Showing papers by "Christopher R. Vakoc published in 2022"
TL;DR: This paper showed that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 is critical in the tuft cell lineage and demonstrated that binding between OCA-T1/POU2AF2/OCA-T2 is essential in tuft-cell-like small-cell lung cancer.
Abstract: Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.
23 citations
TL;DR: This paper showed that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 is critical in the tuft cell lineage and demonstrated that binding between OCA-T1/POU2AF2/OCA-T2 is essential in tuft-cell-like small-cell lung cancer.
Abstract: Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.
19 citations
TL;DR: It is shown that SMARCA4, the catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, is essential for the proliferation, migration and invasion of DMG cells and tumor growth in patient-derived DMG xenograft models.
Abstract: Patients with diffuse midline gliomas-H3K27-altered (DMG) display a dismal prognosis. However, the molecular mechanisms underlying DMG tumorigenesis remain poorly defined. Here we show that SMARCA4, the catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, is essential for the proliferation, migration and invasion of DMG cells and tumor growth in patient-derived DMG xenograft models. SMARCA4 co-localizes with SOX10 at gene regulatory elements (GRE) to control the expression of genes involved in cell growth and extracellular matrix (ECM). Moreover, SMARCA4 chromatin binding is reduced upon depletion of SOX10 or H3.3K27M, a mutation occurring in about 60% DMG tumors. Furthermore, the SMARCA4 occupancy at enhancers marked by both SOX10 and H3K27 acetylation is reduced the most upon depleting the H3.3K27M mutation. Taken together, our results support a model in which epigenome reprogramming by H3.3K27M creates a dependence on SMARCA4-mediated chromatin remodeling to drive gene expression and the pathogenesis of H3.3K27M DMG.
6 citations
TL;DR: In this paper , the authors generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens and demonstrate that a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorsial approach to profile genetic interactions of multiple genes.
Abstract: Abstract Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.
6 citations
TL;DR: In this article , a GBM-specific epigenetic mechanism was described in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex.
Abstract: Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)—the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM. BRD8 is identified as a specific epigenetic vulnerability for glioblastomas that harbour wild-type p53.
5 citations
TL;DR: In this article , the lineage-specifying transcription factors FoxA1 and FoxA2 (FoxA1/2) control identity in NKX2-1/TTF1-negative lung adenocarcinoma (LUAD) and their role in this disease has not been systematically investigated.
5 citations
TL;DR: In this paper , the authors used both high throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1), a GPCR not previously known to interact with DOPA, nor to affect melanoma pathobiology.
5 citations
TL;DR: In this paper , the authors generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens and demonstrate that a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorsial approach to profile genetic interactions of multiple genes.
Abstract: Abstract Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.
5 citations
TL;DR: In this article , the authors show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures.
Abstract: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.
3 citations
TL;DR: Using single-cell RNA sequencing, this work identifies Spdef as a factor required for tumorigenesis in pancreatic cancer cells of epithelial and mucinous nature and discovers multiple tumor cell populations distinguished by their differentiation state.
Abstract: Pancreatic ductal adenocarcinoma (PDA) samples reveal extensive cellular heterogeneity. Using single-cell RNA sequencing, we uncover multiple tumor cell populations distinguished by their differentiation state and associated with different stages of tumor progression in a mouse model of PDA. We identify Spdef as a factor required for tumorigenesis in pancreatic cancer cells of epithelial and mucinous nature. By comparative analysis of cell differentiation states in mice and humans, we find that the Spdef program is highly expressed by human PDAs of the classical subtype. Mouse and human PDA cells expressing elevated levels of Spdef are dependent upon this transcription factor for tumor progression in vivo. The tumor-promoting function of Spdef is recapitulated by two Spdef target genes that regulate protein folding and endoplasmic reticulum activity, Agr2 and Ern2/Ire1β. These findings offer insights into the factors controlling differentiation states in PDA and identify new vulnerabilities in the most common subtype of pancreatic cancer.
3 citations
TL;DR: In this paper , SIRT6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion, and after helminths infection, IEC SIRT 6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance.
Abstract: Abstract Dynamic regulation of intestinal epithelial cell (IEC) differentiation is crucial for both homeostasis and the response to helminth infection. SIRT6 belongs to the NAD + -dependent deacetylases and has established diverse roles in aging, metabolism and disease. Here, we report that IEC Sirt6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion. Conversely, after helminth infection, IEC SIRT6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance. Mechanistically, Sirt6 ablation causes elevated Socs3 expression, and subsequently attenuated tyrosine 641 phosphorylation of STAT6 in IECs. Notably, intestinal epithelial overexpression of constitutively activated STAT6 (STAT6vt) in mice is sufficient to induce the expansion of tuft and goblet cell linage. Furthermore, epithelial STAT6vt overexpression remarkedly reverses the defects in intestinal epithelial remodeling caused by Sirt6 ablation. Our results reveal a novel function of SIRT6 in regulating intestinal epithelial remodeling and mucosal type 2 immunity in response to helminth infection.
TL;DR: Vakoc et al. as mentioned in this paper used genetic screens performed in cancer cell lines to identify a tuft cell variant of small cell lung cancer, which is characterized by high expression of, and addiction to, POU2F3.
Abstract:
Using genetic screens performed in cancer cell lines, we previously identified a tuft cell variant of small cell lung cancer. This novel tumor entity is characterized by high expression of, and addiction to, POU2F3, which encodes a POU homeodomain transcription factor. Subsequent work by several laboratories has revealed tuft cell tumors exist in several different anatomical sites (e.g., thymus, prostate, GI tract), indicating a broader relevance of this cell lineage in human cancer. In my presentation, I will present our efforts to reveal transcriptional mechanisms in tuft cell lung tumors, focusing on the identification of OCA-T1 and OCA-T2 as coactivators of POU2F3.
Citation Format: Christopher R. Vakoc. Transcriptional dependencies of tuft cell lung tumors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA011.
TL;DR: In this paper , the authors used domain-focused CRISPR screening and identified the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells.