BET Family Bromodomain Inhibitor Research Articles

Discovery of N-Ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-oxo-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET Bromodomain Inhibitor with Selectivity for the Second Bromodomain.

The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (∼40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).

Abstract DDT01-05: ABBV-744: A first-in-class highly BDII-selective BET bromodomain inhibitor

Abstract The BET family of proteins consists of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two distinct bromodomains (BDI and BDII). ABBV-075, like other first generation BET family bromodomain inhibitors currently under clinical development, binds to each of the 8 bromodomains with similar affinity and inhibits the proliferation of cancer cells that represent a wide range of tumor types. We hypothesized that selectively targeting specific subsets of the BET bromodomain might abolish this broad spectrum profile such that only the tumor types that are highly addicted to the subtype specific BET bromodomain-mediated transcription would remain sensitive to these selective agents. The BDII bromdomains are highly conserved across BET family members (>70% identity), suggesting that the generation of compounds that are selective for the four BDII bromodomains relative to the four BDI bromodomains might be achievable. Structure-based design targeting the Asp144/His 437 and Ile146/Val439 sequence differences (BRD4 BDI/BDII numbering) helped to guide the identification of structural analogs of ABBV-075 demonstrating greater than 300X selectivity for BRD4 BDII over BRD4 BDI. Further elaboration ultimately resulted in the discovery of BDII-selective compounds with improved oral bioavailability and the identification of the clinical asset ABBV-744. In striking contrast to the broad range of cell growth inhibition effected by pan BET inhibitor ABBV-075, the anti-proliferative activity for BDII-selective ABBV-744 was largely but not exclusively restricted to AML and androgen receptor (AR) positive prostate cancer cell line models, including models of Enzalutamide resistance. In vivo, doses of ABBV-744 at fractions of its MTD induced tumor growth inhibition in AML and prostate cancer xenograft models that were comparable to those observed with ABBV-075 when dosed at its MTD. Together, these in vitro and in vivo results provide proof of concept that highly selective BDII BET family bromodomain inhibitors may have improved tolerability relative to pan-BET inhibitors while maintaining tumor growth inhibition in AML and AR positive prostate cancer. 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: Warren Kati. ABBV-744: A first-in-class highly BDII-selective BET bromodomain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT01-05.

Abstract 1668: NHWD-870, a novel BET family bromodomain inhibitor targeting BRD4, proved to be effective and promising for treatment of small cell lung cancer

Abstract Background, Small molecule inhibitors targeting bromodomain and extraterminal domain (BET) protein is promising for cancer therapy. Previous study show that BRD4 was unregulated in different kind of cancer, with leukemia included, and BETi (BET inhibitors), such as JQ1(+) show evidence to inhibit the proliferation of cancer in vitro and vivo. However, there is no powerful data to evaluate the expression of BRD4, phosphorylation of BRD4 (p-BRD4) and the effectiveness of BETi in small cell lung cancer (SCLC). According to our previous study, BRD4 was highly expressed in 34 SCLC patients (70%) and correlated with poor prognosis in. Method, More than 120 tissue samples and survival data of SCLC patients in Hunan Cancer Hospital were collected. Immunohistochemistry was conducted to evaluate the expression of BRD4, p-BRD4 and the prognosis of these SCLC patients. Based on our previous finding, we design and modify series small molecular compound with carbocycles shape named NHWDs. We use the MTT in SCLC cell lines to evaluate the activity and selected the best. And then potential pathway was demonstrated with Array and Western blot. Finally, the activity of small compounds free base and HCL formation were conducted by xenograft and Patient Derived Xenograft (PDX) with or without double Platinum chemotherapy patients of etoposide and cisplatin resistant. Result, In 120 SCLC formalin-fixed specimens, BRD4 and p-BRD4 was highly expressed in SCLC and correlated with the poor prognosis of SCLC patients. All the compounds, free base and HCL formation which design and modify by ourselves were potent and selective BET family bromodomain inhibitor, not only binds bromodomains of BRD2/3/4/T, but the p-BRD4. Compare with JQ1(+) and other molecular compounds, NHWD-870 free base and NHWD-870-HCL have the best effectiveness and powerful anti-cancer in SCLC, and the IC50 was 1.579nM. NHWD-870 exhibited robust single agent activity in cell viability assay across cell lines in vitro and xenografts of SCLC in vivo by downregulating MYC. Consistent with its broad spectrum of activities in invo, NHWD-870 and NHWD-870-HCL have potent tumor suppressive efficacies in PDX model of SCLC. Monotherapy and combining with antiangiogenesis, such as bevacizumab and apatinib, NHWD-870 and NHWD-870-HCL proved to be more effective in SCLC cell lines and PDX in patients who show resistant to double Platinum chemotherapy with etoposide and cisplatin. Conclusion, NHWD-870 shows to be a powerful anti-cancer drug in SCLC. Further basic and clinical research should be conducted from bench to beside. Citation Format: Yongchang Zhang, Fang Wu, Nong Yang. NHWD-870, a novel BET family bromodomain inhibitor targeting BRD4, proved to be effective and promising for treatment of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1668.

