Arid1a Loss Enhances Disease Progression in a Murine Model of Osteosarcoma.

Osteosarcoma is an aggressive bone malignancy, molecularly characterized by acquired genome complexity and frequent loss of TP53 and RB1. Obtaining a molecular understanding of the initiating mutations of osteosarcomagenesis has been challenged by the difficulty of parsing between passenger and driver mutations in genes. Here, a forward genetic screen in a genetic mouse model of osteosarcomagenesis initiated by Trp53 and Rb1 conditional loss in pre-osteoblasts identified that Arid1a loss contributes to OS progression. Arid1a is a member of the canonical BAF (SWI/SNF) complex and a known tumor suppressor gene in other cancers. We hypothesized that the loss of Arid1a increases the rate of tumor progression and metastasis. Phenotypic evaluation upon in vitro and in vivo deletion of Arid1a validated this hypothesis. Gene expression and pathway analysis revealed a correlation between Arid1a loss and genomic instability, and the subsequent dysregulation of genes involved in DNA DSB or SSB repair pathways. The most significant of these transcriptional changes was a concomitant decrease in DCLRE1C. Our findings suggest that Arid1a plays a role in genomic instability in aggressive osteosarcoma and a better understanding of this correlation can help with clinical prognoses and personalized patient care.

Optogenetic stimulation of mouse Hoxb8 microglia in specific regions of the brain induces anxiety, grooming, or both.

Previously, we have shown that either disruption of the Hoxb8 gene or ablation of a microglial subpopulation, Hoxb8 microglia, results in mice exhibiting both chronic anxiety and OCSD-like behavior, compulsive pathological hair pulling (trichotillomania), to the point of showing lesions at the sites of overgrooming. Herein we show, that optogenetic stimulation of Hoxb8 microglia in specific regions of the brain induces elevated anxiety, grooming or both. Optogenetic stimulation of Hoxb8 microglia within the dorsomedial striatum (DMS) or the medial prefrontal cortex (mPFC) induces grooming, whereas stimulation of Hoxb8 microglia in the basolateral amygdala (BLA) or central amygdala (CeA) produces elevated anxiety. Optogenetic stimulation of Hoxb8 microglia in the ventral CA1 region of the hippocampus (vCA1) induces both behaviors as well as freezing. In vitro we directly demonstrate that optogenetic stimulation of Hoxb8 microglia in specific regions of the brain activate neighboring neural activity through the induction of the c-fos-immediate early response. These experiments connect outputs from optogenetically stimulated Hoxb8 microglia, within specific regions of the brain, to the activation of neurons and neural circuits that in turn enable induction of these behaviors. These experiments suggest that Hoxb8 microglia are likely to be among, or the main, first responders to signals that evoke these behaviors. The same regions of the brain (DMS, mPFC, BLA, CeA and vCA1) have previously been defined at the neuronal level, by optogenetics, to control anxiety in mice. Intriguingly, the optogenetic experiments in microglia suggest that the two populations of microglia, canonical non-Hoxb8 and Hoxb8 microglia, function in opposition rather than in parallel to each other, providing a biological reason for the presence of two microglial subpopulations in mice.

Lrig1 expression identifies quiescent stem cells in the ventricular-subventricular zone from postnatal development to adulthood and limits their persistent hyperproliferation.

BACKGROUND: We previously identified Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) as a marker of long-term neurogenic stem cells in the lateral wall of the adult mouse brain. The morphology of the stem cells thus identified differed from the canonical B1 type stem cells, raising a question about their cellular origin. Thus, we investigated the development of these stem cells in the postnatal and juvenile brain. Furthermore, because Lrig1 is a known regulator of quiescence, we also investigated the effect(s) of its deletion on the cellular proliferation in the lateral wall. METHODS: To observe the development of the Lrig1-lineage stem cells, genetic inducible fate mapping studies in combination with thymidine analog administration were conducted using a previously published Lrig1T2A-iCreERT2 mouse line. To identify the long-term consequence(s) of Lrig1 germline deletion, old Lrig1 knock-out mice were generated using two different Lrig1 null alleles in the C57BL/6J background. The lateral walls from these mice were analyzed using an optimized whole mount immunofluorescence protocol and confocal microscopy. RESULTS: We observed the Lrig1-lineage labeled cells with morphologies consistent with neurogenic stem cell identity in postnatal, juvenile, and adult mouse brains. Interestingly, when induced at postnatal or juvenile ages, morphologically distinct cells were revealed, including cells with the canonical B1 type stem cell morphology. Almost all of the presumptive stem cells labeled were non-proliferative at these ages. In the old Lrig1 germline knock-out mice, increased proliferation was observed compared to wildtype littermates without concomitant increase in apoptosis. CONCLUSIONS: Once set aside during embryogenesis, the Lrig1-lineage stem cells remain largely quiescent during postnatal and juvenile development until activation in adult age. The absence of premature proliferative exhaustion in the Lrig1 knock-out stem cell niche during aging is likely due to a complex cascade of effects on the adult stem cell pool. Thus, we suggest that the adult stem cell pool size may be genetically constrained via Lrig1.

