Spaceflight induces changes in gene expression profiles linked to insulin and estrogen.

Organismal adaptations to spaceflight have been characterized at the molecular level in model organisms, including Drosophila and C. elegans. Here, we extend molecular work to energy metabolism and sex hormone signaling in mice and humans. We found spaceflight induced changes in insulin and estrogen signaling in rodents and humans. Murine changes were most prominent in the liver, where we observed inhibition of insulin and estrogen receptor signaling with concomitant hepatic insulin resistance and steatosis. Based on the metabolic demand, metabolic pathways mediated by insulin and estrogen vary among muscles, specifically between the soleus and extensor digitorum longus. In humans, spaceflight induced changes in insulin and estrogen related genes and pathways. Pathway analysis demonstrated spaceflight induced changes in insulin resistance, estrogen signaling, stress response, and viral infection. These data strongly suggest the need for further research on the metabolic and reproductive endocrinologic effects of space travel, if we are to become a successful interplanetary species.

Spatial multi-omics of human skin reveals KRAS and inflammatory responses to spaceflight.

Spaceflight can change metabolic, immunological, and biological homeostasis and cause skin rashes and irritation, yet the molecular basis remains unclear. To investigate the impact of short-duration spaceflight on the skin, we conducted skin biopsies on the Inspiration4 crew members before (L-44) and after (R + 1) flight. Leveraging multi-omics assays including GeoMx™ Digital Spatial Profiler, single-cell RNA/ATAC-seq, and metagenomics/metatranscriptomics, we assessed spatial gene expressions and associated microbial and immune changes across 95 skin regions in four compartments: outer epidermis, inner epidermis, outer dermis, and vasculature. Post-flight samples showed significant up-regulation of genes related to inflammation and KRAS signaling across all skin regions. These spaceflight-associated changes mapped to specific cellular responses, including altered interferon responses, DNA damage, epithelial barrier disruptions, T-cell migration, and hindered regeneration were located primarily in outer tissue compartments. We also linked epithelial disruption to microbial shifts in skin swab and immune cell activity to PBMC single-cell data from the same crew and timepoints. Our findings present the inaugural collection and examination of astronaut skin, offering insights for future space missions and response countermeasures.

Direct RNA sequencing of astronaut blood reveals spaceflight-associated m6A increases and hematopoietic transcriptional responses.

The advent of civilian spaceflight challenges scientists to precisely describe the effects of spaceflight on human physiology, particularly at the molecular and cellular level. Newer, nanopore-based sequencing technologies can quantitatively map changes in chemical structure and expression at single molecule resolution across entire isoforms. We perform long-read, direct RNA nanopore sequencing, as well as Ultima high-coverage RNA-sequencing, of whole blood sampled longitudinally from four SpaceX Inspiration4 astronauts at seven timepoints, spanning pre-flight, day of return, and post-flight recovery. We report key genetic pathways, including changes in erythrocyte regulation, stress induction, and immune changes affected by spaceflight. We also present the first m6A methylation profiles for a human space mission, suggesting a significant spike in m6A levels immediately post-flight. These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome's dynamic response to spaceflight.

Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

Telomeric RNA (TERRA) increases in response to spaceflight and high-altitude climbing.

Telomeres are repetitive nucleoprotein complexes at chromosomal termini essential for maintaining genome stability. Telomeric RNA, or TERRA, is a previously presumed long noncoding RNA of heterogeneous lengths that contributes to end-capping structure and function, and facilitates telomeric recombination in tumors that maintain telomere length via the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. Here, we investigated TERRA in the radiation-induced DNA damage response (DDR) across astronauts, high-altitude climbers, healthy donors, and cellular models. Similar to astronauts in the space radiation environment and climbers of Mt. Everest, in vitro radiation exposure prompted increased transcription of TERRA, while simulated microgravity did not. Data suggest a specific TERRA DDR to telomeric double-strand breaks (DSBs), and provide direct demonstration of hybridized TERRA at telomere-specific DSB sites, indicative of protective TERRA:telomeric DNA hybrid formation. Targeted telomeric DSBs also resulted in accumulation of TERRA foci in G2-phase, supportive of TERRA's role in facilitating recombination-mediated telomere elongation. Results have important implications for scenarios involving persistent telomeric DNA damage, such as those associated with chronic oxidative stress (e.g., aging, systemic inflammation, environmental and occupational radiation exposures), which can trigger transient ALT in normal human cells, as well as for targeting TERRA as a therapeutic strategy against ALT-positive tumors.

Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment.

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Telomeres and aging: on and off the planet!

Improving human healthspan in our rapidly aging population has never been more imperative. Telomeres, protective "caps" at the ends of linear chromosomes, are essential for maintaining genome stability of eukaryotic genomes. Due to their physical location and the "end-replication problem" first envisioned by Dr. Alexey Olovnikov, telomeres shorten with cell division, the implications of which are remarkably profound. Telomeres are hallmarks and molecular drivers of aging, as well as fundamental integrating components of the cumulative effects of genetic, lifestyle, and environmental factors that erode telomere length over time. Ongoing telomere attrition and the resulting limit to replicative potential imposed by cellular senescence serves a powerful tumor suppressor function, and also underlies aging and a spectrum of age-related degenerative pathologies, including reduced fertility, dementias, cardiovascular disease and cancer. However, very little data exists regarding the extraordinary stressors and exposures associated with long-duration space exploration and eventual habitation of other planets, nor how such missions will influence telomeres, reproduction, health, disease risk, and aging. Here, we briefly review our current understanding, which has advanced significantly in recent years as a result of the NASA Twins Study, the most comprehensive evaluation of human health effects associated with spaceflight ever conducted. Thus, the Twins Study is at the forefront of personalized space medicine approaches for astronauts and sets the stage for subsequent missions. We also extrapolate from current understanding to future missions, highlighting potential biological and biochemical strategies that may enable human survival, and consider the prospect of longevity in the extreme environment of space.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

ArCH: improving the performance of clonal hematopoiesis variant calling and interpretation.

MOTIVATION: The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. RESULTS: Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. AVAILABILITY AND IMPLEMENTATION: The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.

Resistance to PRMT5-targeted therapy in mantle cell lymphoma.

ABSTRACT: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma, and patients who relapse on targeted therapies have poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme essential for B-cell transformation, drives multiple oncogenic pathways and is overexpressed in MCL. Despite the antitumor activity of PRMT5 inhibition (PRT-382/PRT-808), drug resistance was observed in a patient-derived xenograft (PDX) MCL model. Decreased survival of mice engrafted with these PRMT5 inhibitor-resistant cells vs treatment-naive cells was observed (P = .005). MCL cell lines showed variable sensitivity to PRMT5 inhibition. Using PRT-382, cell lines were classified as sensitive (n = 4; 50% inhibitory concentration [IC50], 20-140 nM) or primary resistant (n = 4; 340-1650 nM). Prolonged culture of sensitive MCL lines with drug escalation produced PRMT5 inhibitor-resistant cell lines (n = 4; 200-500 nM). This resistant phenotype persisted after prolonged culture in the absence of drug and was observed with PRT-808. In the resistant PDX and cell line models, symmetric dimethylarginine reduction was achieved at the original PRMT5 inhibitor IC50, suggesting activation of alternative resistance pathways. Bulk RNA sequencing of resistant cell lines and PDX relative to sensitive or short-term-treated cells, respectively, highlighted shared upregulation of multiple pathways including mechanistic target of rapamycin kinase [mTOR] signaling (P < 10-5 and z score > 0.3 or < 0.3). Single-cell RNA sequencing analysis demonstrated a strong shift in global gene expression, with upregulation of mTOR signaling in resistant PDX MCL samples. Targeted blockade of mTORC1 with temsirolimus overcame the PRMT5 inhibitor-resistant phenotype, displayed therapeutic synergy in resistant MCL cell lines, and improved survival of a resistant PDX.

Elevated A-to-I RNA editing in COVID-19 infected individuals.

