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human Breast tumor Notch gene coverage annotations.

S1: NOTCH gene rearrangement in breast cancer cell lines and human breast tumors. S2: NOTCH gene rearrangement and sensitivity to MRK-003. S3: HES4 expression is associated with NOTCH gene-rearrangement or activating mutation in NOTCH. S4: HES4 expression is a poor prognostic marker in TNBC. S5: Proliferation inhibition by combination therapy with MRK-003 and ERK inhibitor (SCH772984). S6: Effect of MRK-003 treatment on CD44+/CD24 high stem-like cell population. S7: N1ICD level correlates with mutation status and is predictive of MRK-003 response in xenograft models.

File includes genomic analysis, cell cycle and cell proliferation analysis.

Table S1: Analysis of Notch gene rearrangements in triple negative breast cancer subtypes. Table S2: Genes whose expression correlates with Notch gene-rearrangement status in cancer cell lines. Table S3: Genes whose expression correlates with Notch gene-rearrangement in triple negative breast cancer. Table S4. Novel mutations identified in the NRR and PEST domains of NOTCH1 in solid tumors. Table S5: Notch NRR and PEST domain mutation frequency. Table S6: NOTCH1 mutational status, N1-ICD levels, MYC expression in Adenoid Cystic Carcinoma models. Table S7: Primary antibodies. Table S8: RT-PCR Primers.

Supplementary Table 1 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Methods and Materials from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure 1 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure Legends 1-2 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure 2 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR+T cells may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrate that deleting the pseudokinase Trib1 shifts TEXtowards CX3CR1+intermediates (TINT) with robust enrichment of KLR+CD8+T cells (TKLR) via clonal T cell expansion. These changes are associated with globally increased KLR gene expression throughout the exhaustion program. Further, Trib1 loss augments anti-PD-L1 blockade to improve viral clearance by expanding the TKLRpopulation. Together, these data identify Trib1 as an important regulator of T cell exhaustion whose targeting enhances the KLR+effector state and improves the response to checkpoint inhibitor therapy.

Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. Trib1 expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage–specific Trib1 deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G+ eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G+ eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.

Chromatin misfolding has been implicated in cancer pathogenesis; yet, its role in therapy resistance remains unclear. Here, we systematically integrated sequencing and imaging data to examine the spatial and linear chromatin structures in targeted therapy-sensitive and -resistant human T cell acute lymphoblastic leukemia (T-ALL). We found widespread alterations in successive layers of chromatin organization including spatial compartments, contact domain boundaries, and enhancer positioning upon the emergence of targeted therapy resistance. The reorganization of genome folding structures closely coincides with the restructuring of chromatin activity and redistribution of architectural proteins. Mechanistically, the derepression and repositioning of the B-lineage-determining transcription factor EBF1 from the heterochromatic nuclear envelope to the euchromatic interior instructs widespread genome refolding and promotes therapy resistance in leukemic T cells. Together, our findings suggest that lineage-determining transcription factors can instruct changes in genome topology as a driving force for epigenetic adaptations in targeted therapy resistance.

Rome et al. show that Trib1 controls effector versus exhausted T cell differentiation and function by restraining effector programming during persistent infection, revealing a new regulatory axis that could be targeted to recover waning T cell responses during chronic disease., In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity., Graphical Abstract

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer–deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes—gender imbalance and splenic marginal zone B-cell lymphoma—emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer–deficient animals., This study reveals that dimerization of the signaling protein Notch contributes in vivo to intestinal homeostasis. Loss of cooperative DNA binding can manifest as Notch gain- or loss-of-function phenotypes. Importantly, mite infestation exacerbates all phenotypes, and triggers marginal zone B cell hyperproliferation in mutant animals, with implications for the role of dust mite allergies in human IBD and cancer.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence paired sites (SPS) located near many Notch-regulated genes. While most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromising viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% DSS. The most striking phenotypes – gender imbalance and splenic marginal zone B cell lymphoma – emerged in combination with dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis, and is consistent with Notch-dependent anti-parasite immune responses being compromised in the dimer deficient animals.HighlightsNotch dimerization has an in vivo role in contributing to intestinal homeostasisLoss of cooperativity can manifest as Notch gain or loss of function phenotypesMite infestation exacerbates all phenotypes, triggers MZB hyperproliferation in mutant animalsMite-infested mutant mice develop SMZL with age

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

Summary Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC , which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.

