BET Family Proteins Research Articles

400 Preclinical studies support combined inhibition of BET family proteins and histone deacetylases as epigenetic therapy for cutaneous T-cell lymphoma

400 Preclinical studies support combined inhibition of BET family proteins and histone deacetylases as epigenetic therapy for cutaneous T-cell lymphoma

Phthalimide conjugations for the degradation of oncogenic PI3K

PI3K/Akt/mTOR pathway is crucial for carcinogenesis and its inhibitors have made a great progress in cancer treatment. However, there is still a great developing space for PI3K inhibitors as the acquired drug resistance hindered their application in clinical. Proteolysis-targeting chimeras (PROTACs) with the potential to handle the challenges faced in drug development could be an alternative therapeutic strategy. Moreover, the past two years have witnessed remarkable advances in the development of phthalimide conjugation as a strategy for the degradation instead of inhibition of the targets, including BET family proteins, Sirtuin 2, CDK 9, Smad 3, and BCR-ABL proteins. Here, we designed and synthesized a series of potential small molecular PROTACs for the degradation of PI3K. Four compounds induced remarkable PI3K degradation and down-regulated the phosphorylation of Akt, S6K and GSK-3β in liver cancer cells HepG2. Furthermore, the representative compound D proved to inhibit tumor cells proliferation by the induction of autophagy instead of apoptosis or cell cycle arrest.

Binding Assays for Bromodomain Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory Disease.

Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates. Foremost among the efforts to target bromodomains are those aimed at identifying compounds that interact with the bromodomain and extra-terminal domain (BET) family of bromodomain-containing proteins (BRD2, BRD3, BRD4, and BRDT). Inhibitors of these proteins are under clinical development for a large variety of oncologic indications. Described in this unit are several assays to assess the binding potency and selectivity within the BET protein family. Included are AlphaScreen, fluorescence polarization, and thermal shift assays. The strengths and weaknesses of each assay are discussed. © 2018 by John Wiley & Sons, Inc.

Abstract P1-09-02: Bromodomain inhibitors for the treatment of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor–positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 (uniquely among BRD family members) was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression (and not other BRD members) was associated with poor recurrence-free survival (p<0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was predominantly Bcl2-dependent. Combination of JQ1 and the Bcl2-inhibitor venetoclax (ABT-199) synergistically killed MDA-MB134V1 cells compared to treatment with JQ1 alone, while combination with the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263) added further synergy. With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice. These results should now be investigated in vivo before a prospective clinical trial. This material is based upon works supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL" and the SFi CDA Award 15/CDA/3438 Citation Format: O'Connor DP, Walsh L, Fan Y, Tarrant F, Chin S-F, Schouten P, Caldas C, Bernards R, Ni Chonghaile T, Gallagher WM. Bromodomain inhibitors for the treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-02.

BET Family Protein BRD4: An Emerging Actor in NFκB Signaling in Inflammation and Cancer.

NFκB (Nuclear Factor-κ-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate regulatory elements called “super enhancers”. BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions.

