Steroid Biosynthesis Pathway Could Be Targeted to Reactivate Anti-tumour Immunity

Guest blog post by Bidesh Mahata.

Bidesh Mahata is a newly recruited group leader in the Division of Immunology, Department of Pathology, University of Cambridge. His laboratory studies the basic principles of immune cell-mediated steroid biosynthesis and its role in regulating inflammation and immunity. The team is committed to finding a way to target this immune cell steroidogenesis and steroid signalling to stimulate anti-tumour immunity (a new approach in cancer immunotherapy.)

“Why do immune cells produce steroids?” is an exciting question. Unfortunately, very little to nothing is known on this topic. This isn’t much of a surprise when we’re still asking “Do such immune cells exist?” or “What are those immune cells?”

Steroidogenesis (the production of steroids) in the adrenal gland, gonads and placenta is well-known. In the past century, the role of systemic steroids in regulating immune cell function has been studied extensively. Steroids are known immunoregulators and synthetic steroids are often used as anti-inflammatory and immunosuppressive drugs. We were surprised when we observed that immune cells, particularly T helper 2 cells, secrete steroids to regulate immune cell function (Mahata et al., Cell Rep, 2014). We believe that this is an important discovery in the area of immunology and endocrinology which needs further study.

To study the functional role of immune cell-mediated steroid biosynthesis you need to block steroid biosynthesis in the immune cell compartment. Ideally, you also need to find a way to track these steroid-producing immune cells. Unfortunately, during 2013-14, there were no available tools to do so. Thanks to WTSI/IMPC/KOMP, we were able to generate two genetically modified mouse models which were very crucial “discovery tools” to study immune cell-mediated steroid biosynthesis. These mouse models included a conditional Cyp11a1 knockout (Cyp11a1 cKO) mouse line which blocks steroid biosynthesis in specific cell types and a steroidogenesis reporter (Cyp11a1-H2B-mCherry) mouse line which allows us to identify steroidogenic cells.

Cyp11a1 is the first rate-limiting enzyme of the steroidogenesis pathway. Using these two discovery tools we studied steroid-producing T cells in cancer. T cells play a vital role in the immune system and can split into two types. T helper cells assist other cells in the immune system, like B cells and cytotoxic T cells, to fight against infections. Cytotoxic T cells, or ‘killer’ T cells, targets abnormalities inside our cells and is therefore very good at killing cancer, tumour or virus-infected cells. In general, T cells are good and protect us from tumours (i.e. anti-tumour immunity) but sometimes this anti-tumour immunity goes wrong. There is a global interest to know the mechanisms that suppress anti-tumour immunity and how this can be reinstated. If we know the anti-tumour immunosuppressive mechanisms, then we can develop immunotherapies by targeting those steps.

In this study, we found that T cell-mediated synthesis of immunosuppressive steroids dysregulates anti-tumour immunity. In other words, tumours induce steroid biosynthesis in T cells to evade anti-tumour immunity. Steroids are immunosuppressive and doctors often prescribe synthetic or pharmaceutical steroids to reduce unwanted hyperactive immune responses. It was a surprise to find that, in some specific cases, tumours may trick immune cells by sending some undiscovered (not fully understood yet) signal to the T cells to secrete steroids. The pathway may be targeted and exploited pharmacologically to reinstate the anti-tumour immunity. Recent success stories suggest that stimulation of the anti-tumour immunity is a new hope in the long-standing war against cancer.

The two mouse models used in our study are extremely useful and may assist in future research in the area of extra-glandular steroid biosynthesis. There are several cell types outside of T cells, known and unknown, with steroidogenic potential. At present we don’t know their physiological and pathological role. The experimental approach shown in our study, including the identification of steroidogenic cells by Cyp11a1-mCherry reporter and targeting by Cyp11a1 cKO, could provide in-depth mechanistic insights into extra-glandular (local) steroidogenic cell types. These steroidogenic cell types could include adipose cells, neuron, osteoblasts, astrocytes, microglia, skin, trophoblast and thymic epithelial cells.

Furthermore, the Cyp11a1-mCherry reporter mouse line can be used as a discovery tool to identify new steroidogenic cell types in infections, and other inflammatory and immunopathological cell types. Their functional role can be dissected by using Cyp11a1 cKO mice using tissue-specific Cre-drivers. We look forward to the coming days to know the biological and pathological importance of extra-glandular steroidogenic cells.

Source Article:

Mahata, B., Pramanik, J., van der Weyden, L. et al. Tumors induce de novo steroid biosynthesis in T cells to evade immunity. Nat Commun 11, 3588 (2020). https://doi.org/10.1038/s41467-020-17339-6

Article image: Brown fat cell rich in mitochondria and having lipid droplets scattered throughout. https://www.scientificanimations.com/ / CC BY-SA

Branched-chain amino acids (BCAAs) are amino acids that have a central carbon atom with a branch of three or more other carbon atoms (otherwise known as aliphatic side chains.) BCAAs includes valine, leucine and isoleucine. They play several key roles in metabolism, such as promoting protein synthesis, neurotransmitter synthesis and the production of energy through glycolysis.

Increased circulating levels of BCAA are linked to obesity, insulin resistance and type 2 diabetes. This link is still unclear – higher BCAA levels should correlate with more energy expenditure which is usually correlated with weight loss.

A recent paper using Pparg-floxed mice supplied by our consortium member the Jackson Laboratory explored BCAA activity in brown fat. Brown adipose tissue (BAT) is well known to be a thermogenic (heat-producing and energy using) organ that helps remove excess glucose from our systems. The thermogenic function of BAT is crucial for survival and metabolic health. Humans usually have the highest amount of BAT during infancy. This is because BAT produces heat and helps infants keep warm whilst their muscles are still underused. As humans grow older and gain more mobility, this thermogenic role is taken over by skeletal muscle and we lose most of our BAT. In adults, BAT helps with cold acclimatisation, not only producing heat but also stimulating the uptake of glucose, lipoproteins and fatty acids.

