Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Disruption of cotranscriptional splicing suggests that RBM39 is a therapeutic target in acute lymphoblastic leukemia.

There are few options for patients with relapse/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus this is a major area of unmet medical need. Here, we reveal that inclusion of a poison exon in RBM39, which could be induced both by CDK9 or CDK9 independent CMGC (cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases) kinase inhibition, is recognized by the nonsense-mediated mRNA decay (NMD) pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors lead to protein downregulation and inhibition of ALL growth, particularly in relapse/refractory B-ALL. Mechanistically, disruption of co-transcriptional splicing by inhibition of CMGC kinases including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), results in alteration of SF3B1 and Pol II association. Disruption of SF3B1 and transcriptional elongation complex alters Pol II pausing, which promotes the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppresses the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong anti-tumor activity in preclinical models. Our data reveal that relapse/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide two potential strategies to target this axis.

Engineering naturally occurring CD7- T cells for the immunotherapy of hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.

Single-Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.

Murine fetal bone marrow does not support functional hematopoietic stem and progenitor cells until birth.

While adult bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) and their extrinsic regulation is well studied, little is known about the composition, function, and extrinsic regulation of the first HSPCs to enter the BM during development. Here, we functionally interrogate murine BM HSPCs from E15.5 through P0. Our work reveals that fetal BM HSPCs are present by E15.5, but distinct from the HSPC pool seen in fetal liver, both phenotypically and functionally, until near birth. We also generate a transcriptional atlas of perinatal BM HSPCs and the BM niche in mice across ontogeny, revealing that fetal BM lacks HSPCs with robust intrinsic stem cell programs, as well as niche cells supportive of HSPCs. In contrast, stem cell programs are preserved in neonatal BM HSPCs, which reside in a niche expressing HSC supportive factors distinct from those seen in adults. Collectively, our results provide important insights into the factors shaping hematopoiesis during this understudied window of hematopoietic development.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse.

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth's Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.

Arid1a mutation suppresses TGF-ß signaling and induces cholangiocarcinoma.

Activating KRAS mutations and functional loss of members of the SWI/SNF complex, including ARID1A, are found together in the primary liver tumor cholangiocarcinoma (CC). How these mutations cooperate to promote CC has not been established. Using murine models of hepatocyte and biliary-specific lineage tracing, we show that Kras and Arid1a mutations drive the formation of CC and tumor precursors from the biliary compartment, which are accelerated by liver inflammation. Using cultured cells, we find that Arid1a loss causes cellular proliferation, escape from cell-cycle control, senescence, and widespread changes in chromatin structure. Notably, we show that the biliary proliferative response elicited by Kras/Arid1a cooperation and tissue injury in CC is caused by failed engagement of the TGF-ß-Smad4 tumor suppressor pathway. We thus identify an ARID1A-TGF-ß-Smad4 axis as essential in limiting the biliary epithelial response to oncogenic insults, while its loss leads to biliary pre-neoplasia and CC.

Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition.

Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

The histone methyltransferase Setd8 alters the chromatin landscape and regulates the expression of key transcription factors during erythroid differentiation.

BACKGROUND: SETD8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. SETD8 and H4K20me1 play a role in a number of essential biologic processes, including cell cycle progression, establishment of higher order chromatin structure, and transcriptional regulation. SETD8 is highly expressed in erythroid cells and erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia, suggesting that it has an erythroid-specific function. The function of SETD8 in the hemopoietic system is poorly understood. The goal of our study was to gain insights into the function of SETD8 during erythroid differentiation. RESULTS: We performed ATAC-seq (assay for transposase-accessible chromatin) on sorted populations of E10.5 Setd8 mutant and control erythroblasts. Accessibility profiles were integrated with expression changes and a mark of heterochromatin (H3K27me3) performed in wild-type E10.5 erythroblasts to further understand the role of SETD8 in erythropoiesis. Data integration identified regions of greater chromatin accessibility in Setd8 mutant cells that co-located with H3K27me3 in wild-type E10.5 erythroblasts suggesting that these regions, and their associated genes, are repressed during normal erythropoiesis. The majority of these more accessible regions were located in promoters and they frequently co-located with the NFY complex. Pathway analysis of genes identified through data integration revealed stemness-related pathways. Among those genes were multiple transcriptional regulators active in multipotent progenitors, but repressed during erythroid differentiation including Hhex, Hlx, and Gata2. Consistent with a role for SETD8 in erythroid specification, SETD8 expression is up-regulated upon erythroid commitment, and Setd8 disruption impairs erythroid colony forming ability. CONCLUSION: Taken together, our results suggest that SETD8 is an important regulator of the chromatin landscape during erythroid differentiation, particularly at promoters. Our results also identify a novel role for Setd8 in the establishment of appropriate patterns of lineage-restricted gene expression during erythroid differentiation.

Hyperacetylated chromatin domains mark cell type-specific genes and suggest distinct modes of enhancer function.

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.