Abstract IA15: Multiple targetable pathways for epigenetic therapy

Abstract Epigenetic reprogramming erases the malignant potential of some transformed cells and the aim of epigenetic therapy is to achieve some degree of reprogramming in-vivo by targeting epigenetic writers/readers. The resultant gene expression response includes reactivation of tumor-suppressor genes and differentiation pathways as well as activation of an immune response program, all of which contribute to eventual clinical activity. The DNA methyltransferase inhibitors azacitidine, decitabine and guadecitabine induce clinically meaningful remissions or improvements in 30-60% of patients with myeloid leukemias and prolong survival compared to standard approaches including chemotherapy. This therapy is accompanied by (i) global and gene specific demethylation, (ii) reactivation of silenced gene expression, (iii) delayed responses that correlate with early epigenetic reactivation and result in clonal elimination and (iv) eventual relapses and resistance the mechanisms of which appear to involve genetic evolution in some cases. Multiple other classes of epigenetic modifiers have now been targeted therapeutically including histone acetylases and deacetylases, histone methylases and demethylases, and readers of the epigenetic marks such as the BET family of proteins. Clinical trials of these drugs are ongoing and have already shown impressive clinical activity in selected cases. Using a parallel approach, we have shown that gene expression directed unbiased phenotypic screens can also uncover new and repurposed drugs for epigenetic therapy (e.g. cardiac glycosides, arsenic trioxide, natural products), and unbiased genetic screens have suggested novel targets as well (SWI/SNF proteins, CHD4 etc.). An even greater promise of epigenetic therapy lies in sensitization to other treatment modalities such as chemotherapy and immunotherapy. Ultimately, epigenetic therapy will be integrated into comprehensive treatment plans aimed long term disease control in many human malignancies. Citation Format: Jean-Pierre Issa. Multiple targetable pathways for epigenetic therapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA15.

Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma is an infiltrative, often high-grade glioma of the brainstem that is not amenable to surgical resection. The current treatment of DIPG by radiation therapy showed dramatically improvement of patient’s condition, however, the tumor recurs rapidly. More and more studies are focused on the genetic and epigenetic drivers of DIPGs, which may provide more and more novel therapy target for DIPG. EZH2 has been proved to be a potential therapeutic target for H3K27M-mutant pediatric gliomas recently. Meanwhile, BET family protein is a hot target in many different types of cancers, including DIPG. In this study, we performed the treatment of both EZH2 and BET inhibitor for DIPG cells.ResultsThe combination of these two inhibitors exhibited better inhibition of the tumor growth both in vitro and in vivo compared to use the inhibitor individually. This inhibition was performed by blocking the proliferation and promoting the cell apoptosis. Meanwhile, combination treatment of these two inhibitors would also affect the epigenetic markers which were abnormal in the tumors of the certain set of genes.ConclusionThus we provided a novel therapy strategy for clinical treatment of DIPG.

BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

We previously reported that induction of the adipocyte-specific gene adiponectin (Adipoq) during 3T3-L1 adipocyte differentiation is closely associated with epigenetic memory histone H3 acetylation on the transcribed region of the gene. We used 3T3-L1 adipocytes and Brd4 heterozygous mice to investigate whether the induction of Adipoq during adipocyte differentiation is regulated by histone acetylation and the binding protein bromodomain containing 4 (BRD4) on the transcribed region. Depletion of BRD4 by shRNA and inhibition by (+)-JQ1, an inhibitor of BET family proteins including BRD4, reduced Adipoq expression and lipid droplet accumulation in 3T3-L1 adipocytes. Additionally, the depletion and inhibition of BRD4 reduced the expression of many insulin sensitivity-related genes, including genes related to lipid droplet accumulation in adipocytes. BRD4 depletion reduced P-TEFb recruitment and histone acetylation on the transcribed region of the Adipoq gene. The expression levels of Adipoq and fatty acid synthesis-related genes and the circulating ADIPOQ protein level were lower in Brd4 heterozygous mice than in wild-type mice at 21 days after birth. These findings indicate that BRD4 regulates the Adipoq gene by recruiting P-TEFb onto acetylated histones in the transcribed region of the gene and regulates adipocyte differentiation by regulating the expression of genes related to insulin sensitivity.

