birabresib (OTX015) / Merck (MSD) - LARVOL DELTA

TMEM132A: a novel susceptibility gene for lung adenocarcinoma combined with venous thromboembolism identified through comprehensive bioinformatic analysis. (PubMed, Front Oncol) - "Pharmacological sensitivity analysis indicated that TMEM132A may serve as a potential target for the therapeutic agents birabresib and abemaciclib. TMEM132A demonstrates diagnostic utility as a predictive biomarker for VTE occurrence in LUAD, suggesting its potential role as a susceptibility gene in this patient cohort."

Journal • Cardiovascular • Lung Adenocarcinoma • Lung Cancer • Non Small Cell Lung Cancer • Oncology • Solid Tumor • Venous Thromboembolism • TIMP1

Bromodomain and Extra-Terminal Family Proteins BRD2, BRD3, and BRD4 Contribute to H19-Dependent Transcriptional Regulation of Cell Adhesion Molecules, Modulating Metastatic Dissemination Program in Prostate Cancer. (PubMed, Noncoding RNA) - "Our study demonstrates that BRD4 epigenetically regulates the H19-mediated transcriptional control of adhesion molecules involved in collective migration and metastatic dissemination. Importantly, these effects are independent of AR status, suggesting that targeting the H19/BRD4 axis may represent a promising therapeutic avenue for advanced PCa."

Journal • Genito-urinary Cancer • Oncology • Prostate Cancer • Solid Tumor • AR • BRD2 • BRD3 • BRD4 • CDH1 • H19

Superior preclinical efficacy of BRG1/BRM inhibitor combined with BET inhibitor or decitabine against MECOM-rearranged AML (AACR 2025) - "Finally, as compared to treatment with each drug alone or vehicle control, in PD xenograft (PDX) models of AML cells with MECOM-r, co-treatment with FHD-286 and decitabine or OTX015 significantly reduced AML burden and improved overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight the promise of FHD-286 treatment alone and in rational combinations in exerting significant anti-AML efficacy against cellular models of AML, especially those with MECOM-r and EVI1 overexpression."

Preclinical • Acute Myelogenous Leukemia • Oncology • CD93 • CDK4 • GATA2 • ITGAM • KIT • MECOM • MYC • PLK1 • SMARCA2 • SMARCA4

ASXL1 mutations in AML are associated with a distinct epigenetic state that results in vulnerabilities to epigenetic-targeted agents (AACR 2025) - "Notably, compared to parental cells, OCIAML3 ASXL1 Y591* cells exhibited reduced sensitivity to standard anti-AML, chemotherapeutic agents, including cytarabine, etoposide and daunorubicin. Our findings confirm the previously published discovery that the presence of mtASXL1 confers an increased sensitivity to BETi inhibitors, e.g., pelabresib or birabresib...Importantly, in the NSG mice engrafted with OCIAML3 ASXL1 Y591*, monotherapy with NEO2734, pelabresib or SEL120 significantly reduced AML burden. Collectively these findings highlight previously uncharacterized biologic effects of the presence of mtASXL1 and support the rationale for further evaluating AML therapies incorporating BETi, HAT-BETi or mediator-kinase inhibitor."

Acute Myelogenous Leukemia • Hematological Malignancies • Oncology • ASXL1 • AURKA • BAP1 • CDK9 • E2F1 • EP300 • FZD5 • HOXA9 • MEIS1 • MYC • PLK1 • SPI1 • TCF7L2

High-risk neuroblastoma: ATRX and TERT as prognostic markers and therapeutic targets. Review and update on the topic. (PubMed, Rev Esp Patol) - "We highlight the promising results of the clinical trial involving the combination of adavosertib and irinotecan, which encourages further clinical trials with adavosertib targeting NB with ATRX mutations. Preclinical results with BET inhibitors (OTX015 and AZD5153) and with 6-thio-2'-deoxyguanosine, targeting NB with TERT mutations, are promising. Both represent future therapeutic targets, emphasizing the need to prioritize research using these models."

Biomarker • Journal • Review • CNS Tumor • Neuroblastoma • Oncology • Solid Tumor • ATRX • MYCN • TERT

Identifying 'Druggable' Targets and Preclinically Overcoming Non-Genetic/Adaptive Resistance to Menin Inhibitors in AML with MLL1r or mtNPM1 (ASH 2024) - "These MITR cells exhibited cross-resistance to other MIs, including ziftomenib and DS1594b...Notably, in a luciferized MLL1r MITR AML PDX model, compared to treatment with each agent alone, co-treatment with FHD-286 and SNDX-5613 or OTX015 for 8-weeks yielded significantly superior survival of the NSG mice (p < 0.05). These findings demonstrate that co-treatment with FHD-286 overcomes in vitro and in vivo MI-resistance, while significantly improving in vivo efficacy of BETi in the cell-line xenograft and PDX models of MITR AML cells. They also show that combinations of epigenetically targeted agents may be effective in MI-sensitive or -resistant AML with MLL1r or mtNPM1."

IO biomarker • Preclinical • Acute Myelogenous Leukemia • Hematological Malignancies • Leukemia • Oncology • BCL2 • BRD4 • CDK6 • CLEC12A • CREBBP • EP300 • FLT3 • HOXA9 • IGF2BP2 • MEF2C • MEIS1 • NPM1 • PBX3 • SMARCA4

Evaluation of the Lethal Activity and Its Mechanism of Tasquinimod in Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase (ASH 2024) - "Importantly, cotreatment with TQ (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax, induced synergistic lethality in advanced MPN-BP cells, represented by delta synergy scores of >1.0 (by the ZIP method)...Co-treatment with TQ and RGFP966 (HDAC3i) induced synergistic lethality in post-MPN sAML cells...These findings demonstrate the pre-clinical efficacy of TQ and/or JAKi or BETi or with novel agents identified here in advanced MPN and MPN-AML cells. They also create the rationale to further interrogate the pre-clinical efficacy of the TQ-based combinations against cellular models of advanced MPN."

IO biomarker • Metastases • Fibrosis • Hematological Malignancies • Immunology • Leukemia • Myelofibrosis • Myeloproliferative Neoplasm • Oncology • BCL2 • BCL2L1 • CALR • CCL2 • CCND1 • CD123 • CD33 • CD34 • CD99 • CDK6 • CDKN1A • CLEC12A • CXCL12 • CXCL8 • CXCR4 • HDAC3 • IL1A • IL3RA • IL6 • ITGAM • JAK2 • MPO • MYC • NLRP3 • NSD2 • S100A8 • S100A9 • TERT • TLR4 • TNFA • TP53

ASXL1 Mutations in AML Are Associated with a Distinct Epigenetic State Which Highlights Vulnerabilities to Specific Epigenetic-Targeted Agents (ASH 2024) - "Notably, compared to parental cells, OCIAML3 ASXL1 Y591* cells exhibited reduced sensitivity to standard anti-AML chemotherapeutic agents, including cytarabine, etoposide and daunorubicin...Our findings also demonstrate and confirm that mtASXL1-expressing AML cells exhibited increased sensitivity to BETi (pelabresib or birabresib)...Importantly, in the NSG mice engrafted with luciferized OCIAML3 ASXL1 Y591*, monotherapy with NEO2734 (5 mg/kg QD), pelabresib (30 mg/kg QD) or SEL120-34A (40 mg/kg QD), compared to vehicle control, significantly reduced AML burden. Collectively these findings highlight previously uncharacterized biologic effects of the presence of mtASXL1 and support the rationale for further evaluating AML therapies incorporating BETi, HAT-BETi or inhibitor of mediator kinase."

Acute Myelogenous Leukemia • Hematological Disorders • Hematological Malignancies • Immunology • Oncology • Targeted Protein Degradation • ASXL1 • AURKA • BAP1 • BRD4 • CD14 • CDK9 • E2F1 • FZD5 • HOXA9 • MEIS1 • MYC • NDUFA2 • PLK1 • SPI1 • TCF7L2

An In Situ Tumor Vaccine Against Triple Negative Breast Cancer. (SABCS 2024) - "The efficacy of OTX015, the top hit from the screen, in combination with RT was validated and compared to each treatment component alone and a chemo-immunotherapy regimen of doxorubicin + paclitaxel + PD1 inhibition. Using an in vivo drug screen, we identified that a short course of tumor-directed RT in combination with pharmacological BET inhibition serves as an in situ tumor vaccine. Our results support that RT + OTX015 offers superior tumor control relative to the current standard-of-care chemo-immunotherapy regimen and elicits immunological memory in a cytotoxic T cell dependent fashion in a pre-clinical model of TNBC."

Breast Cancer • Oncology • Solid Tumor • Triple Negative Breast Cancer • CD8

The KLF16/MYC feedback loop is a therapeutic target in bladder cancer. (PubMed, J Exp Clin Cancer Res) - "Our study revealed the crucial role of the KLF16/MYC regulatory axis in modulating tumor growth and chemotherapy sensitivity in BLCA, suggesting that combining bromodomain inhibitors, such as OTX015 or ABBV-744, with DDP or gemcitabine could be a promising therapeutic intervention for BLCA patients."