Yoneshiro et al simulated cold exposure to look at how temperature affected BAT and BCAA levels.

How does Cold Stimulus Affect BCAA uptake?

The researchers performed a metabolite analysis on healthy male human subjects, dividing them into those with high BAT activity and low BAT activity. They used a cold stimulus of 19 Celsius, a temperature point that stimulates BAT thermogenesis without triggering skeletal muscle shivering.

The cold exposure stimulated lipolysis (the breakdown of fat) and led to a significant increase in circulating fatty acids but did not change blood glucose levels. Valine (Val) levels were significantly reduced in the high BAT subjects but not in the low BAT subjects. As Val concentration dropped, BAT activity increased. A similar response was also seen for Leucine (Leu) concentration but not for any other amino acids. Val, Leu and Isoleucine (Ile) reduction after cold exposure was also seen in obese mice.

Yoneshiro et al then tracked Leu uptake in various tissue types. After cold acclimatisation, they found a robust increase in BAT and a modest increase in inguinal white adipose tissue (WAT) in mice. Uptake was also seen in liver and heart tissues but no significant change was seen in these organs. BAT also displayed high Val oxidation compared to different types of WAT in both mice and humans.

BCAA oxidation can increase fatty acid oxidation which can reduce the risk of obesity. It is the oxidation of BCAAs that promote energy production and protein synthesis, particularly in muscle tissues. In BAT, BCAA is primarily oxidised in the mitochondria, the organelle within cells that produces energy for all cell functions. How BCAAs are transported into the mitochondria, and the transporter that facilitates this, is still unknown, as well as how BAT exactly utilises BCAAs.

How does BCAA Uptake in BAT Affect Metabolism?

As BAT is already known for the metabolic clearance of glucose, Yoneshiro et al investigated whether BAT also contributes to the clearance of BCAAs. They generated a mouse model without functioning (ablated) BAT and compared them to controls. After cold exposure, they found that the BCAA concentration was significantly reduced in control mice but not in the BAT-ablated mice, suggesting BAT plays a role in clearing BCAAs.

The researchers next examined how BCAA catabolism functioned within BAT after clearance, as well as how this affected energy homeostasis.

The branched-chain α-keto acid dehydrogenase (BCKDH) complex is found in the mitochondria and catalyses key oxidation reactions involved in energy production. To examine the extent to which BCAA catabolism in BAT regulates energy homeostasis, Yoneshiro et al produced another mouse model in which BCAA oxidation is impaired specifically in BAT. They deleted the Bckdha gene which produces a subunit of BCKDH, impairing BCKDH’s function. In this model (Bckdha^UCP1-KO), they saw that there was no difference in BAT mass and thermogenic gene expression but the core-body temperature was significantly lower after cold exposure compared to controls. Thermogenesis in BAT was impaired (but not in other tissues) and the mice had higher circulating BCAA levels. This suggests BCAA oxidation is vital for both BAT thermogenesis and BCAA clearance.

Further examination on the use of BCAA within BAT was performed using Leu tracing, stimulation of brown adipocytes with noradrenaline and cold exposure. The researchers found that acute cold exposure activates BCAA oxidation in the TCA cycle, whereas chronic cold promotes other lipogenesis (fat production) processes.

Yoneshiro et al also wanted to find to what extent the BCAA oxidation impairment affected whole-body metabolism for the Bckdha^UCP1-KO mice. When fed a high-fat diet, the impaired mice gained significantly more body weight compared to controls, particularly in terms of adipose tissue and liver mass. The impaired mice “exhibited increased systemic glucose intolerance and insulin resistance” as well as reduced glucose oxidation and fatty oxidation.

How Does the Mitochondria Take Up BCAAs?

The big question now was: how do cells take up BCAAs into the mitochondria? The researchers found that members of the SLC25A protein family would be promising candidates due to including many mitochondrial amino acid transporters. Transcriptome analysis found high expression levels for the already known SLC25A20 and SLC25A22 in mouse and human BAT but also two uncharacterised members: SLC25A39 and SLC25A44. Only SLC25A44 had increased mRNA expression after cold exposure and also showed positive correlations with UCP1 and BCKDHA mRNA expression. SLC25A44 was localised to the mitochondria and was more highly expressed in BAT than other metabolic tissues.

The researchers needed to determine the function of SLC25A44 so they generated brown adipocytes with ablated SLC25A44 (Slc25a44-KO.) Val and Leu uptake was significantly reduced, unlike other amino acids. This response was also found using shRNAs to deplete SLC25A44 whereas ectopic expression in Neuro2a cells restored Val and Leu uptake. Similar responses were also found in cell-free systems.

To determine the role of SLC25A44 in BCAA catabolism, the researchers selectively knocked down (reduction in the expression) SLC25A44 in BAT in mice (Slc25a44^BAT-KD.) The KD mice had larger lipid droplets in their brown adipocytes and impaired BAT thermogenesis. SLC25A44-deficient mice (Slc25a44-KD) mirrored this response as well as higher levels of triglycerides and lower Val oxidation in BAT. Core body temperature was also significantly lower compared to controls after cold exposure but plasma BCAA levels were not lowered. Together, this suggested that “SLC25A44 is the primary BCAA transporter in BAT [and] is required for cold-stimulated BAT thermogenesis and systemic BCAA clearance in vivo.”

Further tests also found that SLC25A44 depletion did not cause a general mitochondrial defect and SLC25A44 depleted adipocytes displayed active mitochondrial respiration.

What Does This Mean for Obesity and Diabetes?