Abstract 3380: Synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition in ovarian clear cell carcinoma

Abstract Introduction: Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Given that ARID1A is inactivated by mutation in over 50% of ovarian clear cell carcinomas, we pursued an ARID1A synthetic lethal screening strategy to identify druggable targets in OCCC. Experimental procedures: We performed synthetic lethal kinome short hairpin (shRNA) screens in a large panel (n=14) of OCCC cell lines having different ARID1A mutation status. Hit validation was performed with isogenic ARID1A ko cell line pairs and in (patient-derived) xenograft mouse models. Summary of the data: We show here that BRD2 inhibition is synthetic lethal with ARID1A mutation in ovarian clear cell cancer cells. Importantly, inhibiting the BET family of proteins, to which BRD2 belongs, with small molecules specifically inhibits proliferation of ARID1A mutated cell lines both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. We demonstrate that ARID1A loss leads to upregulation of the WNT ligand WNT10B, possibly causing a WNT dependency in the ARID1A mutant lines. BET inhibitors cause a reduction in WNT10B expression and WNT target genes such as MYC, JUN and WISP1, providing a potential explanation for the observed synthetic lethal interaction with ARID1A loss. Conclusions: Our study uncovered a new synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition, suggesting a new treatment strategy for ARID1A mutant ovarian clear cell carcinomas. Citation Format: Katrien Berns, Joseph J. Caumanns, E Marielle Hijmans, Annemiek Gennissen, Bastiaan Evers, Bea A. Wisman, Gert Jan Meersema, Cor Lieftink, Roderick L. Beijersbergen, Hiroaki Itamochi, Ate G. van der Zee, Steven de Jong, René Bernards. Synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3380. doi:10.1158/1538-7445.AM2017-3380

Abstract 4674: Epigenetic modulators for the treatment of invasive lobular carcinoma breast cancer

Abstract Invasive lobular carcinoma (ILC) is a breast cancer subtype comprising 10% of breast tumors. The majority of ILC (90%) are estrogen receptor (ER)-positive and therefore candidates for endocrine therapy. Unfortunately, de novo resistant to endocrine therapies occurs in 33% of women and a further 40% will relapse on treatment. Therefore, novel therapeutic targets are required for ILC. Deregulated transcription is a recurring theme in cancer, which can be due to epigenetic events. The bromdomain & extra-terminal domain (BET) family of proteins (BRD2, BRD3, BRD4, BRDT) function as chromatin readers that bind acetylated lysine residues on histones and regulate transcription. We performed RNA-Sequencing analysis on 61 primary ILC samples and found that high expression of BRD3 is associated with poor survival in ILC (log rank test, p=0.037). We validated this finding, that high expression of BRD3 is associated with poor survival, in a second cohort of 99 ILC primary samples from the METABRIC dataset (log rank test, p=0.0157). Next, we tested if ILC cell lines were sensitive to BET inhibition using the small molecule inhibitor JQ1, which inhibits all BET family proteins. JQ1 downregulates growth and survival genes in ILC cell lines including MYC, ER and BCL-XL. Pathway analysis following RNA sequencing revealed that JQ1 targets the apoptotic and Wnt signalling pathways in ILC cell lines. Interestingly, JQ1 inhibited the cell growth in all ILC cell lines tested, however apoptosis was only induced in two ILC cell lines. Furthermore, ILC cell lines which were relatively resistant to JQ1-induced apoptosis expressed both the BCL-2 and BCL-XL anti-apoptotic proteins. This led us to assess the combination of JQ1 and the BH3 mimetics, ABT-199 and ABT-263. ABT-199 is a selective small molecule inhibitor of BCL-2, whereas ABT-263 is an inhibitor of BCL-2, BCL-XL and BCL-W proteins. We found the combination of JQ1 and ABT-263, but not the combination of JQ1 & ABT-199, to be synergistic and enhance apoptosis in ILC cell lines. This is in accordance with BH3 profiling of ILC cell lines which indicated that ILC cell lines are dependent on BCL-2/ BCL-XL proteins for cell survival. As JQ1 is a pan-BET family inhibitor, we also wish to determine which BET protein is responsible for sensitivity to JQ1. Following knockdown of each BET protein in the CAMA-1 cell line we found that BRD3 and BRD4 were responsible for loss of cell viability. Future work will include determining the specific role of BRD3 in ILC and the effectiveness of the JQ1 and ABT-263 combination in vivo. Our work suggests that inhibition of BET proteins in combination with BH3 mimetics may be a rational therapeutic combination for ILC. Citation Format: Louise Walsh, Bruce Moran, Sudipto Das, Finbarr Tarrant, Philip Schouten, Suet-Feung Chin, Rene Bernards, Carlos Caldas, William Gallagher, Tríona Ní Chonghaile*, Darran O'Connor*. Epigenetic modulators for the treatment of invasive lobular carcinoma breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4674. doi:10.1158/1538-7445.AM2017-4674

Abstract 5062: Development of a first in class inhibitor of BET bromodomains and dopamine receptor 2

Abstract ConverGene has developed a first-in-class dual-active small molecule inhibitor that i) inhibits BET family of bromodomain-containing proteins, and ii) antagonizes dopamine receptor D2 (DRD2). BET protein family includes BRD4, an epigenetic reader protein that mediates expression of MYC oncogene. Thus, BRD4 is considered as a cancer therapeutic target to indirectly suppress MYC expression. In addition to being a therapeutic target for psychiatric diseases, DRD2 is emerging as a potential therapeutic target in neuroendocrine tumors, subsets of pancreatic ductal adenocarcinoma and small cell lung cancer. Our lead compound showed high activity in a binding test against BRD4 (Ki = 34 nM); exhibited high bioavailability upon oral administration; profoundly suppressed MYC expression both in vitro and in vivo; inhibited growth of AML and solid tumor cells in xenograft models; potently inhibited both isoforms of DRD2 (IC50 0.1 µM); and interfered with DRD2/β-arrestin/Akt pathway in vitro. Therefore, our BRD4/DRD2 dual-active compounds may hold promise as a novel class of therapeutics that interferes with both cancer growth and maintenance by simultaneously interfering with MYC and DRD2 pathways. We currently are investigating these dual-active compounds in multiple in vitro and in vivo models and expect to report the outcomes at the AACR Annual Meeting in 2017. Citation Format: Makoto Yoshioka, Jay Chauhan, Steven Fletcher, Jeffrey W. Strovel. Development of a first in class inhibitor of BET bromodomains and dopamine receptor 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5062. doi:10.1158/1538-7445.AM2017-5062

Abstract 1515: Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7)

Abstract Persistent androgen receptor (AR) signaling is key to the development and progression of metastatic castration resistant prostate cancer (mCRPC). This is in part due to expression of constitutively active AR splice variants like AR variant 7 (ARV7), confering resistance to current anti-androgens including enzalutamide (E) and abiraterone (A). To improve the outcome for patients with mCRPC, new therapeutic strategies to overcome AR and ARV7 oncogenic signaling are urgently required. The inhibition of co-factors modulating AR signaling are currently being investigated as novel strategies to treat mCRPC. One promising candidate is BRD4, a member of the BET protein family, that binds the AR on androgen response elements and facilitates the recruitment of the transcriptional machinery. BET inhibitors (BETi) have been shown to regulate AR and ARV7 signalling, however, the exact mechanism of ARV7 regulation remains unclear. As BETi are currently being explored in clinical trials of unstratified patients with mCRPC, we investigated their potential mechanism of action in CRPC cell lines, patient derived organoids (PDOs) and a patient derived mouse xenograft (PDX). Here we demonstrate that nuclear expression of BRD4 and ARV7 increases as patients develop resistance to E and/or A and inhibition of BRD4 by BETi is sufficient to block AR and ARV7 signalling in mCRPC. Both inhibition of BRD4 by BETi and genetic knockdown of BRD4 reduced the growth of CRPC cell lines and led to down-regulation of AR and ARV7 at the mRNA and protein level. To further investigate whether BETi is sufficient to inhibit ARV7 activity in patients with mCRPC, we treated patient derived organoids (PDOs) and a mouse xenograft (PDX) grown from metastatic biopsies of patients resistant to E and/or A with BETi. In this study 5 out of 10 PDOs were sensitive to BETi. Consistent with the cell culture experiments, BETi treatment of the PDX led to down-regulation of both ARV7 mRNA and protein expression. Mechanistically, BETi might inhibit pre-mRNA splicing of AR resulting in the observed decrease of ARV7 expression. However, RNAseq analysis of the ARV7 expressing CRPC cell line LNCaP95 demonstrated an increase in total splicing events including skipped exons, retained introns, mutually exclusive exons, alternative 5’ splice site and alternative 3’ splice site after BETi treatment. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being down-regulated by BET inhibition including one yet uncharacterized factor that is crucial for ARV7 expression in LNCaP95 cells. Based on our results we propose a model that BETi mediated inhibition of this novel ARV7-mRNA splicing factor may lead to decreased splicing and subsequent expression of ARV7 at both mRNA and protein level; providing a novel approach to target ARV7 in mCRPC. Citation Format: Jonathan Welti, Adam Sharp, Ines Figueiredo, Wei Yuan, Daniel Nava Rodrigues, Veronica S. Gil, Eleanor Knight, Jian Ning, Jeff Francis, Antje Neeb, Gunther Boysen, Amanda Swain, Johann S. de Bono. Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1515. doi:10.1158/1538-7445.AM2017-1515