Abstract 1150: Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice

Abstract Acute myeloid leukemia (AML) co-clinical modeling has been optimized with peripheral blood mononuclear cells (PBMCs) collected from low volume (14 mL) patient samples to establish an algorithm for efficiently co-clinically modeling AML patients. Methods: PBMCs were ficoll gradient purified and viably cryopreserved. Intrahepatic (i.h.) inoculation of AML PBMCs in neonate NOD/Shi-scid-IL2rγnull (NOG) mice and intravenous (i.v.) inoculation in both juvenile NOG mice and juvenile human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice (NOG-EXL) were evaluated. Bone marrow (BM) aspirates, splenocytes and PBMCs from mice were evaluated by fluorescence-activated cell sorting (FACS) at 12 weeks post AML inoculation for engraftment as determined by % ratio of human CD33+ cells to total CD45+ cells (human + murine cells). Humerus bones from inoculated animals were also evaluated by human CD33 immunohistochemistry (IHC). Results: Cells from 2/6 AML patient samples (CTG-2224 and CTG-2357) successfully engrafted into neonate mice. Animals were dosed with vehicle, cytarabine, ABBV-075 (clinical trial-staged BET family bromodomain (BD) inhibitor), or ABBV-744 (a preclinical BDII selective inhibitor) and evaluated for tumor burden six weeks post drug treatment initiation. ABBV-075 and ABBV-744 treated animals had lower tumor burden in the CTG-2224 model, 17% (p<0.05) and 4% (p<0.01); respectively. Similar trends, albeit at lower engraftment (10%), were observed for both models in spleen and blood compartments. In an effort to improve engraftment efficiency NOG-EXL mice were evaluated as hosts for engraftment with a patient PBMC sample (CTG-2357) and compared with juvenile NOG mice. Efficiency in the number of mice engrafted (8/8 vs. 3/7) and the extent of BM engraftment (51% vs. 14%) following a 0.5 X 106 PBMC inoculation was improved with the NOG-EXL mice. Furthermore, a patient inoculum (CTG-2241) that had previously shown no engraftment in juvenile NOG mice exhibited a 92% take rate with 93% BM tumor burden in NOG-EXL mice. Discussion: Prior advances in AML modeling show engraftment in IL2rγnul mouse models including i.h. and i.v. inoculation of BM clinical isolates in neonate NSG mice, i.v. inoculation of leukapheresis clinical isolates in juvenile NSG and NSG-SGM3 mice, and i.v. inoculation of BM aspirates in MISTRG mice. Each approach has limitations in terms of tedious neonate mice husbandry, ease of clinical sample collection with respect to clinical patient compliance, and mouse strain availability. Here we examined the feasibility of efficiently engrafting AML regardless of mutation or clinical stage from 14 ml PBMC samples. Results showed that i.v. inoculation of AML clinical samples in juvenile NOG-EXL mice was advantageous. This research is prerequisite for conducting translational co-clinical in vivo pharmacology studies for ABBV-075. Citation Format: Neal C. Goodwin, Daniel H. Albert, Angela M. Davies, Jenny Rowe, Gerold Feuer, Michael Boyiadzis, Kathleen A. Dorritie, Maria Mancini, Regina Gandour-Edwards, Warren M. Kati, Mark D. McKee, Keith F. McDaniel, David J. Frost. Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1150.

Abstract LB-261: Targeting BET Family Bromodomain with ABBV-075 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models

Abstract Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABBV-075 is a potent and selective BET family bromodomain inhibitor that entered phase I clinical trials. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH, Cancer Res 2017). In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 blockade by venetoclax in combination with BET inhibitor ABBV-075 in primary AML samples. First, anti-leukemia activity of venetoclax and ABBV-075 was examined in 12 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death (71.2 ± 29.3 %), as compared to the single agent treatment (51.4 ± 32.2 % in venetoclax 10 nM group and 32.8 ± 21.7% in ABBV-075 20 nM group). ABBV-075 inhibited cell proliferation in the majority of AML cases (51.3 ± 30.9 %) and the cell growth suppression was more profound in the combination group (87.9 ± 18.8 %, p<0.01). Most importantly, three of 12 patients were resistant to venetoclax, but two of 3 were sensitive to ABBV-075 or ABBV-075/venetoclax combination. We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to venetoclax and the combination with ABBV-075 were characterized by high level of BCL2 and mid-low level of MCL1 expression. In samples with low level of AR, IL1R1expression and high CCND1 expression, the combination of venetoclax and ABBV-075 was synergistic in inducing AML cell death. To test the efficacy in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutation in NSG mice. Upon engraftment, mice were randomized to receive vehicle, single agent venetoclax at 50 mg/kg for 21 days, ABBV-075 at 0.5 mg/kg for 21 days; or venetoclax at 50 mg/kg plus ABBV-075 at 0.5 mg/kg for 21 days. After 21 days treatment, flow cytometry data demonstrated significantly reduced leukemia burden in treated groups (7.9 ± 5.9 % in venetoclax group and 12.2 ± 5.1 % in ABBV-075 group) compared to controls (33.0 ± 12.2 %), more prominently in the combination group (1.9 ± 0.8 %). In summary, combinatorial blockade of BET family bromodomain and BCL-2 pathways promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Ongoing studies will evaluate efficacy of this combination therapy in 10 additional primary human AML xenografts and elucidate mechanisms of synergy. Citation Format: Tianyu Cai, Vinitha Mary Kuruvilla, Tamar Uziel, Qi Zhang, Lina Han, Antonio Cavazos, Yu Shen, Marina Konopleva. Targeting BET Family Bromodomain with ABBV-075 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-261.

Abstract 931: Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor

Abstract The BET family of proteins consists of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two distinct bromodomains (BDI and BDII). ABBV-075, like other first generation BET family bromodomain inhibitors currently under clinical development, binds to each of the 8 bromodomains with similar affinity and inhibits the proliferation of cancer cells that represent a wide range of tumor types. We hypothesized that selectively targeting specific subsets of the BET bromodomain might abolish this broad spectrum profile such that only the tumor types that are highly addicted to the subtype specific BET bromodomain-mediated transcription would remain sensitive to these selective agents. Both BDI and BDII are highly conserved across BET family members (> 70% identity), suggesting that the generation of compounds that are selective for the BDI bromodomains or the BDII bromodomains might be achievable. Structure-based design targeting the Asp144/His 437 and Ile146/Val439 sequence differences (BRD4 BDI/BDII numbering) led to the identification of structural analogs of ABBV-075 demonstrating greater than 100X selectivity for BRD4 BDII over BRD4 BDI. Further elaboration led to compounds with improved BDII-selectivity and oral bioavailability and the identification of the clinical asset ABBV-744. Disclosures: All authors are employees or former 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: George S. Sheppard, Le Wang, Steven D. Fidanze, Lisa A. Hasvold, Dachun Liu, John K. Pratt, Chang H. Park, Mai-Ha Bui, Emily J. Faivre, Xiaoli Huang, Xiaoyu Lin, Denise M. Wilcox, Yu Shen, Daniel H. Albert, Terrance J. Magoc, Ganesh Rajaraman, Warren M. Kati, Keith F. McDaniel. Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 931.

CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation

BackgroundThe reported antitumor activity of the BET family bromodomain inhibitors has prompted the development of inhibitors against other bromodomains. However, the human genome encodes more than 60 different bromodomains and most of them remain unexplored.ResultsWe report that the bromodomains of the histone acetyltransferases CREBBP/EP300 are critical to sustain the proliferation of human leukemia and lymphoma cell lines. EP300 is very abundant at super-enhancers in K562 and is coincident with sites of GATA1 and MYC occupancy. In accordance, CREBBP/EP300 bromodomain inhibitors interfere with GATA1- and MYC-driven transcription, causing the accumulation of cells in the G0/G1 phase of the cell cycle. The CREBBP/CBP30 bromodomain inhibitor CBP30 displaces CREBBP and EP300 from GATA1 and MYC binding sites at enhancers, resulting in a decrease in the levels of histone acetylation at these regulatory regions and consequently reduced gene expression of critical genes controlled by these transcription factors.ConclusionsOur data shows that inhibition of CREBBP/EP300 bromodomains can interfere with oncogene-driven transcriptional programs in cancer cells and consequently hold therapeutic potential.

CBFβ-SMMHC Inhibition Disrupts Enhancer Chromatin Dynamics and Represses MYC Transcriptional Program in Inv(16) Leukemia

Chromatin complexes regulate gene expression in normal and malignant hematopoiesis. The significance of transcription factor deregulation on the control of epigenetic dynamics in leukemia is poorly understood. The leukemia fusion CBFβ-SMMHC is expressed in inv(16) acute myeloid leukemia (AML), and deregulates the activity of the transcription factor RUNX1. This fusion protein blocks myeloid differentiation, expands pre-leukemic myeloid progenitor cells, and drives AML development. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in an acute RUNX1 release, inducing apoptosis in inv(16) AML cells and delaying leukemia latency in mice. However, the mechanism by which AI-10-49 induces apoptosis is unknown. In this study, we utilize pharmacologic, genomic and genetic approaches to demonstrate a RUNX-dependent mechanism by which CBFβ-SMMHC maintains leukemia survival, and that this mechanism can be pharmacologically targeted in the treatment of inv(16) AML.Gene Set Enrichment Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 identified the deregulation of a MYC signature, including cell cycle, ribosome biogenesis and metabolism, and the reduction of MYC transcript levels (10-fold). MYC shRNA knockdown induced apoptosis of inv(16) AML cells, and MYC expression rescued AI-10-49 induced apoptosis. Furthermore, mouse leukemia cells transduced with Myc shRNAs showed significant delay in leukemic latency upon transplantation, validating the requirement of MYC in inv(16) AML maintenance in vivo. Similarly, pharmacologic inhibition of MYC activity, using a combined treatment with AI-10-49 and the BET-family bromodomain inhibitor JQ1, revealed a strong synergy in inv(16) AML cells and significant delay in leukemia latency in mice. Analysis of chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) revealed that AI-10-49 treatment increased RUNX1 occupancy at three MYC distal enhancer elements (including two new enhancers: +0.18 Mb and +0.5 Mb, and the E3 enhancer of the BDME superenhancer at +1.7 Mb) downstream from MYC TSS but not at the MYC TSS or the T-cell leukemia associated N-ME enhancer. The open-chromatin and active nature of these regions was further validated by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and H3K27Ac ChIP-seq.Since the MYC locus is occupied by a number of tumor-specific enhancers, we analyzed the enhancer interactions in a 4 Mb region (-1Mb to +3 Mb of MYC TSS) by chromosome conformation capture carbon copy (5C) in inv(16) AML cells. The 5C analysis revealed that the three enhancers are physically connected with each other and with the MYC promoter. Again, the T-cell ALL associated N-ME enhancer was not active in these cells. Furthermore, the six-hour AI-10-49 treatment had not altered these interactions, indicating that MYC repression was probably not due to changes in open chromatin but to alterations in regulatory factors. Therefore, we analyzed the occupancy of chromatin complex components at the three enhancers, N-ME and MYC TSS regions, utilizing ChIP-quantitative PCR. We demonstrate that AI-10-49 treatment results in the repression of the SWI/SNF complex component BRG1 and recruitment of polycomb-repressive complex 1 (PRC1) component RING1B at the three MYC enhancers. This switch was associated with a reduction of active enhancer mark H3K4me1 and an increase of the repressive mark H3K27me3 at these sites. Finally, deletion of a small region in each of these enhancers surrounding the RUNX1 binding site (41 bp, 67 bp and 295 bp) by genome editing (CRISPR/Cas9) reduced MYC transcript levels by 50% and the viability of inv(16) AML cells in vitro, indicating that each one of these enhancers plays a critical role in regulating MYC levels and sustaining the survival of inv(16) AML cells.Our results demonstrate that CBFβ-SMMHC regulates MYC levels by repression of RUNX1 activity at three distal MYC enhancers in inv(16) AML cells. AI-10-49 treatment induces an acute release of RUNX1, increases RUNX1 occupancy at the distal enhancers, and disrupts enhancer chromatin dynamics which in turn induces apoptosis by repressing MYC. Furthermore, this study suggests that combined treatment of inv(16) AML with AI-10-49 and BET-family inhibitors may represent a promising targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

