Research vision workshopping: Peer mentoring to support the transition to independence.

The transition to independence requires shared enthusiasm for one's research goals from broad audiences. In this commentary, we describe the use of "research vision workshopping" within peer mentoring networks. We contend that this approach is broadly useful for the development and refinement of research visions for the academic job search.

Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin.

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.

Modeling diverse genetic subtypes of lung adenocarcinoma with a next-generation alveolar type 2 organoid platform.

Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD.

Spatial genomics enables multi-modal study of clonal heterogeneity in tissues.

The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations1-4 as well as the makeup of the tumour microenvironment5,6. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.

Genetic studies reveal an unexpected negative regulatory role for Jak2 in thrombopoiesis.

JAK inhibitor treatment is limited by the variable development of anemia and thrombocytopenia thought to be due to on-target JAK2 inhibition. We evaluated the impact of Jak2 deletion in platelets (PLTs) and megakaryocytes (MKs) on blood counts, stem/progenitor cells, and Jak-Stat signaling. Pf4-Cre-mediated Jak2 deletion in PLTs and MKs did not compromise PLT formation but caused thrombocytosis, and resulted in expansion of MK progenitors and Lin(-)Sca1(+)Kit+ cells. Serum thrombopoietin (TPO) was maintained at normal levels in Pf4-Cre-positive Jak2(f/f) mice, consistent with reduced internalization/turnover by Jak2-deficient PLTs. These data demonstrate that Jak2 in terminal megakaryopoiesis is not required for PLT production, and that Jak2 loss in PLTs and MKs results in non-autonomous expansion of stem/progenitors and of MKs and PLTs via dysregulated TPO turnover. This suggests that the thrombocytopenia frequently seen with JAK inhibitor treatment is not due to JAK2 inhibition in PLTs and MKs, but rather due to JAK2 inhibition in stem/progenitor cells.

Senescent fibroblasts in the tumor stroma rewire lung cancer metabolism and plasticity.

Senescence has been demonstrated to either inhibit or promote tumorigenesis. Resolving this paradox requires spatial mapping and functional characterization of senescent cells in the native tumor niche. Here, we identified senescent p16 Ink4a + cancer-associated fibroblasts with a secretory phenotype that promotes fatty acid uptake and utilization by aggressive lung adenocarcinoma driven by Kras and p53 mutations. Furthermore, rewiring of lung cancer metabolism by p16 Ink4a + cancer-associated fibroblasts also altered tumor cell identity to a highly plastic/dedifferentiated state associated with progression in murine and human LUAD. Our ex vivo senolytic screening platform identified XL888, a HSP90 inhibitor, that cleared p16 Ink4a + cancer-associated fibroblasts in vivo. XL888 administration after establishment of advanced lung adenocarcinoma significantly reduced tumor burden concurrent with the loss of plastic tumor cells. Our study identified a druggable component of the tumor stroma that fulfills the metabolic requirement of tumor cells to acquire a more aggressive phenotype.

Smarca4 Inactivation Promotes Lineage-Specific Transformation and Early Metastatic Features in the Lung.

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein-positive cells within the lung in a cell type-dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. SIGNIFICANCE: We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution.This article is highlighted in the In This Issue feature, p. 275.

Protocol for single-cell ATAC sequencing using combinatorial indexing in mouse lung adenocarcinoma.

Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples. For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020).

Epigenomic State Transitions Characterize Tumor Progression in Mouse Lung Adenocarcinoma.

Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression.

Loss of BAP1 function leads to EZH2-dependent transformation.

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Mining the epigenetic landscape in ALL.

The significance of epigenomic aberrations in cancer development has been underscored by the discovery of mutations in key chromatin modifiers, most notably in hematological malignancies. A new study of pediatric acute lymphoblastic leukemia (ALL) demonstrates the usefulness of mapping global epigenetic signatures and applying these data in a framework to identify and characterize underlying somatic genetic alterations in human cancers.

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo.

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis.

JAK2 the future: therapeutic strategies for JAK-dependent malignancies.

The Janus kinase (JAK) proteins are a family of intracellular nonreceptor tyrosine kinases involved in cytokine signaling via the JAK-STAT (signal transducers and activators of transcription) pathway. Genetic studies have identified somatic JAK2(V617F) mutations and other mutant alleles that activate JAK-STAT signaling in most patients with myeloproliferative neoplasms (MPNs). As a result, JAK inhibitors have been developed to treat various malignancies and have been shown to be efficacious in both preclinical and clinical settings. However, available ATP-competitive JAK (type I) inhibitors are associated with dose-dependent toxicities, and do not yet reduce disease burden in MPN patients. Recent studies suggest that genetic and epigenetic mechanisms can cause insensitivity to type I JAK inhibitors. Novel therapies include the development of type II JAK inhibitors and the use of alternative strategies to abrogate JAK-STAT signaling, perhaps with histone deacetylase (HDAC) and heat shock protein 90 (HSP90) inhibitors. These innovative therapies may translate to treatment of other diseases that are dependent on JAK signaling, including B-precursor acute lymphoblastic leukemia (B-ALL).

ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.

Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.

Loss of the tumor suppressor BAP1 causes myeloid transformation.

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.