Given the current status of coronavirus disease 2019 (COVID-19) as a global pandemic, it is of high priority to gain a deeper understanding of the disease's development and how the virus impacts its host. Adenosine (A)-to-Inosine (I) RNA editing is a post-transcriptional modification, catalyzed by the ADAR family of enzymes, that can be considered part of the inherent cellular defense mechanism as it affects the innate immune response in a complex manner. It was previously reported that various viruses could interact with the host's ADAR enzymes, resulting in epigenetic changes both to the virus and the host. Here, we analyze RNA-seq of nasopharyngeal swab specimens as well as whole-blood samples of COVID-19 infected individuals and show a significant elevation in the global RNA editing activity in COVID-19 compared to healthy controls. We also detect specific coding sites that exhibit higher editing activity. We further show that the increment in editing activity during the disease is temporary and returns to baseline shortly after the symptomatic period. These significant epigenetic changes may contribute to the immune system response and affect adverse outcomes seen in post-viral cases.

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Intra-host variation in the spike S1/S2 region of a feline coronavirus type-1 in a cat with persistent infection.

Feline coronavirus type 1 (FCoV-1) is widely known for causing feline infectious peritonitis (FIP), a systemic infection that is often fatal, with the virus known as the FIPV biotype. However, subclinical disease also occurs, in which cats may not show signs and intermittently shed the virus, including in feces, possibly for long periods of time. This virus is known as the FECV biotype. Progression of FECV to FIPV has been linked to several genomic changes, however a specific region of the viral spike protein at the interface of the spike S1 and S2 domains has been especially implicated. In this study, we followed a cat (#576) for six years from 2017, at which time FCoV-1 was detected in feces and conjunctival swabs, until 2022, when the animal was euthanized based on a diagnosis of alimentary small cell lymphoma. Over this time period, the cat was clinically diagnosed with inflammatory bowel disease and chronic rhinitis, and cardiac problems were also suspected. Using hybridization capture targeting the spike (S) gene of FCoV followed by next-generation sequencing, we screened 27 clinical samples. We detected FCoV-1 in 4 samples taken in 2017 (intestine and nasal tissue, feces, and conjunctiva), and 3 samples taken in 2022 (feces, and intestinal and heart tissue), but not in fecal samples taken in 2019 and 2020. Next, we focused on the S1/S2 region within S, which contains the furin cleavage site (FCS), a key regulator of viral transmission and pathogenesis. We show that the FCoV-1 variants obtained from feces in 2017 and 2022 were identical, while the ones from conjunctiva (2017), heart (2022), and intestine (2017 and 2022) were distinct. Sequence comparison of all the variants obtained showed that most of the non-synonymous changes in the S1/S2 region occur within the FCS. In the heart, we found two variants that differed by a single nucleotide, resulting in distinct FCS motifs that differ in one amino acid. It is predicted that one of these FCS motifs will down-regulate spike cleavability. The variant from the conjunctiva (2017) had a 6-nucleotide in-frame insertion that resulted in a longer and more exposed S1/S2 loop, which is predicted to be more accessible to the furin protease. Our studies indicate that FCoV-1 can independently persist in the gastrointestinal tract and heart of a cat over a long period of time without evidence of typical FIP signs, with intermittent viral shedding from the gastrointestinal and respiratory tracts.

PRMT5 supports multiple oncogenic pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with an overall poor prognosis, particularly for patients that progress on targeted therapies. Novel, more durable treatment options are needed for patients with MCL. Protein arginine methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and posttranslational modification of cell cycle regulators, DNA repair genes, components of prosurvival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here, we report on the antitumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in xenografts derived from patients with MCL. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of B-cell receptor-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and wild-type TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.

IDH2 and TET2 mutations synergize to modulate T Follicular Helper cell functional interaction with the AITL microenvironment.

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.

Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas.

Mutations affecting enhancer chromatin regulators CREBBP and KMT2D are highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other's stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8 + T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. SIGNIFICANCE: Although CREBBP and KMT2D have similar enhancer regulatory functions, they are paradoxically co-mutated in lymphomas. We show that their combined loss causes specific disruption of super-enhancers driving immune synapse genes. Importantly, this leads to reduction of CD8 cells in lymphomas, linking super-enhancer function to immune surveillance, with implications for immunotherapy resistance.

Distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells.

Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.