Selective BET bromodomain inhibition as an antifungal therapeutic strategy

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

Correction: Altered regulation and expression of genes by BET family of proteins in COPD patients.

[This corrects the article DOI: 10.1371/journal.pone.0173115.].

Altered regulation and expression of genes by BET family of proteins in COPD patients.

BackgroundBET proteins (BRD2, BRD3, BRDT and BRD4) belong to the family of bromodomain containing proteins, which form a class of transcriptional co-regulators. BET proteins bind to acetylated lysine residues in the histones of nucleosomal chromatin and function either as co-activators or co-repressors of gene expression. An imbalance between HAT and HDAC activities resulting in hyperacetylation of histones has been identified in COPD. We hypothesized that pan-BET inhibitor (JQ1) treatment of BET protein interactions with hyperacetylated sites in the chromatin will regulate excessive activation of pro-inflammatory genes in key inflammatory drivers of alveolar macrophages (AM) in COPD.Methods and findingsTranscriptome analysis of AM from COPD patients indicated up-regulation of macrophage M1 type genes upon LPS stimulation. Pan-BET inhibitor JQ1 treatment attenuated expression of multiple genes, including pro-inflammatory cytokines and regulators of innate and adaptive immune cells. We demonstrated for the first time that JQ1 differentially modulated LPS-induced cytokine release from AM or peripheral blood mononuclear cells (PBMC) of COPD patients compared to PBMC of healthy controls. Using the BET regulated gene signature, we identified a subset of COPD patients, which we propose to benefit from BET inhibition.ConclusionsThis work demonstrates that the effects of pan-BET inhibition through JQ1 treatment of inflammatory cells differs between COPD patients and healthy controls, and the expression of BET protein regulated genes is altered in COPD. These findings provide evidence of histone hyperacetylation as a mechanism driving chronic inflammatory changes in COPD.

