Search

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell–specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4+ inflammatory cells, F4/80+/Gr-1+ (myeloid-derived suppressor cells), CD4+/Foxp3+ (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFα, IFNγ, and IL1β following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.

Supplementary Figure Legend, Tables 1-2 from Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

Supplementary Figure 1 from Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

The biological purpose of long non-coding RNAs (lncRNAs) is poorly understood. Haploinsufficient mutations in HNF1A homeobox A (HNF1A), encoding a homeodomain transcription factor, cause diabetes mellitus. Here, we examine HASTER, the promoter of an lncRNA antisense to HNF1A. Using mouse and human models, we show that HASTER maintains cell-specific physiological HNF1A concentrations through positive and negative feedback loops. Pancreatic β cells from Haster mutant mice consequently showed variegated HNF1A silencing or overexpression, resulting in hyperglycaemia. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. We demonstrate that the HASTER promoter DNA, rather than the lncRNA, modulates HNF1A promoter-enhancer interactions in cis and thereby regulates HNF1A transcription. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. They also show that disruption of a mammalian lncRNA promoter can cause diabetes mellitus. This research was supported by the Medical Research Council (MR/L02036X/1), Wellcome Trust (WT101033), European Research Council (Advanced Grant 789055), Spanish Ministry of Science and Innovation (BFU2014-54284-R and RTI2018-095666-B-I00) and National Institute for Health Research Imperial Biomedical Research Centre. Work in the Centre for Genomic Regulation was supported by the Centres de Recerca de Catalunya programme, Generalitat de Catalunya, Centro de Excelencia Severo Ochoa (CEX2020-001049) and by the Spanish Ministry of Science and Innovation aid to the European Molecular Biology Laboratory partnership. We thank the University of Barcelona School of Medicine animal facility, Center for Genomic Regulation and Imperial College London genomics units, Imperial College High Performance Computing Service, as well as K. Kaestner (University of Pennsylvania), M. Gannon (Vanderbilt University) and D. Tuveson (Cold Spring Harbor Laboratory) for the Cre lines. We thank J. Valcarcel and T. Graf (Center for Genomic Regulation) for comments on the manuscript, F. X. Real (Spanish National Cancer Research Centre) for insights and support, V. Grau and C. Roth for technical support and members of J.F.’s laboratory for valuable discussions.

Intestinal ischemia induces mucosal damage while simultaneously activating intestinal stem cells (ISCs), which subsequently regenerate the damaged intestinal epithelium. However, whether paracrine factors secreted from vascular endothelial cells (ECs) - blood and lymphatic ECs (BECs and LECs, respectively) – regulate ISC-mediated regeneration have yet to be elucidated. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in regeneration of the small intestine after ischemia-reperfusion (I/R) injury. EC- and LEC-specific deletions of Foxc1, Foxc2, or both in mice augment I/R-induced intestinal damage by causing defects in vascular regrowth, expression of the chemokine CXCL12 and the Wnt activator R- spondin 3 in BECs and LECs, respectively, and activation of Wnt signaling in ISCs. Treatment with CXCL12 and R-spondin 3 rescues the I/R-induced intestinal damage in EC- and LEC-Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.

