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Figure S1. Protein sequence alignment of HPyV6, HPyV7 and MCPyV small T antigens. Figure S2. Expression of HPyV sT induces p53-dependent senescence in human fibroblasts. Figure S3. Proliferation of BJ fibroblasts with expression of HPyV sT. Figure S4. Analysis of RNA-Seq from HPyV6, HPyV7, and MCPyV sT expressing BJ fibroblasts. Figure S5. Time course and extended analysis of additional SASP gene expression in BJ cells after sT expression. Figure S6. MCPyV sT shRNA confirms role of sT in SASP gene expression. Figure S7. Impact of LSD motif of MCPyV sT on gene expression and proliferation. Figure S8. MCPyV st, but not HPyV6/7 sT, stabilizes and activates c-Myc in an LSD motif dependent manner. Figure S9. Chromatin remodeling signatures induced by MCPyV sT. Figure S10. Mechanism of ncNF-κB and SASP induction by MCPyV sT. Figure S11. ncNF-κB signaling is required for SASP and EZH2 expression and affects cell proliferation. Figure S12. PyV sT conditioned media is not sufficient for growth in low serum. Figure S13. ST expression in VP-MCC lines and tumors. Figure S14. Impact of ncNF-κB inhibition on VP-MCC and VN-MCC. Figure S15. Summary Figure.

Primers used for cloning and qRT-pCR

Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB signaling prevented VP-MCC cell growth in vitro and in xenografts. We identify MCPyV sT–induced ncNF-κB signaling as an essential tumorigenic pathway in MCC.Implications:This work is the first to identify the activation of ncNF-κB signaling by any polyomavirus and its critical role in MCC tumorigenesis.

Somatic mutations in non-malignant tissues accumulate with age and insult, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate mutations found in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to non-alcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss ofMboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side-by-side. Thisin vivotracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion ofBcl6, Tbx3,orSmyd2resulted in protection against NASH. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.Highlights:MosaicMboat7mutations that increase lipotoxicity lead to clonal disappearance in NASH.In vivo screening can identify genes that alter hepatocyte fitness in NASH.MosaicGpammutations are positively selected due to reduced lipogenesis.In vivo screening of transcription factors and epifactors identified new therapeutic targets in NASH.

SummaryHematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency, but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illuminating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.

Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB signaling prevented VP-MCC cell growth in vitro and in xenografts. We identify MCPyV sT–induced ncNF-κB signaling as an essential tumorigenic pathway in MCC. Implications: This work is the first to identify the activation of ncNF-κB signaling by any polyomavirus and its critical role in MCC tumorigenesis.

The dynamic regulation of β-cell abundance is poorly understood. Since chromatin remodeling plays critical roles in liver regeneration, these mechanisms could be generally important for regeneration in other tissues. Here, we show that the ARID1A mammalian SWI/SNF complex subunit is a critical regulator of β-cell regeneration. Arid1a is highly expressed in quiescent β-cells but is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice are protected against streptozotocin-induced diabetes. Cell-type and temporally specific genetic dissection show that β-cell-specific Arid1a deletion can potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets reveal increased neuregulin-ERBB-NR4A signaling. Chemical inhibition of ERBB or NR4A1 blocks increased regeneration associated with Arid1a loss. Mammalian SWI/SNF (mSWI/SNF) complex activity is a barrier to β-cell regeneration in physiologic and disease states.

SWI/SNF chromatin remodelers play critical roles in development and cancer. The causal links between SWI/SNF complex disassembly and carcinogenesis are obscured by redundancy between paralogous components. Canonical cBAF-specific paralogs ARID1A and ARID1B are synthetic lethal in some contexts, but simultaneous mutations in both ARID1s are prevalent in cancer. To understand if and how cBAF abrogation causes cancer, we examined the physiologic and biochemical consequences of ARID1A/ARID1B loss. In double knockout liver and skin, aggressive carcinogenesis followed de-differentiation and hyperproliferation. In double mutant endometrial cancer, add-back of either induced senescence. Biochemically, residual cBAF subcomplexes resulting from loss of ARID1 scaffolding were unexpectedly found to disrupt polybromo containing pBAF function. 37 of 69 mutations in the conserved scaffolding domains of ARID1 proteins observed in human cancer caused complex disassembly, partially explaining their mutation spectra. ARID1-less, cBAF-less states promote carcinogenesis across tissues, and suggest caution against paralog-directed therapies for ARID1-mutant cancer.