Defining the Hoxb8 cell lineage during murine definitive hematopoiesis.

Previously, we have demonstrated that a subpopulation of microglia, known as Hoxb8 microglia, is derived from the Hoxb8 lineage during the second wave (E8.5) of yolk sac hematopoiesis, whereas canonical non-Hoxb8 microglia arise from the first wave (E7.5). Hoxb8 microglia have an ontogeny distinct from non-Hoxb8 microglia. Dysfunctional Hoxb8 microglia cause the acquisition of chronic anxiety and an obsessive-compulsive spectrum-like behavior, trichotillomania, in mice. The nature and fate of the progenitors generated during E8.5 yolk sac hematopoiesis have been controversial. Herein, we use the Hoxb8 cell lineage reporter to define the ontogeny of hematopoietic cells arising during the definitive waves of hematopoiesis initiated in the E8.5 yolk sac and aorta-gonad-mesonephros (AGM) region. Our murine cell lineage analysis shows that the Hoxb8 cell lineage reporter robustly marks erythromyeloid progenitors, hematopoietic stem cells and their progeny, particularly monocytes. Hoxb8 progenitors and microglia require Myb function, a hallmark transcription factor for definitive hematopoiesis, for propagation and maturation. During adulthood, all immune lineages and, interestingly, resident macrophages in only hematopoietic/lymphoid tissues are derived from Hoxb8 precursors. These results illustrate that the Hoxb8 lineage exclusively mirrors murine definitive hematopoiesis.

The clear cell sarcoma functional genomic landscape.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control.

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors.

Lrig1 expression prospectively identifies stem cells in the ventricular-subventricular zone that are neurogenic throughout adult life.

BACKGROUND: Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) regulates stem cell quiescence. As a marker, it identifies stem cells in multiple organs of the mouse. We had detected Lrig1 expression in cultured Id1high neural stem cells obtained from the lateral walls lining the lateral ventricles of the adult mouse brain. Thus, we investigated whether Lrig1 expression also identifies stem cells in that region in vivo. METHODS: Publicly available single cell RNA sequencing datasets were analyzed with Seurat and Monocle. The Lrig1+ cells were lineage traced in vivo with a novel non-disruptive co-translational Lrig1T2A-iCreERT2 reporter mouse line. RESULTS: Analysis of single cell RNA sequencing datasets suggested Lrig1 was highly expressed in the most primitive stem cells of the neurogenic lineage in the lateral wall of the adult mouse brain. In support of their neurogenic stem cell identity, cell cycle entry was only observed in two morphologically distinguishable Lrig1+ cells that could also be induced into activation by Ara-C infusion. The Lrig1+ neurogenic stem cells were observed throughout the lateral wall. Neuroblasts and neurons were lineage traced from Lrig1+ neurogenic stem cells at 1 year after labeling. CONCLUSIONS: We identified Lrig1 as a marker of long-term neurogenic stem cells in the lateral wall of the mouse brain. Lrig1 expression revealed two morphotypes of the Lrig1+ cells that function as long-term neurogenic stem cells. The spatial distribution of the Lrig1+ neurogenic stem cells suggested all subtypes of the adult neurogenic stem cells were labeled.

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression.

Histone deacetylases (HDACs) are promising targets for cancer therapy, although their individual actions remain incompletely understood. Here, we identify a role for HDAC2 in the regulation of MDM2 acetylation at previously uncharacterized lysines. Upon inactivation of HDAC2, this acetylation creates a structural signal in the lysine-rich domain of MDM2 to prevent the recognition and degradation of its downstream substrate, MCL-1 ubiquitin ligase E3 (MULE). This mechanism further reveals a therapeutic connection between the MULE ubiquitin ligase function and tumor suppression. Specifically, we show that HDAC inhibitor treatment promotes the accumulation of MULE, which diminishes the t(X; 18) translocation-associated synovial sarcomagenesis by directly targeting the fusion product SS18-SSX for degradation. These results uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response.

Silencing of retrotransposon-derived imprinted gene RTL1 is the main cause for postimplantational failures in mammalian cloning.

Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.

Two distinct ontogenies confer heterogeneity to mouse brain microglia.

Hoxb8 mutant mice show compulsive behavior similar to trichotillomania, a human obsessive-compulsive-spectrum disorder. The only Hoxb8 lineage-labeled cells in the brains of mice are microglia, suggesting that defective Hoxb8 microglia caused the disorder. What is the source of the Hoxb8 microglia? It has been posited that all microglia progenitors arise at embryonic day (E) 7.5 during yolk sac hematopoiesis, and colonize the brain at E9.5. In contrast, we show the presence of two microglia subpopulations: canonical, non-Hoxb8 microglia and Hoxb8 microglia. Unlike non-Hoxb8 microglia, Hoxb8 microglia progenitors appear to be generated during the second wave of yolk sac hematopoiesis, then detected in the aorto-gonad-mesonephros (AGM) and fetal liver, where they are greatly expanded, prior to infiltrating the E12.5 brain. Further, we demonstrate that Hoxb8 hematopoietic progenitor cells taken from fetal liver are competent to give rise to microglia in vivo Although the two microglial subpopulations are very similar molecularly, and in their response to brain injury and participation in synaptic pruning, they show distinct brain distributions which might contribute to pathological specificity. Non-Hoxb8 microglia significantly outnumber Hoxb8 microglia, but they cannot compensate for the loss of Hoxb8 function in Hoxb8 microglia, suggesting further crucial differences between the two subpopulations.

