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Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non-standardized behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/ 6J backgrounds generated in a standardized battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently used group sizes, a single retest of a significant result conforming to the commonly used P < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P-value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies. [ABSTRACT FROM AUTHOR]

Oncogenic KRAS mutations are well-described functionally and are known to drive tumorigenesis. Recent reports describe a significant prevalence of KRAS allelic imbalances or gene dosage changes in human cancers, including loss of the wild-type allele in KRAS mutant cancers. However, the role of wild-type KRAS in tumorigenesis and therapeutic response remains elusive. We report an in vivo murine model of colorectal cancer featuring deletion of wild-type Kras in the context of oncogenic Kras. Deletion of wild-type Kras exacerbates oncogenic KRAS signalling through MAPK and thus drives tumour initiation. Absence of wild-type Kras potentiates the oncogenic effect of KRASG12D, while incidentally inducing sensitivity to inhibition of MEK1/2. Importantly, loss of the wild-type allele in aggressive models of KRASG12D-driven CRC significantly alters tumour progression, and suppresses metastasis through modulation of the immune microenvironment. This study highlights the critical role for wild-type Kras upon tumour initiation, progression and therapeutic response in Kras mutant CRC., (© 2024. The Author(s).)

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete Eif4a1 , Eif4b or Eif4h in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death., (© 2023 Screen et al.)

Barth syndrome (BTHS) is caused by mutations in tafazzin resulting in deficits in cardiolipin remodeling that alter major metabolic processes. The tafazzin gene is encoded on the X chromosome, and therefore BTHS primarily affects males. Female carriers are typically considered asymptomatic, but age-related changes have been reported in female carriers of other X-linked disorders. Therefore, we examined the phenotype of female mice heterozygous for deletion of the tafazzin gene ( Taz -HET) at 3 and 12 months of age. Food intakes, body masses, lean tissue and adipose depot weights, daily activity levels, metabolic measures, and exercise capacity were assessed. Age-related changes in mice resulted in small but significant genotype-specific differences in Taz -HET mice compared with their female Wt littermates. By 12 months, Taz -HET mice weighed less than Wt controls and had smaller gonadal, retroperitoneal, and brown adipose depots and liver and brain masses, despite similar food consumption. Daily movement, respiratory exchange ratio, and total energy expenditure did not vary significantly between the age-matched genotypes. Taz -HET mice displayed improved glucose tolerance and insulin sensitivity at 12 months compared with their Wt littermates but had evidence of slightly reduced exercise capacity. Tafazzin mRNA levels were significantly reduced in the cardiac muscle of 12-month-old Taz -HET mice, which was associated with minor but significant alterations in the heart cardiolipin profile. This work is the first to report the characterization of a model of female carriers of heterozygous tafazzin deficiency and suggests that additional study, particularly with advancing age, is warranted.

Barth Syndrome, a rare X-linked disorder affecting 1:300,000 live births, results from defects in Tafazzin, an acyltransferase that remodels cardiolipin and is essential for mitochondrial respiration. Barth Syndrome patients develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists, and downstream transcriptional effects of Tafazzin dysfunction are incompletely understood. To identify novel, cell-specific, pathological pathways that mediate heart dysfunction, we performed single-nucleus RNA-sequencing (snRNA-seq) on wild-type (WT) and Tafazzin -knockout (Taz-KO) mouse hearts. We determined differentially expressed genes (DEGs) and inferred predicted cell-cell communication networks from these data. Surprisingly, DEGs were distributed heterogeneously across the cell types, with fibroblasts, cardiomyocytes, endothelial cells, macrophages, adipocytes and pericytes exhibiting the greatest number of DEGs between genotypes. One differentially expressed gene was detected for the lymphatic endothelial and mesothelial cell types, while no significant DEGs were found in the lymphocytes. A Gene Ontology (GO) analysis of these DEGs showed cell-specific effects on biological processes such as fatty acid metabolism in adipocytes and cardiomyocytes, increased translation in cardiomyocytes, endothelial cells and fibroblasts, in addition to other cell-specific processes. Analysis of ligand-receptor pair expression, to infer intercellular communication patterns, revealed the strongest dysregulated communication involved adipocytes and cardiomyocytes. For the knockout hearts, there was a strong loss of ligand-receptor pair expression involving adipocytes, and cardiomyocyte expression of ligand-receptor pairs underwent reorganization. These findings suggest that adipocyte and cardiomyocyte mitochondria may be most sensitive to mitochondrial Tafazzin deficiency and that rescuing adipocyte mitochondrial dysfunction, in addition to cardiomyocyte mitochondrial dysfunction, may provide therapeutic benefit in Barth Syndrome patients.