P2.15-001 NHWD-870, a Novel BET Family Bromodomain Inhibitor Targeting BRD2/3/4, Proved to Be Effective and Promising for Treatment of Small Cell Lung Cancer

P2.15-001 NHWD-870, a Novel BET Family Bromodomain Inhibitor Targeting BRD2/3/4, Proved to Be Effective and Promising for Treatment of Small Cell Lung Cancer

Abstract 1382: A novel BET family bromodomain inhibitor NHWD-870 represents a promising therapeutic agent for a broad spectrum of cancers

Abstract Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. NHWD-870 is a potent and selective BET family bromodomain inhibitor and only binds bromodomains of BRD2/3/4/T. NHWD-870 exhibited robust single agent activity in cell viability assay across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to NHWD-870 indicated that NHWD-870 manifested diverse mechanisms of action in different cancer settings. These include: 1) inhibition of tumor cell growth by downregulating the PDGFRβ, MEK1/2 and STAT1/MYC signaling in tumor cells; 2) inhibition of tumor angiogenesis by decreasing PDGF production in tumor cells and the PDGFRβ and MEK1/2 signaling in endothelial cells. Consistent with its broad spectrum of activities in vitro, NHWD-870 has potent tumor suppressive efficacies in xenograft mouse models of small cell lung cancer, triple negative breast cancer and ovarian cancer. These results support its further development for diverse solid tumor indications in the clinic. Citation Format: Nenghui Wang, Mingzhu Yin, Qin Yan. A novel BET family bromodomain inhibitor NHWD-870 represents a promising therapeutic agent for a broad spectrum of cancers [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 1382. doi:10.1158/1538-7445.AM2017-1382

Preclinical Characterization of BET Family Bromodomain Inhibitor ABBV-075 Suggests Combination Therapeutic Strategies.

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.

A novel BET family bromodomain inhibitor NHWD‐870 represents a promising therapeutic agent for a broad spectrum of cancers

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. NHWD‐870 is a potent and selective BET family bromodomain inhibitor and only binds bromodomains of BRD2/3/4/T. NHWD‐870 exhibited robust single agent activity in cell viability assay across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to NHWD‐870 indicated that NHWD‐870 manifested diverse mechanisms of action in different cancer settings. These include: 1) inhibition of tumor cell growth by downregulating the PDGFRβ, MEK1/2 and STAT1/MYC signaling in tumor cells; 2) inhibition of tumor angiogenesis by decreasing PDGF production in tumor cells and the PDGFRβ and MEK1/2 signaling in endothelial cells. Consistent with its broad spectrum of activities in vitro, NHWD‐870 has potent tumor suppressive efficacies in xenograft mouse models of small cell lung cancer, triple negative breast cancer and ovarian cancer. These results support its further development for diverse solid tumor indications in the clinic.

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 4718: ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications

Abstract Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 clinical trials. It binds bromodomains of BRD2/4/T with similar affinities (Ki of 1-2.2 nM), but exhibits roughly 10-fold weaker potency towards BRD3 (Ki of 12.2 nM). ABBV-075 is highly selective for 18 bromodomain proteins tested (Kd > 1 μM; more than 600-fold selectivity vs. BRD4) and has moderate activity towards CREBBP (Kd = 87 μM; 54-fold selectivity vs. BRD4). ABBV-075 exhibited robust single agent activity in cell viability assays across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to ABBV-075 indicated that ABBV-075 manifested diverse mechanisms of action in different cancer settings. These include 1): disruption of cell cycle control leading to G1 arrest followed by senescence, 2) inhibition of oncogenesis drivers leading to apoptosis and 3) potentially targeting tumor microenvironment to provide additional therapeutic benefit. Consistent with its broad spectrum of activities in vitro, ABBV-075 has comparable or superior efficacies to standard of care agents in flank xenograft mouse models of non-small-cell and small cell lung cancers, pancreatic, breast, prostate, head & neck cancers, multiple myeloma, diffuse large B cell lymphoma and leukemia. These results support the development of ABBV-075 in diverse hematological malignancies and solid tumor indications. Citation Format: Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Warder Scott, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise M. Wilcox, Terry Magoc, Fritz G. Buchanan, Paul Tapang, George S. Sheppard, Le Wang, Steve D. Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Paul Hessler, Tamar Uziel, Lloyd Lam, Ganesh Rajaraman, Guowei Fang, Steven W. Elmore, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications. [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 4718.

Abstract 3077: Potent inhibition of bromodomain-containing BET family with ABBV-075 induces robust antitumor efficacy in preclinical models of breast cancer and exhibits in vitro synergy with doxorubicin

Abstract Breast cancer diagnosisis highly prevalent in the US, with an estimated 231,840 new cases occurring in 2015. Targeted therapies, such as tamoxifen or Trastuzumab (herceptin) are available to woman whose tumors are estrogen receptor (ER) positive or HER2 positive, respectively. However, women with triple negative breast cancer or relapsed metastatic disease have limited treatment options. ABBV-075 is a novel BET family bromodomain inhibitor currently under phase I clinical investigation for a wide spectrum of cancer indications. Here we show that ABBV-075 exhibits broad anti-proliferative activity across cell lines representing ER positive, HER2+, and triple negative breast cancer. Notably, ABBV-075 induced G1 arrest and growth inhibition of breast cancer cell lines regardless of ER or RB status. This result suggests that ABBV-075 may provide therapeutic benefit to a broader patient population relative to the recently approved cdk4/6 inhibitor, palbociclib. The G1 arrest mechanism of ABBV-075 was efficacious in breast cancer xenograft studies, where significant tumor growth inhibition was observed. Additionally, ABBV-075 exhibited synergy in ER positive cell lines with doxorubicin, a TOPO2 inhibitor and DNA intercalating agent prescribed to ER positive patients refractory to hormone therapies. Intriguingly, the mechanism by which ABBV-075 and doxorubicin interact was independent of the TOP2-trapping activity of doxorubicin and instead required DNA intercalation. Together, these results demonstrate the potential of ABBV-075 as a treatment option across the different subtypes of breast cancer and in combination with doxorubicin in patients with ER positive disease Citation Format: Emily J. Faivre, Xiaoyu Lin, Denise M. Wilcox, Xiaoli Huang, Aparna Sarthy, Terry Magoc, Daniel H. Albert, Guowei Fang, Saul H. Rosenberg, Keith F. McDaniel, Warren M. Kati, Yu Shen. Potent inhibition of bromodomain-containing BET family with ABBV-075 induces robust antitumor efficacy in preclinical models of breast cancer and exhibits in vitro synergy with doxorubicin. [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 3077.

Abstract 3085: The BET family bromodomain inhibitor ABBV-075 targets multiple pathogenesis factors in multiple myeloma and exhibits robust in vivo efficacies as a single agent and in combination with bortezomib

Abstract Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins represent a potential new class of cancer therapeutic agents. ABBV-075 is a potent and selective BET family bromodomain inhibitor that was recently advanced to Phase 1 studies. Here we report the in vitro and in vivo characterization of ABBV-075 in preclinical models of multiple myeloma. ABBV-075 exhibited significant anti-proliferative activities and triggered robust apoptosis across many multiple myeloma cell lines, including cell lines that were engineered to become resistant to bortezomib. As reported for other BET inhibitors, ABBV-075 potently inhibited super-enhancer regulated potential cancer drivers such as Myc, IRF8 etc. In addition to directly impacting multiple myeloma cell growth and survival, ABBV-075 also diminished stroma cell derived IL-6 secretion and prevented HUVEC cell proliferation in vitro. Given the importance of angiogenesis and the survival factor IL-6 for multiple myeloma pathogenesis, ABBV-075 could potentially produce significant therapeutic benefits in myeloma by simultaneously targeting multiple critical pathogenesis mechanisms for this disease. ABBV-075 caused significant tumor growth delay in the OPM2 flank tumor model as a monotherapy. Combining ABBV-075 with Bortezomib provided further therapeutic benefit compared with the treatment of either of the two agents individually. Taken together, our results suggest that ABBV-075 could be a promising option to explore for the treatment of multiple myeloma. Citation Format: Tamar Uziel, Xiaoyu Lin, Emily Faivre, Aparna Sarthy, Daniel H. Albert, Leiming Li, Denise Wilcox, Xiaoli Huang, Terry Magoc, Lloyd Lam, Steven W. Elmore, Keith McDaniel, Warren Kati, Yu Shen. The BET family bromodomain inhibitor ABBV-075 targets multiple pathogenesis factors in multiple myeloma and exhibits robust in vivo efficacies as a single agent and in combination with bortezomib. [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 3085.

Abstract 4738: The BET family bromodomain inhibitor ABBV-075 is a promising therapeutic agent for acute myeloid leukemia and myelodysplastic syndrome

Abstract Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins exhibit interesting activities in preclinical models of various cancer indications, and several of these inhibitors are currently under clinical investigation. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 studies. Upon characterizing cellular responses to BET inhibitors across a large panel of cancer cell lines, we identified AML/MDS as one of the few cancer indications where BET inhibitors triggered robust apoptosis in cancer cell lines and in patient-derived cancer cells. The induction of apoptosis by BET inhibitors in AML/MDS cells may be partly attributed to their abilities to down regulate Bcl-XL and Bcl-2, and combining BET inhibitors with the Bcl-2 inhibitor venetoclax (ABT-199) resulted in robust cytotoxicity in AML/MDS cells. ABBV-075 exhibited significant antitumor efficacy as a monotherapy in flank xenograft models of AML and MDS. Furthermore, combining low doses of ABBV-075 with the standard of care agent azacitidine in the SKM1 model led to significant tumor regression, and the combination regimen was better tolerated than BETi monotherapy at doses that produced a similar degree of therapeutic benefit. Expression profiling of SKM1 tumors from mice treated with the ABBV-075/azacitidine combination or each of these agents as monotherapies revealed that ABBV-075 and azacitidine regulated a common set of biologic pathways. The combination of ABBV-075/azacitidine led to a more robust impact on these common pathways, which may partially contribute to the enhanced efficacy of the ABBV-075/azacitidine combination in the SKM-1 model. Citation Format: Mai H. Bui, Xiaoyu Lin, Xiaoli Huang, Leiming Li, Aparna Sarthy, Daniel Albert, Terry Magoc, Lloyd Lam, Paul Hessler, Tamar Uziel, Steven Elmore, Keith McDaniel, Warren Kati, Yu Shen. The BET family bromodomain inhibitor ABBV-075 is a promising therapeutic agent for acute myeloid leukemia and myelodysplastic syndrome. [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 4738.

Abstract 4695: Functional group elaboration of a low molecular weight fragment to yield the novel BET family bromodomain inhibitor ABBV-075

Abstract Phenotypic cell-based screening assays combined with affinity chromatography and mass-spectrometry identified the BET family of bromodomains as potential targets for blocking proliferation in a variety of cancer cell lines. Lead-finding investigations included screening a library of compounds with an average molecular weight of 225 for binding to the 13C-labeled 2nd bromodomain of BRD4 using 2-dimensional NMR. A pyridazinone fragment emerged from this effort that possessed weak binding affinity (Kd = 130 uM). The binding affinity was improved by roughly 100,000-fold through an X-ray structure enabled medicinal chemistry program that included moving to a pyrrolopyridone core along with judicious placement of additional functional groups. Antiproliferative potencies strongly correlated with potencies in a cell-based target engagement assay, suggesting that the antiproliferative effects resulted from the inhibition of BRD4/BET protein function. In vitro metabolite ID studies in rat liver microsomes helped identify sites of oxidative metabolism. The addition of fluorine atoms at these locations improved rat in vitro microsomal stability that translated to low in vivo clearance and high oral exposure in rat. The final molecule, ABBV-075, exhibited long half-lives and low unbound clearances in rat, mouse, dog and monkey. ABBV-075 demonstrated significant tumor growth inhibition in mouse flank xenograft studies representing diverse hematological and solid tumor malignancies and recently entered Phase I clinical studies. 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: Keith McDaniel, Le Wang, George Sheppard, Steve Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Robert Mantei, Chang Park, Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Scott Warder, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise Wilcox, Rongqi Wang, Terry Magoc, Ganesh Rajaraman, Andrew Petros, Sanjay Panchal, Chaohong Sun, Guowei Fang, Steven W. Elmore, Saul Rosenberg, Yu Shen, Warren Kati. Functional group elaboration of a low molecular weight fragment to yield the novel BET family bromodomain inhibitor ABBV-075. [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 4695.