The SS18-SSX Oncoprotein Hijacks KDM2B-PRC1.1 to Drive Synovial Sarcoma.

Synovial sarcoma is an aggressive cancer invariably associated with a chromosomal translocation involving genes encoding the SWI-SNF complex component SS18 and an SSX (SSX1 or SSX2) transcriptional repressor. Using functional genomics, we identify KDM2B, a histone demethylase and component of a non-canonical polycomb repressive complex 1 (PRC1.1), as selectively required for sustaining synovial sarcoma cell transformation. SS18-SSX1 physically interacts with PRC1.1 and co-associates with SWI/SNF and KDM2B complexes on unmethylated CpG islands. Via KDM2B, SS18-SSX1 binds and aberrantly activates expression of developmentally regulated genes otherwise targets of polycomb-mediated repression, which is restored upon KDM2B depletion, leading to irreversible mesenchymal differentiation. Thus, SS18-SSX1 deregulates developmental programs to drive transformation by hijacking a transcriptional repressive complex to aberrantly activate gene expression.

Paracrine osteoprotegerin and ß-catenin stabilization support synovial sarcomagenesis in periosteal cells.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5-expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of ß-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2-transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX-driven oncogenesis in cells as differentiated as preosteoblasts.

The Influential Role of BCL2 Family Members in Synovial Sarcomagenesis.

Synovial sarcomas are deadly soft tissue malignancies associated with t(X;18) balanced chromosomal translocations. Expression of the apoptotic regulator BCL2 is prominent in synovial sarcomas and has prompted the hypothesis that synovial sarcomagenesis may depend on it. Herein, it is demonstrated that Bcl2 overexpression enhances synovial sarcomagenesis in an animal model. Furthermore, we determined increased familial clustering of human synovial sarcoma patients with victims of other BCL2-associated malignancies in the Utah Population Database. Conditional genetic disruption of Bcl2 in mice also led to reduced sarcomagenesis. Pharmacologic inhibition specific to BCL2 had no demonstrable efficacy against human synovial sarcoma cell lines or mouse tumors. However, targeting BCLxL in human and mouse synovial sarcoma with the small molecule BH3 domain inhibitor, BXI-72, achieved significant cytoreduction and increased apoptotic signaling. Thus, the contributory role of BCL2 in synovial sarcomagenesis does not appear to render it as a therapeutic target, but mitochondrial antiapoptotic BCL2 family members may be.Implications: The association of BCL2 expression with synovial sarcoma is found to fit with a subtle, but significant, impact of its enhanced presence or absence during early tumorigenesis. However, specific pharmacologic inhibition of BCL2 does not demonstrate a persistent dependence in fully developed tumors. Conversely, inhibition of the BCL2 family member BCLxL resulted in nanomolar potency against human synovial sarcoma cell lines and 50% tumor reduction in a genetically engineered mouse model. Mol Cancer Res; 15(12); 1733-40. ©2017 AACR.

Mouse fitness measures reveal incomplete functional redundancy of Hox paralogous group 1 proteins.

Here we assess the fitness consequences of the replacement of the Hoxa1 coding region with its paralog Hoxb1 in mice (Mus musculus) residing in semi-natural enclosures. Previously, this Hoxa1B1 swap was reported as resulting in no discernible embryonic or physiological phenotype (i.e., functionally redundant), despite the 51% amino acid sequence differences between these two Hox proteins. Within heterozygous breeding cages no differences in litter size nor deviations from Mendelian genotypic expectations were observed in the outbred progeny; however, within semi-natural population enclosures mice homozygous for the Hoxa1B1 swap were out-reproduced by controls resulting in the mutant allele being only 87.5% as frequent as the control in offspring born within enclosures. Specifically, Hoxa1B1 founders produced only 77.9% as many offspring relative to controls, as measured by homozygous pups, and a 22.1% deficiency of heterozygous offspring was also observed. These data suggest that Hoxa1 and Hoxb1 have diverged in function through either sub- or neo-functionalization and that the HoxA1 and HoxB1 proteins are not mutually interchangeable when expressed from the Hoxa1 locus. The fitness assays conducted under naturalistic conditions in this study have provided an ultimate-level assessment of the postulated equivalence of competing alleles. Characterization of these differences has provided greater understanding of the forces shaping the maintenance and diversifications of Hox genes as well as other paralogous genes. This fitness assay approach can be applied to any genetic manipulation and often provides the most sensitive way to detect functional differences.

Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells.

Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. Stem Cells Translational Medicine 2017;6:340-351.