Background and Aims
Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug ...targets that specifically inhibit the growth of HCC cells.
Approach and Results
We used a CRISPR library targeting ~2,000 druggable genes to perform a high‐throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR‐mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S‐phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate.
Conclusions
Our findings uncover the role of ADSL‐mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. ...To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E HCoV-229E, HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.
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•SARS-CoV-2 host protein interactome CRISPR screens for SARS-CoV-2 and three coronaviruses•Parallel CRISPR screens uncover unique and shared coronavirus host factors•Numbers of interacting host proteins and functional interactors are not proportional•Identified SARS-CoV-2 host factors are expressed in relevant cells in the human airway
Building upon a published SARS-CoV-2 protein interactome, Hoffmann et al. use a custom CRISPR library to determine which of these interacting host proteins are essential for infection by SARS-CoV-2 virus as well as three seasonal coronaviruses. These factors represent potential targets to combat COVID-19 and perhaps future coronavirus outbreaks.
Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular ...mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of
in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally,
cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition.
Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of ...systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.
The interplay between metabolism and chromatin signaling is implicated in cancer progression. However, whether and how metabolic reprogramming in tumors generates chromatin vulnerabilities remain ...unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway, which drives aggressive and chemo-resistant disease. Using a chromatin-focused CRISPR screen, we report that NRF2 activation sensitizes LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDACs). This association is observed across cultured cells, mouse models, and patient-derived xenografts. Integrative epigenomic, transcriptomic, and metabolomic analysis demonstrates that HDAC inhibition causes widespread redistribution of H4ac and its reader protein, which transcriptionally downregulates metabolic enzymes. This results in reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest NRF2 activation as a potential biomarker for effective repurposing of HDAC inhibitors to treat solid tumors.
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•NRF2-active cells are sensitive to class I histone deacetylase inhibition•HDAC inhibition causes genomic redistribution of histone acetylation and BRD4•Epigenomic reprogramming leads to downregulation of metabolic genes•HDAC inhibition suppresses metabolic pathways critical for NRF2-active cell growth
Abnormal activation of the NRF2 antioxidant response pathway in lung cancer drives aggressive and chemo-resistant disease. Karagiannis et al. report that inhibition of histone deacetylases has an antiproliferative effect in this cancer context through extensive changes in chromatin, metabolic gene expression, and suppression of amino acid and nucleotide metabolic pathways.
Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor ...MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.
Reactivation of p53 in established tumors typically results in one of two cell fates, cell cycle arrest or apoptosis, but it remains unclear how this cell fate is determined. We hypothesized that ...high mitochondrial priming prior to p53 reactivation would lead to apoptosis, while low priming would lead to survival and cell cycle arrest. Using a panel of Kras-driven, p53 restorable cell lines derived from genetically engineered mouse models of lung adenocarcinoma and sarcoma (both of which undergo cell cycle arrest upon p53 restoration), as well as lymphoma (which instead undergo apoptosis), we show that the level of mitochondrial apoptotic priming is a critical determinant of p53 reactivation outcome. Cells with high initial priming (e.g., lymphomas) lacked sufficient reserve antiapoptotic capacity and underwent apoptosis after p53 restoration. Forced BCL-2 or BCL-XL expression reduced priming and resulted in survival and cell cycle arrest. Cells with low initial priming (e.g., lung adenocarcinoma and sarcoma) survived and proceeded to arrest in the cell cycle. When primed by inhibition of their antiapoptotic proteins using genetic (BCL-2 or BCL-XL deletion or BAD overexpression) or pharmacologic (navitoclax) means, apoptosis resulted upon p53 restoration in vitro and in vivo. These data demonstrate that mitochondrial apoptotic priming is a key determining factor of cell fate upon p53 activation. Moreover, it is possible to enforce apoptotic cell fate following p53 activation in less primed cells using p53-independent drugs that increase apoptotic priming, including BH3 mimetic drugs.
Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense ...mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by
) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing
occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the
tumor suppressor locus. Disruption of
cooperates with
overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the
locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated
expression increases its binding to the
locus and co-activates gene transcription. Endogenous
restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in
and
were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting
activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.
Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and ...genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin-modifying complexes that dictates response to Menin-MLL inhibitors. MLL1-Menin safeguards leukemia survival by impeding the binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the Menin-MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1-Menin and MLL3/4-UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials.
Menin-MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin-MLL inhibitor-resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses. This article is highlighted in the In This Issue feature, p. 1.
Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide; yet, patients with HCC have limited treatment options. There is an urgent need to identify new drug targets that ...specifically inhibit the growth of HCC cells. Here, we used a newly-engineered CRISPR library targeting ~2,000 druggable genes to perform a high throughput screen, and identified adenylosuccinate lyase (ADSL) – a key enzyme involved in the
de novo
purine synthesis pathway – as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting adenosine monophosphate (AMP) production. In the absence of ADSL, the growth of liver tumors is retarded
in vivo
. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest, not by inducing DNA damage, but by impairing mitochondrial function. Using HCC patient data, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. In conclusion, our findings uncover the role of ADSL-mediated
de novo
purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for HCC patients.
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