Mitochondrial 10-formyltetrahydrofolate (10-formyl-THF) is utilized by three mitochondrial enzymes to produce formate for nucleotide synthesis, NADPH for antioxidant defense, and formyl-methionine (fMet) to initiate mitochondrial mRNA translation. One of these enzymes-aldehyde dehydrogenase 1 family member 2 (ALDH1L2)-produces NADPH by catabolizing 10-formyl-THF into CO 2 and THF. Using breast cancer cell lines, we show that reduction of ALDH1L2 expression increases ROS levels and the production of both formate and fMet. Both depletion of ALDH1L2 and direct exposure to formate result in enhanced cancer cell migration that is dependent on the expression of the formyl-peptide receptor (FPR). In various tumor models, increased ALDH1L2 expression lowers formate and fMet accumulation and limits metastatic capacity, while human breast cancer samples show a consistent reduction of ALDH1L2 expression in metastases. Together, our data suggest that loss of ALDH1L2 can support metastatic progression by promoting formate and fMet production, resulting in enhanced FPR-dependent signaling., Competing Interests: Declaration of interests K.H.V. is on the board of directors and is a shareholder of Bristol Myers Squibb and is on the scientific advisory board (with stock options) of PMV Pharma, RAZE Therapeutics, Volastra Pharmaceuticals, and Kovina Therapeutics. She is on the scientific advisory board (SAB) of Ludwig Cancer and is a co-founder and consultant of Faeth Therapeutics. She has been in receipt of research funding from Astex Pharmaceuticals and AstraZeneca and contributed to the CRUK Cancer Research Technology filing of patent application WO/2017/144877. C.S. acknowledges grant support from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx, Inc., collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical, and Personalis. He is an AstraZeneca advisory board member, chief investigator for the AZ MeRmaiD 1 and 2 clinical trials, co-chief investigator of the NHS Galleri trial funded by GRAIL, and a paid member of GRAIL’s SAB. He receives consultant fees from Achilles Therapeutics (also a SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, China Innovation Center of Roche (CICoR), formerly the Roche Innovation Center – Shanghai, Metabomed (until July 2022), and the Sarah Cannon Research Institute. C.S. has received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana. C.S. had stock options in Apogen Biotechnologies and GRAIL until June 2021 and currently has stock options in Epic Bioscience and Bicycle Therapeutics and has stock options and is co-founder of Achilles Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Barth syndrome is an X-linked disorder caused by loss-of-function mutations in Tafazzin (TAZ), an acyltransferase that catalyzes remodeling of cardiolipin, a signature phospholipid of the inner mitochondrial membrane. Patients develop cardiac and skeletal muscle weakness, growth delay and neutropenia, although phenotypic expression varies considerably between patients. Taz knockout mice recapitulate many of the hallmark features of the disease. We used mouse genetics to test the hypothesis that genetic modifiers alter the phenotypic manifestations of Taz inactivation. We crossed TazKO/X females in the C57BL6/J inbred strain to males from eight inbred strains and evaluated the phenotypes of first-generation (F1) TazKO/Y progeny, compared to TazWT/Y littermates. We observed that genetic background strongly impacted phenotypic expression. C57BL6/J and CAST/EiJ[F1] TazKO/Y mice developed severe cardiomyopathy, whereas A/J[F1] TazKO/Y mice had normal heart function. C57BL6/J and WSB/EiJ[F1] TazKO/Y mice had severely reduced treadmill endurance, whereas endurance was normal in A/J[F1] and CAST/EiJ[F1] TazKO/Y mice. In all genetic backgrounds, cardiolipin showed similar abnormalities in knockout mice, and transcriptomic and metabolomic investigations identified signatures of mitochondrial uncoupling and activation of the integrated stress response. TazKO/Y cardiac mitochondria were small, clustered and had reduced cristae density in knockouts in severely affected genetic backgrounds but were relatively preserved in the permissive A/J[F1] strain. Gene expression and mitophagy measurements were consistent with reduced mitophagy in knockout mice in genetic backgrounds intolerant of Taz mutation. Our data demonstrate that genetic modifiers powerfully modulate phenotypic expression of Taz loss-of-function and act downstream of cardiolipin, possibly by altering mitochondrial quality control., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Barth syndrome (BTHS) is an X-linked mitochondrial disease caused by mutations in the gene encoding for tafazzin ( TAZ ), a key enzyme in the remodeling of cardiolipin. Mice with a germline deficiency in Taz have been generated ( Taz -KO) but not yet fully characterized. We performed physiological assessments of 3-, 6-, and 12-month-old male Taz -KO mice, including measures of perinatal survival, growth, lifespan, gross anatomy, whole-body energy and substrate metabolism, glucose homeostasis, and exercise capacity. Taz -KO mice displayed reduced viability, with lower-than-expected numbers of mice recorded at 4 weeks of age, and a shortened lifespan due to disease progression. At all ages, Taz -KO mice had lower body weights compared with wild-type ( Wt ) littermates despite similar absolute food intakes. This finding was attributed to reduced adiposity and diminutive organs and tissues, including heart and skeletal muscles. Although there were no differences in basal levels of locomotion between age-matched genotypes, indirect calorimetry studies showed higher energy expenditure measures and respiratory exchange ratios in Taz -KO mice. At the youngest age, Taz -KO mice had comparable glucose tolerance and insulin action to Wt mice, but while these measures indicated metabolic impairments in Wt mice with advancing age that were likely associated with increasing adiposity, Taz -KO mice were protected. Comparisons across the three age-cohorts revealed a significant and more severe deterioration of exercise capacity in Taz -KO mice than in their Wt littermate controls. The Taz -KO mouse model faithfully recapitulates important aspects of BTHS, and thus provides an important new tool to investigate pathophysiological mechanisms and potential therapies.

The pro-tumourigenic role of epithelial TGFβ signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFβ signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFβ signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFβ signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFβ signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits., (© 2022. The Author(s).)

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al. , Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are suboptimal in vivo because of poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled noninvasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol- or CCl 4 -induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1 + ) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

Current therapeutic options for treating colorectal cancer have little clinical efficacy and acquired resistance during treatment is common, even following patient stratification. Understanding the mechanisms that promote therapy resistance may lead to the development of novel therapeutic options that complement existing treatments and improve patient outcome. Here, we identify RAC1B as an important mediator of colorectal tumourigenesis and a potential target for enhancing the efficacy of EGFR inhibitor treatment. We find that high RAC1B expression in human colorectal cancer is associated with aggressive disease and poor prognosis and deletion of Rac1b in a mouse colorectal cancer model reduces tumourigenesis. We demonstrate that RAC1B interacts with, and is required for efficient activation of the EGFR signalling pathway. Moreover, RAC1B inhibition sensitises cetuximab resistant human tumour organoids to the effects of EGFR inhibition, outlining a potential therapeutic target for improving the clinical efficacy of EGFR inhibitors in colorectal cancer.

BRAF V600E is the most common driver mutation in human cutaneous melanoma and is frequently accompanied by loss of the tumor-suppressing phosphatase PTEN. Recent evidence suggests a co-operative role for RAC1 activity in BRAF V600E -driven melanoma progression and drug resistance. However, the underlying molecular mechanisms and the role of RAC1 downstream targets are not well-explored. In this study, we examine the role of the NCKAP1 subunit of the pentameric cytoskeletal SCAR/WAVE complex, a major downstream target of RAC1, in a mouse model of melanoma driven by BRAF V600E ;PTEN loss. The SCAR/WAVE complex is the major driver of lamellipodia formation and cell migration downstream of RAC1 and depends on NCKAP1 for its integrity. Targeted deletion of Nckap1 in the melanocyte lineage delayed tumor onset and progression of a mutant Braf;Pten loss‒driven melanoma mouse model. Nckap1-depleted tumors displayed fibrotic stroma with increased collagen deposition concomitant with enhanced immune infiltration. Nckap1 loss slowed proliferation and tumor growth, highlighting a role in cell-cycle progression. Altogether, we propose that NCKAP1-orchestrated actin polymerization is essential for tumor progression and maintenance of tumor tissue integrity in a mutant Braf/Pten loss‒driven mouse model for melanoma., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo , constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities., (© 2021 Humpton et al.; Published by Cold Spring Harbor Laboratory Press.)