The researchers proposed the following model:

“in addition to glucose and fatty acids, cold stimuli potently increase mitochondrial BCAA uptake and oxidation in BAT, leading to enhanced BCAA clearance in the circulation. This process requires SLC25A44, a mitochondrial BCAA transporter in brown adipocytes. In turn, defective BCAA catabolism in BAT results in impaired BCAA clearance and thermogenesis, leading to the development of diet-induced obesity and glucose intolerance.”

The researchers highlighted the implications these findings have for our understanding of obesity and diabetes. There is ongoing evidence that incompletely oxidised intermediates resulting from BCAA oxidation can cause insulin resistance. Lowering circulating BCAA levels in rats via inhibition of kinase BDK or overexpression of phosphatase PPM1K has also been found to improve glucose tolerance, regardless of body-weight. Reduced BCAA oxidation and the subsequent accumulation of BCAAs can cause inhibition of insulin signalling. This study suggests impaired BAT activity in obesity and diabetes reduces systemic BCAA clearance. Active and well-functioning BAT acts as a significant metabolic filter for circulating BCAA and protects against obesity and insulin resistance.

The researchers finally suggest that utilising SLC25A44 to enhance BCAA catabolism could improve BCAA clearance. This, in turn, could assist with glucose homeostasis and metabolic diseases such as obesity and diabetes.

Source:

Yoneshiro, T., Wang, Q., Tajima, K. et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature 572, 614–619 (2019). https://doi.org/10.1038/s41586-019-1503-x

Scientists worldwide are still attempting to define ageing and mortality through research. Debate on ageing continues to vary widely and includes theories involving conception, birth, the nadir of mortality, puberty, completion of development and much more. Many of these theories lack a quantitative nature, making it difficult to scientifically measure ageing, causing further debate within the ageing research community.

Researchers recently utilised IMPC data to concretely define ageing. They argued against current theories that ageing begins at the onset of reproduction (around puberty age) or at the nadir of mortality and highlight the inaccuracy of using ‘mortality patterns’ to define ageing. Elvira et al propose measuring ageing as:

‘the accumulation of damage such as mutations, methylation changes, protein oxidation and other deleterious changes with age.’

Explaining the U-Curve of Mortality

Previous work ties mortality closely with ageing. As we grow older, we gradually grow frailer which eventually leads to death – this is mortality. However, the pattern of mortality presents as a U-shaped curve with high mortality rates in very early life and again in later life. The lowest point of the U-curve, the nadir, is still being debated. Some researchers argue the nadir is at puberty age whilst Elvira et al define the nadir specifically at age nine, significantly before the onset of reproduction age.

This curve is in opposition to Elvira et al’s accumulation of damage hypothesis which would present as a slow, gradual incline in mortality instead of the observed U-shaped curve. They therefore further investigated the U-curve, particularly the causes for the early-age mortality spike. Data analysis showed that age-related disease patterns (such as heart disease, infection, cancer and sepsis) were also U-shaped with the nadir consistently before the age of puberty at around age nine. Causes of mortality specific to childhood, however, did not show a U curve and presented as an age-related decrease with the maximum at birth. The question was: why were the age-related disease patterns spiking at an early age?

Examining Mortality with Quantitative Biomarkers: Somatic Mutations

To examine the nature of high early-life mortality and its relationship to ageing, they analysed the behaviour of quantitative biomarkers of ageing. They first focused on the age-associated accumulation of mutations in somatic tissues – all cells that are not associated with the production of gametes and in which any mutations are not heritable. The researchers previously found that this accumulation of somatic mutations can be assessed by following age-related mutations in cancers. Unlike the U-curve of mortality, somatic mutations increased with age throughout the lifetime for the cancer types analysed.

Due to this lack of accumulation of somatic mutations in early-life, the researchers suggest that early-life mortality is not related to ageing. They further suggested that whilst early-life mortalities may appear phenotypically similar to age-related mortality causes (heart disease, cancer, infection), this is only the surface level appearance. These cases of early-life mortality were not being caused by somatic mutations and, therefore, must have another non-age-related cause.

DNA Methylation as a Clock for Aging

They next examined DNA methylation changes as another quantitative biomarker of ageing. Methylation, the addition of methyl groups to DNA molecules, is a natural mechanism used to suppress the transcription of certain genes. It’s a method used in vital biological processes such as X-chromosome inactivation and embryonic development. DNA methylation, and other epigenetic ‘clocks’, are great for measuring the biological age of tissues. The researchers analysed the pace of this clock in both humans and mice, finding for both organisms that DNA methylation increased gradually in a similar manner to age-related somatic mutations, apart from early life where it increased quickly before slowing down.

Studying Knockout Lethality Using IMPC Data

Lastly, they analysed patterns of knockout lethality using IMPC datasets. They found that:

“most lethal-knockout mice are characterised by abnormal survival before and at mid-gestation; lethality is reduced dramatically afterward, including the period after birth.”

They concluded that mortality during development can be explained partly by parental genotype. Enrichment analysis found that mortality during early-gestation is associated with the regulation of transcription and translation, chromosomal organisation, and regulation of the cell cycle and regulation of organogenesis at mid-gestation. They suggested that these mortality rates act as active negative selection against heterozygous loss of gene function.

They also noted that: early-life mortality is associated with aneuploidy and other chromosomal abnormalities, that maternal age correlates with spontaneous abortion rates and how abnormal karyotypes account for 75% of spontaneous abortions in older mothers but only 35% in younger mothers. This further highlights the role of parental genotype in early-life mortality but also shows that a substantial fraction of early-life mortality, particularly with younger mothers, could be unrelated to maternal genotype.

In Conclusion

Elvira et al divided mortality into two processes:

“(1) age-related deleterious molecular changes, [corresponding] to the aging process and (2) damaging mutations, chromosomal abnormalities, and chance of failure, [corresponding] to developmental mortality (early-life mortality).”

They propose that mortality doesn’t represent ageing in early-life but shows a strong negative selection against deleterious alleles. Instead of using the inaccurate biomarker of mortality, ageing is better represented and measured by the sum of deleterious changes over time.

They summarised that ageing begins during development, after which it “runs unnoticed in the shadow of high early-life mortality.” After age nine, ageing becomes the dominant factor in human mortality.

IMPC and Aging

Many age-related diseases and phenotypes may not occur until later life stages. To examine these phenotypes, selected mice at the IMPC enter our ageing pipeline, called our late adult pipeline, starting at 52 weeks or later. Our most recent data release 11 was our first release to include late adult data and we’re going to include more in our future releases. You can view more information about this data and the progress we’re making on our late adult data focus page.

Source:

Kinzina, E., Podolskiy, S., Dmitriev, S. & Gladyshev, V. Patterns of Aging Biomarkers, Mortality, and Damaging Mutations Illuminate the Beginning of Aging and Causes of Early-Life Mortality. Cell Reports 29(13), 4276-4284 (2019) https://doi.org/10.1016/j.celrep.2019.11.091

A study investigating the role of Arf1 shows promising results for new therapeutic strategies involving DAMP-mediated anti-tumour immunity to target cancer stem cells. This method of inducting an anti-tumour response could be another frontier in anti-tumour immune therapy.

“[This could be] the first demonstration that endogenous metabolic stress elicits anti-tumour immune responses by inducing DAMPs in rodent tumor models.”

Cancer stem cells (CSCs) are found in tumours and, like other stem cell types, have the ability to give rise to many different cell types. Their affinity for self-renewal, differentiation, tumourigenicity and their possible resistance to traditional treatments like chemotherapy suggests they could be responsible for treatment resistance, tumour metastasis, reoccurrence, evasion and patient death. CSCs, therefore, prove a good target for future tumour therapies.

CSCs have a unique metabolism. Leukaemia stem cells have been shown to rely on amino acids for oxidative phosphorylation and some CSC-enriched disseminated tumour cells obtain energy from fatty acids. The researchers’ previous work with drosophila found that the COPI/Arf1-mediated lipolysis pathway selectively sustains and transforms stem cells and that targeting this pathway by knocking out Arf1 kills stems cells via necrosis.

In this study, the researchers investigated the Arf1-mediated lipid metabolism pathway’s role in sustaining CSCs in mice and how disrupting this pathway would affect tumour growth and number. IMPC Arf1 embryonic stem cells were used in this study.

Does the Arf1 Lipolysis Pathway Sustain CSCs?

The researchers used the Lgr5-CreERT2/Apc^f/f (Lgr5/Apc) mouse model, one known to be a good model for CSCs, and knocked out Arf1 to produce Lgr5-CreERT2/Arf1^f/f/Apc^f/f mice. They found a dramatic reduction in stem cell tumour numbers and a significant increase in lifespan for the Lgr4/Arf1/Apc mice. After examining intestinal cells, they found abnormal mitochondria, poor cristae, vacuoles, degeneration and necrosis.

They then took mouse colon cancer CT26 cells and human liver cancer Huh-7 cells and treated them with Arf1 inhibitors, BFA and GCA. The treatment dramatically increased lipid droplet accumulation in both cell types.

Deletion of Arf1 from the ventral foregut endoderm with Foxa3-Cre resulted in significantly underdeveloped livers and lethality at embryonic day 15.5. Further study found that it also caused a significant reduction in hepatoblast markers Hnf4α and Hnf6, lipid droplet accumulation and necrotic death of hepatoblasts.

“The Arf1-regulated lipolysis pathway selectively sustains stem cells, progenitors and cells enriched with CSCs in mice and that disrupting the pathway in these cells results in lipid droplet accumulation, mitochondrial defects and cell necrosis.”

Arf1-KO’s Effect on Intestinal CSCs

Arf1 depleted mice showed accumulation of CD3+, CD8+ and CD4+, evidence of a T-cell immune response, with dendritic cells (DCs) and inflammatory DCs also being significantly increased. Upon examining the population of lamina propria lymphocytes within the GI tract, they found that certain T-cells (CD8/4+ with and without IFN-γ+) were all significantly increased. These changes were not seen in other areas within the GI tract nor were these changes observed in other key immune cell sites, including the spleen, draining lymph nodes and inguinal lymph nodes.

T-cell-related chemokines (CCL5, CXCL10 and CCL22) were upregulated in the Lgr5/Arf1/Apc model. Real-time PCR showed elevated expressions of IFN-γ, perforin, granzyme A and B and IL-1β. The accumulated evidence suggested that:

“knocking out Arf1 in Lgr5+ stem cells triggers T cell infiltration and activation, leading to CSC death and prolonged survival of Lgr5/Arf1/Apc mice.”

Arf1-KO’s Effect on Liver CSCs

The researchers induced liver tumours using the Tet-o-MYC/LAP-tTA system before adding the Arf1 inhibitors GCA or BFA to mouse food, dramatically reducing the number of liver tumours and extending the lifespan of the mice. Selective depletion of Arf1 in Axin2+ liver cells or CSCs also produced this effect.

DCs, B cells and both CD4+ and CD8+ T-cells were significantly increased in the liver, as were many T-cell-related cytokines (CXCL10, CXCL11 and CCL22.) Real-time PCR showed elevated expression levels for the same molecules as in the intestine. Real-time PCR also, however, showed a significant decrease in PD-L1 expression in several mouse models and cell types.