Notch receptors influence cellular behavior by participating in a seemingly simple signaling pathway, but outcomes produced by Notch signaling are remarkably varied depending on signal dose and cell context. Here, after briefly reviewing new insights into physiologic mechanisms of Notch signaling in healthy tissues and defects in Notch signaling that contribute to congenital disorders and viral infection, we discuss the varied roles of Notch in cancer, focusing on cell autonomous activities that may be either oncogenic or tumor suppressive.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting homeostatic proliferation of mature lymphocytes. Although this process operates in multiple clinically relevant settings, including after bone marrow transplantation and chemotherapy as well as in aging individuals, little is known about its underlying molecular mechanisms and functional consequences. In mice, mature naïve B cells include mostly follicular B (FoB) cells in spleen and lymph nodes, as well as specialized marginal zone B (MZB) cells in the spleen with innate-like functions and a capacity for rapid plasma cell differentiation. To identify mechanisms controlling the size and composition of the peripheral B cell pool, we developed a mouse model in which highly purified CTV-labelled B6-CD45.1 FoB cells were adoptively transferred intravenously to lymphopenic B6-CD45.2 Rag2-/- mice, lacking mature B and T cells, or control B6-CD45.2 lymphoid-replete recipients. Within two days after transfer of CD19+ CD93neg CD21int CD23high B6-CD45.1 FoB cells (106, >99% purity), CD45.1+ donor-derived B cells had upregulated expression of surface IgM (sIgM) and CD21 in the spleen of Rag2-/- recipients, but not in wild type control recipients. At day 4 and day 8, the majority of transferred B cells remained sIgMhi and CD21hi in Rag2-/- recipients and gradually showed decreased CD23 and sIgD as well as increased CD1d expression, consistent with loss of their FoB phenotype and acquisition of a characteristic MZB cell phenotype (sIgMhisIgDlow CD21hi CD1dhi CD23low). These phenotypic changes were followed by a burst of proliferation (CTV dilution) between day 4 and day 8 after transfer. Immunofluorescence microscopy of host Rag2-/- spleen sections at day 2 post-transfer identified clusters of transferred B cells localized around CD169+ macrophages at the white pulp/red pulp interface close to the marginal sinus, with subsequent proliferation in this area. We next investigated if Notch signaling regulates this transdifferentiation process, by analogy to its role in normal MZB cell homeostasis. Treatment of Rag2-/- recipients with blocking antibodies against Delta-like-1 Notch ligands (anti-DLL1) or Notch2 receptors (anti-NRR2) completely inhibited lymphopenia-induced FoB to MZB cell conversion and proliferation. To identify the cellular source of DLL1 Notch ligands, we studied transferred B cells in Rag2-/-;Ccl19-Cre+;Dll1f/f recipients, lacking Dll1 expression in all Ccl19-Cre+ fibroblastic reticular cells and their progeny. In these mice as compared to Rag2-/- recipients, FoB to MZB transdifferentiation was almost completely abrogated and transferred cells no longer clustered with marginal sinus-associated macrophages. Thus, stromal DLL1 Notch ligands are critical to regulate the size and composition of the splenic peripheral B cell pool in lymphopenic mice through DLL1/Notch2 interactions. Furthermore, FoB cells are not locked in their FoB cell fate, as commonly assumed, but are instead endowed with plastic transdifferentiation potential in response to DLL1/Notch2-mediated signals that function as a sensor of B cell lymphopenia. We speculate that these adaptive physiological functions of the Notch signaling pathway play an important role in the homeostasis of mature B cells within their stromal microenvironment, and that they can be hijacked during malignant transfomation in Notch-dependent mature B cell lymphomas such as CLL and marginal zone lymphoma. Disclosures Siebel: Genentech: Current Employment. Maillard:Allogene: Consultancy; Regeneron: Consultancy; Genentech: Consultancy.

