Citations

BET bromodomain inhibitors are efficacious as anti-inflammatory compounds, but clinical translation is hampered by dose-limiting toxicities from non-selective inhibition. We herein report compounds 16 and 26, based on previously reported 4-methyl-1,2,3-triazole-based inhibitor, DW34. 16 has a BRD4 BD1 Ki of 10 nM, and up to 13–38-fold selectivity for BRD4 BD1 over other BD2 domains, and an improved hERG affinity of 4.24 µM over DW34. 26, A biscyclopropyl analog of 16 showed further enhancement of BD1 selectivity over BD2 (26–203-fold), but exhibited more potent hERG inhibition. Compound 16 had improved liver and plasma clearance over DW34, with good biodistribution to the liver, and had an intracellular target-engagement affinity Ki of 92 nM, with reduction of Ccl2, Cxcl1, AST, and ALT in in vitro and in vivo mouse models of liver inflammation. 16 represents a useful lead for treating inflammatory liver diseases, with improved specificity, drug-like properties, and reduced hERG affinity.

Abstract Introduction Cancer immunotherapy has transformed the treatment landscape by leveraging the immune system to eradicate malignant cells. Among immune effectors, CD8+ T cells play a pivotal role in tumor clearance through recognition and destruction of cancer cells. To effectively counter the rapid proliferation and continuous evolution of cancer, robust effector function of cytotoxic CD8+ T cells is essential. The expression of effector genes in CD8+ T cells is tightly regulated, ensuring that cytotoxic molecules are expressed only at appropriate times and in relevant contexts. We previously reported that upregulation of the gene NKG7 following immunotherapy is critical for clinical response and demonstrated that NKG7 expression is both necessary and sufficient for T cell cytotoxicity. However, the mechanisms governing NKG7 expression in CD8+ T cells remain poorly defined. Methods We performed a cytokine screen to identify potential drivers of NKG7 expression at both the transcriptional and protein levels in cell lines and healthy donor peripheral blood mononuclear cells (PBMC). We employed quantitative RT-PCR, flow cytometry, and Western blotting to determine and validate the expression level of NKG7 and relevant signal molecules. We validated the signal pathways identified in single cell RNAseq data analysis in PD-1 therapy-responsive CD8+ T cells. Results In PBMC derived CD8+ T cells, we found that IL-2 increases NKG7 mRNA levels, correlating with a dose dependent increase in NKG7 protein expression. IL-2—induced NKG7 expression is associated with elevated levels of T-bet, a transcription factor essential for effector T cell differentiation. Consistent with this, scRNA-seq analysis revealed enrichment of the IL-2 signaling pathway in CD8+ T cells responsive to anti—PD-1 therapy. Conclusion Our results suggest that NKG7 is upregulated by IL-2 and T-bet signaling. Due to the critical role of this signaling axis during effector CD8+ T cell differentiation, NKG7 expression may be determined during this phase. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract In both type 1 and type 2 diabetes (T1D and T2D), insulin-producing β cells undergo progressive dysfunction due to inflammation, leading to impaired glucose responsiveness, dedifferentiation, and cell loss. While bile acid (BA) dysregulation under diabetic conditions is known to influence metabolic and inflammatory pathways, its mechanistic role in β cell regulation remains incompletely defined 1–3 . Here we show that bile acid sensor Farnesoid X receptor (FXR) and Bromodomain and Extra-Terminal motif (BET) signaling cooperatively regulates β cell inflammatory response and β cell identity. We identified the physiological protein-protein interaction between FXR and the bromodomain-containing protein 4 (BRD4) as a regulatory axis that protects against β cell dysfunction. We show that FXR activation by Fexaramine (Fex) together with BRD4 inhibition by JQ1 synergistically suppressed IL-1β-induced inflammation while also improving β cell identity and insulin secretion in both db/db model and high-fat diet (HFD) plus multi low-dose streptozotocin (MLD-STZ) model of diabetes. Importantly, this cooperative effect is abolished in β cell–specific FXR knockout (βFXRKO) mice, establishing that FXR is required for the functional synergy between these pathways in vivo . Mechanistically, structure-guided modeling and mutational analyses identified a direct interaction between FXR and the BD2 domain of BRD4, depending on specific lysine acetylation sites. Additionally, inhibition of the BD2 domain of BET combined with FXR activation markedly improved β cell survival in human T1D and T2D models established from human pluripotent stem cell (hPSC)-derived islet-like organoids (HILOs). Collectively, these findings establish a BA–bromodomain axis as a transcriptional interface linking metabolic signaling and chromatin regulation, and highlight FXR–BET targeting as a promising strategy to counter progressive β cell failure in diabetes.