“Arf1 inhibition may down-regulate PD-L1 through reducing the AP-1/C-Jun pathway…PD-L1 reduction could be partially responsible for the activation of infiltrated T cells that enhances the anti-tumour effect of Arf1 ablation”

Defining the Effect: T-cell Activation, DC Infiltration and DAMPs

In order to confirm that the effect relied on an adaptive immune response, they used anti-CD4 and anti-CD8 antibodies to deplete T-cells. In response, tumour numbers dramatically increased and lifespans were shortened. They then additionally knocked out either Rag1 or IFN-γ in the Lgr5/Arf1/Apc mice. Both caused survival times to significantly decrease. They concluded that the anti-tumour effects were due to an induction of a T-cell-dependant immune response.

Knocking out Arf1 had an effect on other aspects of the immune system. They found Arf1 inhibition may also induce inflammasome-mediated cell necrosis/pyroptosis. Arf1 inhibition or ablation also induced key DAMPS like Calr, HMGBI, ERp46 and LAMP1, as well as ER-stress markers. It was concluded that Arf1 inhibition triggers ER stress and, as a result, induces DAMPS and DC infiltration, enhancing the T-cell infiltration.

Through tests with a series of inhibitors, including the ATPase inhibitor ARL, it was concluded that the anti-tumour effect worked via ATP and HMGBI. Arf1 knockdown was not directly cytotoxic to tumour cells and, together with the above conclusions, the anti-tumour effect was DCs-ATP-IFN-γ-mediated T-cell immunity. Knockdown also prevented tumour metastasis.

Using Arf1-KO Cells for Vaccination

Vaccination with knockout Arf1 cells also proved successful in protecting animals from developing tumours. The vaccination was effective not only against melanoma but also against a variety of histologic types like colon cancer and breast carcinomas. Arf1 inhibition triggered a localised T-cell immune response that then attacked cancer throughout the body.

Simultaneous inhibition of T-cell checkpoint receptor PD-1 with Arf1 ablation had a synergistic effect and further reduced tumour numbers.

Upon analysing public datasets on human cancer, the researchers found Arf1 was amplified or overexpressed in the majority of cancer types examined. The cases with lower expression levels of Arf1 had significantly better survival probabilities for a number of cancer types, suggesting Arf1 is a negative prognostic factor.

How it All Happens

The researchers proposed a model:

“a small number of active anti-CSC T cells penetrate the tumor and attack…; as a result…the CTLs produce cytokines and re-stimulate the local environment to convert an immunosuppressive to an immunostimulatory environment;…[reawakening] the pre-existing inactive anti-tumor T cells [and recruiting] new anti-tumor T cells; these additional T cells are directed against tumour antigens other than the DAMPs to amplify the effects for destroying the bulk tumors; these active T cells may then migrate to other tumor sites and become memory T cells to produce widespread and durable antitumor effects.”

If results like the above are reproducible in humans, DAMP-mediated anti-tumour immune therapy could be a breakthrough for the treatment of reoccurring tumours, particularly those with high Arf1 expression. When used in conjunction with other already effective treatments or with the addition of successful checkpoint blockades, this method could prove even more effective.

Source:

Wang, G., Xu, J., Zhao, J. et al. Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice. Nat Commun11, 220 (2020). https://doi.org/10.1038/s41467-019-14046-9

With a contribution from the IMPC, recent research, published in Nature Communications, has identified 198 genes associated with brain morphogenesis in mice – 83% of these constitute genes newly implicated in brain architecture.

Brain development and morphogenesis is critical to higher-order cognition, but our knowledge of its biological basis is at best incomplete, and at worst, severely lacking. Previous studies have demonstrated that there is a significant genetic influence on brain morphology. However knowledge of which genes influence brain morphology is limited, and this presents an important problem in developmental biology. By identifying 198 genes that are associated with mouse brain morphogenesis the study provides a complementary resource to human genetic studies and predicts that many more genes could be involved in mammalian brain morphogenesis.

Binnaz Yalcin, corresponding author of the study says “This study aims at understanding the genetics of brain anatomy in the mouse. It provides a wealth of novel knowledge about which genes control the size and the shape of the brain, and a foundation on which neurobiologists can build to further study how exactly these genes control the brain anatomy”.

“I have no doubt that this resource will help medical geneticists working on ultra-rare human neurodevelopmental disorders

The researchers obtained brain samples of 1566 mutant mouse lines from the Sanger Institute Mouse Genetics Project, a partner of the IMPC. Using a histological pipeline, 118 brain morphological parameters were analysed, covering brain size, commissures, ventricles, cortex, subcortex and cerebellum. To detect neuroanatomical phenotypes (NAPs), the researchers used PhenStat, a statistical method developed by the IMPC for the identification of abnormal phenotypes. 198 genes associated with neuroanatomical phenotypes (NAP genes) were subsequently identified. The vast majority of these genes (94%) have never previously been associated with brain anatomy in mice.

Interestingly, unique human orthologs were identified for 173 of the identified mouse NAP genes. Whilst 17% of human unique orthologs of mouse NAP genes are known loci for cognitive dysfunction, 83% constitute a vast number of genes that are newly implicated in brain architecture. This dataset may therefore help in improving clinical interpretation.

Yalcin states “I have no doubt that this resource will help medical geneticists working on ultra-rare human neurodevelopmental disorders who sometimes struggle to determine the genetic mutation responsible for the underlying disease, when for example there is only one patient world-wide. So when a mutation is found in a patient’s genome and that patient exhibits the same phenotype than the mouse, a molecular diagnosis can finally be made.”

This study would not have been possible without the IMPC

The identified NAP genes converge into a small number of groups of functionally similar genes participating in shared cellular pathways. Disruption of genes within the same module can yield a similar pattern of neuroanatomical abnormalities, revealing interesting neurodevelopmental pathway/phenotype relationships. For example, the study indicates that mechanisms confined to sub-cellular compartments as subtle as dendritic spines can translate into major anatomical features.

The study represents the largest atlas of the link between genetic mutation and its associated neuroanatomical features yet, and contributes a wealth of new knowledge on the genetics of brain morphogenesis. The authors were keen to note the contribution of the IMPC towards their work, with Yalcin commenting that “This study would not have been possible without the IMPC and is a tribute to the remarkable work of the people involved in this consortium.”

The IMPC is aiming to design and produce a genome-wide mouse strain resource of human disease-associated coding variants associated with rare disease that can be used for validation of putative functional variants and insight into disease mechanism(s). To find out more, click here.

Original publication: Large-scale neuroanatomical study uncovers 198 gene associations in mouse brain morphogenesis

Members of the IMPC Consortium at CNR-Monterotondo (Italy) have used embryonic stem (ES) cells produced as part of the IMPC project, to engineer a Ccdc151 ‘knockout’ mouse model that is valuable for the study of human ciliopathies, gaining significant insight into this set of rare conditions.

Primary ciliary dyskinesia (PCD) is a rare, genetic disorder that results in chronic respiratory tract infections, abnormally orientated internal organs and infertility. The root cause of these symptoms are dysfunctional cilia and flagella, which if functioning correctly, are finger-like projections on the surface of cells that act to clear mucus and debris by coordinated beating. In addition, normal function of cilia is required for migration of egg and sperm cells.

Ccdc151 is a gene that is known to be associated with PCD, and here, researchers have engineered a mouse model, using embryonic stem cells from the IMPC, in which the gene is deleted. Common features of PCD in human patients such as left-right body asymmetry and dysfunctional spermatogenesis were detected in these Ccdc151-knockout mice. “The Ccdc151-knockout mouse model faithfully recapitulates several features of human PCD disease” remark Francesco Chiani and Tiziana Orsini, co-first authors of the paper. “The availability of this animal model will allow researchers to further dissect the mechanisms by which pathological conditions develop in different organs”.

This animal model will be useful for studying mechanisms underlying hydrocephalus, a condition whose treatment has not changed for decades.

The researchers also observed that Ccdc151-knockouts develop severe hydrocephalus – a condition in which cerebrospinal fluid accumulates in the brain, causing increased pressure inside the skull and potentially leading to brain damage and other complications. This is the first example of hydrocephalus caused by loss of function in the Ccdc151 gene. Although hydrocephalus is rarely seen in human patients with PCD, having a mouse model that exhibits all of the features of hydrocephalus could be useful for researchers. “This animal model will be useful for studying mechanisms underlying hydrocephalus, a condition whose treatment has not changed for decades”, said the lead authors. It is hoped that further studies may help to uncover other genes that interact with Ccdc151 that lead to the development of hydrocephalus.

this micro-CT imaging methodology could be applied to facilitate studies on gene expression directly in the intact brain

The researchers made extensive use of X-ray micro-CT 3D imaging to study the hydrocephalic brains of Ccdc151 knockout mice – a technique that uses X-rays to create a virtual 3D model of a target object. Additionally, a novel micro-CT method was used to study expression of the Ccdc151 gene in the brain. This novel method is based on the generation of a molecular signal within the mouse brain. The authors remarked that “this micro-CT imaging methodology could be applied to facilitate studies on gene expression directly in the intact brain carrying the lacZ reporter gene, which is widely used as a reporter gene in mouse models.”

The link between Ccdc151 and ciliary function is now clear, but the mechanism by which the Ccdc151 protein contributes is less so. “The precise mechanism by which Ccdc151 protein accomplishes its function is unknown and will be addressed in future research”, says Chiani. The Ccdc151-knockout mouse model, generated via IMPC resources, could be “instrumental to dissect the role of the motile cilia in diverse physiological processes during development, adult life and aging”.

In addition to providing biological resources that can contribute to research such as this, the IMPC is curating a catalogue of mammalian gene function, with phenotyping data for knockout mouse models such as Ccdc151.

Research paper: Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia

Quotes taken from interview with Disease Models & Mechanism: First person – Francesco Chiani and Tiziana Orsini

CLOVES syndrome is a rare condition that is characterised by tissue overgrowth and vascular abnormalities, caused by mosaic gain-of-function mutations in the PIK3CA gene. The way in which CLOVES syndrome manifests itself is highly variable but common features include fatty overgrowths, vascular anomalies, kidney problems and spinal-related symptoms. The condition has no specific treatment and a low survival rate. It is one of a number of conditions that can be grouped under the umbrella of PIK3CA-related overgrowth syndromes (PROS).

By initially undertaking research on a mouse model, and subsequently with human patients, it has been shown by Dr Guillaume Canaud and his team at the Necker-Enfants Malades Hospital in Paris, that BYL719 (an inhibitor of PIK3CA, currently undergoing clinical trials for treating PIK3CA dependent tumours) can prevent and improve organ dysfunction and can improve disease symptoms in patients suffering from CLOVES syndrome.

A mouse model of CLOVES syndrome

The researchers generated mice that express a PIK3CA transgene upon the administration of tamoxifen, mimicking the activity of human CLOVES syndrome. These mice showed similar symptoms to human sufferers of CLOVES, with MRI revealing scoliosis, kidney cysts and muscle abnormalities. Subsequent histological examination revealed further organ abnormalities including additional kidney problems, and abnormalities in the liver and spleen. Furthermore, a high level of cell proliferation was observed in all of the affected organs.

Rapamycin or BYL719?

Rapamycin has previously shown evidence of improving vascular malformations, and when tested on the mouse model of CLOVES syndrome it improved survival rate. However, it did not improve organ abnormalities and did not significantly reduce tumour growth. In contrast, mice treated with BYL719 were found to have preserved tissues and normal vessels. Importantly, BYL719 administration strongly reduced cell proliferation in all affected organs. Withdrawal of BYL719 led to the recurrence of tumours within four weeks, suggesting that continuous administration of BYL719 could relieve the symptoms of CLOVES syndrome.