Abstract P6-11-06: Bromodomain inhibitors represent a rational therapeutic option for the treatment of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor–positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression was associated with poor recurrence-free survival (p<0.01, HR=3.16, CI 1.24-8.03). Using a two ILC cell lines (SUM44PE and MDA-MB134VI) we found that ILC cells were relatively resistant to the anti-estrogen therapies tamoxifen and fulvestrant compared to those derived from IDC. Next, we tested whether the ILC cell lines were sensitive to BET protein inhibition using the pan-BET family inhibitor JQ1. Interestingly, while JQ1 inhibited cell growth in both ILC cell lines tested, apoptosis was only induced in SUM44PE cells, while MDA-MB134VI cells exhibited G1 arrest. Dynamic BH3 profiling was used to dissect the underlying anti-apoptotic dependencies in each ILC cell type and showed that in the JQ1-resistant MDA-MB134VI cells, survival was predominantly Bcl2-dependent. Combination of JQ1 and the Bcl2-inhibitor venetoclax (ABT-199) synergistically killed MDA-MB134V1 cells compared to treatment with JQ1 alone, while combination with the Bcl2/Bcl-Xl/Bcl-W inhibitor navitoclax (ABT-263) added further synergy . With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice. These results should now be investigated in vivo before a prospective clinical trial. "This material is based upon works supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL". Citation Format: O'Connor DP, Walsh L, Tarrant F, Chin S-F, Schouten P, Linn S, Bernards R, Caldas C, Gallagher WM, ni Chonghaile T. Bromodomain inhibitors represent a rational therapeutic option for the treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-06.

HEXIM1 as a Robust Pharmacodynamic Marker for Monitoring Target Engagement of BET Family Bromodomain Inhibitors in Tumors and Surrogate Tissues.

An increasing number of BET family protein inhibitors have recently entered clinical trials. It has been reported that attempts of monitoring target engagement of the BET bromodomain inhibitor OTX015 using literature-described putative pharmacodynamic markers, such as c-Myc, BRD2, etc., failed to detect pharmacodynamic marker responses in AML patients treated at active dose and those with clinical responses. Here, we report the identification and characterization of HEXIM1 and other genes as robust pharmacodynamic markers for BET inhibitors. Global gene expression profiling studies were carried out using cancer cells and surrogate tissues, such as whole blood and skin, to identify genes that are modulated by BET family proteins. Candidate markers were further characterized for concentration- and time-dependent responses to the BET inhibitor ABBV-075 in vitro and in vivo HEXIM1 was found to be the only gene that exhibited robust and consistent modulation by BET inhibitors across multiple cancer indications and surrogate tissues. Markers such as SERPINI1, ZCCHC24, and ZMYND8 were modulated by ABBV-075 and other BET inhibitors across cancer cell lines and xenograft tumors but not in blood and skin. Significant downregulation of c-Myc, a well-publicized target of BET inhibitors, was largely restricted to hematologic cancer cell lines. Incorporating well-characterized pharmacodynamic markers, such as HEXIM1 and other genes described here, can provide a better understanding of potential efficacy and toxicity associated with inhibiting BET family proteins and informs early clinical decisions on BET inhibitor development programs. Mol Cancer Ther; 16(2); 388-96. ©2016 AACR.

Abstract IA31: Phthalimide conjugation as a strategy for targeted protein degradation

Abstract Ligand-induced target protein destabilization is an efficacious therapeutic strategy in cancer, but discovery in this field has historically been serendipitous given the lack of rational design principles. Building on the molecular logic of immunomodulatory drugs (“IMiDs”), we innovated a facile chemical solution for targeted protein degradation using bifunctional small molecules capable of recruiting the E3 ligase cereblon (CRBN). These bifunctional molecules induce immediate, and complete proteasomal degradation of their target proteins and thus represent a novel class of small molecule therapeutics with an entirely uncharted and unique molecular pharmacology. Focusing our initial efforts on degraders of the BET family of proteins, we observed that they exhibit superior efficacy than BET inhibitors through unknown mechanisms. In order to dissect and mechanistically explain that differential molecular pharmacology, we used optimized small-molecule degraders and kinetic measures of chromatin structure and function to unveil an unrecognized, essential role for BRD4 in global control of transcriptional pause-release. Targeted BET degradation nucleates a collapse in the transcriptional core regulatory circuitry and thus enabled us to exploit transcriptional addictions in T-Cell acute lymphoblastic leukemia (T-ALL) that were intractable using competitive BET bromodomain inhibitors. Citation Format: Georg E. Winter, Dennis L. Buckley, James E. Bradner. Phthalimide conjugation as a strategy for targeted protein degradation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA31.