Abstract B21: Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC

Abstract The RAF-MEK-ERK signaling pathway regulates the proliferation of Triple Negative Breast Cancer (TNBC). Although MEK inhibition by trametinib in TNBC cell lines and mouse models inhibits growth, the response is not durable and the tumor develops resistance. The lack of durability of targeted kinase inhibitors is a common problem in many tumor types due to induction of alternative bypass signaling pathways that lead to resistance. We have profiled the response of TNBC to MEK inhibition using trametinib in order to identify adaptive signaling pathways which drive resistance. We used a chemical proteomics method [multiplexed inhibitor beads coupled to mass spectrometry (MIB/MS)], to quantify global changes in kinase activity coupled with transcriptional changes in response to trametinib using RNAseq. Our studies revealed that adaptive resistance to trametinib occurs through increased expression and activation of multiple receptor tyrosine kinases, leading to reactivation of ERK. These adaptive kinases are heterogeneous across TNBC including PDGFRβ, VEGFR2, DDR1, AXL, KIT and FGFR2. There is no single targeted kinase inhibitor able to block all the adaptive kinases induced and activated, making the design of combination targeted kinase inhibitor therapies impractical. We therefore hypothesized an alternative therapeutic approach was required to inhibit adaptive reprogramming. Our goal was to block the initial transcriptional response to trametinib which upregulates adaptive kinase expression. Several studies in the lab determined that we could block adaptive kinase expression by inhibiting BET family bromodomain proteins that interact with acetylated histones and facilitate the formation of transcriptional regulatory complexes. Our work demonstrated that BET family bromodomain inhibitors (JQ1 and iBET151), synergize with trametinib to prevent TNBC growth in short-term (4 days) and long-term (4 weeks) growth assays. Using MIB/MS we found that combination trametinib/BET bromodomain inhibitor treatment blocks upregulation of adaptive kinase expression and activation. RNAseq demonstrated that BET bromodomain inhibitors synergize with trametinib to block the induction of kinases expressed as part of the adaptive bypass response. ChIP-seq of the BET bromodomain protein BRD4 and H3K27ac, a marker of active enhancers, showed BRD4 is recruited to super-enhancer regions proximal to adaptive response kinases as early as 4hr following trametinib treatment. Furthermore, the recruitment of BRD4 to enhancer regions is maintained in cells made resistant to trametinib by constant culture in low-dose drug. In sum, these results suggest that BRD4 recruitment to enhancer regions, following trametinib treatment, leads to the formation of transcriptional complexes which maintain adaptive gene expression. Ongoing studies seek to understand the mechanism by which BRD4 is recruited to super-enhancers following trametinib treatment in TNBC. Mouse xenograft studies also show promising evidence of enhanced tumor growth suppression in vivo using combination trametinib/iBET151 treatment. The results presented provide a novel treatment strategy to block adaptive resistance and have the potential to lead to durable clinical responses of TNBC. Citation Format: Samantha M. Miller, Jon S. Zawistowski, Timothy J. Stuhlmiller, Noah Sciaky, Charlene M. Santos, David B. Darr, Gary L. Johnson. Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B21.

Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons

Recently, direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine, but applications could be limited by the genetic manipulation involved. Here, we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules, with a yield of up to >90% being TUJ1-positive after 16 days of induction. After a further maturation stage, these chemically induced neurons (CiNs) possessed neuron-specific expression patterns, generated action potentials, and formed functional synapses. Mechanistically, we found that a BET family bromodomain inhibitor, I-BET151, disrupted the fibroblast-specific program, while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall, our findings provide a "proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation, through disruption of cell-specific programs and induction of an alternative fate.