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 (NGF) 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. we apologize to those authors whose work could not be cited due to size restrictions. We thank M. S. Soengas and the members of her laboratory for melanoma cells, primary melanocyte preparations and helpful discussions. We thank M. Detmar and S. Proulx for the mouse B16-F1R2 cell line. We are grateful to M. Yañez-Mo and M. Valés for antibodies against sEV markers. We thank D. Grela and A. Escobar from IESMAT for their support with the Zetasizer analysis. We thank G. Roncador, L. Maestre and J. L. Martinez Torrecuadrada for their help with the development and characterization of anti-NGFR antibodies and C. Villarroya Beltri for her help in flow cytometry analysis. This work was funded by the Starr Cancer Consortium (B.J.M., D.L. and H.P.), the US NIH (R01-CA169416), the Nancy C. and Daniel P. Paduano Foundation, the Children’s Cancer and Blood Foundation (H.P. and D.L.), the Melanoma Research Alliance, the Feldstein Foundation, RETOS SAF2017-82924-R (AEI/10.13039/501100011033/FEDER-UE), the Fundación Ramón Areces, the Fundación Bancaria ‘la Caixa’ (HR18-00256), ATRES-MEDIA AXA Foundation (CONSTANTES Y VITALES, una iniciativa de laSexta y Fundación AXA) and the Fundación Científica AECC (LABAE19027PEIN, GCB15152978SOEN-HP) (H.P.), the Malcolm Hewitt Wiener Foundation, the AHEPA Fifth District Cancer Research Foundation, the Hartwell Foundation and the Manning Foundation (D.L.). We are also grateful for the support of the Translational Network for the Clinical Application of Extracellular Vesicles (TeNTaCLES), RED2018-102411-T (AEI/10.13039/501100011033), the Ramón y Cajal Programme, the FERO Foundation, Comunidad of Madrid 2017-T2/BMD6026 (L.N.) and La Caixa Foundation (ID100010434, fellowship LCF/BQ/ES17/11600007) (A.H.-B.). The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII. No

Exogenous administration of lymphangiogenic growth factors is widely used to study changes in lymphatic function in pathophysiology. However, this approach can result in off-target effects, thereby generating conflicting data. To circumvent this issue, we modulated intracellular VEGF-C signaling by conditionally knocking out the lipid phosphatase PTEN using the Vegfr3 promoter to drive the expression of Cre-lox in lymphatic endothelial cells (LECs). PTEN is an intracellular brake that inhibits the downstream effects of the activation of VEGFR3 by VEGF-C. Activation of Cre-lox recombination in adult mice resulted in an expanded functional lymphatic network due to LEC proliferation that was independent of lymphangiogenic growth factor production. Furthermore, compared with lymphangiogenesis induced by VEGF-C injection, LECPTEN animals had mature, nonleaky lymphatics with intact cell-cell junctions and reduced local tissue inflammation. Last, compared with wild-type or VEGF-C-injected mice, LECPTEN animals had an improved capacity to resolve inflammatory responses. Our findings indicate that intracellular modulation of lymphangiogenesis is effective in inducing functional lymphatic networks and has no off-target inflammatory effects.

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell–specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4+ inflammatory cells, F4/80+/Gr-1+ (myeloid-derived suppressor cells), CD4+/Foxp3+ (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFα, IFNγ, and IL1β following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated to 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 knockouts we report here that, whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild microcephaly during development, lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our novel mouse model of MCPH8 suggests that Cep135 deficiency results in centriole duplication, TP53 activation and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death, but not microcephaly, and leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that microcephaly 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.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a Kras FSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras +/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras +/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences. We thank Marta San Roman, Raquel Villar, and Nuria Cabrera for excellent technical assistance; Mayte Lamparero and Isabel Blanco (Animal Facility) for mouse work; the Histopathology Unit for processing of mouse tissues; Lola Martinez (Flow Cytometry Unit) for her help with flow cytometry analyses; Diego Megias and Manuel Perez (Confocal Microscopy Unit) for assistance with confocal microscopy; and the Mouse Genome Editing Unit for support with the generation of the mouse strains described here. We also thank Ignacio Perez de Castro (Instituto de Salud Carlos III, Madrid, Spain) for sharing the EGFP-KRAS4B plasmid and Orlando Dominguez (Genomics Unit) and Pedro P. Lopez-Casas (Clinical Research Program) for their advice on exome sequencing. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1), and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM); a grant from the CRIS Cancer Foundation (to M.B.); and a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00, to M.B. and M.M.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S. was supported by predoctoral contract "Severo Ochoa" (BES-2016-079096) from the SpanishMinistry of Science, Innovation and Universities. G.P. was a recipient of a "Young Ph.D." grant from the Government of the Community of Madrid. F.F.-G. was supported by a formacion de profesorado universitario (FPU) fellowship from the Spanish Ministry of Science, Innovation and Universities. Sí

