Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

H3K36 dimethylation shapes the epigenetic interaction landscape by directing repressive chromatin modifications in embryonic stem cells.

Epigenetic modifications on the chromatin do not occur in isolation. Chromatin-associated proteins and their modification products form a highly interconnected network, and disturbing one component may rearrange the entire system. We see this increasingly clearly in epigenetically dysregulated cancers. It is important to understand the rules governing epigenetic interactions. Here, we use the mouse embryonic stem cell (mESC) model to describe in detail the relationships within the H3K27-H3K36-DNA methylation subnetwork. In particular, we focus on the major epigenetic reorganization caused by deletion of the histone 3 lysine 36 methyltransferase NSD1, which in mESCs deposits nearly all of the intergenic H3K36me2. Although disturbing the H3K27 and DNA methylation (DNAme) components also affects this network to a certain extent, the removal of H3K36me2 has the most drastic effect on the epigenetic landscape, resulting in full intergenic spread of H3K27me3 and a substantial decrease in DNAme. By profiling DNMT3A and CHH methylation (mCHH), we show that H3K36me2 loss upon Nsd1-KO leads to a massive redistribution of DNMT3A and mCHH away from intergenic regions and toward active gene bodies, suggesting that DNAme reduction is at least in part caused by redistribution of de novo methylation. Additionally, we show that pervasive acetylation of H3K27 is regulated by the interplay of H3K36 and H3K27 methylation. Our analysis highlights the importance of H3K36me2 as a major determinant of the developmental epigenome and provides a framework for further consolidating our knowledge of epigenetic networks.

TETonic shift: biological roles of TET proteins in DNA demethylation and transcription.

In many organisms, the methylation of cytosine in DNA has a key role in silencing 'parasitic' DNA elements, regulating transcription and establishing cellular identity. The recent discovery that ten-eleven translocation (TET) proteins are 5-methylcytosine oxidases has provided several chemically plausible pathways for the reversal of DNA methylation, thus triggering a paradigm shift in our understanding of how changes in DNA methylation are coupled to cell differentiation, embryonic development and cancer.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

Distinctive aspects of the placental epigenome and theories as to how they arise.

The placenta has a methylome dramatically unlike that of any somatic cell type. Among other distinctions, it features low global DNA methylation, extensive "partially methylated domains" packed in dense heterochromatin and methylation of hundreds of CpG islands important in somatic development. These features attract interest in part because a substantial fraction of human cancers feature the exact same phenomena, suggesting parallels between epigenome formation in placentation and cancer. Placenta also features an expanded set of imprinted genes, some of which come about by distinctive developmental pathways. Recent discoveries, some from far outside the placental field, shed new light on how the unusual placental epigenetic state may arise. Nonetheless, key questions remain unresolved.

Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

RAD: a web application to identify region associated differentially expressed genes.

SUMMARY: With the advance of genomic sequencing techniques, chromatin accessible regions, transcription factor binding sites and epigenetic modifications can be identified at genome-wide scale. Conventional analyses focus on the gene regulation at proximal regions; however, distal regions are usually less focused, largely due to the lack of reliable tools to link these regions to coding genes. In this study, we introduce RAD (Region Associated Differentially expressed genes), a user-friendly web tool to identify both proximal and distal region associated differentially expressed genes (DEGs). With DEGs and genomic regions of interest (gROI) as input, RAD maps the up- and down-regulated genes associated with any gROI and helps researchers to infer the regulatory function of these regions based on the distance of gROI to differentially expressed genes. RAD includes visualization of the results and statistical inference for significance. AVAILABILITY AND IMPLEMENTATION: RAD is implemented with Python 3.7 and run on a Nginx server. RAD is freely available at https://labw.org/rad as online web service. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Mutant p53: one name, many proteins.

There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function.

Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX.

TET (ten-eleven-translocation) proteins are Fe(ii)- and α-ketoglutarate-dependent dioxygenases that modify the methylation status of DNA by successively oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, potential intermediates in the active erasure of DNA-methylation marks. Here we show that IDAX (also known as CXXC4), a reported inhibitor of Wnt signalling that has been implicated in malignant renal cell carcinoma and colonic villous adenoma, regulates TET2 protein expression. IDAX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The IDAX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. Unexpectedly, IDAX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the IDAX CXXC domain, suggesting that IDAX recruits TET2 to DNA before degradation. IDAX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against IDAX increases TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 is also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3, respectively, as previously unknown regulators of caspase activation and TET enzymatic activity.

Tet proteins influence the balance between neuroectodermal and mesodermal fate choice by inhibiting Wnt signaling.

TET-family dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and oxidized methylcytosines in DNA. Here, we show that mouse embryonic stem cells (mESCs), either lacking Tet3 alone or with triple deficiency of Tet1/2/3, displayed impaired adoption of neural cell fate and concomitantly skewed toward cardiac mesodermal fate. Conversely, ectopic expression of Tet3 enhanced neural differentiation and limited cardiac mesoderm specification. Genome-wide analyses showed that Tet3 mediates cell-fate decisions by inhibiting Wnt signaling, partly through promoter demethylation and transcriptional activation of the Wnt inhibitor secreted frizzled-related protein 4 (Sfrp4). Tet1/2/3-deficient embryos (embryonic day 8.0-8.5) showed hyperactivated Wnt signaling, as well as aberrant differentiation of bipotent neuromesodermal progenitors (NMPs) into mesoderm at the expense of neuroectoderm. Our data demonstrate a key role for TET proteins in modulating Wnt signaling and establishing the proper balance between neural and mesodermal cell fate determination in mouse embryos and ESCs.

Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin.

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

Arabidopsis AtMORC4 and AtMORC7 Form Nuclear Bodies and Repress a Large Number of Protein-Coding Genes.

The MORC family of GHKL ATPases are an enigmatic class of proteins with diverse chromatin related functions. In Arabidopsis, AtMORC1, AtMORC2, and AtMORC6 act together in heterodimeric complexes to mediate transcriptional silencing of methylated DNA elements. Here, we studied Arabidopsis AtMORC4 and AtMORC7. We found that, in contrast to AtMORC1,2,6, they act to suppress a wide set of non-methylated protein-coding genes that are enriched for those involved in pathogen response. Furthermore, atmorc4 atmorc7 double mutants show a pathogen response phenotype. We found that AtMORC4 and AtMORC7 form homomeric complexes in vivo and are concentrated in discrete nuclear bodies adjacent to chromocenters. Analysis of an atmorc1,2,4,5,6,7 hextuple mutant demonstrates that transcriptional de-repression is largely uncoupled from changes in DNA methylation in plants devoid of MORC function. However, we also uncover a requirement for MORC in both DNA methylation and silencing at a small but distinct subset of RNA-directed DNA methylation target loci. These regions are characterized by poised transcriptional potential and a low density of sites for symmetric cytosine methylation. These results provide insight into the biological function of MORC proteins in higher eukaryotes.

TET proteins and 5-methylcytosine oxidation in hematological cancers.

DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting, and X-chromosome inactivation. Cancer cells display highly dysregulated DNA methylation profiles characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands that presumably lead to genome instability and aberrant expression of tumor suppressor genes or oncogenes. The recent discovery of ten-eleven-translocation (TET) family dioxygenases that oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in DNA has led to profound progress in understanding the mechanism underlying DNA demethylation. Among the three TET genes, TET2 recurrently undergoes inactivating mutations in a wide range of myeloid and lymphoid malignancies. TET2 functions as a bona fide tumor suppressor particularly in the pathogenesis of myeloid malignancies resembling chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) in human. Here we review diverse functions of TET proteins and the novel epigenetic marks that they generate in DNA methylation/demethylation dynamics and normal and malignant hematopoietic differentiation. The impact of TET2 inactivation in hematopoiesis and various mechanisms modulating the expression or activity of TET proteins are also discussed. Furthermore, we also present evidence that TET2 and TET3 collaborate to suppress aberrant hematopoiesis and hematopoietic transformation. A detailed understanding of the normal and pathological functions of TET proteins may provide new avenues to develop novel epigenetic therapies for treating hematological malignancies.