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.

Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle–promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.

Human milk-derived extracellular vesicles (HMEVs) are key components in breast milk, promoting infant health and development. Maternal conditions could affect HMEV cargo; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study investigated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. The median duration from SARS-CoV-2 test positivity to milk collection was 3 months. After defatting and casein micelle disaggregation, HMEVs were isolated from milk samples of nine mothers with prenatal SARS-CoV-2 and six controls by sequential centrifugation, ultrafiltration, and qEV-size exclusion chromatography. The presence of HMEV was confirmed via transmission electron microscopy. Nanoparticle tracking analysis demonstrated particle diameters of 1 × 10¹¹ particles per mL of milk. Western immunoblots detected ALIX, CD9, and HSP70, supporting the presence of HMEVs in the isolates. Cargo from thousands of HMEVs were analyzed using a multi-omics approach, including proteomics and microRNA sequencing, and predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming, mucosal tissue development, and immunomodulation. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.

Glioblastoma multiforme (GBM), a common type of brain cancer, has a very poor prognosis. In general, viable GBM cells exhibit elevated phosphatidylserine (PS) on their membrane surface compared to healthy cells. We have developed a drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), that selectively targets cancer cells by honing in on this surface PS. To examine whether SapC-DOPS, a stable, blood–brain barrier-penetrable nanovesicle, could be an effective delivery system for precise targeted therapy of radiation, we iodinated several carbocyanine-based fluorescent reporters with either stable iodine (¹²⁷I) or radioactive isotopes (¹²⁵I and ¹³¹I). While all of the compounds, when incorporated into the SapC-DOPS delivery system, were taken up by human GBM cell lines, we chose the two that best accumulated in the cells (DiI (22,3) and DiD (16,16)). Pharmacokinetics were conducted with ¹²⁵I-labeled compounds and indicated that DiI (22,3)-SapC-DOPS had a time to peak in the blood of 0.66 h and an elimination half-life of 8.4 h. These values were 4 h and 11.5 h, respectively, for DiD (16,16)-SapC-DOPS. Adult nude mice with GBM cells implanted in their brains were treated with ¹³¹I-DID (16,16)-SapC-DOPS. Mice receiving the radionuclide survived nearly 50% longer than the control groups. These data suggest a potential novel, personalized treatment for a devastating brain disease.

Reliable pregnancy diagnostics would be beneficial for monitoring polar bear (Ursus maritimus) populations both in situ and ex situ, but currently there is no method of non-invasive pregnancy detection in this species. Recent reports in several carnivore species described the identification of fecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. The objective of the current analysis was to utilize an unbiased, antibody-free, label-free method for the identification and quantification of fecal proteins to determine if differences associated with pregnancy are detectable in polar bears. Protein was extracted from fecal samples (n = 48) obtained from parturient (n = 6) and non-parturient (n = 6) profiles each at four timepoints: pre-breeding season, embryonic diapause, early placental pregnancy, and mid-placental pregnancy. Protein was prepared and analyzed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system. A total of 312 proteins was identified and quantified; however, coefficients of variation (CV) were high for both abundance ratio variability (384.8 ± 61.0% SEM) and within group variability (86.8 ± 1.5%). Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology’s limitations, but rather, are reflective of true variation that exists among samples.