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., M.N.S. was funded by a Leverhulme Trust early career fellowship. Work in the laboratory of M.Z.-G. was funded by the Wellcome Trust (098287/Z/12/Z) and the European Research Council (ERC) (669198). I.C. was funded by the Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and European Social Fund. I.A. and P.S. were supported by the Fondation pour la Recherche Medicale (DEQ20170336757), Infrastructure Nationale en Biologie et Santé INGESTEM (ANR-11-INBS-0009), IHU-B CESAME (ANR-10-IBHU-003), LabEx REVIVE (ANR-10-LABX-73), LabEx DEVweCAN (ANR-10-LABX-0061) and LabEx CORTEX (ANR-11-LABX-0042) of University of Lyon within the programme ‘Investissements d’Avenir’ (ANR-11-IDEX-0007). Research by J.P., S.M. and C.B.-A. was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness (SAF2013-44267-R) and by the CNIO. Work in the laboratory of D.F. was funded by the Institut National du Cancer (PLBIO10-068 and PLBIO15-005) and the Ligue National Contre le Cancer (EL2018.LNCC/DF). Work in the laboratory of N.M. was funded by the ERC, under the European Union Horizon 2020 research and innovation programme (StG-2014–640525_REGMAMKID), the Spanish Association Against Cancer (AECC/LABAE16006), Carlos III Health Institute (Red TerCel, CardioCel, RD16/0011/0027), Ministry of Economy and Competitiveness (MINECO) projects SAF2017–89782-R, SAF2015–72617-EXP and RYC-2014–16242, and the CERCA/Government of Catalonia (2017 SGR 1306). Work in the laboratory of S.O. was funded by SAF2013–44866-R from MINECO Spain. Work in the laboratory of M.F.F. was funded by Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892, to M.F.F. and A.F.F.); the ISCIII-Subdireccion General de Evaluación y Fomento de la Investigación and Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131, to A.F.F.); IUOPA (to M.I.S.); and the Asturias Regional Government (GRUPIN14–052, to M.F.F.). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Work in the laboratory of M.S. was funded by the CNIO, the IRB and by grants from Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), Botin Foundation, Banco Santander (Santander Universities Global Division), laCaixa Foundation and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).

Full differentiation potential along with self‐renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already‐established murine and human PSCs. Short exposure to miR‐203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human–mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR‐203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

Autosomal Recessive Primary Microcephaly (MCPH) is a rare disease associated to proteins involved in centrosomal and spindle dynamics including Cep135 (MCPH8). Although Cep135 has been associated to centriolar assembly, the mechanisms associated to the pathogenesis underlying MCPH8 mutations are unclear. By using a series of CRISPR/Cas9-edited murine Cep135 alleles, we report here that lack of Cep135 results in perinatal lethality accompanied by significant microcephaly in a dosis-dependent manner. Cep135 deficiency, but not that of other centrosomal microcephaly proteins such as Aspm or Cdk5rap2, induces centrosome duplication defects, and perturbed centriole structure and dynamics. Whereas other cell types are able to quickly adapt to these defects, neural progenitors display a prolonged response leading to chromosomal instability and cell death in later developmental stages. Genetic ablation of Trp53 in these mutant embryos prevents apoptotic cell death but does not rescue the microcephaly induced by Cep135 loss. These results suggest that microcephaly can arise from the lack of adaptation to centriole defects in neural progenitors of the developing neocortex in a p53-independent manner.