Abstract O.A. Shariq: None. K.E. Lines: None. K.A. English: None. I. Rioja: Employee; Self; GlaxoSmithKline. A. Selberherr: None. H. Ferry: None. A.L. Oberg: None. R. Prinjha: Employee; Self; GlaxoSmithKline. D. Richards: None. R.V. Thakker: None. Background: Effective treatments for metastatic pancreatic neuroendocrine tumors (PNETs) are scarce. We previously showed that inhibiting bromodomain and extra-terminal (BET) epigenetic reader proteins via non-selective targeting of their two bromodomains (BD1 and BD2) reduces proliferation in preclinical PNET models. However, the clinical relevance of BET proteins in PNETs and functional differences between BD1/BD2 are unclear. Here, we characterised BET expression in human PNETs and performed efficacy and mechanistic studies using next-generation BD1- (BD1i) and BD2-selective (BD2i) BET inhibitors. Methods: BET expression was evaluated by immunohistochemistry in functioning and non-functioning human PNETs (n=52) vs normal adjacent islets (n=18). RNA sequencing (RNA-seq) and gene set enrichment analyses (GSEA) were performed in an independent PNET cohort (n=46) to identify biological pathways upregulated in metastatic vs non-metastatic tumors. Effects of BD1i and BD2i on viability, apoptosis, and cell cycle progression were assessed in three PNET cell lines (BON1, QGP1, MIN6). Early (6h) and late (48h) transcriptional responses to BET inhibition were examined in vitro using nascent RNA-seq (SLAM-seq) and standard RNA-seq, respectively. Epigenomic responses were explored using chromatin immunoprecipitation sequencing (ChIP-seq) for the histone mark H3K27ac. Effects of BD1i and BD2i on viability were evaluated in ex vivo neoplastic pancreatic islets derived from Men1L/L/RIP2-Cre mice. Results: BET proteins (BRD2, BRD3 and BRD4) were overexpressed by 1.5-2.5-fold in human PNETs vs islets (p<0.01). GSEA showed enrichment of BET-regulated gene sets in metastatic tumors (normalized enrichment score 1.31, adjusted p=0.04). BD2i marginally affected cell viability (IC50 55-90 µM), apoptosis, and cell cycle profile, while BD1i had a moderate impact on all phenotypes (IC50 1-1.5 µM). Synergistic effects were observed when BD1i+BD2i were combined (IC50 0.2-0.5 µM). RNA-seq and SLAM-seq revealed transcriptional changes that were minimal with BD2i (n=73 and n=10 differentially expressed genes, respectively), modest with BD1i (n=354 and n=60 genes), and greatest with BD1i+BD2i combined (n=1739 and n=375 genes). ChIP-seq showed a reduction in H3K27ac enrichment at promoters and super-enhancers of genes involved in BET protein-regulated pathways (e.g. cell cycle progression), with the greatest decrease observed with BD1i+BD2i combined. In ex vivo cultured neoplastic islets, viability was reduced by <30% with BD2i (p<0.005), ∼40% with BD1i (p<0.0001), and ∼60% with BD1i+BD2i (p<0.0001). Conclusions: Our studies confirm BET proteins as clinically-relevant targets in human PNETs and show that dual (BD1+BD2) inhibition has synergistic efficacy. These findings have important implications for the clinical application of next-generation epigenetic modulators. Sunday, June 2, 2024

BET bromodomain inhibitors demonstrate significant promise as anti-inflammatory agents targeting a variety of inflammatory disorders. However, clinical data has demonstrated that the administration of non-selective BET bromodomain inhibitors has led to significant dose-limiting toxicity in clinical settings. More selective inhibitors of the second BET bromodomains, referred to as pan-D2 inhibitors, are better tolerated, however their efficacy varies significantly depending on disease indication. Here, we use three orally bioavailable inhibitors, 1–3, that are either selective for the first N-terminal bromodomain of BRD4 (BRD4-D1 selective), or pan-D1 biased, for assessing their cellular and in vivo efficacy and safety profile in two models of inflammatory liver disease. We compare their effects to known pan-D2 and pan-BET inhibitors. Our results show that pan-D1 biased inhibitor, 3, is as efficacious as the pan-BET inhibitor I-BET151 in reducing inflammation from LPS-induced and drug-induced toxicity, whereas pan-D2 inhibitors are less effective. BRD4-D1 selective inhibitors, 1 and 2, are also efficacious; however, new inhibitors with improved cellular target engagement will be necessary to more thoroughly assess their effects. Finally, BRD4-D1 selective inhibitors are better tolerated in a preclinical model of thrombocytopenia than pan-D1 biased inhibitors, while gastrointestinal (GI) toxicity may be a BRD4-driven effect. These results highlight the importance of assessing specific BET protein and bromodomain functions due to their varying roles in diverse disease models.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer 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 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Abstract Purpose: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. Experimental Design: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. Results: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. Conclusions: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.