed by usage statistics and qualitative interviews; preliminary efficacy was informed by patient-generated sleep data. RESULTS: Twelve participants enrolled and, on average, were 75.8 years of age. Ten participants completed the study and logged in most days. Most participants reported a positive overall experience, and interviews revealed successful and independent program management and completion. There were significant changes on all baseline to post-assessment sleep measures, including clinically meaningful improvements on the Insomnia Severity Index (13.5 to 8.3, p < 0.01), sleep efficiency, wake after sleep onset, and sleep onset latency (ps < 0.02). There was no statistically significant change in cognitive measures (p > 0.05). CONCLUSION: This study supports that older adults with cognitive impairment can independently complete CBT-I via the Internet and achieve clinical sleep improvements.

Genomes for Kids: The Scope of Pathogenic Mutations in Pediatric Cancer Revealed by Comprehensive DNA and RNA Sequencing.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor-normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. SIGNIFICANCE: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers. This article is highlighted in the In This Issue feature, p. 2945.

Phase separation drives aberrant chromatin looping and cancer development.

The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.

Prospective KIR genotype evaluation of hematopoietic cell donors is feasible with potential to benefit patients with AML.

Donor KIR and recipient HLA combinations that minimize inhibition and favor activation of the NK repertoire are associated with improved outcomes after allogeneic hematopoietic cell transplantation (HCT) in patients with myeloid neoplasia. We prospectively evaluated a weighted donor ranking algorithm designed to prioritize HLA-compatible unrelated donors (URDs) with weak inhibitory KIR3DL1/HLA-Bw4 interaction, followed by donors with nontolerized activating KIR2DS1, and finally those with KIR centromeric B haplotype. During donor evaluation, we performed KIR genotyping and ranked 2079 URDs for 527 subjects with myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). Among all patients, 394 (75%) had at least 1 KIR-advantageous donor, and 263 (50%) underwent HCT. In patients with AML, KIR3DL1 weak inhibition provided protection from relapse. Compared with KIR3DL1-Weak Inhibiting donors, KIR3DL1-Noninteracting donors were associated with increased risk of relapse (HR, 2.97; 95% CI, 1.33-6.64; P = .008) and inferior event-free survival (EFS; HR, 2.14; 95% CI, 1.16-3.95; P = .015). KIR3DL1-Strong Inhibiting donors were associated with HR, 1.65 (95% CI, 0.66-4.08; P = .25) for AML relapse and HR, 1.6 (95% CI, 0.81-3.17; P = .1) for EFS when compared with the use of KIR3DL1-weak inhibiting donors. Donor KIR2DS1/HLA-C1 status and centromeric KIR haplotype-B content were not associated with decreased risk of AML relapse. There was no benefit to KIR-based donor selection in patients with MDS. This study demonstrates that donor KIR typing is feasible, and prioritization of donors with certain KIR3DL1 genotypes may confer a protection from relapse after HCT in patients with AML.

Cellular effects of nicotine salt-containing e-liquids.

"Pod-based" e-cigarettes such as JUUL are currently the most prevalent electronic nicotine delivery systems (ENDS) in the United States. JUUL-type ENDS utilize nicotine salts protonated with benzoic acid rather than freebase nicotine. However, limited information is available on the cellular effects of these products. Cytoplasmic Ca2+ is a universal second messenger that controls many cellular functions including cell growth and cell death. Of note, dysregulation of cell Ca2+ homeostasis has been linked with several disease processes including autoimmune disease and several types of cancer. We exposed HEK293T cells and THP-1 macrophage-like cells to different JUUL e-liquids. We evaluated their effects on cellular viability and Ca2+ signaling by measuring fluorescence from calcein-AM/propidium iodide and Fluo-4, respectively. E-liquid autofluorescence was used to look for e-liquid permeation into cells. To identify the mechanisms behind the Ca2+ responses, different inhibitors of Ca2+ channels and phospholipase C signaling were used. JUUL e-liquids caused significant cytotoxic effects, with "Mint" flavor being the most cytotoxic. The Mint flavored e-liquid also caused a significant elevation in cytoplasmic Ca2+ . Using autofluorescence, the permeation of JUUL e-liquids into live cells was confirmed, indicating that intracellular organelles are directly exposed to e-liquids. Further studies identified the endoplasmic reticulum as being the source of e-liquid-induced changes in cytoplasmic Ca2+ . Nicotine salt-based e-liquids cause cytotoxicity and elevate cytoplasmic Ca2+ , indicating that they can exert biological effects beyond what would be expected with nicotine alone. These effects are flavor-dependent, and we propose that flavored e-liquids be reassessed for potential lung toxicity.