The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries. We thank Cristina Villa del Campo, Ester de la Cruz, and other members of the Torres group for stimulating discussions and suggestions. We thank members of the Microscopy, Genomics, Bioinformatics, Transgenesis, and Animal Facility CNIC units for excellent support. This work was supported by grants PGC2018-096486-B-I00 and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Science, Innovation, and Universities and grant S2017/BMD3875 from the Comunidad de Madrid to M.T., grant ITN-CARDIONET-ref.289600 from EU to G.L., and by grant BFU2015-71376-R to S.O. The CNIC is supported by the Ministerio de Ciencia, Innovacio´ n y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work was also supported by NIH grant RO1HL073402 to G.O. and by AHA grant 18CDA34110356 to X.L. Sí

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding Lysine‐specific demethylase 6A, carries somatic mutations in PDAC. Here, we show that pancreas‐specific Hnf1a null mutant transcriptomes phenocopy those of Kdm6a mutations, and both defects synergize with Kras G12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic, and biochemical studies to show that HNF1A recruits KDM6A to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates differentiated acinar cell programs, and indirectly suppresses oncogenic and epithelial–mesenchymal transition genes. We also identify a subset of non‐classical PDAC samples that exhibit the HNF1A/KDM6A‐deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A deficiency promotes PDAC. They also connect the tumor‐suppressive role of KDM6A deficiency with a cell‐specific molecular mechanism that underlies PDAC subtype definition., Cooperation between a transcription factor and an epigenetic modifier reveals how defective transcriptional control can contribute to development of pancreatic ductal adenocarcinoma.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs.

The crosstalk between cancer cells and the lymphatic vasculature has long been proposed to define competency for metastasis. Nevertheless, the discovery of selective blockers of lymphovascular niches has been compromised by the paucity of experimental systems for whole-body analyses of tumor progression. Here we present immunocompetent and immunodeficient mouse models for live imaging of melanoma-induced neolymphangiogenesis (driven by Vegfr3) as a cost-effective platform for drug screeningin vivo. Spatio-temporal analyses in autochthonous melanomas and patient-derived xenografts identified double stranded RNA mimics (dsRNA nanoplexes) as potent repressors of lymphangiogenesis and metastasis. Mechanistically, dsRNA nanoplexes were found to suppress lymphangiogenic drivers in both tumor cells and their associated lymphatic vasculature (via MIDKINE and Vegfr3, respectively). This dual inhibitory action, driven by type I interferon, was not shared by FDA-approved antimelanoma treatments or by lymphangiogenic blockers in clinical testing. These results underscore the power of Vegfr3-lymphoreporters for pharmacological testing in otherwise aggressive cancers.RELEVANCEAlthough tumor-induced lymphangiogenesis has long been associated with metastasis, selective targeting of this process has been compromised by the paucity of experimental platforms for whole-body imaging of tumor progression and drug response. Here we present animal models engineered for spatio-temporal analyses of neolymphangiogenesis in clinically relevant autochthonous melanomas and patient-derived xenografts, and identify a unique action of double stranded-RNA nanoplexes as potent repressors of lymphatic dissemination and metastatic relapse.

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression ofde novoDNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

Summary The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells., Graphical Abstract, Highlights • Mammalian haploid cell cultures become progressively enriched in diploid cells • DAB, a precursor of Taxol, facilitates the maintenance of haploidy • DAB selects for cells with lower ploidy in mixed cultures of mammalian cells • Statins accelerate the gradual loss of haploid cells in culture, Cultures of haploid animal cell lines become progressively enriched in diploid cells. By conducting a chemical screen in HAP1 cells, Olbrich et al. identify compounds that facilitate the maintenance of haploid cells and a general strategy to select for cells with lower ploidy in mixed cultures of mammalian cells.

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding the histone demethylase UTX, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutations phenocopy Utx deficient mutations, and both synergize with KrasG12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic and biochemical studies to show that HNF1A recruits UTX to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates a differentiation program, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. Finally, we identify a subset of non-classical PDAC samples that exhibit the HNF1A/UTX-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A-deficiency promotes PDAC. They also connect the tumor suppressive role of UTX deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.

By whole exome sequencing, we recently identified a missense mutation (p.R703C) in the human ATP4a gene, which encodes the proton pump responsible for gastric acidification. This mutation causes an aggressive familial type I gastric neuroendocrine tumor in homozygous individuals. Affected individuals show an early onset of the disease, characterized by gastric hypoacidity, hypergastrinemia, iron-deficiency anemia, gastric intestinal metaplasia and, in one case, an associated gastric adenocarcinoma. Total gastrectomy was performed as the definitive treatment in all affected individuals. We now describe the generation and characterization of a knockin mouse model for the ATP4aR703C mutation to better understand the tumorigenesis process. Homozygous mice recapitulated most of the phenotypical alterations that were observed in human individuals, strongly suggesting that this mutation is the primary alteration responsible for disease development. Homozygous mice developed premalignant condition with severe hyperplasia, dysplasia and glandular metaplasia in the stomach. Interestingly, gastric acidification in homozygous mice, induced by treatment with 3% HCl acid in the drinking water, prevented (if treated from birth) or partially reverted (if treated during adulthood) the development of glandular metaplasia and dysplasia in the stomach and partially rescued the abnormal biochemical parameters. We therefore suggest that, in this model, achlorhydria contributes to tumorigenesis to a greater extent than hypergastrinemia. Furthermore, our mouse model represents a unique and novel tool for studying the pathologies associated with disturbances in gastric acid secretion., Summary: Gastric pathologies in an ATP4a knockin mouse model of a mutation responsible for the development of gastric neuroendocrine tumors in humans are prevented and reverted by adding HCl to drinking water.

One recurring theme in drug development is to exploit synthetic lethal properties as means to preferentially damage the DNA of cancer cells. We and others have previously developed inhibitors of the ATR kinase, shown to be particularly genotoxic for cells expressing certain oncogenes. In contrast, the mechanisms of resistance to ATR inhibitors remain unexplored. We report here on a genome-wide CRISPR-Cas9 screen that identified CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A-deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A-deficient cells. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells and reveals genetic interactions that could be used for their rational use. We thank the laboratories of Feng Zhang and Kosuke Yusa for sharing all CRISPR-related plasmids used here through Addgene (plasmids 42230, 50946, and 50947) and Edna Fonseca for her comments on the manuscript. Research was funded by Fundacion Botin, Banco Santander, through its Santander Universities Global Division and by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2011-23753 and SAF2014-57791-REDC), Fundacio La Marato de TV3, the Howard Hughes Medical Institute, and the European Research Council (ERC-617840) to O.F.-C.; by a PhD fellowship from La Caixa Foundation to C.M.-R.; by grants from MINECO to S.R. (RYC2011-09242 and SAF2013-49147P, this last project co-financed with European FEDER funds); and by a grant from MINECO (SAF2013-44866-R) to S.O. Sí

MEK inhibition in combination with a glycogen synthase kinase-3β (GSK3β) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs. We thank Cian Lynch, Jorge Monsech, and Diego Megias for their help with microarray, ChIP-seq, and high-throughput microscopy analyses. We also thank Dr. Manuel Serrano and Dr. André Nussenzweig for their input on the manuscript, and Dr. Diego Sanz for his support throughout the project. C.M.-R. was funded by a PhD fellowship from La Caixa Foundation, T.O. was funded by a PhD fellowship from the Boehringer Ingelheim Fonds, and S.R. was funded by a Ramon y Cajal contract (RYC-2011-09242). Research was funded by Fundación Botín and Banco Santander through its Santander Universities Global Division; grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-23753 and SAF2014- 57791-REDC; these projects were cofinanced with European Fonds Européen de Développement Économique et Régional [FEDER] funds), Fundació La Marato de TV3, Howard Hughes Medical Institute, and the European Research Council (ERC- 617840) to O.F.-C.; and grants from the Spanish Ministryof Economy and Competitiveness (SAF2013-49147-P and SAF2016-80874-P; these projects were cofinanced with European FEDER funds) to S.R. Author contributions: C.M.-R. and S.R. conducted most of the experiments; T.O. helped with the characterization of RASlox/lox mESCs and with ERF localization studies; E.L. helped with ChIP-seq experiments; M.D., S.O., and M.B. contributed to the work on RAS-deficient cells; M.V.-S. provided technical help; O.D. helped with genomics experiments and bioinformatics analysis; and S.R. and O.F.-C. coordinated the study and wrote the manuscript. Sí