BYL719 leads to huge improvements in patients with CLOVES syndrome

BYL719 was initially administered to two patients suffering with CLOVES syndrome, who both, after being treated with BYL719, showed dramatic and rapid improvement in their condition. There was a major reduction of vascular tumour abnormalities and overgrowths in addition to improved renal function and a significantly increased quality of life in both patients. The only observed side-effect was hyperglycaemia, which was able to be controlled by a controlled diet.

On the basis of these initial results, Canaud and his team were given permission to treat 17 additional patients with CLOVES syndrome by administering BYL719. The 14 children and 3 adults all showed substantial clinical improvement. A reduction in size of vascular tumours was observed in all of the patients, as well as a drastic reduction in metabolic activity of affected areas. In addition to an improvement to skin capillary abnormalities and scoliosis, all patients reported decreased tiredness. The growth of the children was not affected during the 6 months of treatment and the only side-effects seen were discrete mouth ulcerations in 3 patients (that ultimately disappeared spontaneously) and the aforementioned hyperglycaemia.

The IMPC is aiming to design and produce a genome-wide mouse strain resource of human disease-associated coding variants associated with rare disease that can be used for validation of putative functional variants and insight into disease mechanism(s). To find out more, click here. 

Research paper: Targeted therapy in patients with PIK3CA-related overgrowth syndrome

Research published in Nature identifies SET domain protein 3 (SETD3) as a physiological actin methyltransferase, and uncovers SETD3’s crucial role in the regulation of smooth muscle contractility and its link to primary dystocia in mammals.

For many years it has been known that actin, essential for a large number of cellular processes such as cell motility and the regulation of DNA transcription, is methylated at the amino acid histidine 73 (His73). His73 methylation is found in several model organisms, but its function for many years had remained unclear. After identifying SETD3 as the methylator of actin His73, researchers sought to discover the purpose of actin His73 methylation, and with the help of some mice, they were successful.

Identifying the function of SETD3

To identify the enzymatic function of SETD3, the researchers performed in vitro methylation assays, which showed that the only potential substrate methylated by SETD3 was β-actin. The researchers were then able to identify the exact location of methylation on actin by SETD3 using tandem mass spectrometry. This turned out to be His73. In order to analyse the catalytic specificity of SETD3, the scientists compared methylation events in human cells both with and without the presence of SETD3. Of the 180 histidine methylated peptides, actin-His73 methylation was the only modification that was altered in the absence of SETD3 – identifying actin-His73 methylation as the primary physiological function of SETD3.

Studying SETD3 deficient mice

To confirm the physiological function of SETD3 in vivo, the researchers obtained mice with one copy of their Setd3 gene knocked out (Setd3^+/-) from the Canadian Mouse Mutant Repository. The mouse strain was made at the Toronto Centre for Phenogenomics, an IMPC member institution, using embryonic stem cells. From this strain, it was possible to generate Setd3 null homozygote mice (Setd3^-/-) i.e. mice with both of their copies of Setd3 knocked out. The methylation of actin wasn’t detected in any tissues obtained from SETD3-deficient mice, however, in tissues that expressed SETD3, the majority of actin was methylated – confirming the role of SETD3 as the actin His73 methyltransferase.

Actin methylation regulates actin polymerisation

Previous studies suggest that the methylation of His73 influences actin polymerization dynamics, and here the researchers observed that methylation promoted actin polymerization kinetics in vitro. To explore this idea further, they obtained mouse embryonic fibroblasts that were positive for actin-His73me and compared them with fibroblasts from Setd3^-/- mice that lacked methylation. Cells containing methylated actin were more efficient at migrating than cells without methylated actin – consistent with the idea that methylation of actin by SETD3 positively regulates the polymerisation of actin.

Uterine cell contraction and primary dystocia

Mice without functioning SETD3 protein are able to survive, despite the cell migration defect observed in their embryonic fibroblasts. The researchers were able to infer therefore, that actin-His73me must have a specialised role that isn’t a necessity for survival. IMPC data for Setd3 identifies several phenotypes associated with Setd3 knockout mice including short tibia, decreased body length and decreased lean body mass. In addition to this, the researchers noticed that litter sizes in female Setd3^-/- mice were significantly smaller than expected. After mating Setd3^-/- females with wild type males, dystocia, normally a rare phenotype, was noted in 8 out of 9 Setd3^-/- mice. The lack of obvious pelvic abnormalities in the Setd3^-/- females mean that the cause of dystocia is most likely genetic. Unlike with wild type mice, early labour could not be induced in Setd3^-/- mice with oxytocin, suggesting a specific requirement for SETD3 in the contraction of the uterus during labour, a process which relies upon correctly functioning actin. This led to the proposal that actin-His73me is linked to uterine cell contraction in the primary dystocia of SETD3 deficient mice.

More IMPC related research: Research Reveals Novel Genetic Influences On Osteoporosis

Research paper covered in this article: SETD3 is an actin histidine methyltransferase that prevents primary dystocia

Recent research, published in nature genetics, identifies novel genetic influences on osteoporosis, with the potential to empower future research on osteoporosis and potentially lead to new drug targets.

Osteoporosis is a condition that develops slowly over several years and leads to increased bone fragility. Fragile bones are more prone to fracture, with hip, wrist and spine bones being particularly vulnerable to breakage. Whilst there are many factors that affect the chances of an individual developing osteoporosis such as low body weight, certain medical conditions and age, the disease is highly heritable – you are much more likely to develop osteoporosis if your family has a history of the condition. By assessing the genetic determinants of bone mineral density (BMD), the most clinically relevant factor for diagnosis, the researchers were able to create an atlas of genetic influences on osteoporosis.