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors1–6. LEPR+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors7–12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin13,14, distinguishes peri-arteriolar LEPR+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR+osteolectin+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin+ cells depleted osteolectin+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing. A peri-arteriolar niche in the bone marrow for osteogenesis and lymphopoiesis is maintained by mechanical stimulation and is depleted during ageing.

SummaryThe dynamic regulation of β-cell abundance is poorly understood. Since chromatin remodeling plays critical roles in liver regeneration, these mechanisms could be generally important for regeneration in other tissues. Here we show that the ARID1A mammalian SWI/SNF complex subunit is a critical regulator of β-cell regeneration. Arid1a is highly expressed in quiescent β-cells but is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice were protected against streptozotocin induced diabetes. Cell-type and temporally specific genetic dissection showed that β-cell specific Arid1a deletion could potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets revealed increased Neuregulin-ERBB-NR4A signaling. Functionally, ERBB3 overexpression in β-cells was sufficient to protect against diabetes, and chemical inhibition of ERBB or NR4A was able to block increased regeneration associated with Arid1a loss. mSWI/SNF complex activity is a barrier to β-cell regeneration in physiologic and disease states.

INTRODUCTION: The liver’s remarkable capacity to maintain proper tissue mass after injury has been known since ancient times. However, there has been considerable debate about the source of new liver cells that contribute to tissue growth, maintenance, and regeneration. Multiple studies have reported that disparate cell populations in the liver serve as rare stem cells, whereas others have proposed that most hepatocytes are similar in their regenerative activity regardless of position or function. Although hepatocytes appear histologically homogeneous, the liver lobule is actually organized into concentric zones, or rings, in which hepatocytes express different metabolic enzymes across the portal vein–to–central vein axis through which blood flows. Recently, single-cell profiling has enriched our understanding of the extraordinary diversity of hepatocytes, but this “zonal” heterogeneity has not been functionally interrogated in the context of tissue homeostasis, because the critical genetic labeling tools have not been available. RATIONALE: Previous efforts to identify the most-regenerative hepatocytes have not definitively resolved fundamental questions about whether regenerative activity is spatially restricted within particular zones or whether rare or common subsets of hepatocytes are responsible. This uncertainty was in part because fate mapping had only been performed on a few hepatocyte subsets and without side-by-side comparisons. We sought to systematically address fundamental questions about the source of new liver cells by generating a panel of 11 new CreER knock-in mouse models that label zonal subpopulations across the liver lobule. By using these tools in tandem with three existing CreER lines, tissue maintenance and regeneration as a function of zonal position were assessed. RESULTS: In contrast to the idea that all hepatocytes across the lobule contribute equally to regeneration, we identified major differences between hepatocytes from different locations. During steady-state homeostasis, zone 1 cells near the portal vein decreased in number over time, as did zone 3 cells near the central vein on the opposite end of the lobule. However, midlobular zone 2 hepatocytes marked by the hepcidin antimicrobial peptide 2 (Hamp2) gene were in large part responsible for homeostatic repopulation. Zone 2 cells were also shelteredfrom toxic injuries affecting either end of the lobule and thus were well positioned to contribute to regeneration after these insults. To define the mechanistic basis of these lineage-tracing results, single-cell and bulk RNA sequencing transcriptomics were used to define genes that were specifically up- or down-regulated in zone 2. We then used in vivo CRISPR knock-out and activation screening to identify functionally important pathways that regulate zone 2 proliferation. These methods revealed that zone 2 repopulation is driven by theinsulin-like growth factor binding protein 2–mechanistic target of rapamycin–cyclin D1 (IGFBP2-mTOR-CCND1) axis. CONCLUSION: Different regions of the liver lobule exhibit differences in their contributions to hepatocyte turnover, and zone 2 is an important source of new hepatocytes during homeostasis and regeneration. These results challenge the idea that stem cells near the portal or central veins have the highest rates of liver repopulation, but they also support the principle that there are important zonal differences in hepatocyte biology. This study reconciles findings from multiple groups and offers a more unified view of hepatocyte repopulation. The identification of zone 2 hepatocytes as a regenerative population has far-reaching implications for the cellular basis of chronic disease pathogenesis, cancer development, and regenerative medicine strategies.