The effects of Notch signaling in hematopoietic stem and progenitor cells remain controversial. In work presented in this issue of Blood, Duarte et al interfere genetically with the Notch transcriptional complex (NTC) in hematopoiesis and demonstrate that canonical Notch signals are dispensable in primitive hematopoietic progenitors, as well as across the myeloid, erythroid (E), and megakaryocyte (Mk) lineages.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is currently incurable with conventional chemotherapy. Some TNBC patients harbor either recurrent somatic gain-of-function mutations or focal amplification in genes coding for Notch receptors. Recent findings suggest that Notch signals, in the absence of Notch mutations are also important to maintain chemotherapy-resistant circulating tumor cells. Importantly, elevated Notch signaling is specifically enriched in the TNBC and is associated with worse survival in this subtype. These findings underscore the importance of Notch signaling in pathogenesis of TNBC and suggest potential new avenue for therapeutic approaches. Despite this, the target genes and oncogenic mechanisms downstream of aberrant Notch signaling are poorly understood. To create a high confidence map of the oncogenic Notch-driven regulome in TNBC, we performed an integrative, comprehensive and functional genome-wide analysis of Notch-driven targets in Notch-mutated TNBC cell lines by combining high-resolution genome-wide chromatin topology data (HiChIP), dynamic mapping of Notch transcriptional complex members and active and repressive chromatin marks (ChIP-seq), as well as evaluation of dynamic transcription (RNAseq) in presence and absence of Notch signals. One important oncogene and critical Notch target in TNBC is MYC. Our integrative analysis identified detailed map of Notch-dependent MYC regulatory network. The TNBC-specific MYC regulatory region, located 5' of MYC, is much more complex compared to other known Notch-driven malignancies and constitutes of a broad domain of active enhancers marked by H3K27ac that are not present in Notch-driven lymphoma and leukemia. Integrative analysis of ChIP-seq and HiChIP revealed multiple enhancers within this broad domain of regulatory elements that are directly bound by the members of Notch transcriptional complex, sensitive to acute changes in Notch activation and that contact MYC promoter via tumor specific chromatin loops. We showed that genetic perturbation of MYC by CRISPR/Cas9 approach was sufficient to kill TNBC cells. Since, MYC is difficult to target pharmacologically an alternative strategy is to use drugs that inhibit factors regulating MYC expression. To this end, elucidating the Notch regulatory landscape, and in particular, the mechanism by which Notch regulates MYC expression in TNBC may reveal new therapeutic strategies to precisely target MYC in Notch-dependent breast cancer. Citation Format: Petrovic J, Zhou Y, Georgakilas G, Vahedi G, Pear WS, Faryabi RB. Triple negative breast cancer-specific chromatin conformation links Notch signal to tumor-specific transcriptional program [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-07.

PURPOSE OF REVIEW: Lineage commitment is governed by instructive and stochastic signals which drive both active induction of the lineage program as well as repression of alternative fates. Eosinophil lineage commitment is driven by the ordered interaction of transcription factors, supported by cytokine signals. This review summaries key findings in the study of eosinophil lineage commitment and examines new data investigating the factors that regulate this process. RECENT FINDINGS: Recent and past studies highlight how intrinsic and extrinsic signals modulate transcription factor network and lineage decisions. Early action of the transcription factors C/EBPα and GATA-1 along with C/EBPε supports lineage commitment and eosinophil differentiation. This process is regulated and enforced by the pseudokinase Trib1, a regulator of C/EBPα levels. The cytokines IL-5 and IL-33 also support early eosinophil development. However, current studies suggest that these cytokines are not specifically required for lineage commitment. SUMMARY: Together, recent evidence suggests a model where early transcription factor activity drives expression of key eosinophil genes and cytokine receptors to prime lineage commitment. Understanding the factors and signals that control eosinophil lineage commitment may guide therapeutic development for eosinophil mediated diseases and provide examples for fate choices in other lineages.

Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1–3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidative-signature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.

The Tribbles (TRIB) family of pseudokinase proteins has been shown to play key roles in cell cycle, metabolic diseases, chronic inflammatory disease, and cancer development. A better understanding of the mechanisms of TRIB pseudokinases could provide new insights for disease development and help promote TRIB proteins as novel therapeutic targets for drug discovery. At the 2nd International Symposium on Tribbles and Diseases held on May 7-9, 2018 in Beijing, China, a group of leading Tribbles scientists reported their findings and ongoing studies about the effects of the different TRIB proteins in the areas of immunity, metabolism, fundamental cell biology and cancer. Here, we summarize important and insightful overviews from 4 keynote lectures, 13 plenary lectures and 8 short talks that took place during this meeting. These findings may offer new insights for the understanding of the roles of TRIB pseudokinases in the development of various diseases. (C) 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. Dr. Zhuowei Hu' s work was supported by National Key R&D Program of China (Grant No. 2017YFA0205400, China), the National Natural Science Foundation of China (Grant Nos. 81530093 and 81773781, China), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant No. 2016-12M-1-007, China) and CAMS Central Public-interest Scientific Institution Basic Research Fund (Grant No. 2017PT3104, China); Dr. Bing Cui' s work was supported by grants of the National Natural Science Foundation of China (Grant No. 81874316, China), the CAMS Innovation Fund for Medical Sciences (Grant No. 2016-I2M-3-008, China); Dr. Patrick A. Eyers' s work was supported by grants of from the BBSRC and NWCR (Grant Nos. 1088 and 1097, UK); Dr. Leonard L. Dobens' s work was supported by grants of NSF (Grant No. IOS-1456023, USA), NIH (Grant No. NIH R21 CA197317, USA); Dr. Nguan Soon Tan' s work was supported by grants of Ministry of Education, Singapore (Grant Nos. MOE2014-T2-1-012 and 2012-T1-001-036, Singapore); Dr. Peter D. Mace' s work was supported by grants from the Health Research Council of New Zealand, and additionally supported by a Rutherford Discovery Fellowship from the New Zealand government administered by the Royal Society of New Zealand; Dr. Wolfgang Link' s work was supported by Fundacao para a Ciencia e a Tecnologia (FCT) Research Center Grant UID/KM/04773/2013 Centre for Biomedical Research 1334, by a research grant from Liga Portuguesa Contra o Cancro NUcleo Regional do Sul (LPCC/NRS, Portugal), Terry Fox, by a FCT 2014 research grant SFRH/BPD/100434/2014 and a ProRegem grant PD/BD/114258/2016 (Portugal); Dr. Endre Kiss-Toth' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU)and Innovation Network and the British Heart Foundation (PG/16/44/32146, UK); Dr. Karen Keeshan' s work was supported by grants from The Howat Foundation Ltd. (UK), Children with Cancer UK, Bloodwise and the Friends of Paul O' Gorman (UK); Dr. Takuro Nakamura' s work was supported by grants of P-CREATE from Japan Agency for Medical Research and Development; Dr. Warren S. Pear' s work was supported by grants from the NIH (NIAID, USA), Alex' s Lemonade Stand Foundation (USA) and the Samuel Waxman Cancer Research Foundation (USA); Yodit Feseha is supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU). Yodit Feseha' s work is conducted in the context of the "Fondation Centaure" (RTRS), which supports a French transplantation research network, the 1HU-Cesti project, the DHU Oncogreffe and the LabEX IGO thanks to French government financial support managed by the National Research Agency via the "Investment into the Future" program (Grant Nos. ANR-10-IBHU-005 and ANR-11-LABX-0016-01, France). The IHU-Cesti project is also supported by Nantes Metropole and Region Pays de la Loire (France); Dr. Jessica Johnston's work was supported by grants of the British Heart Foundation (PG/16/44/32146, UK); Dr. Arkatiz Carracedo' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Marcel Scheideler' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Zabran llyas' s work was supported by a joint Ph.D studentship beween the A* Star Institute and the University of Sheffield (UK); Dr. Robert C. Bauer s work was supported by funding from the National Institutes of Health National Heart, Lung, and Blood Institute (R01HL141745, USA); Dominika Grzesik' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Juan Salamanca-Viloria's work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Xiaoxi Lv' s work was supported by the National Natural Science Foundation of China (Grant No. 81503128, China) and CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-008, China); Dr. Yishi Jin' s work was supported by National Institute of Health (NS R01-035546, USA); Dr. Ke Li' s work was supported by the National Natural Science Foundation of China (Grant No. 81400140, China), and CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-011, China); Dr. Guillermo Velasco' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Jose M. Lizcano's work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) and Fondo Europeo de desarrollo Regional (FEDER) (Grant No. INNPACTO/IPT-2012-0614-010000, Spain); Dr. Xiaowei Zhang' s work was supported by the National Natural Science Foundation of China (Grant Nos. 81400286 and 81530093, China), and the CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-010, China); Dr. Fang Hua' s work was supported by the National Natural Science Foundation of China (Grant Nos. 81472717 and 81673474, China), Beijing Natural Science Foundation (Grant No. 7162133, China), and the CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-007, China); Dr. Jiaojiao Yu' s work was supported by the National Natural Science Foundation of China (Grant No. 81703564, China); Dr. Jinmei Yu' s work was supported by the National Natural Science Foundation of China (Grant No. 81603129, China). In addition, the Meeting Organizing Committee was greatly thankful to PerkinElmer and Innovent Biologics for their generous financial supports, as well as Dr. Zhuowei Hu' s laboratory members for their assistance in preparing and organizing the conference.

Transcriptional programs contribute to phenotypic and functional cell states. While elucidation of cell state heterogeneity and its role in biology and pathobiology has been advanced by studying single cell level measurements, the underlying assumptions of current analytical methods limit the identification and exploration of cell clades. Unlike other methods, which produce a single uni-layer partition of cells ignoring echelons of cell states, we presentTooManyCells, a software consisting of a suite of graph-based tools for efficient, global, and unbiased identification and visualization of cell clades while maintaining and presenting the relationship between cell states.TooManyCellsprovides a set of tools based on a matrix-free efficient divisive hierarchical spectral clustering algorithm wholly different from the prevalent Louvain-based methods.BirchBeer, the visualization component ofTooManyCells, introduces a new approach for single cell analysis that is built on a concept intentionally orthogonal to the widely used dimensionality reduction methods. Together, this suite of tools provide a paradigm shift in the analysis and interpretation of single cell data by enabling simultaneous comparisons of cell states at context-and application-dependent scales. A byproduct of this shift is the immediate detection and visualization of rare populations that outperforms previous algorithms as demonstrated by applying these tools to existing single cell RNA-seq data sets from various mouse organs.

SUMMARY Emerging single-cell epigenomic assays are used to investigate the heterogeneity of chromatin activity and its function. However, identifying cells with distinct regulatory elements and clearly visualizing their relationships remains challenging. To this end, we introduce TooManyPeaks to address the need for the simultaneous study of chromatin state heterogeneity in both rare and abundant subpopulations. Our analyses of existing data from three widely used single-cell assays for transposase-accessible chromatin using sequencing (scATAC-seq) show the superior performance of TooManyPeaks in delineating and visualizing pure clusters of rare and abundant subpopulations. Furthermore, the application of TooManyPeaks to new scATAC-seq data from drug-naive and drug-resistant leukemic T cells clearly visualizes relationships among these cells and stratifies a rare “resistant-like” drug-naive sub-clone with distinct cis-regulatory elements., Graphical Abstract, In brief Schwartz et al. present TooManyPeaks, a suite of algorithms to explore and visualize heterogeneity of regulatory elements by using single-cell ATAC-seq data. Using TooManyPeaks’s functionalities, they find evidence that heterogeneity of the chromatin accessibility state contributes to the propensity of Notch-mutated T leukemic cells to develop resistance to Notch inhibitors.

COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. We report here structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β-propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β-propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.