Abstract 3059: Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry

Abstract As part of a multi-year technology integration strategy to identify unprecedented targets, AbbVie has committed to building broad-endpoint profiling assays to enable phenotypic screening campaigns and compound prioritization. Early on, phenotypic cell-based screening employing a panel of protein-fragment complementation assays (PCAs) identified A-1107604 as a hit with potent activity against a subset of endpoints. While its activity profile had some commonalities with known anti-cancer agents, the overall profile of PCAs that were significantly and concomitantly modulated represented a unique signature. Further analysis revealed A-1107604 to have potent and selective activity in a panel of human tumor cell lines. Inhibitor affinity capture from cellular lysates coupled with mass spectrometry identified the BET family of proteins as the putative cellular targets of A-1107604. Binding was localized to the bromodomain of the target proteins using affinity capture-protease digestion and was confirmed by thermal shift assay, solution binding and X-ray crystallography. This binding was found to be highly selective when A-1107604 was counter-screened against a 150-member kinase panel and an 80-member receptor panel. To correlate target affinity with cellular efficacy, a series of analogs were prepared with affinities spanning 3 orders magnitude. Affinity for BRD4, a BET family member, strongly correlated with efficacy in human tumor cell lines. Finally, A-1107604 was evaluated in human tumor xenograft models where it demonstrated significant tumor growth inhibition. This discovery effort laid the foundation for our BET inhibitor program. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Scott E. Warder, Shaun M. McLoughlin, T. Matthew Hansen, Paul L. Richardson, Denise M. Wilcox, Sadiya N. Addo, Hua Tang, Chaohong Sun, Andrew M. Petros, Sanjay C. Panchal, Chang H. Park, M. Shannon Duggan, Melanie J. Patterson, F. Greg Buchanan, Dong Cheng, Heather M. Davis, David J. Calderwood, Steven W. Elmore, Yu Shen. Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3059.

Abstract 4711: Broad anti-tumor activity of a novel BET bromodomain inhibitor

Abstract Inhibitors against the bromodomain and extra terminal domain (BET) family of proteins have been pursued as promising oncology agents based on growing understanding of epigenetic control of disease processes. Through scaffold-based and crystallography-guided drug design, we discovered PLX51107, a potent and selective small molecule inhibitor of the BET family bromodomains. PLX51107 is structurally unrelated to the benzodiazepines such as JQ1, I-BET762, and OTX015 and other published BET inhibitors. PLX51107 exhibits low nanomolar potency in blocking interactions mediated by the four BET family proteins BRD2, BRD3, BRD4, and BRDT. Pharmacologic inhibition of BET proteins by PLX51107 suppresses the transcription of genes essential for tumor growth and survival and leads to selective killing of cancer cell lines across a broad range of hematologic malignancies (e.g. leukemia, lymphoma and multiple myeloma). A subset of solid tumors (e.g. melanoma and SCLC) is also sensitive to growth inhibition by the BET inhibitor PLX51107. Novel biomarkers in these diseases have been identified. PLX51107 is well tolerated and has sufficient potency and oral bioavailability to demonstrate in vivo efficacy in animal models of a variety of tumor types, representing both hematologic and solid tumors of diverse genetic backgrounds. In combination studies, PLX51107 showed potential to improve the efficacy (response rates and duration of response) of other anticancer treatments without increased toxicity. These results support further development of PLX51107 as an epigenetic-based therapy for a variety of cancer indications. Citation Format: Yan Ma, Ben Powell, Jiazhong Zhang, Ying Zhang, Heidi Carias, Ullrich Schwertschlag, Gaston Habets, Prabha Ibrahim, Wayne Spevak, Chao Zhang, Gideon Bollag. Broad anti-tumor activity of a novel BET bromodomain inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4711.