Identifying new pathways that regulate mammalian regeneration is challenging due to the paucity of in vivo screening approaches. We employed pooled CRISPR knockout and activation screening in the regenerating liver to evaluate 165 chromatin regulatory proteins. Both screens identified the imitation-SWI chromatin remodeling components Baz2a and Baz2b, not previously implicated in regeneration. In vivo sgRNA, siRNA, and knockout strategies against either paralog confirmed increased regeneration. Distinct BAZ2-specific bromodomain inhibitors, GSK2801 and BAZ2-ICR, resulted in accelerated liver healing after diverse injuries. Inhibitor-treated mice also exhibited improved healing in an inflammatory bowel disease model, suggesting multi-tissue applicability. Transcriptomics on regenerating livers showed increases in ribosomal and cell cycle mRNAs. Surprisingly, CRISPRa screening to define mechanisms showed that overproducing Rpl10a or Rpl24 was sufficient to drive regeneration, whereas Rpl24 haploinsufficiency was rate limiting for BAZ2 inhibition-mediated regeneration. The discovery of regenerative roles for imitation-SWI components provides immediate strategies to enhance tissue repair.

Despite considerable advances in defining the somatic driver mutations underlying myeloid malignancies, including the myeloproliferative neoplasms (MPNs), a significant heritable component for these diseases remains poorly understood. While common genetic variant association studies have been valuable, they fail to explain the majority of heritable variation. We reasoned that rare variant association studies could provide a valuable complementary approach to identify additional inherited risk factors. We therefore utilized exome sequencing data from 166,953 UK Biobank participants and performed a gene-based burden analysis for germline genetic variants conferring risk for acquiring a myeloid malignancy. CTR9, which encodes a key component of the PAF1 transcription elongation complex, was among the significant genes identified (SKAT-O p-value = 5.47x10 -7). The deleterious variants in CTR9 collectively exhibit a 9.6 (95%CI = 4.86-19.04) increased odds of acquiring a myeloid malignancy and this risk was largely driven by the MPNs. We replicated this association in an independent cohort of 211 MPN patients using external controls. We could show through structural and biochemical analyses that the identified deleterious variants perturbed assembly of the PAF1 complex but did not display dominant negative activity. Given that increased hematopoietic stem cell (HSC) self-renewal has been shown to predispose to the risk of acquiring MPNs, we sought to define whether CTR9 perturbation could alter HSC self-renewal or function. We achieved predominantly heterozygous loss-of-function in human hematopoietic stem and progenitor cells (HSPCs) by titrating Cas9 ribonucleoprotein delivery with several independent guide RNAs. Partial loss of CTR9 in HSPCs resulted in expansion of phenotypic long-term HSCs (LT-HSCs) and more differentiated short-term HSCs (ST-HSCs). We additionally could show through single cell RNA-sequencing (scRNA-seq) that there was an expansion of molecularly defined HSCs upon partial loss of CTR9. The observed increase in HSCs appeared paradoxical, given that the PAF1 complex has been suggested to be crucial for HSC maintenance. To explore how the observed HSC expansion with CTR9 perturbation may arise, as well as given known interactions between the PAF1 complex and the competing transcriptional super elongation complex (SEC), we examined whether SEC target genes in HSCs, such as mid to posterior HOXA genes, may be activated with partial CTR9 loss. Remarkably, we observed a significant enrichment for hematopoietic SEC target genes upon CTR9 perturbation in HSCs by gene set enrichment analysis (normalized enrichment score = 3.29, p-value < 0.001). In light of these findings suggesting that SEC activity may be increased with partial CTR9 loss-of-function, as occurs in individuals harboring myeloid malignancy variants, we sought to functionally validate these observations. Using the inhibitors of the SEC, including SR-0813 that targets MLLT3 or with an inhibitor of CDK9, we noted rescue of the CTR9-mediated expansion of phenotypic LT- and ST-HSCs without a significant impact on the bulk HSPC population. To further elucidate underlying mechanisms, we performed immunoprecipitation of PAF1 or SEC component MLLT3 in HSPCs with control or CTR9 editing. While we continued to pull down all PAF1 complex components with PAF1, we also noted pulldown of MLLT3, which increased with CTR9 editing. MLLT3 immunoprecipitation revealed selective pulldowns of PAF1 and CDC73, which also increased with CTR9 editing. These findings show how PAF1 complex components PAF1 and CDC73 interact with and stimulate SEC activity. Our findings reveal how CTR9 usually restricts this activity and constrains transcriptional elongation to limit HSC self-renewal. We functionally validated these findings through selective editing of different PAF1 complex components in HSPCs: we observed reduced HSCs upon editing of PAF1 and CDC73, but increases with editing of other PAF1 complex components. Our findings collectively demonstrate a mechanism by which a previously undefined myeloid malignancy predisposition occurs. We demonstrate that CTR9 loss-of-function stimulates SEC activity and thereby results in HSC expansion to confer risk for acquiring MPNs and other myeloid malignancies. Disclosures Armstrong: Neomorph Inc: Consultancy, Current holder of individual stocks in a privately-held company; Imago Biosciences: Consultancy; Vitae/Allergan Pharma: Consultancy; Cyteir Therapeutics: Consultancy; C4 Therapeutics: Consultancy; OxStem Oncology: Consultancy; Accent Therapeutics: Consultancy; Mana Therapeutics: Consultancy; Janssen: Research Funding; Novartis: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Sankaran: Ensoma: Consultancy; Forma: Consultancy; Cellarity: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy.

BACKGROUND & AIMS: Thirty percent to 90% of hepatocytes contain whole-genome duplications, but little is known about the fates or functions of these polyploid cells or how they affect development of liver disease. We investigated the effects of continuous proliferative pressure, observed in chronically damaged liver tissues, on polyploid cells. METHODS: We studied Rosa-rtTa mice (controls) and Rosa-rtTa;TRE-short hairpin RNA mice, which have reversible knockdown of anillin, actin binding protein (ANLN). Transient administration of doxycycline increases the frequency and degree of hepatocyte polyploidy without permanently altering levels of ANLN. Mice were then given diethylnitrosamine and carbon tetrachloride (CCl(4)) to induce mutations, chronic liver damage, and carcinogenesis. We performed partial hepatectomies to test liver regeneration and then RNA-sequencing analyses to identify changes in gene expression. Lineage tracing was used to rule out repopulation from non-hepatocyte sources. We imaged dividing hepatocytes to estimate the frequency of mitotic errors during regeneration. We also performed whole-exome sequencing of 54 liver nodules from patients with cirrhosis to quantify aneuploidy, a possible outcome of polyploid cell divisions. RESULTS: Liver tissues from control mice given CCl(4) had significant increases in ploidy compared with livers from uninjured mice. Mice with knockdown of ANLN had hepatocyte ploidy above physiologic levels and developed significantly fewer liver tumors after administration of diethylnitrosamine and CCl(4) compared with control mice. Increased hepatocyte polyploidy was not associated with altered regenerative capacity or tissue fitness, changes in gene expression, or more mitotic errors. Based on lineage-tracing experiments, non-hepatocytes did not contribute to liver regeneration in mice with increased polyploidy. Despite an equivalent rate of mitosis in hepatocytes of differing ploidies, we found no lagging chromosomes or micronuclei in mitotic polyploid cells. In nodules of human cirrhotic liver tissue, there was no evidence of chromosome-level copy number variations. CONCLUSIONS: Mice with increased polyploid hepatocytes develop fewer liver tumors following chronic liver damage. Remarkably, polyploid hepatocytes maintain the ability to regenerate liver tissues during chronic damage without generating mitotic errors, and aneuploidy is not commonly observed in cirrhotic livers. Strategies to increase numbers of polypoid hepatocytes might be effective in preventing liver cancer.

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

Gene fusions and their encoded products (fusion RNAs and proteins) are viewed as one of the hallmarks of cancer. Traditionally, they were thought to be generated solely by chromosomal rearrangements. However, recent discoveries of trans-splicing and cis-splicing events between neighboring genes, suggest that there are other mechanisms to generate chimeric fusion RNAs without corresponding changes in DNA. In addition, chimeric RNAs have been detected in normal physiology, complicating the use of fusions in cancer detection and therapy. On the other hand, "intergenically spliced" fusion RNAs represent a new repertoire of biomarkers and therapeutic targets. Here, we review current knowledge on chimeric RNAs and implications for cancer detection and treatment, and discuss outstanding questions for the advancement of the field.

Summary Normal tissues accumulate genetic changes with age, but it is unknown if somatic mutations promote clonal expansion of non-malignant cells in the setting of chronic degenerative diseases. Exome sequencing of diseased liver samples from 82 patients revealed a complex mutational landscape in cirrhosis. Additional ultra-deep sequencing identified recurrent mutations in PKD1, PPARGC1B, KMT2D, and ARID1A. The number and size of mutant clones increased as a function of fibrosis stage and tissue damage. To interrogate the functional impact of mutated genes, a pooled in vivo CRISPR screening approach was established. In agreement with sequencing results, examination of 147 genes again revealed that loss of Pkd1, Kmt2d, and Arid1a promoted clonal expansion. Conditional heterozygous deletion of these genes in mice was also hepatoprotective in injury assays. Pre-malignant somatic alterations are often viewed through the lens of cancer, but we show that mutations can promote regeneration, likely independent of carcinogenesis.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be tumor suppressive. In some hepatocellular carcinoma patients, ARID1A was highly expressed in primary tumors but not in metastatic lesions, suggesting that ARID1A can be lost after initiation. Mice with liver-specific homozygous or heterozygous Arid1a loss were resistant to tumor initiation while ARID1A overexpression accelerated initiation. In contrast, homozygous or heterozygous Arid1a loss in established tumors accelerated progression and metastasis. Mechanistically, gain of Arid1a function promoted initiation by increasing CYP450-mediated oxidative stress, while loss of Arid1a within tumors decreased chromatin accessibility and reduced transcription of genes associated with migration, invasion, and metastasis. In summary, ARID1A has context-dependent tumor-suppressive and oncogenic roles in cancer.

mRNA-mediated protein replacement represents a promising concept for the treatment of liver disorders. Children born with fumarylacetoacetate hydrolase (FAH) mutations suffer from Hepatorenal Tyrosinemia Type 1 (HT-1) resulting in renal dysfunction, liver failure, neurological impairments, and cancer. Protein replacement therapy using FAH mRNA offers tremendous potential to cure HT-1, but is currently hindered by the development of effective mRNA carriers that can function in diseased livers. Structure-guided, rational optimization of 5A2-SC8 mRNA-loaded dendrimer lipid nanoparticles (mDLNPs) increases delivery potency of FAH mRNA, resulting in functional FAH protein and sustained normalization of body weight and liver function in FAH-/- knockout mice. Optimization using luciferase mRNA produces DLNP carriers that are efficacious at mRNA doses as low as 0.05 mg kg-1 in vivo. mDLNPs transfect > 44% of all hepatocytes in the liver, yield high FAH protein levels (0.5 mg kg-1 mRNA), and are well tolerated in a knockout mouse model with compromised liver function. Genetically engineered FAH-/- mice treated with FAH mRNA mDLNPs have statistically equivalent levels of TBIL, ALT, and AST compared to wild type C57BL/6 mice and maintain normal weight throughout the month-long course of treatment. This study provides a framework for the rational optimization of LNPs to improve delivery of mRNA broadly and introduces a specific and viable DLNP carrier with translational potential to treat genetic diseases of the liver.

Gene fusions and their products (RNA and protein) were once thought to be unique features to cancer. However, chimeric RNAs can also be found in normal cells. Here, we performed, curated and analyzed nearly 300 RNA-Seq libraries covering 30 different non-neoplastic human tissues and cells as well as 15 mouse tissues. A large number of fusion transcripts were found. Most fusions were detected only once, while 291 were seen in more than one sample. We focused on the recurrent fusions and performed RNA and protein level validations on a subset. We characterized these fusions based on various features of the fusions, and their parental genes. They tend to be expressed at higher levels relative to their parental genes than the non-recurrent ones. Over half of the recurrent fusions involve neighboring genes transcribing in the same direction. A few sequence motifs were found enriched close to the fusion junction sites. We performed functional analyses on a few widely expressed fusions, and found that silencing them resulted in dramatic reduction in normal cell growth and/or motility. Most chimeras use canonical splicing sites, thus are likely products of 'intergenic splicing'. We also explored the implications of these non-pathological fusions in cancer and in evolution. NCI [CA190713]; American Cancer Society [126405-RSG-14-065-01-RMC] NCI [CA190713]; American Cancer Society [Research Scholar Grant 126405-RSG-14-065-01-RMC to H.L.]; St. Baldrick's V Scholar. Funding for open access charge: NCI [CA190713].