Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight.

Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space.

A Second Space Age Spanning Omics, Platforms, and Medicine Across Orbits.

The recent acceleration of commercial, private, and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit (LEO), concomitant with the highest-ever number of crewed missions entering space and preparations for exploration-class (>1 year) missions. Such rapid advancement into space from many new companies, countries, and space-related entities has enabled a"Second Space Age." This new era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, encompassing multi-omic, single-cell, and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring, and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics (PGx), as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this review, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration (NASA), Japan Aerospace Exploration Agency (JAXA), European Space Agency (ESA), and other space agencies, and also detail the commercial spaceflight sector's (e.g. SpaceX, Blue Origin, Axiom, Sierra Space) entrance into aerospace medicine and space biology, the first aerospace medicine biobank, and the myriad upcoming missions that will utilize these tools to ensure a permanent human presence beyond LEO, venturing out to other planets and moons.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. Methods: In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). Results and discussion: A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

Multi-ancestry epigenome-wide analyses identify methylated sites associated with aortic augmentation index in TOPMed MESA.

Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.

Multi-ancestry epigenome-wide analyses identify methylated sites associated with aortic augmentation index in TOPMed MESA.

Despite the prognostic value of arterial stiffness (AS) and pulsatile hemodynamics (PH) for cardiovascular morbidity and mortality, epigenetic modifications that contribute to AS/PH remain unknown. To gain a better understanding of the link between epigenetics (DNA methylation) and AS/PH, we examined the relationship of eight measures of AS/PH with CpG sites and co-methylated regions using multi-ancestry participants from Trans-Omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA) with sample sizes ranging from 438 to 874. Epigenome-wide association analysis identified one genome-wide significant CpG (cg20711926-CYP1B1) associated with aortic augmentation index (AIx). Follow-up analyses, including gene set enrichment analysis, expression quantitative trait methylation analysis, and functional enrichment analysis on differentially methylated positions and regions, further prioritized three CpGs and their annotated genes (cg23800023-ETS1, cg08426368-TGFB3, and cg17350632-HLA-DPB1) for AIx. Among these, ETS1 and TGFB3 have been previously prioritized as candidate genes. Furthermore, both ETS1 and HLA-DPB1 have significant tissue correlations between Whole Blood and Aorta in GTEx, which suggests ETS1 and HLA-DPB1 could be potential biomarkers in understanding pathophysiology of AS/PH. Overall, our findings support the possible role of epigenetic regulation via DNA methylation of specific genes associated with AIx as well as identifying potential targets for regulation of AS/PH.

Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes.

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.

Measurable residual disease by flow cytometry in acute myeloid leukemia is prognostic, independent of genomic profiling.

Measurable residual disease (MRD) assessment provides a potent indicator of the efficacy of anti-leukemic therapy. It is unknown, however, whether integrating MRD with molecular profiling better identifies patients at risk of relapse. To investigate the clinical relevance of MRD in relation to a molecular-based prognostic schema, we measured MRD by flow cytometry in 189 AML patients enrolled in ECOG-ACRIN E1900 trial (NCT00049517) in morphologic complete remission (CR) (28.8 % of the original cohort) representing 44.4 % of CR patients. MRD positivity was defined as ≥ 0.1 % of leukemic bone marrow cells. Risk classification was based on standard cytogenetics, fluorescence-in-situ-hybridization, somatic gene analysis, and sparse whole genome sequencing for copy number ascertainment. At 84.6 months median follow-up of patients still alive at the time of analysis (range 47.0-120 months), multivariate analysis demonstrated that MRD status at CR (p = 0.001) and integrated molecular risk (p = 0.0004) independently predicted overall survival (OS). Among risk classes, MRD status significantly affected OS only in the favorable risk group (p = 0.002). Expression of CD25 (α-chain of the interleukin-2 receptor) by leukemic myeloblasts at diagnosis negatively affected OS independent of post-treatment MRD levels. These data suggest that integrating MRD with genetic profiling and pre-treatment CD25 expression may improve prognostication in AML.

Harnessing the power of sphingolipids: Prospects for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Validation of CRISPR targeting for proliferation and cytarabine resistance control genes in the acute myeloid leukemia cell line MOLM-13.

Acute myeloid leukemia patients with FMS-like tyrosine kinase 3-internal tandem duplications and mixed lineage leukemia-protein AF9 fusion proteins suffer from poor clinical outcomes. The MOLM-13 acute myeloid leukemia cell line harbors both of these abnormalities and is used in CRISPR experiments to identify disease drivers. However, experimental observations may be biased or inconclusive in the absence of experimentally validated positive control genes. We validated sgRNAs for knockdown of TP53 for cell proliferation and for DCK knockdown and CDA upregulation for cytarabine resistance control genes in MOLM-13 cells. We have provided a detailed CRISPR protocol applicable to both gene knockdown or activation experiments and downstream leukemic phenotype analyses. Inclusion of these controls in CRISPR experiments will enhance the capacity to identify novel myeloid leukemia drivers in MOLM-13 cells.

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions.

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

Frequent somatic TET2 mutations in chronic NK-LGL leukemia with distinct patterns of cytopenias.

Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher's exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch's t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Chemotherapy Induces Senescence-Like Resilient Cells Capable of Initiating AML Recurrence.

Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo. This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo, and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia.This article is highlighted in the In This Issue feature, p. 1307.

Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers.

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight.