Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/ß-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Luminal Rank loss decreases cell fitness leading to basal cell bipotency in parous mammary glands.

Rank signaling pathway regulates mammary gland homeostasis and epithelial cell differentiation. Although Rank receptor is expressed by basal cells and luminal progenitors, its role in each individual cell lineage remains unclear. By combining temporal/lineage specific Rank genetic deletion with lineage tracing techniques, we found that loss of luminal Rank reduces the luminal progenitor pool and leads to aberrant alveolar-like differentiation with high protein translation capacity in virgin mammary glands. These Rank-deleted luminal cells are unable to expand during the first pregnancy, leading to lactation failure and impairment of protein synthesis potential in the parous stage. The unfit parous Rank-deleted luminal cells in the alveoli are progressively replaced by Rank-proficient cells early during the second pregnancy, thereby restoring lactation. Transcriptomic analysis and functional assays point to the awakening of basal bipotency after pregnancy by the induction of Rank/NF-κB signaling in basal parous cell to restore lactation and tissue homeostasis.

miR-203 drives breast cancer cell differentiation.

A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Pseudoalignment tools as an efficient alternative to detect repeated transposable elements in scRNAseq data.

MOTIVATION: Transposable elements (TE) have played a major role in configuring the structures of mammalian genomes through evolution. In normal conditions, the expression of these elements is repressed by different epigenetic regulation mechanisms such as DNA methylation, histone modification and regulation by small RNAs. TE re-activation is associated with stemness potential acquisition, regulation of innate immunity and disease, such as cancer. However, the vast majority of current knowledge in the field is based on bulk expression studies, and very little is known on cell-type- or state-specific expression of TE-derived transcripts. Therefore, cost-efficient single-cell-resolution TE expression analytical approaches are needed. RESULTS: We have implemented an analytical approach based on pseudoalignment to consensus sequences to incorporate TE expression information to scRNAseq data. AVAILABILITY AND IMPLEMENTATION: All the data and code implemented are available as Supplementary data and in: https://github.com/jmzvillarreal/kallisto_TE_scRNAseq. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells.

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.

Folliculin-interacting protein FNIP2 impacts on overweight and obesity through a polymorphism in a conserved 3' untranslated region.

BACKGROUND: Overweight and obesity are defined by an anomalous or excessive fat accumulation that may compromise health. To find single-nucleotide polymorphisms (SNPs) influencing metabolic phenotypes associated with the obesity state, we analyze multiple anthropometric and clinical parameters in a cohort of 790 healthy volunteers and study potential associations with 48 manually curated SNPs, in metabolic genes functionally associated with the mechanistic target of rapamycin (mTOR) pathway. RESULTS: We identify and validate rs2291007 within a conserved region in the 3'UTR of folliculin-interacting protein FNIP2 that correlates with multiple leanness parameters. The T-to-C variant represents the major allele in Europeans and disrupts an ancestral target sequence of the miRNA miR-181b-5p, thus resulting in increased FNIP2 mRNA levels in cancer cell lines and in peripheral blood from carriers of the C allele. Because the miRNA binding site is conserved across vertebrates, we engineered the T-to-C substitution in the endogenous Fnip2 allele in mice. Primary cells derived from Fnip2 C/C mice show increased mRNA stability, and more importantly, Fnip2 C/C mice replicate the decreased adiposity and increased leanness observed in human volunteers. Finally, expression levels of FNIP2 in both human samples and mice negatively associate with leanness parameters, and moreover, are the most important contributor in a multifactorial model of body mass index prediction. CONCLUSIONS: We propose that rs2291007 influences human leanness through an evolutionarily conserved modulation of FNIP2 mRNA levels.

Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism.

Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9-RAGE-NF-κB-JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis.

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma.

CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

MicroRNAs Targeting HIF-2α, VEGFR1 and/or VEGFR2 as Potential Predictive Biomarkers for VEGFR Tyrosine Kinase and HIF-2α Inhibitors in Metastatic Clear-Cell Renal Cell Carcinoma.

