MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-ß (TGF-ß) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

Zyxin contributes to coupling between cell junctions and contractile actomyosin networks during apical constriction.

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and ß-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved.

Disparities in contemporary human papilloma virus vaccination uptake among adult women living in the United States: An All of Us Research Program study.

OBJECTIVE: Age-adjusted rates of new cervical cancer diagnoses in the United States have remained stable despite increasing availability of Human Papilloma Virus (HPV) vaccination. As it is well established that sociodemographic factors drive cervical cancer care inequity, we aimed to evaluate their impact on catch-up HPV vaccination rates in adults. METHODS: The All of Us (AoU) Research Program is a longitudinal cohort study sponsored by the National Institutes of Health. All participants ages 18-47 assigned female sex at birth enrolled between May 2018 and April 2023 were included in this analysis. Primary outcome was receipt of HPV vaccination. Bivariable and multivariable tests were used to examine associations. RESULTS: A total of 113,344 participants were identified in the AoU program, with 53 % (n = 60,594) self-identifying as a racial or ethnic minority. Only 3575 participants (3.2 %) were documented as having received HPV vaccination. Median age of vaccination was 26 and participants ages 18-27 were more likely to be vaccinated. Participants without health insurance (OR = 0.32, 95 % CI 0.26-0.40), stable employment (OR = 0.85, 95 % CI 0.79-0.91), and those who reported lower income (OR = 0.87, 95 % CI 0.79-0.97) were significantly less likely to have received HPV vaccination. Participants who described cost as a barrier to healthcare were also less likely to have received HPV vaccination (OR = 0.82, 95 % CI 0.73-0.93). CONCLUSION: Sociodemographic factors including low income, lack of health insurance, and lack of stable employment were all associated with lower likelihood of catch-up HPV vaccination among adult women living in the United States.

Cervical squamous cell carcinoma outcomes across continents: A retrospective study.

OBJECTIVE: To assess the influence of geographies and race on the survival outcomes in patients diagnosed with cervical squamous cell carcinoma (CSCC) across three continents. METHODS: This multicontinental retrospective study was conducted in 8 hospitals across Asia, Europe, and North America (NA). Clinicopathologic data of 595 patients with presumed early stages of CSCC, treated surgically, with curative intent was collected. Descriptive analysis and Cox regression models were produced. RESULTS: A total of 595 patients, consisting of 445 (74.8 %) white, 75 (12.6 %) Blacks, and 75 (12.6 %) Asian patients were included. Geographical distribution comprised 69 % of patients from NA, 22 % from Europe, and 9 % from Asia. The median age at diagnosis was 46 years. The median overall survival (OS) and relapse-free survival (RFS) were 22.09 years and 21.19 years, respectively. Patient characteristics varied significantly across geographical regions, except for consensus tumor grade. Patients in Europe from middle-income countries with limited CC screening had a substantially higher risk of death than those in NA (HR, 1.79; 95 % CI, 1.13 to 2.79; p = 0.015). Patients from single center in Japan had higher risk of relapse than those from the four heterogeneous NA centers (sub-distribution hazard ratio, 2.19; 95 % CI, 1.22 to 3.95; p = 0.009), although OS did not differ significantly. Race remained statistically insignificant for survival outcomes across the three continents but seemed to influence survival outcomes in NA centers. CONCLUSION: Our study highlights impact of geographies and races on CSCC survival outcomes, emphasizing the need of considering these factors when developing targeted interventions against CSCC.

Malignant Pleural Effusions in the Era of Immunotherapy and Antiangiogenic Therapy.

Malignant pleural effusions (MPE) have historically been associated with a poor prognosis, and patients often require a series of invasive procedures and hospitalizations that significantly reduce quality of life at the terminus of life. However, advances in the management of MPE have coincided with the era of immunotherapies, and to a lesser extent, antiangiogenic therapies for the treatment of lung cancer. Landmark studies have shown these drugs to improve overall survival and progression-free survival in patients with lung cancer, but a paucity of phase III trial data exists for the impact of immune checkpoint inhibitors (ICI) on lung cancers associated with MPE. This review will focus on the leading studies investigating the impact of ICI and antiangiogenic therapies in patients with lung cancer and MPE. The diagnostic and prognostic values of vascular endothelial growth factor and endostatin expression levels in malignancy will also be discussed. These advancements are changing the paradigm of MPE management from palliation to treatment for the first time since 1767 when MPE was first reported. The future holds the promise of durable response and extended survival in patients with MPE.

Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia.

Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.

Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML.

There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy.

Delirium detection by a novel bispectral electroencephalography device in general hospital.

AIM: Delirium is common and dangerous among elderly inpatients; yet, it is underdiagnosed and thus undertreated. This study aimed to test the diagnostic characteristics of a noninvasive point-of-care device with two-channel (bispectral) electroencephalography (EEG) for the screening of delirium in the hospital. METHODS: Patients admitted to the University of Iowa Hospitals and Clinics were assessed for the presence of delirium with a clinical assessment, the Confusion Assessment Method for Intensive Care Unit and Delirium Rating Scale. Subsequently, we obtained a 10-min bispectral EEG (BSEEG) recording from a hand-held electroencephalogram device during hospitalization. We performed power spectral density analysis to differentiate between those patients with and without delirium. RESULTS: Initially 45 subjects were used as a test dataset to establish a cut-off. The BSEEG index was determined to be a significant indicator of delirium, with sensitivity 80% and specificity 87.7%. An additional independent validation dataset with 24 patients confirmed the validity of the approach, with a sensitivity of 83.3% and specificity of 83.3%. CONCLUSION: In this pilot study, the BSEEG method was able to distinguish delirious patients from non-delirious patients. Our data showed the feasibility of this technology for mass screening of delirium in the hospital.

Nuclear factor-kappa B regulates pain and COMT expression in a rodent model of inflammation.

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund's adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures.

The Clonal Hematopoiesis-associated Gene Srcap Plays an Essential Role in Hematopoiesis.

Somatic mutations arising in hematopoietic stem cells (HSCs) may provide the latter with a fitness advantage, allowing the mutant HSC to clonally expand. Such mutations have been recurrently identified in the chromatin modifier, SRCAP, in both non-malignant and leukemic clones, suggesting that this gene plays a significant role in hematopoiesis. We generated a conditional Srcap loss of function murine model and determined the consequences of hematopoietic-specific loss of this gene. We show that Srcap is essential for normal fetal liver erythropoiesis and monocytopoiesis. In Srcap deficient fetal livers, the number of phenotypic HSCs is similar to that of controls, but these HSCs exhibit a profound repopulating defect. Likewise, conditional deletion of Srcap during adult hematopoiesis results in a rapid loss of HSCs. Loss of Srcap is associated with evidence of increased DNA damage in HSCs and lineage-restricted progenitors as assessed by y-H2AX expression. Consistent with this finding, we observed strong transcriptional upregulation of the p53 pathway in Srcap deficient erythroid precursors. Collectively our data highlight the importance of Srcap in maintaining HSC function and supporting hematopoietic differentiation and suggests that it plays an essential role in maintaining genomic integrity.

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Adipose microenvironment promotes hypersialylation of ovarian cancer cells.

