Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

ACE2-EGFR-MAPK signaling contributes to SARS-CoV-2 infection.

SARS-CoV-2 triggered the most severe pandemic of recent times. To enter into a host cell, SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2). However, subsequent studies indicated that other cell membrane receptors may act as virus-binding partners. Among these receptors, the epidermal growth factor receptor (EGFR) was hypothesized not only as a spike protein binder, but also to be activated in response to SARS-CoV-2. In our study, we aim at dissecting EGFR activation and its major downstream signaling pathway, the mitogen-activated signaling pathway (MAPK), in SARS-CoV-2 infection. Here, we demonstrate the activation of EGFR-MAPK signaling axis by the SARS-CoV-2 spike protein and we identify a yet unknown cross talk between ACE2 and EGFR that regulated ACE2 abundance and EGFR activation and subcellular localization, respectively. By inhibiting the EGFR-MAPK activation, we observe a reduced infection with either spike-pseudotyped particles or authentic SARS-CoV-2, thus indicating that EGFR serves as a cofactor and the activation of EGFR-MAPK contributes to SARS-CoV-2 infection.

Increased osteoclastogenesis contributes to bone loss in the Costello syndrome Hras G12V mouse model.

RAS GTPases are ubiquitous GDP/GTP-binding proteins that function as molecular switches in cellular signalling and control numerous signalling pathways and biological processes. Pathogenic mutations in RAS genes severely affect cellular homeostasis, leading to cancer when occurring in somatic cells and developmental disorders when the germline is affected. These disorders are generally termed as RASopathies and among them Costello syndrome (CS) is a distinctive entity that is caused by specific HRAS germline mutations. The majority of these mutations affect residues 12 and 13, the same sites as somatic oncogenic HRAS mutations. The hallmarks of the disease include congenital cardiac anomalies, impaired thriving and growth, neurocognitive impairments, distinctive craniofacial anomalies, and susceptibility to cancer. Adult patients often present signs of premature aging including reduced bone mineral density and osteoporosis. Using a CS mouse model harbouring a Hras G12V germline mutation, we aimed at determining whether this model recapitulates the patients' bone phenotype and which bone cells are driving the phenotype when mutated. Our data revealed that Hras G12V mutation induces bone loss in mice at certain ages. In addition, we identified that bone loss correlated with an increased number of osteoclasts in vivo and Hras G12V mutations increased osteoclastogenesis in vitro. Last, but not least, mutant osteoclast differentiation was reduced by treatment in vitro with MEK and PI3K inhibitors, respectively. These results indicate that Hras is a novel regulator of bone homeostasis and an increased osteoclastogenesis due to Hras G12V mutation contributes to bone loss in the Costello syndrome.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Senescence in RASopathies, a possible novel contributor to a complex pathophenoype.

Senescence is a biological process that induces a permanent cell cycle arrest and a specific gene expression program in response to various stressors. Following studies over the last few decades, the concept of senescence has evolved from an antiproliferative mechanism in cancer (oncogene-induced senescence) to a critical component of physiological processes associated with embryonic development, tissue regeneration, ageing and its associated diseases. In somatic cells, oncogenic mutations in RAS-MAPK pathway genes are associated with oncogene-induced senescence and cancer, while germline mutations in the same pathway are linked to a group of monogenic developmental disorders generally termed RASopathies. Here, we consider that in these disorders, senescence induction may result in opposing outcomes, a tumour protective effect and a possible contributor to a premature ageing phenotype identified in Costello syndrome, which belongs to the RASopathy group. In this review, we will highlight the role of senescence in organismal homeostasis and we will describe the current knowledge about senescence in RASopathies. Additionally, we provide a perspective on examples of experimentally characterised RASopathy mutations that, alone or in combination with various stressors, may also trigger an age-dependent chronic senescence, possibly contributing to the age-dependent worsening of RASopathy pathophenotype and the reduction of lifespan.

Pancreatic Ductal Organoids React Kras Dependent to the Removal of Tumor Suppressive Roadblocks.

Pancreatic ductal adenocarcinoma (PDAC) is still the Achilles heel in modern oncology, with an increasing incidence accompanied by a persisting high mortality. The developmental process of PDAC is thought to be stepwise via precursor lesions and sequential accumulation of mutations. Thereby, current sequencing studies recapitulate this genetic heterogeneity in PDAC and show besides a handful of driver mutations (KRAS, TP53) a plethora of passenger mutations that allow to define subtypes. However, modeling the mutations of interest and their effects is still challenging. Interestingly, organoids have the potential to recapitulate in vitro, the in vivo characteristics of the tissue they originate from. Here, we could establish and develop tools allowing us to isolate, culture, and genetically modify ductal mouse organoids. Transferred to known effectors in the IPMN-PDAC sequence, we could reveal significantly increased proliferative and self-renewal capacities for PTEN and RNF43 deficiency in the context of oncogenic KRASG12D in mouse pancreatic organoids. Overall, we were able to obtain promising data centering ductal organoids in the focus of future PDAC research.