Aortic intramural hematoma (IMH) can evolve toward reabsorption, dissection or aneurysm. Hypertension is the most common predisposing factor in IMH and aneurysm patients, and the hypertensive mediator angiotensin-II induces both in mice. We have previously shown that constitutive deletion of Rcan1 isoforms prevents Angiotensin II-induced aneurysm in mice. Here we generate mice conditionally lacking each isoform or all isoforms in vascular smooth muscle cells, endothelial cells, or ubiquitously, to determine the contribution to aneurysm development of Rcan1 isoforms in vascular cells. Surprisingly, conditional Rcan1 deletion in either vascular cell-type induces a hypercontractile phenotype and aortic medial layer disorganization, predisposing to hypertension-mediated aortic rupture, IMH, and aneurysm. These processes are blocked by ROCK inhibition. We find that Rcan1 associates with GSK-3β, whose inhibition decreases myosin activation. Our results identify potential therapeutic targets for intervention in IMH and aneurysm and call for caution when interpreting phenotypes of constitutively and inducibly deficient mice., CNIC is supported by the Spanish Ministerio de Ciencia, Investigación y Universidades (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Support was also provided by grants from MINECO (grants SAF2013- 45258P and SAF2017-88881R (M.R.C.), SAF2015-636333R (J.M.R.), PI15/01224 (J.F.N.), and BIO2015-67580-P (J.V.)), Comunidad de Madrid (grant AORTASANA-CM; B2017/ BMD-3676 (M.R.C. and J.M.R.)), CSIC (M.R.C.), Fundacion La Marato TV3 (grant 20151330 (J.M.R.)), The Marfan Foundation Faculty grant 2017 (J.M.R.), and the CIBERCV of Ministerio de Sanidad (grants CB16/11/00264 (J.M.R.) and CB16/11/00277 (J.V.)). S.V. and P.S.Y.-L. hold FPI fellowships SVP-2013-067777 and BES-2016-076637, respectively. The cost of this publication was paid in part with FEDER funds.

Summary The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity., Graphical Abstract, Highlights • RPAP1 is an RNA Pol II regulator, conserved from plants to mammals • RPAP1 depletion erases cell identity gene expression, triggering de-differentiation • Mechanistically, RPAP1 is critical for the Mediator-RNA Pol II interaction • RPAP1 preferentially contributes to enhancer-driven gene transcription, Lynch et al. report a regulator of RNA Pol II called RPAP1, displaying functional conservation from plants to mammals. RPAP1 is required to establish and maintain cell identity. Mechanistically, RPAP1 is critical for the Mediator-RNA Pol II interaction, thereby preserving normal transcription at enhancer-driven genes.

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging. We thank María Otero for expert assistance with histological analy-sis. We are indebted to Konrad Hochedlinger for Sox2‐TK ES cells. J.M.V. is a Xunta de Galicia predoctoral fellow. A.F. is an FPU pre- doctoral fellow from MECD. M.C. is a "Miguel Servet II" investigator (CPII16/00015) Sí

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells.

Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated in vivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication. We thank all members of the DNA Replication and Chromosome Dynamics Groups for discussions, S. Ruiz and I. Blanco for their help with mouse work, B. Urcelay for technical support, M. Udriste for assistance with IHC quantifications, U. Cronin and D. Martınezfor assistance with flow cytometryexperiments, M. Canamero for the initial histopathology analyses, and O. Fernandez-Capetillo for comments on the manuscript. Research was supported by MINECO grants BFU2013-49153-P, BFU2016-80402-R, and CSD2007- 00015 to J.M. and MINECO predoctoral fellowships to S.M. and S.B Sí

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis., M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (project SAF2014-56868-R), the Asociación Española Contra el Cáncer (AECC), the Worldwide Cancer Research, an Established Investigator Award from the Melanoma Research Alliance (MRA), and a L’Oréal Paris USA-MRA Team Science Award for Woman in Scientific Research. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P and P.O.-R are funded by grants FIS 2014/1737, 11/02568 and FIS 2014/01784, 11/1759, respectively, from the Spanish Ministry of Health. F.M. is funded by the AMIT Project/CDTI/CENIT Programme (MICINN), S.O. by SAF2013-44866-R (MINECO), and J.J.B.-C. by an NCI K22CA196750 grant and the TCI Young Scientist Cancer Research Award JJR Fund (P30 CA196521). J.D.M. is the recipient of a postdoctoral fellowship from the ARC Foundation and E.R.-F. from Fundación Científica de la Asociación Española Contra el Cáncer. D.C.-W. is the recipient of a predoctoral fellowship from Fundación La Caixa, and M.C.-A. and X.C. are recipients of the Immutrain Marie Skłodowska-Curie ITN Grant.

Summary The NAD-dependent deacetylase SIRT1 is involved in chromatin silencing and genome stability. Elevated SIRT1 levels in embryonic stem cells also suggest a role for SIRT1 in pluripotency. Murine SIRT1 attenuates telomere attrition in vivo and is recruited at telomeres in induced pluripotent stem cells (iPSCs). Because telomere elongation is an iPSC hallmark, we set out to study the role of SIRT1 in pluripotency in the setting of murine embryonic fibroblasts reprogramming into iPSCs. We find that SIRT1 is required for efficient postreprogramming telomere elongation, and that this effect is mediated by a c-MYC-dependent regulation of the mTert gene. We further demonstrate that SIRT1-deficient iPSCs accumulate chromosomal aberrations and show a derepression of telomeric heterochromatin. Finally, SIRT1-deficient iPSCs form larger teratomas that are poorly differentiated, highlighting a role for SIRT1 in exit from pluripotency. In summary, this work demonstrates a role for SIRT1 in the maintenance of pluripotency and modulation of differentiation., Highlights • SIRT1 is required for mTERT-dependent telomere elongation after reprogramming • Sirt1−/− xenografts are larger than wild-type and display delayed differentiation • SIRT1 regulates TERRA transcription and contributes to maintain genomic stability • SIRT1 deficiency impairs telomeric chromatin remodeling upon reprogramming, SIRT1 is a multifunctional protein that also plays a role in pluripotency and telomere dynamics. Blasco and colleagues demonstrate that SIRT1, although not directly involved in the reprogramming of somatic cells, is required for telomere elongation taking place after reprogramming. Moreover, SIRT1 maintains genomic stability upon long-term cell culture and modulates the differentiation potential of iPSCs.

Although lymph node transplantation has been shown to improve lymphatic function, the mechanisms regulating lymphatic vessel reconnection and functional status of lymph nodes remains poorly understood.The authors developed and used LacZ lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred lymph nodes. In addition, the authors analyzed lymphatic function, expression of vascular endothelial growth factor (VEGF)-C, maintenance of T- and B-cell zone, and anatomical localization of lymphatics and high endothelial venules.Reporter mice were specific and highly sensitive in identifying lymphatic vessels. Lymph node transfer was associated with rapid return of lymphatic function and clearance of technetium-99 secondary to a massive infiltration of recipient mouse lymphatics and putative connections to donor lymphatics. T- and B-cell populations in the lymph node were maintained. These changes correlated with marked increases in the expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomical proximity to high endothelial venules.Transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and maintain T- and B-cell populations. This process correlates with increased endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Anatomical proximity of newly formed lymphatics and high endothelial venules supports the hypothesis that lymph node transfer can improve lymphedema by exchanges with the systemic circulation.