Metastatic clear-cell renal cell carcinoma (m-ccRCC) is characterized by increased hypoxia-induced factor (HIF)-2α and vascular endothelial growth factor receptor (VEGFR)-dependent angiogenesis through loss of function of the von Hippel-Lindau protein. VEGFR tyrosine kinase inhibitors (VEGFR-TKIs) are a cornerstone of m-ccRCC treatment, and new treatments targeting HIF-2α are currently under investigation. However, predictive biomarkers for these treatments are lacking. In this retrospective cohort study including 109 patients treated with VEGFR-targeted therapies as first-line treatment, we aimed to study the possible predictive function of microRNAs (miRNAs) targeting HIF-2α, VEGFR1 and VEGFR2. We selected miRNAs inversely correlated with HIF-2α, VEGFR1 and/or VEGFR2 expression and with predicted target sites in the respective genes and subsequently studied their impact on therapeutic outcomes. We identified four miRNAs (miR-34c-5p, miR-221-3p, miR-222-3p and miR-3529-3p) inversely correlated with VEGFR1 and/or VEGFR2 expression and associated with tumor shrinkage and progression-free survival (PFS) upon treatment with VEGFR-TKIs, highlighting the potential predictive value of these miRNAs. Moreover, we identified three miRNAs (miR-185-5p, miR-223-3p and miR-3529-3p) inversely correlated with HIF-2α expression and associated with tumor shrinkage and PFS upon treatment with VEGFR-TKIs. These three miRNAs can have a predictive value not only upon treatment with VEGFR-TKIs but possibly also upon treatment with the upcoming HIF-2α inhibitor belzutifan.

Compi: a framework for portable and reproducible pipelines.

Compi is an application framework to develop end-user, pipeline-based applications with a primary emphasis on: (i) user interface generation, by automatically generating a command-line interface based on the pipeline specific parameter definitions; (ii) application packaging, with compi-dk, which is a version-control-friendly tool to package the pipeline application and its dependencies into a Docker image; and (iii) application distribution provided through a public repository of Compi pipelines, named Compi Hub, which allows users to discover, browse and reuse them easily. By addressing these three aspects, Compi goes beyond traditional workflow engines, having been specially designed for researchers who want to take advantage of common workflow engine features (such as automatic job scheduling or logging, among others) while keeping the simplicity and readability of shell scripts without the need to learn a new programming language. Here we discuss the design of various pipelines developed with Compi to describe its main functionalities, as well as to highlight the similarities and differences with similar tools that are available. An open-source distribution under the Apache 2.0 License is available from GitHub (available at https://github.com/sing-group/compi). Documentation and installers are available from https://www.sing-group.org/compi. A specific repository for Compi pipelines is available from Compi Hub (available at https://www.sing-group.org/compihub.

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that - whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development - lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.

Inhibition of Rag GTPase signaling in mice suppresses B cell responses and lymphomagenesis with minimal detrimental trade-offs.

B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC. Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagCQ119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagCQ119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagCQ119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells.

MicroRNAs Possibly Involved in the Development of Bone Metastasis in Clear-Cell Renal Cell Carcinoma.

Bone metastasis in clear-cell renal cell carcinoma (ccRCC) leads to substantial morbidity through skeletal related adverse events and implicates worse clinical outcomes. MicroRNAs (miRNA) are small non-protein coding RNA molecules with important regulatory functions in cancer development and metastasis. In this retrospective analysis we present dysregulated miRNA in ccRCC, which are associated with bone metastasis. In particular, miR-23a-3p, miR-27a-3p, miR-20a-5p, and miR-335-3p specifically correlated with the earlier appearance of bone metastasis, compared to metastasis in other organs. In contrast, miR-30b-3p and miR-139-3p were correlated with less occurrence of bone metastasis. These miRNAs are potential biomarkers and attractive targets for miRNA inhibitors or mimics, which could lead to novel therapeutic possibilities for bone targeted treatment in metastatic ccRCC.

Short and dysfunctional telomeres sensitize the kidneys to develop fibrosis.

Accumulation of short telomeres is a hallmark of aging. Mutations in telomerase or telomere-binding proteins lead to telomere shortening or dysfunction and are at the origin of human pathologies known as 'telomere syndromes', which are characterized by loss of the regenerative capacity of tissues and fibrotic pathologies. Here, we generated two mouse models of kidney fibrosis, either by combining telomerase deficiency to induce telomere shortening and a low dose of folic acid, or by conditionally deleting Trf1, a component of the shelterin telomere protective complex, from the kidneys. We find that short telomeres sensitize the kidneys to develop fibrosis in response to folic acid and exacerbate the epithelial-to-mesenchymal transition (EMT) program. Trf1 deletion in kidneys led to fibrosis and EMT activation. Our findings suggest that telomere shortening or dysfunction may contribute to pathological, age-associated renal fibrosis by influencing the EMT program.

Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state.

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.

Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases.

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF-MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF-MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.