New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Evidence for a mouse origin of the SARS-CoV-2 Omicron variant

The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting the possibility of host-jumping. The molecular spectrum (i.e., the relative frequency of the twelve types of base substitutions) of mutations acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients, but was highly consistent with spectra associated with evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.

Imaging pheochromocytoma in small animals: preclinical models to improve diagnosis and treatment

Pheochromocytomas (PCCs) and paragangliomas (PGLs), together referred to as PPGLs, are rare chromaffin cell-derived tumors. They require timely diagnosis as this is the only way to achieve a cure through surgery and because of the potentially serious cardiovascular complications and sometimes life-threatening comorbidities that can occur if left untreated. The biochemical diagnosis of PPGLs has improved over the last decades, and the knowledge of the underlying genetics has dramatically increased. In addition to conventional anatomical imaging by CT and MRI for PPGL detection, new functional imaging modalities have emerged as very useful for patient surveillance and stratification for therapy. The availability of validated and predictive animal models of cancer is essential for translating molecular, imaging and therapy response findings from the bench to the bedside. This is especially true for rare tumors, such as PPGLs, for which access to large cohorts of patients is limited. There are few animal models of PPGLs that have been instrumental in refining imaging modalities for early tumor detection, as well as in identifying and evaluating novel imaging tracers holding promise for the detection and/or treatment of human PPGLs. The in vivo PPGL models mainly include xenografts/allografts generated by engrafting rat or mouse cell lines, as no representative human cell line is available. In addition, there is a model of endogenous PCCs (i.e., MENX rats) that was characterized in our laboratory. In this review, we will summarize the contribution that various representative models of PPGL have given to the visualization of these tumors in vivo and we present an example of a tracer first evaluated in MENX rats, and then translated to the detection of these tumors in human patients. In addition, we will illustrate briefly the potential of ex vivo biological imaging of intact adrenal glands in MENX rats.

Global analysis of human-to-mouse intercellular RNA transfer in cell culture

Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer- and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

Quantification of alternative 3′UTR isoforms from single cell RNA-seq data with scUTRquant

Although half of human genes use alternative polyadenylation (APA) to generate mRNA isoforms that encode the same protein but differ in their 3′UTRs, most single cell RNA-sequencing (scRNA-seq) pipelines only measure gene expression. Here, we describe an open-access pipeline, called scUTRquant (https://github.com/Mayrlab/scUTRquant), that measures gene and 3′UTR isoform expression from scRNA-seq data obtained from known cell types in any species. scUTRquant-derived gene and 3′UTR transcript counts were validated against standard methods which demonstrated their accuracy. 3′UTR isoform quantification was substantially more reproducible than previous methods. scUTRquant provides an atlas of high-confidence 3′ end cleavage sites at single-nucleotide resolution to allow APA comparison across mouse datasets. Analysis of 120 mouse cell types revealed that during differentiation genes either change their expression or they change their 3′UTR isoform usage. Therefore, we identified thousands of genes with 3′UTR isoform changes that have previously not been implicated in specific biological processes.

Transcription modulates chromatin dynamics and locus configuration sampling

In living cells the 3D structure of gene loci is dynamic, but this is not revealed by 3C and FISH experiments in fixed samples, leaving a significant gap in our understanding. To overcome these limitations we applied the "highly predictive heteromorphic polymer" (HiP-HoP) model, validated by experiments, to determine chromatin fibre mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects the movement of 40 kbp regions, we observed that the motion of smaller 1 kbp regions depends strongly on local disruption to chromatin fibre structure marked by H3K27 acetylation. This also significantly influenced locus configuration dynamics by modulating promoter-enhancer loops associated with protein bridging. Importantly these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible conformations of loci within minutes, generating wide dynamic variability of gene loci structure within single cells. Experiments inhibiting transcription change chromatin fibre structure subtly, yet we predict they should substantially affect mobility. This combination of simulation and experimental validation provide a novel insight and mechanistic model to explain how transcriptional activity influences chromatin structure and gene dynamics.

836 Murine fecal microbiota transfer models colonize human microbes selectively and reveal transcriptional pathways associated with response to neoadjuvant checkpoint inhibitors

BackgroundHuman gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in murine models using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, including identification of colonizing species associated with murine outcomes, details of murine cohorts, and statistical methods. Thus, the reproducibility and robustness of ICI murine models remain uncertain.MethodsTo investigate gut microbial species that impact ICI responses, we performed human to germ-free (GF) mouse FMT using pre-treatment stools from a pathologic lung cancer responder (R) and a pathologic lung cancer non-responder (NR) after neoadjuvant anti-PD-1 and anti-CTLA4 treatment, followed by implantation of the mice with syngeneic tumors and anti-PD-L1 treatment. Cohorts of GF mice varied by sex, age and syngeneic cell line implanted. To identify relevant microbes, murine tumor progressors (MT-P) and non-progressors (MT-NP) to anti-PD-L1 were classified based on tumor growth curves, 16S rRNA sequencing of human and mouse stools was performed, and data was statistically corrected for mouse characteristics using a generalized linear model. RNA sequencing was performed to assess transcriptional changes in murine tumors.ResultsR-FMT mice yielded a greater anti-tumor response in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on the mouse cell line, sex, and individual experiment. Microbiota analysis revealed a shared presence of the most highly abundant taxa between the human inocula and mice, however low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T cell and NK cell-related pathways in responding tumors, irrespective of FMT source, and enrichment of these cell types were confirmed by immunohistochemistry.ConclusionsThis study identifies several human intestinal microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine models.Ethics ApprovalAll studies in this abstract have been approved by Johns Hopkins University Animal Care and Use and Johns Hopkins Medicine Institutional Review Board.

Trastuzumab does not bind rat or mouse ErbB2/neu: implications for selection of non-clinical safety models for trastuzumab-based therapeutics

Purpose Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody–drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu. Methods Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo. Results Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected. Conclusions These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.