BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.
Antineoplastic Agents , Breast Neoplasms , Bromodomain Containing Proteins , Cell Cycle Proteins , Cell Proliferation , Transcription Factors , Humans , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Animals , Cell Proliferation/drug effects , Peptides/pharmacology , Peptides/chemistry , Cell Line, Tumor , Mice , Protein Domains , Mice, Nude , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm is closely associated with coronavirus disease 2019 (COVID-19) severity and lethality. However, drugs that are effective against inflammation to treat lethal COVID-19 are still urgently needed. Here, we constructed a SARS-CoV-2 spike protein-specific CAR, and human T cells infected with this CAR (SARS-CoV-2-S CAR-T) and stimulated with spike protein mimicked the T-cell responses seen in COVID-19 patients, causing cytokine storm and displaying a distinct memory, exhausted, and regulatory T-cell phenotype. THP1 remarkably augmented cytokine release in SARS-CoV-2-S CAR-T cells when they were in coculture. Based on this "two-cell" (CAR-T and THP1 cells) model, we screened an FDA-approved drug library and found that felodipine, fasudil, imatinib, and caspofungin were effective in suppressing the release of cytokines, which was likely due to their ability to suppress the NF-κB pathway in vitro. Felodipine, fasudil, imatinib, and caspofungin were further demonstrated, although to different extents, to attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in a SARS-CoV-2-infected Syrian hamster model, which were also linked to their suppressive role in inflammation. In summary, we established a SARS-CoV-2-specific CAR-T-cell model that can be utilized as a tool for anti-inflammatory drug screening in a fast and high-throughput manner. The drugs identified herein have great potential for early treatment to prevent COVID-19 patients from cytokine storm-induced lethality in the clinic because they are safe, inexpensive, and easily accessible for immediate use in most countries.
COVID-19 , Receptors, Chimeric Antigen , Humans , SARS-CoV-2/metabolism , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Caspofungin , Felodipine , Cytokine Release Syndrome/drug therapy , Inflammation , Cytokines/metabolism
A tremendous amount of proteomic and phosphoproteomic data has been produced over the years with the development of mass spectrometry techniques, providing us with new opportunities to explore and understand the proteome and phosphoproteome as well as the function of proteins and protein phosphorylation sites. However, a lack of powerful tools that we can utilize to explore these valuable data limits our understanding of the proteome and phosphoproteome, particularly in diseases such as cancer. To address these unmet needs, we established CPPA (Cancer Proteome and Phosphoproteome Atlas), a web tool to mine abnormalities of the proteome and phosphoproteome in cancer based on published data sets. All analysis results are presented in CPPA with a flexible web interface to provide key customization utilities, including general analysis, differential expression profiling, statistical analysis of protein phosphorylation sites, correlation analysis, similarity analysis, survival analysis, pathological stage analysis, etc. CPPA greatly facilitates the process of data mining and therapeutic target discovery by providing a comprehensive analysis of proteomic and phosphoproteomic data in normal and tumor tissues with a simple click, which helps to unlock the precious value of mass spectrometry data by bridging the gap between raw data and experimental biologists. CPPA is currently available at https://cppa.site/cppa.
Neoplasms , Proteome , Humans , Proteome/metabolism , Proteomics , Data Mining , Mass Spectrometry , Phosphorylation , Phosphoproteins/metabolism
Estrogen and estrogen receptor alpha (ERα)-induced gene transcription is tightly associated with ERα-positive breast carcinogenesis. ERα-occupied enhancers, particularly super-enhancers, have been suggested to play a vital role in regulating such transcriptional events. However, the landscape of ERα-occupied super-enhancers (ERSEs) as well as key ERα-induced target genes associated with ERSEs remain to be fully characterized. Here, we defined the landscape of ERSEs in ERα-positive breast cancer cell lines, and demonstrated that bromodomain protein BRD4 is a master regulator of the transcriptional activation of ERSEs and cognate ERα target genes. RET, a member of the tyrosine protein kinase family of proteins, was identified to be a key ERα target gene of BRD4-regulated ERSEs, which, in turn, is vital for ERα-induced gene transcriptional activation and malignant phenotypes through activating the RAS/RAF/MEK2/ERK/p90RSK/ERα phosphorylation cascade. Combination therapy with BRD4 and RET inhibitors exhibited additive effects on suppressing ERα-positive breast cancer both in vitro and in vivo, comparable with that of standard endocrine therapy tamoxifen. Furthermore, combination therapy re-sensitized a tamoxifen-resistant ERα-positive breast cancer cell line to tamoxifen treatment. Taken together, our data uncovered the critical role of a super-enhancer-associated positive feedback loop constituting BRD4/ERα-RET-ERα in ERα-positive breast cancer, and suggested that targeting components in this loop would provide a new therapeutic avenue for treating ERα-positive breast cancer in the clinic.
Breast Neoplasms , Estrogen Receptor alpha , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens , Feedback, Physiological , Female , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Proto-Oncogene Proteins c-ret/therapeutic use , Tamoxifen/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism
Glioblastoma (GBM) is the deadliest brain malignancy without effective treatments. Here, we reported that epidermal growth factor receptor-targeted chimeric antigen receptor T cells (EGFR CAR-T) were effective in suppressing the growth of GBM cells in vitro and xenografts derived from GBM cell lines and patients in mice. However, mice soon acquired resistance to EGFR CAR-T cell treatment, limiting its potential use in the clinic. To find ways to improve the efficacy of EGFR CAR-T cells, we performed genomics and transcriptomics analysis for GBM cells incubated with EGFR CAR-T cells and found that a large cohort of genes, including immunosuppressive genes, as well as enhancers in vicinity are activated. BRD4, an epigenetic modulator functioning on both promoters and enhancers, was required for the activation of these immunosuppressive genes. Accordingly, inhibition of BRD4 by JQ1 blocked the activation of these immunosuppressive genes. Combination therapy with EGFR CAR-T cells and JQ1 suppressed the growth and metastasis of GBM cells and prolonged survival in mice. We demonstrated that transcriptional modulation by targeting epigenetic regulators could improve the efficacy of immunotherapy including CAR-T, providing a therapeutic avenue for treating GBM in the clinic.
Azepines/administration & dosage , Brain Neoplasms/therapy , Cell Cycle Proteins/metabolism , ErbB Receptors/immunology , Glioblastoma/therapy , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/metabolism , Transcription Factors/metabolism , Triazoles/administration & dosage , Animals , Azepines/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Combined Modality Therapy , Epigenesis, Genetic/drug effects , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Mice , Neoplasm Metastasis , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , Xenograft Model Antitumor Assays
OBJECTIVES: Triple-negative breast cancer (TNBC) is well known for its strong invasiveness, rapid recurrence and poor prognosis. Immunotherapy, including chimeric antigen receptor-modified T (CAR-T) cells, has emerged as a promising tool to treat TNBC. The identification of a specific target tumor antigen and the design of an effective CAR are among the many challenges of CAR-T therapy. METHODS: We reported that epidermal growth factor receptor (EGFR) is highly expressed in TNBC and consequently designed an optimal third generation of CAR targeting EGFR. The efficacy of primary T lymphocytes infected with EGFR CAR lentivirus (EGFR CAR-T) against TNBC was evaluated both in vitro and in vivo. The signalling pathways activated in tumor and EGFR CAR-T cells were revealed by RNA sequencing analysis. RESULTS: Third-generation EGFR CAR-T cells exerted potent and specific suppression of TNBC cell growth in vitro, whereas limited cytotoxicity was observed towards normal breast epithelial cells or oestrogen receptor-positive breast cancer cells. This capability was further demonstrated in vivo in a xenograft mouse model, with minimal off-tumor cytotoxicity. Mechanistically, in vitro stimulation with TNBC cells induced the expansion of naïve-associated EGFR CAR-T cells and enhanced their persistence. Furthermore, EGFR CAR-T cells activated the interferon γ, granzyme-perforin-PARP and Fas-FADD-caspase signalling pathways in TNBC cells. CONCLUSION: We demonstrate that EGFR is a relevant immunotherapeutic target in TNBC, and EGFR CAR-T exhibits potent and specific antitumor activity against TNBC, suggesting the potential of this third-generation EGFR CAR-T as an immunotherapy tool to treat TNBC in the clinic.