GWAS identifies gene loci linked to bone mineral density and fracture risk

Using UK Biobank data, the researchers carried out a genome-wide association study (GWAS), with the aim of identifying the common genetic variants associated with estimated bone mineral density (eBMD). The study found 518 genome-wide significant loci, with 301 of these being novel. Additionally, they undertook a GWAS aimed at identifying genetic loci linked with fracture risk. The results revealed 13 loci associated with risk of fracture. As might be expected, genetic variation associated with risk of fracture was also found to be associated with BMD i.e. alleles associated with low eBMD increased fracture risk.

Causal genes linked to genetic loci

Genetic associations in humans, however, rarely result in improved clinical care, usually due to a lack of identification of causal genes at the associated loci. With this in mind, the researchers tested the DNA locations associated with BMD and fracture risk, to see which features of the DNA linked to genes that are known to influence bone biology in humans. This resulted in a set of ‘Target Genes’ that are known to have an effect on bone density and strength in humans – prioritising genes at associated loci for functional testing.

Using mouse models for in-depth analysis

The study initially used mouse models to successfully test the validity of the identified Target Genes, with outlier phenotypes more frequent in mice with disruptions to 126 Target Genes compared with 526 unselected knockout lines. The in vivo and in vitro data produced thus far in the study converged to identify DAAM2 as a highly credible and novel osteoporosis gene.

After discovering that inducing a double strand break in the DAAM2 gene severely impairs mineralization in human bone cells, the researchers used mice obtained from the Wellcome Trust/Sanger Institute (generated as part of the IMPC, using ES cells) for in-depth characterisation of DAAM2. Mice with both versions of their DAAM2 gene disrupted (Daam2^tm1a/tm1a) exhibited reduced vertebral bone mineral content accompanied by a small reduction in femur length.  Perhaps most significantly, the mutant mice also had markedly reduced bone strength. The increased cortical porosity observed in both male and female Daam2^tm1a/tm1a mutants, along with the abnormal bone composition and structure exhibited, allowed the researchers to conclude that this decreased bone strength in Daam2^tm1a/tm1a mice was not simply a result of abnormal bone turnover. If DAAM2 proves to be a viable drug target – it could result in a complementary therapeutic strategy for the prevention and treatment of osteoporosis.

The goal of the IMPC is to knock out and phenotype all 20,000 or so genes in the mouse genome, potentially providing major insights into unexplored areas of the mammalian genome. To order IMPC mice, click on the shopping cart on the top part of the gene page of interest or scroll down to “Order Mouse and ES Cells”.

IMPC Data

Details on more phenotype associations for DAAM2 can be found on the IMPC Daam2 gene page. Various phenotype association data can be found such as that shown below.

Research Paper: An atlas of genetic influences on osteoporosis in humans and mice

The inhibition of alkaptonuria by the chemical nitisinone in a mouse model led to a full-scale clinical trial funded by the European Commission – the results of which are expected to produce medicine for a condition for which there was previously no treatment available.

Alkaptonuria (AKU) is caused by a deficiency of homogentisate 1,2 dioxygenase, an enzyme that is required for breaking down the two amino acids tyrosine and phenylalanine. The lack of a functional copy of this enzyme results in a vast increase in circulating concentration of homogentisic acid (HGA), leading to a darkening of the urine upon exposure to air.

High levels of homogentisic acid within the human body eventually leads to ochronosis – a progressive pigmentation of connective tissues and eventually severe joint disease, which can become fatal later in life.

Whilst AKU is a serious condition, for many years it was not seen as prevalent enough among the population to be lucrative for drug developers. However, physicians had long suspected that nitisinone, a chemical that was first developed for use as a weed-killer, could be used to treat AKU – nitisinone was being used effectively to treat tyrosinemia (a condition that results from disruption to the same metabolic pathway as AKU).

There was a small-scale clinical trial run in the US between 2005 and 2008, but too few patients were recruited and the clinical endpoint (the success of the treatment) was defined very narrowly. No positive results were obtained.

In order for a clinical trial to succeed, a more reliable endpoint would have to be suggested, and this required more knowledge of how the disease developed. With this in mind, funding obtained from Britain’s Big Lottery Fund went to a research team at the University of Liverpool to develop a mouse model of AKU.

A colony of Hgd-/- (the genetic equivalent of human AKU) mice were bred by the research team in Liverpool and the mice were established as a model of the plasma biochemistry of AKU and its associated arthropathy. The concentration of plasma HGA was found to be 0.149 ± 0.019 mM, whilst HGA levels in WT mice were below the level of UV detection. The researchers were also able to determine that pigmentation of connective tissue increased linearly with increasing age. Perhaps most importantly though, a significant difference was found between HGA concentrations before and after treatment with nitisinone. Levels of plasma HGA dropped by 88% – a figure that was maintained over the mouse lifetime, with joint tissue from nitisinone-treated AKU mice showing no pigmented tissue.

The results of this mouse model study led to a full-scale clinical trial, funded by the European Commission, with use of nitisinone as a treatment for AKU expected to be approved across Europe once the trial comes to an end.

The aim of the IMPC is to generate similar knock out lines – on a much broader scale, with the goal of producing knock out mutations in embryonic stem cells for 20,000 known and predicted mouse genes, and determining the function of each of these genes. The mouse’s genetic similarity to humans (95% at the gene level) means that data generated by the IMPC could be a powerful tool as we seek to understand the genetic basis of human disease. As our knowledge of rare genetic diseases increases in line with rapid technological advancement, the desire for treatments targeted to small groups of sufferers with these diseases will also likely increase – the case involving alkaptonuria is likely to be just one instance of a mouse model proving to be a driver behind new treatments.

Research article: Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone