Multiple Model Systems Research Articles

A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreatic cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multi-drug therapies, providing a potential strategy for overcoming chemoresistance.

Comparison of mouse models of microbial experience reveals differences in microbial diversity and response to vaccination.

Specific pathogen-free (SPF) laboratory mice dominate preclinical studies for immunology and vaccinology. Unfortunately, SPF mice often fail to accurately model human responses to vaccination and other immunological perturbations. Several groups have taken different approaches to introduce additional microbial experience to SPF mice to better model human immune experience. How these different models compare is unknown. Here, we directly compare three models: housing SPF mice in a microbe-rich barn-like environment (feralizing), adding wild-caught mice to the barn-like environment (fer-cohoused), or cohousing SPF mice with pet store mice in a barrier facility (pet-cohoused); the two latter representing different murine sources of microbial transmission. Pet-cohousing mice resulted in the greatest microbial exposure. Feralizing alone did not result in the transmission of any pathogens tested, while fer-cohousing resulted in the transmission of several picornaviruses. Murine astrovirus 2, the most common pathogen from pet store mice, was absent from the other two model systems. Previously, we had shown that pet-cohousing reduced the antibody response to vaccination compared with SPF mice. This was not recapitulated in either the feralized or fer-cohoused mice. These data indicate that not all dirty mouse models are equivalent in either microbial experience or immune responses to vaccination. These disparities suggest that more cross model comparisons are needed but also represent opportunities to uncover microbe combination-specific phenotypes and develop more refined experimental models. Given the breadth of microbes encountered by humans across the globe, multiple model systems may be needed to accurately recapitulate heterogenous human immune responses.IMPORTANCEAnimal models are an essential tool for evaluating clinical interventions. Unfortunately, they can often fail to accurately predict outcomes when translated into humans. This failure is due in part to a lack of natural infections experienced by most laboratory animals. To improve the mouse model, we and others have exposed laboratory mice to microbes they would experience in the wild. Although these models have been growing in popularity, these different models have not been specifically compared. Here, we directly compare how three different models of microbial experience impact the immune response to influenza vaccination. We find that these models are not the same and that the degree of microbial exposure affects the magnitude of the response to vaccination. These results provide an opportunity for the field to continue comparing and contrasting these systems to determine which models best recapitulate different aspects of the human condition.

Abstract A155: The TGF-β/BMP signaling cascades induce opposing fates in breast cancer and medulloblastoma through chromatin and cell cycle modulation

Abstract The transforming growth factor β (TGF-β) superfamily comprises more than 30 structurally-related proteins, including TGF-β1, TGF-β2, TGF-β3 and bone morphogenesis proteins (BMPs). TGF-β/BMP ligands bind to their respective kinase receptors culminating paradoxically in either tumor-promoting or suppressing outcomes. The former fate is attributed mainly to the induction of the epithelial-mesenchymal transition and microenvironmental remodeling by TGF-β/BMP, whereas the latter outcome is a consequence of various antiproliferative mechanisms. To gain a broader perspective, we studied the fates of different cancer models, including breast cancer and medulloblastoma, either challenged with TGF-β/BMP ligands or depleted of essential transcription factors. In 3D cultures, TGF-β stimulation enhances the genome-wide chromatin accessibility of normal-like and transformed breast epithelial models. Concomitantly, we observed a dramatic increase in accessible regions mapped to transcription start sites and, to less extent, at annotated enhancers. The corresponding transcriptome-wide analyses indicated positive modulation of DNA replication and repair associated with resistance to cyclin-dependent kinase inhibition in breast cancer organoids. Nevertheless, such TGF-β-induced remodeling was partly dependent on the activity of the transcription factor SOX4. Further implementation of whole transcriptome spatial profiling in medulloblastoma PDX tissues identified several cell cycle-related pathways and chromatin remodeling terms that negatively associate with activated BMP signaling. In this context, the stem cell capacity of medulloblastoma spheroids was significantly inhibited by BMP stimulation in vitro. Moreover, we uncovered a regulatory network that maintains the transcriptional activation of BMP receptor type I (ACVR1) through the LHX2 transcription factor that maintains an active chromatin landscape at the ACVR1 proximal promoter. The depletion of LHX2 resulted in reduced tumor formation capacity of medulloblastoma in vivo. Taken together, our extensive studies across multiple model systems suggest that the TGF-β superfamily remodels the chromatin state to alter the cell cycle progression and, subsequently, affect cancer stemness and cell fate. Citation Format: Mohamad Moustafa Ali, Yae Ohata, Carl-Henrik Heldin, Aristidis Moustakas. The TGF-β/BMP signaling cascades induce opposing fates in breast cancer and medulloblastoma through chromatin and cell cycle modulation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A155.

Mitotic Dysregulation Sensitizes Malignant Stem Cells to CHK1 Inhibition in SF3B1-Mutant Myeloid Neoplasms

Myelodysplastic syndromes (MDS) arise from the acquisition of driver mutations in hematopoietic stem cells (HSCs). Splicing factor SF3B1 mutations are recurrent initiating lesions, found in up to 30% of patients with MDS. SF3B1 mutations are associated with aberrant splicing marked by recognition of alternative 3' splice sites (a3'ss). However, the causal role of mis-spliced genes in disease pathogenesis remains largely unexplored. Although SF3B1 mutations and downstream mis-spliced genes provide attractive therapeutic targets, targeted therapies remain unavailable. Here, we developed a novel method of precise gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) to introduce an SF3B1 K700E mutation at the endogenous locus, enabling stoichiometric expression of mutant protein. An intronic fluorescent marker was introduced upstream of the mutation for isolation of edited cells. Global splicing analysis in SF3B1 K700E knock-in HSPCs recapitulated canonical targets identified in SF3B1-mutant (SF3B1m) MDS patients. Annotation of a3'ss mis-spliced genes showed profound enrichment of gene ontology (GO) terms related to cell cycle regulation, mitotic checkpoint signaling, and chromosome segregation. Consistently, cell cycle analysis of SF3B1m HSCs revealed accumulation of cells in G2/M phase, suggesting a previously unidentified stall in G2/M progression driven by SF3B1 mutations. To identify mis-spliced genes that cause this G2/M delay, we compared a3'ss mis-spliced genes in the cell cycle GO category across multiple model systems. BUBR1 and CDC27 were consistently mis-spliced in all datasets, resulting in reduced mRNA and protein levels. BUBR1 is a mitotic kinase involved in the spindle assembly checkpoint and chromosome alignment, and CDC27 is a component of the anaphase-promoting complex. Knockdown of BUBR1 or CDC27 in normal CD34 + HSPCs recapitulated G2/M delay. BUBR1 and CDC27 are thus attractive therapeutic targets to impair cell cycle progression of SF3B1m cells. While there are no direct inhibitors of BUBR1 kinase, BUBR1 is phosphorylated by CHK1 during mitosis, and its expression is highly correlated with response to a selective CHK1 inhibitor prexasertib in a large panel of cancer cell lines (Blosser et al., Oncotarget 2020). Indeed, SF3B1m K562 cells and CD34 + HSPCs were highly sensitized to prexasertib. Knockdown of BUBR1 or CDC27 in wild-type cells induced CHK1 phosphorylation, especially upon prexasertib treatment, and sensitized normal cells to prexasertib. These data indicate that mis-splicing of BUBR1 or CDC27 delays G2/M progression leading to CHK1 activation, which sensitizes SF3B1m cells to prexasertib. To determine if CHK1 inhibition can eradicate SF3B1m HSCs, we leveraged our gene editing system to test drug responses in CD34 +CD133 +CD38 - primary immunophenotypic HSCs. Prexasertib selectively eliminated SF3B1m HSCs, including high-risk SF3B1/ RUNX1 and SF3B1/ STAG2 genotypes, while control AAVS1-edited HSCs were maintained. Furthermore, prexasertib depleted CD34 + cells in primary SF3B1m MDS patient samples. To test prexasertib efficiency in vivo, we transplanted NSGS mice with iPSC-derived HSPCs reprogrammed from a patient with high-risk MDS and SF3B1/ RUNX1 co-mutations, and treated with a standard preclinical regimen. While all the vehicle-treated mice showed consistent engraftment, none of the prexasertib-treated mice were engrafted. Together, these data indicate that prexasertib selectively targets SF3B1m HSCs in vitro and eradicates SF3B1m MDS in vivo. In conclusion, we developed a precise gene editing strategy of human HSCs to identify prexasertib as a promising therapy for SF3B1m myeloid neoplasms, and implicate the mitotic function of CHK1 as a SF3B1m sensitivity. The safety and toxicity profiles displayed in early phase clinical trials make prexasertib a suitable agent for further clinical investigation in SF3B1m MDS and its advanced stages.

C9orf72 proline-arginine dipeptide repeats disrupt the proteasome and perturb proteolytic activities.

The hexanucleotide G4C2 repeat expansion in C9orf72 is the most frequent genetic cause of familial amyotrophic lateral sclerosis (ALS). Aberrant translation of this hexanucleotide sequence leads to production of 5 dipeptide repeats (DPRs). One of these DPRs is proline-arginine (polyPR), which is found in C9orf72-expanded ALS (C9ALS) patient brain tissue and is neurotoxic across multiple model systems. PolyPR was previously reported to bind and impair proteasomes in vitro. Nevertheless, the clinical relevance of the polyPR-proteasome interaction and its functional consequences in vivo are yet to be established. Here, we aim to confirm and functionally characterize polyPR-induced impairment of proteolysis in C9ALS patient tissue and an in vivo model system. Confocal microscopy and immunofluorescence studies on both human and Drosophila melanogaster brain tissues revealed sequestration of proteasomes by polyPR into inclusion-like bodies. Co-immunoprecipitation in D. melanogaster showed that polyPR strongly binds to the proteasome. In vivo, functional evidence for proteasome impairment is further shown by the accumulation of ubiquitinated proteins along with lysosomal accumulation and hyper-acidification, which can be rescued by a small-molecule proteasomal enhancer. Together, we provide the first clinical report of polyPR-proteasome interactions and offer in vivo evidence proposing polyPR-induced proteolytic dysfunction as a pathogenic mechanism in C9ALS.

Atom counting based on Voronoi averaged STEM intensities using a crosstalk correction scheme

If quantitative scanning transmission electron microscopy is used for very precise thickness measurements with atomic resolution, it is commonly referred to as »atom counting«. Due to scattering and the finite probe extent the signal recorded in one atomic column is dependent not only on its own height but also on the height of its neighbours. Especially for thicker specimens this crosstalk effect can have significant impact on the measured intensity. If it is not appropriately accounted for in the evaluation, it can result in a deterioration of accuracy that impedes the possibility of actual atom counting. However, as the number of possible neighbour configurations can be excessively large, a comprehensive consideration of all in the evaluation reference is neigh impossible. This work proposes a method that allows for the a-posteriori reduction of crosstalk during the evaluation by algebraic means. Based on a parametric model, which is described in detail in the article, the crosstalk is expressed by an invertible matrix. Applying the inverted matrix to the measurement yields crosstalk corrected intensity values with very little computational effort. These can subsequently be evaluated by direct comparison to simple reference data. The working principle of the method is presented on the example of crystalline gold. The crosstalk parametrisation is found by fitting a model to sets of specifically created multislice simulations. The parameters are given for both aberration corrected and uncorrected STEM. Subsequently the abilities and potential of the technique are assessed in simulative studies on multiple model systems including gold nanoparticles. Overall a significant and robust improvement of the attainable precision can be demonstrated making the proposed method a promising tool for reference-based atom counting.

Exploiting the fibroblast growth factor receptor-1 vulnerability to therapeutically restrict the MYC-EZH2-CDKN1C axis-driven proliferation in Mantle cell lymphoma

Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global “unified” analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.

Mast Cell Deficiency in Mice Attenuates Insulin Phenolic Preservative-Induced Inflammation.

One major obstacle that limits the lifespan of insulin infusion pumps is surmounting the tissue site reaction at the device implantation site. All commercial insulin formulations contain insulin phenolic preservatives (IPPs) designed to ensure insulin protein stability and prolong shelf-life. However, our laboratory demonstrated that these preservatives are cytotoxic and induce inflammation. Mature mast cells (MCs) reside in cutaneous tissue and are one of the first responders to an epidermal breach. Upon activation, MCs release proinflammatory and immunomodulatory prepacked mediators that exacerbate these inflammatory reactions. Thus, we hypothesized that once the epidermis is breached, cutaneous MCs are triggered inciting the inflammatory response to IPP-induced inflammation. This hypothesis was pursued utilizing our modified in vivo mouse air pouch model, including a c-kit dependent (C57BL/6J-kitW-sh/W-sh) and a c-kit independent (Cpa3-Cre; Mcl-1fl/fl) MC-deficient mouse model. Leukocytes were quantified in the mouse air pouch lavage fluid following flow cytometry analysis for IPP infusion under three different states, insulin-containing phenolic preservatives (Humalog®), insulin preservatives alone, and normal saline as a control. The air pouch wall was assessed using histopathological evaluations. Flow cytometry analysis demonstrated a statistically significant difference in inflammatory cell recruitment for both MC-deficient mouse models when compared to the control strain including infused control saline. Significantly less inflammation was observed at the site of infusion for the MC-deficient strains compared to the control strain. Overall, concordant results were obtained in both mouse types, C57Bl6-kitW-sh/W-sh and Cpa3-Cre; Mcl-1fl/fl. These findings in multiple model systems support the conclusion that MCs have important or possible unique roles in IPP-induced inflammation.

Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems.

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.

Abstract ND01: ABBV-319: A first-in-class Glucocorticoid Receptor Modulator (GRM) agonist ADC for the treatment of B-cell malignancies

Abstract ABBV-319 is a novel CD19-targeting multi-modal antibody drug conjugate (ADC) for the treatment of B-cell malignancies. The ADC is engineered to have 3 distinct mechanisms of action to maximize anti-cancer activity while reducing systemic toxicities: (1) targeted GRM payload delivery, (2) signaling inhibition of CD19/BCR and (3) enhanced ADCC via afucosylation. Unlike traditional cytotoxic ADCs, the GRM payload is a high potency agonist (sub nM EC50) of the intracellular glucocorticoid receptor (GR) which then traffics to the nucleus resulting in sustained GR-driven transcriptional events leading to cancer cell death. CD19 is a clinically validated B-cell target (approved antibodies, ADCs, CARTs) known to be highly expressed even following multiple lines of therapy, making it an appropriate target for this novel approach. The CD19 antibody in ABBV-319 is an optimized high affinity IgG1 that can inhibit CD19/BCR downstream signaling events including AKT, leading to inhibition of growth in certain B-cell lines. Given ADCC/ADCP is a highly effective mechanism in B-cell malignancies (e.g., rituximab, tafasitamab) the CD19 antibody was engineered to have increased FcγR binding via afucosylation, that retains its enhanced ADCC activity as a GRM drug conjugate. By engineering the molecule in this way, the multi-modal ADC is not solely reliant on one mechanism of action for activity and therefore has the potential for broader activity. Sustained regressions/cures have been observed in multiple B-cell malignancy xenograft model systems (cell-line, PDX, humanized) after a single ADC dose that compares favorably against approved benchmark antibodies, systemic steroids and ADCs. ABBV-319 targets clinically validated mechanisms of action that are known to combine well with multiple standard-of-care agents. This combination potential together with its favorable safety profile, suggests ABBV-319 will be a highly effective combination treatment for B-cell malignancies. ABBV-319 was well tolerated in cynomolgus monkey studies with favorable drug-like properties and antibody-like pharmacokinetics, enabling a high mg/kg clinical starting dose. A first in-human Phase 1 clinical trial evaluating ABBV-319 in B-cell malignancies (DLBCL, FL, CLL) was recently initiated in late 2022 (clinicaltrials.gov, NCT05512390). Citation Format: James W. Purcell. ABBV-319: A first-in-class Glucocorticoid Receptor Modulator (GRM) agonist ADC for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND01.

Joint representation: Modeling a phenomenon with multiple biological systems

Biologists often study particular biological systems as models of a phenomenon of interest even if they already know that the phenomenon is produced by diverse mechanisms and hence none of those systems alone can sufficiently represent it. To understand this modeling practice, the present paper provides an account of how multiple model systems can be used to study a phenomenon that is produced by diverse mechanisms. Even if generalizability of results from a single model system is significantly limited, generalizations concerning specific aspects of mechanisms often hold across certain ranges of biological systems, which enables multiple model systems to jointly represent such a phenomenon. Comparing mechanisms that operate in different biological systems as examples of the same phenomenon also facilitates characterization and investigation of individual mechanisms. I also compare my account with two existing accounts of the use of multiple model systems and argue that my account is distinct from and complementary to them.

Multiple sclerosis plasma IgG aggregates induce complement-dependent neuronal apoptosis

Grey matter pathology is central to the progression of multiple sclerosis (MS). We discovered that MS plasma immunoglobulin G (IgG) antibodies, mainly IgG1, form large aggregates (>100 nm) which are retained in the flow-through after binding to Protein A. Utilizing an annexin V live-cell apoptosis detection assay, we demonstrated six times higher levels of neuronal apoptosis induced by MS plasma IgG aggregates (n = 190, from two cohorts) compared to other neurological disorders (n = 116) and healthy donors (n = 44). MS IgG aggregate-mediated, complement-dependent neuronal apoptosis was evaluated in multiple model systems including primary human neurons, primary human astrocytes, neuroblastoma SH-SY5Y cells, and newborn mouse brain slices. Immunocytochemistry revealed the co-deposition of IgG, early and late complement activation products (C1q, C3b, and membrane attack complex C5b9), as well as active caspase 3 in treated neuronal cells. Furthermore, we found that MS plasma cytotoxic antibodies are not present in Protein G flow-through, nor in the paired plasma. The neuronal apoptosis can be inhibited by IgG depletion, disruption of IgG aggregates, pan-caspase inhibitor, and is completely abolished by digestion with IgG-cleaving enzyme IdeS. Transmission electron microscopy and nanoparticle tracking analysis revealed the sizes of MS IgG aggregates are greater than 100 nm. Our data support the pathological role of MS IgG antibodies and corroborate their connection to complement activation and axonal damage, suggesting that apoptosis may be a mechanism of neurodegeneration in MS.

Enhanced fitness of SARS-CoV-2 B.1.617.2 Delta variant in ferrets

Competition assays were conducted in vitro and in vivo to examine how the Delta (B.1.617.2) variant displaced the prototype Washington/1/2020 (WA/1) strain. While WA/1 virus exhibited a moderately increased proportion compared to that in the inoculum following co-infection in human respiratory cells, Delta variant possessed a substantial in vivo fitness advantage as this virus becoming predominant in both inoculated and contact animals. This work identifies critical traits of the Delta variant that likely played a role in it becoming a dominant variant and highlights the necessities of employing multiple model systems to assess the fitness of newly emerged SARS-CoV-2 variants.

Abstract 3285: Targeting TBK1 to overcome resistance to cancer immunotherapy

Abstract Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. We identified the innate immune kinase TANK-binding kinase 1 (TBK1) as a candidate immune evasion gene in a pooled genetic screen. Using a suite of genetic and pharmacologic tools across multiple experimental model systems, we confirm a role for TBK1 as an immune evasion gene. Targeting TBK1 enhances response to PD-1 blockade by lowering the cytotoxicity threshold to effector cytokines (TNFα/IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumor models. Cancer cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNFα/IFNγ in a JAK/STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy. Citation Format: Yi Sun, Or-Yam Revach, Seth Anderson, Anne Jenney, Caitlin E. Mills, Payal Tiwari, Robert T. Manguso, Russell William Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3285.

Xuanfei Baidu Decoction regulates NETs formation via CXCL2/CXCR2 signaling pathway that is involved in acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening symptoms in Coronavirus Disease 2019 (COVID-19) patients. Xuanfei Baidu Decoction (XFBD) is a recommend first-line traditional Chinese medicine (TCM) formula therapeutic strategy for COVID-19 patients. Prior studies demonstrated the pharmacological roles and mechanisms of XFBD and its derived effective components against inflammation and infections through multiple model systems, which provided the biological explanations for its clinical use. Our previous work revealed that XFBD inhibited macrophages and neutrophils infiltration via PD-1/IL17A signaling pathway. However, the subsequent biological processes are not well elucidated. Here, we proposed a hypothesis that XFBD can regulate the neutrophils-mediated immune responses, including neutrophil extracellular traps (NETs) formation and the generation of platelet-neutrophil aggregates (PNAs) after XFBD administration in lipopolysaccharide (LPS)-induced ALI mice. The mechanism behind it was also firstly explained, that is XFBD regulated NETs formation via CXCL2/CXCR2 axis. Altogether, our findings demonstrated the sequential immune responses of XFBD after inhibiting neutrophils infiltration, as well as shedding light on exploiting the therapy of XFBD targeting neutrophils to ameliorate ALI during the clinical course.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n=43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

HiConfidence: a novel approach uncovering the biological signal in Hi-C data affected by technical biases.

The chromatin interaction assays, particularly Hi-C, enable detailed studies of genome architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mechanisms mediated by epigenetics. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach, HiConfidence, eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not substantially influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and topologically associating domains. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases, that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in histone acetylation pattern affect chromatin organization in Drosophila melanogaster S2 cells. The method is freely available at GitHub: https://github.com/victorykobets/HiConfidence.

The REPAIR Project, a Deep-Underground Radiobiology Experiment Investigating the Biological Effects of Natural Background Radiation: The First 6 Years.

In 2017, a special edition of Radiation Research was published [Oct; Vol. 188 4.2 (https://bioone.org/journals/radiation-research/volume-188/issue-4.2)] which focused on a recently established radiobiology project within SNOLAB, a unique deep-underground research facility. This special edition included original articles, reviews and commentaries relevant to the research goals of this new project which was titled Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR). These research goals were founded in understanding the biological effects of terrestrial and cosmic natural background radiation (NBR). Since 2017, REPAIR has evolved into a sub-NBR radiobiology research program which investigates these effects using multiple model systems and various biological endpoints. This paper summarizes the evolution of the REPAIR project over the first 6-years including its experimental scope and capabilities as well as research accomplishments.

A whole-genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors.

The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yeast and haploid stem cell screening, we demonstrate that a single cellular pathway, namely porphyrin (heme) biosynthesis, is required for the cytotoxicity of Artemisinins. Genetic or pharmacological modulation of porphyrin production is sufficient to alter its cytotoxicity in eukaryotic cells. Using multiple model systems of human brain tumor development, such as cerebral glioblastoma organoids, and patient-derived tumor spheroids, we sensitize cancer cells to dihydroartemisinin using the clinically approved porphyrin enhancer and surgical fluorescence marker 5-aminolevulinic acid, 5-ALA. A combination treatment of Artemisinins and 5-ALA markedly and specifically killed brain tumor cells in all model systems tested, including orthotopic patient-derived xenografts invivo. These data uncover the critical molecular pathway for Artemisinin cytotoxicity and a sensitization strategy to treat different brain tumors, including drug-resistant human glioblastomas.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite thesuccess of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.