T cells provide a fundamental role in cell-mediated immunity, but during chronic antigen exposure such as in the case of cancer, T cells begin to lose effector function and undergo exhaustion. Our goal is to find a target within T cells that could be manipulated to prolong T-cell function and reduce tumor growth. Initial experiments focused on the pseudokinase Trib1, as data suggest that Trib1 acts as a negative regulator of T-cell activation. T cells isolated from mice in which Trib1 was specifically ablated within the T-cell compartment produced more IL-2 compared to wild-type T cells. T cells lacking Trib1 also proliferated more than wild-type T cells. In mice lacking Trib1 in grafted tumor models, tumor growth was reduced. These observations suggest that Trib1 restricts T-cell activation and function. Trib1 is a pseudokinase that is thought to act as a scaffolding molecule to facilitate protein interactions. To understand the mechanism by which Trib1 regulates T-cell function, we performed a proteomic screen to identify interacting partners. The screen was performed in Jurkat cells, an immortalized human T-cell line, expressing a FLAG/strep tagged Trib1. A FLAG pulldown was performed to isolate Trib1, and interacting partners were identified by mass spectrometry. The screen revealed two interacting partners: RFWD2, an E3 ubiquitin ligase also known as COP1, and DET1, a binding partner of COP1. These results provide evidence that Trib1 interacts with COP1 in T cells and suggest that this interaction might be important in the regulation of T-cell function and the regression of tumor growth. Note:This abstract was not presented at the conference. Citation Format: Franklin Iheanacho, Sarah J. Stein, Kelly S. Rome, Martha S. Jordan, Warren S. Pear. Uncovering the mechanism of Trib1 in cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A55.

Chromatin loops enable transcription factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors, such as active forms of Notch, can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3-dimensional (3D) organization of the cancer genome is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B-cell lymphoma, we show that far beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes through establishing new long-range regulatory interactions. Moreover, a large fraction of Notch-promoted regulatory loops forms highly interacting enhancer and promoter spatial clusters, termed “3D cliques”. Loss-and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types.

Interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and CD4(+) inducible regulatory T cells (iTreg cells) emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 cell development. Here we found that IL-1 was needed to fully override RA-mediated expression of the transcription factor Foxp3 and induce protective TH17 cell responses. By repressing expression of the negative regulator SOCS3 dependent on the transcription factor NF-κB, IL-1 increased the amplitude and duration of phosphorylation of the transcription factor STAT3 induced by TH17-polarizing cytokines, which led to an altered balance in the binding of STAT3 and STAT5 to shared consensus sequences in developing T cells. Thus, IL-1 signaling modulated STAT activation downstream of cytokine receptors differently to control the TH17 cell-iTreg cell developmental fate.

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer–binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and have been linked to the Notch responsiveness of a few genes. To assess the overall contribution of SPSs to Notch-dependent gene regulation, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay and applied insights from these in vitro studies to Notch-“addicted” T cell acute lymphoblastic leukemia (T-ALL) cells. We found that SPSs contributed to the regulation of about a third of direct Notch target genes. Although originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5 expression. Our work provides a general method for identifying SPSs in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells.

Hair follicles: Secret to prevent scars? Although some animals easily regenerate limbs and heal broken flesh, mammals are generally not so gifted. Wounding can leave scars, which are characterized by a lack of hair follicles and cutaneous fat. Plikus et al. now show that hair follicles in both mice and humans can convert myofibroblasts, the predominant dermal cell in a wound, into adipocytes (see the Perspective by Chan and Longaker). The hair follicles activated the bone morphogenetic protein (BMP) signaling pathway and adipocyte transcription factors in the myofibroblast. Thus, it may be possible to reduce scar formation after wounding by adding BMP. Science , this issue p. 748 ; see also p. 693

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including MYC and HES1 , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

During chronic infection, the magnitude of the antiviral T cell response is determined by the balance between T cell effector (TEFF) function and T cell exhaustion. While CD8 T cell exhaustion has been extensively characterized, how TEFF responses are regulated during chronic antigen exposure is less clear. We identify Tribbles Pseudokinase 1 (Trib1) as a central regulator of the TEFF response to chronic infection. We show that Trib1 expression is elevated in activated T cells and in human or mouse T cells following chronic infection. In chronically infected mice, T cell-specific deletion of Trib1 enhanced CD4 and CD8 TEFF expansion, persistence and function and reduced viral load. Mechanistically, we show that Trib1 functions as a negative regulator of signaling downstream of the T cell receptor (TCR), whereby Trib1 expression disrupts TCR signaling complexes. These data support a model of negative feedback regulation whereby Trib1 restrains TCR signaling and downstream function. Together, our work shows that Trib1 functions to restrain the magnitude and duration of the TEFF response, at least in part through the disruption of TCR signaling complexes, and reveals a new regulatory node downstream of TCR signaling that is critical during chronic infection. In light of growing interest in checkpoint blockade therapies that act to overcome exhaustion during chronic antigen exposure, our work reveals an important new avenue for enhancing the efficacy of current T cell reactivation strategies during chronic disease.

The main oncogenic driver in T-lymphoblastic leukemia is NOTCH1, which activates genes by forming chromatin-associated Notch transcription complexes. Gamma-secretase-inhibitor treatment prevents NOTCH1 nuclear localization, but most genes with NOTCH1-binding sites are insensitive to gamma-secretase inhibitors. Here, we demonstrate that fewer than 10% of NOTCH1-binding sites show dynamic changes in NOTCH1 occupancy when T-lymphoblastic leukemia cells are toggled between the Notch-on and -off states with gamma-secretase inhibiters. Dynamic NOTCH1 sites are functional, being highly associated with Notch target genes, are located mainly in distal enhancers, and frequently overlap with RUNX1 binding. In line with the latter association, we show that expression of IL7R, a gene with key roles in normal T-cell development and in T-lymphoblastic leukemia, is coordinately regulated by Runx factors and dynamic NOTCH1 binding to distal enhancers. Like IL7R, most Notch target genes and associated dynamic NOTCH1-binding sites cooccupy chromatin domains defined by constitutive binding of CCCTC binding factor, which appears to restrict the regulatory potential of dynamic NOTCH1 sites. More remarkably, the majority of dynamic NOTCH1 sites lie in superenhancers, distal elements with exceptionally broad and high levels of H3K27ac. Changes in Notch occupancy produces dynamic alterations in H3K27ac levels across the entire breadth of superenhancers and in the promoters of Notch target genes. These findings link regulation of superenhancer function to NOTCH1, a master regulatory factor and potent oncoprotein in the context of immature T cells, and delineate a generally applicable roadmap for identifying functional Notch sites in cellular genomes.

Although it is well recognized that haematopoietic stem cells (HSCs) develop from a specialized population of endothelial cells known as haemogenic endothelium, the regulatory pathways that control this transition are not well defined. Here we identify Sox17 as a key regulator of haemogenic endothelial development. Analysis of Sox17-GFP reporter mice revealed that Sox17 is expressed in haemogenic endothelium and emerging HSCs and that it is required for HSC development. Using the mouse embryonic stem cell differentiation model, we show that Sox17 is also expressed in haemogenic endothelium generated in vitro and that it plays a pivotal role in the development and/or expansion of haemogenic endothelium through the Notch signalling pathway. Taken together, these findings position Sox17 as a key regulator of haemogenic endothelial and haematopoietic development.

Summary Group 2 innate lymphoid cells (ILC2) are innate lymphocytes that confer protective type 2 immunity during helminth infection and are also involved in allergic airway inflammation. Here we report that ILC2 development required T cell factor 1 (TCF-1, the product of the Tcf7 gene), a transcription factor also implicated in T cell lineage specification. Tcf7 −/− mice lack ILC2, and were unable to mount ILC2-mediated innate type 2 immune responses. Forced expression of TCF-1 in bone marrow progenitors partially bypassed the requirement for Notch signaling in the generation of ILC2 in vivo. TCF-1 acted through both GATA-3-dependent and GATA-3-independent pathways to promote the generation of ILC2. These results are reminiscent of the critical roles of TCF-1 in early T cell development. Hence, transcription factors that underlie early steps of T cell development are also implicated in the development of innate lymphoid cells.