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CRISPR screening identifies synergy between BET and mTOR inhibitors in cholangiocarcinoma through impaired serine glycine one carbon metabolism [RNA-seq]

ABSTRACT: Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify novel drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV-825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tied together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248632 | GEO | 2024/02/01

REPOSITORIES: GEO

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CRISPR screening identifies synergy between BET and mTOR inhibitors in cholangiocarcinoma through impaired serine glycine one carbon metabolism [CHIP-seq]

Project description:Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify novel drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV-825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tied together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

2024-02-01 | GSE248633 | GEO

Novel BET protein Proteolysis Targeting Chimera (BET-PROTAC) exerts superior lethal activity than Bromodomain Inhibitor (BETi) against post-myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Project description:The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Whereas the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reversed Phase Protein Array and mass cytometry ‘CyTOF’ analyses demonstrated that ARV-825 caused greater perturbations in mRNA and protein expressions than OTX015 in sAML cells. Specifically, compared to OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, pSTAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared to OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against the established and PD-CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

2017-04-06 | GSE93578 | GEO

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BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against Mantle Cell Lymphoma cells

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2019-04-26 | GSE98268 | GEO

RUNX1 knockdown and BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mtRUNX1 AML cells

Project description:Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins which undermines the growth and survival of AML cells. However, BETi treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4, which potentially compromises the activity of BETi in AML cells. Unlike BETi, BET-PROTAC (proteolysis-targeting chimera) ARV-825 recruits and utilize an E3-ubiquitin ligase to effectively degrade BETPs in AML cells. BET-PROTACs induce more apoptosis than BETi of mtRUNX1 AML cells. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi. It was noted that treatment with BETi or BET-PROTAC caused significant and sustained depletion of RUNX1 in AML cells. We also determined the effects of global depletion of RUNX1 in mtRUNX1 expressing AML OCI-AML5 cells. We observed an overlap in the signature of RUNX1 knockdown by shRNA with that of OTX015 and ARV-825 in OCI-AML5 cells.

2020-08-11 | GSE119261 | GEO

Effect of PROTAC-mediated BRD4 degradation in acute myeloid leukemia [OCI-AML3]

Project description:BRD4, a member of the BET family of histone readers, binds to acetylated lysine of histone H3 and promotes assembly of super-enhancer complexes that drive expression of key oncogenes in acute myeloid leukemia (AML) and other cancers. ARV-825 is a proteolysis-targeting chimera (PROTAC) that targets BRD4 for CRBN-mediated ubiquitination and degradation. We treated the AML cell line OCI-AML3, as well as primary tumor cells from a case of AML, with ARV-825 in vitro. At low concentrations, ARV-825 caused profound and sustained reduction in BRD4 protein levels. This caused reduction in transcription of key genes including MYC, anti-apoptotic BCL2 and BCLXL, PIM1, and CD44. Downstream effects included loss of CXCR4 surface expression and mitochondrial respiration; increased reactive oxygen species; toxicity to AML cells in vitro; and efficacy vs. OCI-AML3 cells in a mouse model of AML.

2019-10-01 | GSE101380 | GEO

Serine biosynthesis is a metabolic vulnerability in IDH2-driven breast cancer progression

Project description:Cancer-specific metabolic phenotypes and their vulnerabilities are one among the viable areas of cancer research. We studied the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses reveal the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration and antioxidant defense. Genome-scale metabolic modeling identified PHGDH and PSAT1 as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction showed patients with IDH2-high/PHGDH-low have better survival than IDH2-high/PHGDH-high. Furthermore, we show the efficacy of PHGDH inhibitors in treating IDH2-high cells in vitro and in in vivo. Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression.

2021-01-04 | PXD023142 | Pride

Bromodomain and extraterminal proteins foster the core transcriptional regulatory programs and confer vulnerability in liposarcoma (ChIP-Seq)

Project description:Purpose: Identification of core transcriptional regulatory programs and bromodomain and extraterminal (BET) protein dependency in liposarcoma (LPS) Methods: ChIP-seq and RNA-seq were performed on LPS cells. Antibodies against H3K27ac, H3K4me1, H3K4me3, RNA-Pol2, BRD2, BRD3, BRD4, FOSL2, pan-RUNX, and DDIT3 (FUS-DDIT3) were used for ChIP-seq assays. The transcriptome responses of LPS141 and MLS402 cells to OTX015 and ARV-825 were compared. Result: We generated genome-wide chromatin-state maps of LPS cells. By charting the super-enhancer structures, we identify a bromodomain and extraterminal (BET) protein-cooperated FUS-DDIT3 function in myxoid LPS and a BET protein-dependent core transcriptional regulatory circuitry consisting of FOSL2, MYC, and RUNX1 in de-differentiated LPS. This study also provides a framework for discovering and targeting of core oncogenic transcriptional programs.

2019-04-01 | GSE111253 | GEO

Bromodomain and extraterminal proteins foster the core transcriptional regulatory programs and confer vulnerability in liposarcoma (RNA-Seq)

2019-04-01 | GSE111252 | GEO

Non-metabolic PHGDH facilitates cMyc to urge advanced liver cancer

Project description:Metabolic dysregulation is an important feature of malignant tumors. Serine synthesis pathway (SSP) reshapes tumor cells to enhance their growth and survival capabilities, especially under serine starvation. However, whether and how SSP is involved in the progression of liver cancer has not been clearly stated. We applied a combination of quantitative proteomics and transcriptomics and discovered the increased expression of PHGDH and PSAT1 in mice liver cancer induced by SB. PHGDH is the first rating-limited enzyme of SSP and the conditional knockout of PHGDH in liver impaired the advanced liver cancer formation. However, the specific inhibition of enzymatic activity of PHGDH in liver has no obvious suppressive effect on advanced liver cancer. Further, we explored a non-metabolic function of PHGDH, which used its ACT domain to bind and regulate cMyc transcriptional activity, then selectively activated genes expression of Lcn2, Got1, Fabp5 and Psat1, indicating PHGDH drives/links multiple metabolic pathways and augmented its function through its non-metabolic role. In addition, the mutant p53 would release the suppression of PHGDH in mice liver cancer model and clinical liver cancer samples. Hence, the mutant p53/PHGDH/cMyc axis could be a potential target to cure advanced liver cancer.

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