Mitochondria constantly adapt to the metabolic needs of a cell. This mitochondrial plasticity is critical to T cells, which modulate metabolism depending on antigen-driven signals and environment. We show here that de novo synthesis of the mitochondrial membrane-specific lipid cardiolipin maintains CD8 + T cell function. T cells deficient for the cardiolipin-synthesizing enzyme PTPMT1 had reduced cardiolipin and responded poorly to antigen because basal cardiolipin levels were required for activation. However, neither de novo cardiolipin synthesis, nor its Tafazzin-dependent remodeling, was needed for T cell activation. In contrast, PTPMT1-dependent cardiolipin synthesis was vital when mitochondrial fitness was required, most notably during memory T cell differentiation or nutrient stress. We also found CD8 + T cell defects in a small cohort of patients with Barth syndrome, where TAFAZZIN is mutated, and in a Tafazzin-deficient mouse model. Thus, the dynamic regulation of a single mitochondrial lipid is crucial for CD8 + T cell immunity., Competing Interests: Declaration of Interests E.L.P. is a SAB member of Immunomet, and E.L.P. and E.J.P. are founders of Rheos Medicines. E.L.P. is Advisory Board member of Cell and Cell Metabolism., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The small GTPase RhoA is a master regulator of signalling in cell-extracellular matrix interactions. RhoA signalling is critical to many cellular processes including migration, mechanotransduction, and is often disrupted in carcinogenesis. Investigating RhoA activity in a native tissue environment is challenging using conventional biochemical methods; we therefore developed a RhoA-FRET biosensor mouse, employing the adaptable nature of intravital imaging to a variety of settings. Mechanotransduction was explored in the context of osteocyte processes embedded in the calvaria responding in a directional manner to compression stress. Further, the migration of neutrophils was examined during in vivo "chemotaxis" in wound response. RhoA activity was tightly regulated during tissue remodelling in mammary gestation, as well as during mammary and pancreatic carcinogenesis. Finally, pharmacological inhibition of RhoA was temporally resolved by the use of optical imaging windows in fully developed pancreatic and mammary tumours in vivo . The RhoA-FRET mouse therefore constitutes a powerful tool to facilitate development of new inhibitors targeting the RhoA signalling axis.

Rationale: Barth syndrome is an X-linked cardiac and skeletal myopathy caused by mutation of the gene Tafazzin ( TAZ ). Currently, there is no targeted treatment for Barth syndrome. Lack of a proper genetic animal model that recapitulates the features of Barth syndrome has hindered understanding of disease pathogenesis and therapeutic development., Objective: We characterized murine germline TAZ knockout mice (TAZ-KO) and cardiomyocyte-specific TAZ knockout mice models and tested the efficacy of adeno-associated virus (AAV)-mediated gene replacement therapy with human TAZ (hTAZ)., Methods and Results: TAZ-KO caused embryonic and neonatal lethality, impaired growth, dilated cardiomyopathy, and skeletal myopathy. TAZ-KO mice that survived the neonatal period developed progressive, severe cardiac dysfunction, and fibrosis. Cardiomyocyte-specific inactivation of floxed Taz in cardiomyocytes using Myh6-Cre caused progressive dilated cardiomyopathy without fetal or perinatal loss. Using both constitutive and conditional knockout models, we tested the efficacy and durability of Taz replacement by AAV gene therapy. Neonatal AAV-hTAZ rescued neonatal death, cardiac dysfunction, and fibrosis in TAZ-KO mice, and both prevented and reversed established cardiac dysfunction in TAZ-KO and cardiomyocyte-specific TAZ knockout mice models. However, both neonatal and adult therapies required high cardiomyocyte transduction (≈70%) for durable efficacy., Conclusions: TAZ-KO and cardiomyocyte-specific TAZ knockout mice recapitulate many of the key clinical features of Barth syndrome. AAV-mediated gene replacement is efficacious when a sufficient fraction of cardiomyocytes are transduced.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (Pten Δ/Δ BRF1 Tg ) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In Pten Δ/Δ BRF1 Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.

Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC-eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.

Mammalian embryos change shape dramatically upon implantation. The cellular and molecular mechanism underlying this transition are largely unknown. Here, we show that this transition is directed by cross talk between the embryonic epiblast and the first extra-embryonic tissue, the trophectoderm. Specifically, we show via visualisation of a Cdx2-GFP reporter line and pharmacologically mediated loss and gain of function experiments that the epiblast provides FGF signal that results in differential fate acquisition in the multipotent trophectoderm leading to the formation of a tissue boundary within this tissue. The trophectoderm boundary becomes essential for expansion of the tissue into a multi-layered epithelium. Folding of this multi-layered trophectoderm induces spreading of the second extra-embryonic tissue, the primitive endoderm. Together, these events remodel the pre-implantation embryo into its post-implantation cylindrical shape. Our findings uncover how communication between embryonic and extra-embryonic tissues provides positional cues to drive shape changes in mammalian development during implantation.

Mitochondria contain cardiolipin (CL), an organelle-specific phospholipid that carries four fatty acids with a strong preference for unsaturated chains. Unsaturation is essential for the stability and for the function of mitochondrial CL. Surprisingly, we found tetrapalmitoyl-CL (TPCL), a fully saturated species, in the testes of humans and mice. TPCL was absent from other mouse tissues but was the most abundant CL species in testicular germ cells. Most intriguingly, TPCL was not localized in mitochondria but was in other cellular membranes even though mitochondrial CL was the substrate from which TPCL was synthesized. During spermiogenesis, TPCL became associated with the acrosome, a sperm-specific organelle, along with a subset of authentic mitochondrial proteins, including Ant4, Suox, and Spata18. Our data suggest that mitochondria-derived membranes are assembled into the acrosome, challenging the concept that this organelle is strictly derived from the Golgi apparatus and revealing a novel function of mitochondria., (© 2019 Ren et al.)

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene , a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data., Competing Interests: SW, MN, AM, YM, DH, IM, CV, JC, PM, TC, YW, JM, HW, DS, KA, TP, MR, PT No competing interests declared, (© 2018, Warren et al.)

Epithelial-resident T lymphocytes, such as intraepithelial lymphocytes (IELs) located at the intestinal barrier, can offer swift protection against invading pathogens. Lymphocyte activation is strictly regulated because of its potential harmful nature and metabolic cost, and most lymphocytes are maintained in a quiescent state. However, IELs are kept in a heightened state of activation resembling effector T cells but without cytokine production or clonal proliferation. We show that this controlled activation state correlates with alterations in the IEL mitochondrial membrane, especially the cardiolipin composition. Upon inflammation, the cardiolipin composition is altered to support IEL proliferation and effector function. Furthermore, we show that cardiolipin makeup can particularly restrict swift IEL proliferation and effector functions, reducing microbial containment capability. These findings uncover an alternative mechanism to control cellular activity, special to epithelial-resident T cells, and a novel role for mitochondria, maintaining cells in a metabolically poised state while enabling rapid progression to full functionality., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

While the use of bioluminescent proteins for molecular imaging is a powerful technology to further our understanding of complex processes, fluorescent labeling with visible light fluorescent proteins such as GFP and RFP suffers from poor tissue penetration and high background autofluorescence. To overcome these limitations, we generated an inducible knock-in mouse model of iRFP713. This model was used to assess Cre activity in a Rosa Cre-ER background and quantify Cre activity upon different tamoxifen treatments in several organs. We also show that iRFP can be readily detected in 3D organoid cultures, FACS analysis and in vivo tumour models. Taken together we demonstrate that iRFP713 is a progressive step in in vivo imaging and analysis that widens the optical imaging window to the near-infrared spectrum, thereby allowing deeper tissue penetration, quicker image acquisition without the need to inject substrates and a better signal to background ratio in genetically engineered mouse models (GEMMs).

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, the level of the initiator methionine tRNA (tRNA i Met ) in stromal fibroblasts has been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNA i Met within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNA i Met gene. We show that increased tRNA i Met in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus, specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53 -/- ) or double (Trp53 -/- ;Brca2 -/- ) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53 -/- ;Brca2 -/- mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53 -/- cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3 + T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118-29. ©2016 AACR., (©2016 American Association for Cancer Research.)

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin., (© 2016 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons Ltd.)

CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rab coupling protein (FIP1C), an effector of the Rab11 GTPases, including Rab25, is amplified and overexpressed in 10% to 25% of primary breast cancers and correlates with poor clinical outcome. Rab25 is also frequently silenced in triple-negative breast cancer, suggesting its ability to function as either an oncogene or a tumor suppressor, depending on the breast cancer subtype. However, the pathobiologic role of FIP family members, such as FIP1C, in a tumor-specific setting remains elusive. In this study, we used ErbB2 mouse models of human breast cancer to investigate FIP1C function in tumorigenesis. Doxycycline-induced expression of FIP1C in the MMTV-ErbB2 mouse model resulted in delayed mammary tumor progression. Conversely, targeted deletion of FIP1C in the mammary epithelium of an ErbB2 model coexpressing Cre recombinase led to accelerated tumor onset. Genetic and biochemical characterization of these FIP1C-proficient and -deficient tumor models revealed that FIP1C regulated E-cadherin (CDH1) trafficking and ZONAB (YBX3) function in Cdk4-mediated cell-cycle progression. Furthermore, we demonstrate that FIP1C promoted lysosomal degradation of ErbB2. Consistent with our findings in the mouse, the expression of FIP1C was inversely correlated with ErbB2 levels in breast cancer patients. Taken together, our findings indicate that FIP1C acts as a tumor suppressor in the context of ErbB2-positive breast cancer and may be therapeutically exploited as an alternative strategy for targeting aberrant ErbB2 expression. Cancer Res; 76(9); 2662-74. ©2016 AACR., (©2016 American Association for Cancer Research.)