Introduction: Ovarian and other peritoneal cancers have a strong tendency to metastasize into the surrounding adipose tissue. This study describes an effect of the adipose microenvironment on upregulation of sialic acid-containing glycans in ovarian cancer (OC). Heterogeneous populations of glycosylated OC tumors converged to a highly sialylated cell state that regulates tumorigenesis in an immune-dependent manner. Methods: We modeled the adipose microenvironment by conditioning growth media with human patient-derived adipose tissue. OC cell lines grown in the presence vs. absence of adipose conditioned media (ACM) were characterized by transcriptomics, western blotting, and chemical biology glycan labeling methods. Fluorescence-activated cell sorting was used to separate adipose-driven upregulation of hypersialylated ("SNA-high") vs. hyposialylated ("SNA-low") OC subpopulations. The two subpopulations were characterized by further transcriptomic and quantitative polymerase chain reaction analyses, then injected into a syngeneic mouse model. Immune system involvement was implicated using wild type and athymic nude mice with a primary endpoint of overall survival. Results: Adipose conditioning resulted in upregulation of sialyltransferases ST3GAL1, ST6GAL1, ST6GALNAC3, and ST8Sia1. In culture, OC cells displayed two distinct sialylated subpopulations that were stable for up to 9 passages, suggesting inherent heterogeneity in sialylation that is maintained throughout cell division and media changes. OC tumors that implanted in the omental adipose tissue exclusively reprogrammed to the highly sialylated subpopulation. In wild type C57BL/6 mice, only the hypersialylated SNA-high subpopulation implanted in the adipose, whereas the hyposialylated SNA-low subpopulation failed to be tumorigenic (p=0.023, n=5). In the single case where SNA-low established a tumor, post-mortem analysis revealed reprogramming of the tumor to the SNA-high state in vivo. In athymic nude mice, both subpopulations rapidly formed tumors, implicating a role of the adaptive immune system. Conclusions: These findings suggest a model of glycan-dependent tumor evolution wherein the adipose microenvironment reprograms OC to a tumorigenic state that resists the adaptive immune system. Mechanistically, adipose factors upregulate sialyltransferases. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancer tumorigenesis and metastasis.

Adipose microenvironment promotes hypersialylation of ovarian cancer cells.

Sialylation, the addition of negatively charged sialic acid sugars to terminal ends of glycans, is upregulated in most cancers. Hypersialylation supports multiple pro-tumor mechanisms such as enhanced migration and invasion, resistance to apoptosis and immune evasion. A current gap in knowledge is the lack of understanding on how the tumor microenvironment regulates cancer cell sialylation. The adipose niche is a main component of most peritoneal cancers' microenvironment. This includes ovarian cancer (OC), which causes most deaths from all gynecologic cancers. In this report, we demonstrate that the adipose microenvironment is a critical regulator of OC cell sialylation. In vitro adipose conditioning led to an increase in both ⍺2,3- and ⍺2,6-linked cell surface sialic acids in both human and mouse models of OC. Adipose-induced sialylation reprogramming was also observed in vivo from intra-peritoneal OC tumors seeded in the adipose-rich omentum. Mechanistically, we observed upregulation of at least three sialyltransferases, ST3GAL1, ST6GAL1 and ST3GALNAC3. Hypersialylated OC cells consistently formed intra-peritoneal tumors in both immune-competent mice and immune-compromised athymic nude mice. In contrast, hyposiaylated OC cells persistently formed tumors only in athymic nude mice demonstrating that sialylation impacts OC tumor formation in an immune dependent manner. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancers.

Adipose-derived exosomal miR-421 targets CBX7 and promotes metastatic potential in ovarian cancer cells.

Background: Chromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression. Results: Using conditioned media from human omental explants, we found that adipose-derived exosomes mediate CBX7 downregulation and enhance migratory potential of human ovarian cancer cells. Further, we identified adipose-derived exosomal miR-421 as a novel regulator of CBX7 expression and the main effector that downregulates CBX7. Conclusion: In this study, we identified miR-421 as a specific signaling pathway in the ovarian tumor microenvironment that can downregulate CBX7 to induce epigenetic change in OC cells, which can drive disease progression. These findings suggest that targeting exosomal miR-421 may curtail ovarian cancer progression.

Adipose-derived exosomal miR-421 targets CBX7 and promotes metastatic potential in ovarian cancer cells.

BACKGROUND: Chromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression. RESULTS: Using conditioned media from human omental explants, we found that adipose-derived exosomes mediate CBX7 downregulation and enhance migratory potential of human ovarian cancer cells. Further, we identified adipose-derived exosomal miR-421 as a novel regulator of CBX7 expression and the main effector that downregulates CBX7. CONCLUSION: In this study, we identified miR-421 as a specific signaling pathway in the ovarian tumor microenvironment that can downregulate CBX7 to induce epigenetic change in OC cells, which can drive disease progression. These findings suggest that targeting exosomal miR-421 may curtail ovarian cancer progression.

A Humanized Animal Model Predicts Clonal Evolution and Therapeutic Vulnerabilities in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. SIGNIFICANCE: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945.

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures.

RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among gamma-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.