Journal • Bladder Cancer • Genito-urinary Cancer • Oncology • Solid Tumor • DUSP1

BET inhibitor and CDK4/6 inhibitor synergistically inhibit breast cancer by suppressing BRD4 stability and DNA damage repair. (PubMed, Transl Oncol) - "We provide the evidence that CDK4/6 inhibitor LY2835219 plus BRD4 inhibitor OTX-015 synergistically inhibits both ER positive and triple-negative breast cancer cells growth in vitro and in vivo. This instability of BRD4 protein in turn enhances the anti-tumor effect of CDK4/6 inhibitor by suppressing transcription of DNA damage repair gene RAD51, and synergistically promotes γ-H2AX accumulation and DNA double-strand breaks. Overall, we demonstrated the potential combined therapeutic value of CDK4/6 and BRD4 inhibitors and elucidated the mechanisms, which may provide a new rational approach for breast cancer patients."

Journal • Breast Cancer • Hormone Receptor Breast Cancer • Hormone Receptor Positive Breast Cancer • Oncology • Solid Tumor • Triple Negative Breast Cancer • BRD4 • ER • RAD51

Preclinical efficacy of CDK7 inhibitor-based combinations against myeloproliferative neoplasms transformed to AML. (PubMed, Blood) - "Co-treatment with SY-5609 and ruxolitinib was synergistically lethal in HEL, SET2 and PD post-MPN-sAML cells.  A CRISPR screen in SET2 and HEL cells revealed BRD4, CBP and p300 as co-dependencies with SY-5609 treatment. Accordingly, co-treatment with SY-5609 and the BETi OTX015 or pelabresib or with the CBP/p300 inhibitor GNE-049 was synergistically lethal in MPN-sAML cells (including those exhibiting TP53 loss). Finally, in the HEL-Luc/GFP xenograft model, compared to each agent alone, co-treatment with SY-5609 and OTX015 reduced post-MPN-sAML burden and improved survival without inducing host toxicity. These findings demonstrate promising preclinical activity of the CDK7i-based combinations with BETi or HATi against advanced-MPNs, including post-MPN-sAML."

Journal • Preclinical • Acute Myelogenous Leukemia • Myeloproliferative Neoplasm • Oncology • BCL2L1 • BRD4 • CASP3 • CASP9 • CCND1 • CDK4 • CDK6 • CDKN1A • ITGAM • MYC • PIM1 • TP53

Unraveling the molecular mechanism of aqueous extract of Sargentodoxa cuneata against ulcerative colitis from serum metabolomics and bioinformatics perspectives. (PubMed, J Chromatogr B Analyt Technol Biomed Life Sci) - "In addition, based on "metabolite-target-disease" network, the serum metabolites cholylleucine, 9,10,13-TriHOME, birabresib, anthramycin methyl ether, trans-hexadec-2-enoyl carnitine, and lucidumol A were found to have the therapeutic potential for UC...AESc corrects serum metabolic disturbances in UC mice, and multiple serum metabolites have therapeutic potential for UC. AESc may treat UC by regulating biological processes such as lipid metabolism, amino acid metabolism, thereby restoring normal physiological function of the intestine."

Journal • Atherosclerosis • Cardiovascular • Dermatology • Dyslipidemia • Gastroenterology • Gastrointestinal Disorder • Immunology • Inflammatory Arthritis • Inflammatory Bowel Disease • Metabolic Disorders • Rheumatoid Arthritis • Rheumatology • Ulcerative Colitis

The bromodomain and extraterminal domain family proteins, BRD2, BRD3 and BRD4, are enrolled in H19-dependent transcriptional regulation of cell adhesion molecules and modulate metastatic dissemination program in prostate cancer (EORTC-NCI-AACR 2024) - "In vitro effect was evaluated by gene expression and proliferation before and after treatment with a specific inhibitor of the BET family of acetyl-lysine recognition motifs, JQ1, with the BET Inhibitor OTX015, with the BET degrader dBET6 or DMSO and enantiomer R-JQ1 as negative control. These data were confirmed in OSCs Conclusions These finding underscore BET proteins involvement in regulation H19/cell adhesion molecules suggesting that BETs might potentially modulate metastatic dissemination program in the early stages of PCa progression, regardless AR status. The opportunity to early modulate this mechanism might prevent establishment of collective metastatic phenotype, thus representing a logical foundation for targeted therapy"

Metastases • Genito-urinary Cancer • Oncology • Prostate Cancer • Solid Tumor • BRD2 • BRD3 • BRD4 • CDH1 • H19

Dual targeting of DNA damage repair pathway by BET and CDK4/6 inhibitors synergistically inhibits breast cancer (EORTC-NCI-AACR 2024) - " According to the proliferation inhibition data of 40 breast cancer cells treated with CDK4/6 inhibitor palbociclib in GDSC (https://www.cancerrxgene.org/) and CCLE (https://depmap.org/portal/) databases, we found that BRD4 expression level was in direct proportion to the IC50 values, suggesting that inhibition of BRD4 may sensitize CDK4/6 inhibitors. Cell proliferation assay and colony formation assay both demonstrated that CDK4/6 inhibitors combined with BRD4 inhibitor OTX-015 exerted marked synergistic antitumor effects in ER+ breast cancer and TNBC cells... Our study strongly suggests that CDK4 inhibition-induced instability of BRD4 enhances the anti-tumor effect of CDK4/6 inhibitors by suppressing transcription of DNA damage repair gene RAD51, and synergistically promotes γ-H2AX accumulation and DNA double-strand breaks. We demonstrate the potential combined therapeutic value of CDK4/6 and BRD4 inhibitors, which may provide a new approach for ER+ and TNBC patients."

Breast Cancer • Estrogen Receptor Positive Breast Cancer • Hormone Receptor Breast Cancer • Hormone Receptor Positive Breast Cancer • Oncology • Solid Tumor • Triple Negative Breast Cancer • BRCA1 • BRD4 • CCNA2 • CHEK1 • ER • RAD51

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer. (PubMed, Cancers (Basel)) - "The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer."

Journal • Bladder Cancer • Genito-urinary Cancer • Oncology • Solid Tumor • Urothelial Cancer • CDKN1A

Sequential Inhibition of PARP and BET as a Rational Therapeutic Strategy for Glioblastoma. (PubMed, Adv Sci (Weinh)) - "Through unbiased transcriptomic and proteomic sequencing, it is discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, beyond the previously reported impact of BET inhibition on homologous recombination repair...In GBM cells with elevated baseline replication stress, the sequential regimen exhibits comparable efficacy to concurrent treatment, protecting normal glial cells with lower baseline replication stress from DNA toxicity and subsequent death. This study provides compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy."

Journal • Brain Cancer • CNS Tumor • Glioblastoma • Oncology • Solid Tumor • Transplantation • HRD

Pharmacological targeting of histone H3K27 acetylation/BRD4-dependent induction of ALDH1A3 for early-phase drug tolerance of gastric cancer. (PubMed, Cancer Res Commun) - "ChIP-PCR and ChIP-seq analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-Fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the BET inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth...Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment."

Journal • Gastric Cancer • Gastrointestinal Cancer • Oncology • Solid Tumor • ALDH1A3 • BRD2 • BRD4

Sequential inhibition of PARP and BET as a rational therapeutic strategy for glioblastoma multiforme (AACR 2024) - "Through unbiased transcriptomic and proteomic sequencing, and employing Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA), and Transcriptome-Proteome Correlation Analysis, we discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, extending beyond the previously known impact of BET inhibition on homologous recombination repair...Our findings provide a broader and deeper understanding of the synergistic interaction between BETi and PARPi than previously recognized. This study offers compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy."

Late-breaking abstract • Brain Cancer • CNS Tumor • Glioblastoma • Oncology • Solid Tumor • HRD

Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1 (AACR 2024) - "In vivo treatment with FHD-286 and OTX015 or SNDX-5613 significantly reduced the AML burden in mice bearing OCI-AML3-MITR xenografts. These findings underscore preclinical activity of epigenetically-targeted agent-based combinations and highlight their promise in overcoming MI resistance in AML with MLL1r or mtNPM1."

Combination therapy • IO biomarker • Acute Myelogenous Leukemia • Hematological Malignancies • Leukemia • Oncology • BCL2 • BRD4 • CDK6 • CLEC12A • CREBBP • EP300 • FLT3 • HOXA9 • IGF2BP2 • MEF2C • MEIS1 • NPM1 • PBX3 • SMARCA2 • SMARCA4

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models. (PubMed, Int J Mol Sci) - "RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus."

Journal • Gastrointestinal Cancer • Hepatology • Oncology • Pancreatic Cancer • Pancreatic Ductal Adenocarcinoma • Solid Tumor • BRD4 • MYC

Notable Efficacy of Co-Treatment with FHD-286, a Dual BRG1/BRM ATP-Ase Inhibitor, and Menin or BET Inhibitor, Decitabine or Venetoclax Against AML with MLL-r or Mutant NPM1 (ASH 2023) - "BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the multi-protein, ATP-dependent, chromatin remodeling BAF complexes that regulate gene transcription. Finally, co-treatment with FHD-286 and OTX015 or SNDX-5613 (oral gavage) was significantly more effective than each drug alone in reducing the AML burden and overall survival of mice engrafted with a separate PDX model of AML cells with mtNPM1 and FLT3-ITD, without significant toxicity. These findings demonstrate the pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against AML with MLL1r or mtNPM1."

Clinical • IO biomarker • Acute Myelogenous Leukemia • BCL2 • BRD4 • CASP3 • CD123 • CD33 • CD99 • CDK4 • CDKN1A • CEBPA • CLEC12A • FLT3 • HEXIM1 • IL3RA • ITGAM • MCL1 • MEF2C • MYC • NPM1 • PBX3 • PLK1 • SMARCA2 • SMARCA4

Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML (ASH 2023) - "Treatment with JAK inhibitor (JAKi), e.g., ruxolitinib, venetoclax or hypomethylating agents alone or in combination are ineffective in improving the poor survival in MPN-sAML...Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and clinical grade SY-5609, dose-dependently (20 to 250 nM) increased % G1 while reducing the % of cell-cycle S phase SET2 and HEL cells...SY-5609 treatment also exerted synergistic lethality with the BETi pelabresib or BD2-selective BETi ABBV-744 or the CBP/p300 inhibitor GNE-049 in MPN-sAML cells...Additionally, compared to each drug or vehicle control, co-treatment with SY-5609 and OTX015 (30 mg/kg/day by oral gavage) reduced more MPN-sAML burden and significantly improved survival in a HEL-Luc/GFP xenograft model without inducing toxicity. These findings demonstrate promising preclinical activity of CDK7 inhibition against the cellular models of MPN-sAML, supporting the rationale to..."

Metastases • Preclinical • Acute Myelogenous Leukemia • Hematological Malignancies • Leukemia • Myeloproliferative Neoplasm • Oncology • ASXL1 • AURKA • BCL2L1 • BRD4 • CALR • CASP9 • CCND1 • CD123 • CD34 • CD99 • CDK1 • CDK4 • CDK6 • CDK9 • CDKN1A • CLEC12A • HEXIM1 • IL3RA • ITGAM • JAK2 • MCL1 • MYC • PIM1 • PLK1 • RUNX1 • SRSF2 • STAT5 • TET2 • TGFB1

Development and Efficacy of a Novel Bromodomain and Extraterminal Domain Degrader K-256 in MYC/BCL2-Related Lymphoma (ASH 2023) - "The GI50 of K-256 in SU-DHL4 and SU-DHL6 was 12.8 nM and 7.50 nM, respectively, and K-256 induced cell death at lower concentrations than existing drugs (vs. JQ1, OTX-015, and ABBV-075, p < 0.0001; vs. dBET6 and ARV-771, p < 0.01)...As expected, the combination of K-256 with venetoclax also demonstrated synergistic effects in PDX cells, similar to those observed in cell lines (CI of 0.515 to 0.762 for inhibiting cell proliferation; 0.085 to 0.995 for inducing apoptosis)... The novel BET degrader, K-256, bound to BET proteins at lower concentrations than existing BET inhibitors and degraders, strongly suppressing MYC expression, primarily via BRD4 degradation. Additionally, K-256 demonstrated superior therapeutic effects in MYC/BCL2-related PDX models both in vitro and in vivo, suggesting that this novel drug could be a promising therapeutic agent for MYC/BCL2-related lymphoma. Its translation to future clinical applications warrants further consideration."

Clinical • IO biomarker • Diffuse Large B Cell Lymphoma • Hematological Malignancies • Lymphoma • Non-Hodgkin’s Lymphoma • Oncology • BCL2 • BRD2 • BRD3 • BRD4 • BRDT • MYC

DW71177: A novel [1,2,4]triazolo[4,3-a]quinoxaline-based potent and BD1-Selective BET inhibitor for the treatment of acute myeloid leukemia. (PubMed, Eur J Med Chem) - "DW-71177 effectively inhibits oncogenes comparable to the pan-BET inhibitor OTX-015, but with a milder impact on housekeeping genes. It efficiently blocks cancer-associated transcriptional changes by targeting genes that are highly enriched with BRD4 and histone acetylation marks, suggesting that BD1-selective targeting could be an effective and safe therapeutic strategy against leukemia."

Journal • Acute Myelogenous Leukemia • Hematological Malignancies • Leukemia • Oncology • BRD4