Bromodomain And Extra-terminal Motif Inhibitor Research Articles

Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

BET-inhibitor DYB-41 reduces pulmonary inflammation and local and systemic cytokine levels in LPS-induced acute respiratory distress syndrome: an experimental rodent study

BackgroundAcute respiratory distress syndrome (ARDS) is a form of respiratory failure stemming from various underlying conditions that ultimately lead to inflammation and lung fibrosis. Bromodomain and Extra-Terminal motif (BET) inhibitors are a class of medications that selectively bind to the bromodomains of BET motif proteins, effectively reducing inflammation. However, the use of BET inhibitors in ARDS treatment has not been previously investigated. In our study, we induced ARDS in rats using endotoxin and administered a BET inhibitor. We evaluated the outcomes by examining inflammation markers and lung histopathology.ResultsNine animals received treatment, while 12 served as controls. In the lung tissue of treated animals, we observed a significant reduction in TNFα levels (549 [149–977] pg/mg vs. 3010 [396–5529] pg/mg; p = 0.009) and IL-1β levels (447 [369–580] pg/mg vs. 662 [523–924] pg/mg; p = 0.012), although IL-6 and IL-10 levels showed no significant differences. In the blood, treated animals exhibited a reduced TNFα level (25 [25–424] pg/ml vs. 900 [285–1744] pg/ml, p = 0.016), but IL-1β levels were significantly higher (1254 [435–2474] pg/ml vs. 384 [213–907] pg/ml, p = 0.049). No differences were observed in IL-6 and IL-10 levels. There were no significant variations in lung tissue levels of TGF-β, SP-D, or RAGE. Histopathological analysis revealed substantial damage, with notably less perivascular edema (3 vs 2; p = 0.0046) and visually more inflammatory cells. However, two semi-quantitative histopathologic scoring systems did not indicate significant differences.ConclusionsThese preliminary findings suggest a potential beneficial effect of BET inhibitors in the treatment of acute lung injury and ARDS. Further validation and replication of these results with a larger cohort of animals, in diverse models, and using different BET inhibitors are needed to explore their clinical implications.

Abstract C109: INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation

Abstract Background: BRD4 is a member of the family of bromodomain and extra-terminal motif (BET) proteins that act as epigenetic readers and regulators of gene transcription. BRD4 has been shown to be significantly upregulated in cancer including melanoma, with preclinical data supporting BRD4 inhibition as a promising therapeutic target. However, clinical translation of BRD4 inhibitors (BRD4i) has remained elusive. Current small molecule inhibitors of BET proteins are not specific to BRD4 as demonstrated by their inhibition of other members of the BET protein family. Additionally, systemic approaches must overcome a narrow therapeutic window. The INTASYL™ platform is a self-delivering RNAi technology that imparts small molecule-like properties to interfering RNAs, providing efficient delivery to target cells without need for specialized formulations or drug delivery systems. PH-894 is an INTASYL compound that silences BRD4 mRNA. PH-894 has been shown to specifically silence BRD4 mRNA and provide robust direct and abscopal antitumor efficacy in vivo when administered intratumorally (IT). Therefore, IT PH-894 holds promise to realize the therapeutic potential of BRD4 inhibition by avoiding toxicities associated with systemic delivery of small molecule BET inhibitors. Here, we explored the impact of PH-894 mediated BRD4 inhibition on antigenicity of melanoma tumor cells. Specifically, expression of the melanoma tumor-associated antigen MART-1 was studied following PH-894 treatment. Methods: SK-MEL-5 melanoma cells were treated for 72 hours with PH-894 or a non-targeting control (NTC) INTASYL compound. After 72 hours (Day 3), PH-894 was washed out and cells were replated in media containing no INTAYSL compound for the duration of the experiment. Cells were collected for analysis of both BRD4 and MART-1 mRNA and protein from Day 3 through Day 7. Results: PH-894 provided robust silencing of BRD4 mRNA and protein in SK-MEL-5 melanoma cells. Furthermore, PH-894 treatment resulted in a statistically significant upregulation of MART-1 mRNA and protein in these cells. BRD4 silencing and MART-1 upregulation persisted through at least 7 days post-treatment with PH-894. Conclusions: This study demonstrates the ability of PH-894 to efficiently silence BRD4 and upregulate MART-1, thereby increasing the antigenicity of melanoma cells. Local treatment with PH-894 is a potential strategy to decrease BRD4 expression and increase immune response to cancer cells while reducing toxicities associated with systemic BETi. This study supports further development of PH-894 for injectable solid tumor indications such as melanoma. Citation Format: Brianna Rivest, Melissa Maxwell, Andrew Boone, Benjamin Cuiffo, Dingxue Yan, James Cardia. INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C109.

BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5.

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNβ1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.

Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins

Targeting aberrant epigenetic programs that drive tumorigenesis is a promising approach to cancer therapy. DNA-encoded library (DEL) screening is a core platform technology increasingly used to identify drugs that bind to protein targets. Here, we use DEL screening against bromodomain and extra-terminal motif (BET) proteins to identify inhibitors with new chemotypes, and successfully identified BBC1115 as a selective BET inhibitor. While BBC1115 does not structurally resemble OTX-015, a clinically active pan-BET inhibitor, our intensive biological characterization revealed that BBC1115 binds to BET proteins, including BRD4, and suppresses aberrant cell fate programs. Phenotypically, BBC1115-mediated BET inhibition impaired proliferation in acute myeloid leukemia, pancreatic, colorectal, and ovarian cancer cells invitro. Moreover, intravenous administration of BBC1115 inhibited subcutaneous tumor xenograft growth with minimal toxicity and favorable pharmacokinetic properties invivo. Since epigenetic regulations are ubiquitously distributed across normal and malignant cells, it will be critical to evaluate if BBC1115 affects normal cell function. Nonetheless, our study shows integrating DEL-based small-molecule compound screening and multi-step biological validation represents a reliable strategy to discover new chemotypes with selectivity, efficacy, and safety profiles for targeting proteins involved in epigenetic regulation in human malignancies.

Reset of Inflammatory Priming of Joint Tissue and Reduction of the Severity of Arthritis Flares by Bromodomain Inhibition.

We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model. BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo. Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs. Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir.

Reversing HIV-1 latency promotes killing of infected cells and is essential for cure strategies; however, no single latency reversing agent (LRA) or LRA combination have been shown to reduce HIV-1 latent reservoir size in persons living with HIV-1 (PLWH). Here, we describe an approach to systematically identify LRA combinations to reactivate latent HIV-1 using genome-wide CRISPR screens. Screens on cells treated with suboptimal concentrations of an LRA can identify host genes whose knockout enhances viral gene expression. Therefore, inhibitors of these genes should synergize with the LRA. We tested this approach using AZD5582, an activator of the noncanonical nuclear factor κB (ncNF-κB) pathway, as an LRA and identified histone deacetylase 2 (HDAC2) and bromodomain-containing protein 2 (BRD2), part of the bromodomain and extra-terminal motif (BET) protein family targeted by BET inhibitors, as potential targets. Using CD4+ T cells from PLWH, we confirmed synergy between AZD5582 and several HDAC inhibitors and between AZD5582 and the BET inhibitor, JQ1. A reciprocal screen using suboptimal concentrations of an HDAC inhibitor as an LRA identified BRD2 and ncNF-κB regulators, especially BIRC2, as synergistic candidates for use in combination with HDAC inhibition. Moreover, we identified and validated additional synergistic drug candidates in latency cell line cells and primary lymphocytes isolated from PLWH. Specifically, the knockout of genes encoding CYLD or YPEL5 displayed synergy with existing LRAs in inducing HIV mRNAs. Our study provides insights into the roles of host factors in HIV-1 reactivation and validates a system for identifying drug combinations for HIV-1 latency reversal.

NUT Carcinoma-An Underdiagnosed Malignancy.

NUT carcinoma (NC) is a rare and highly aggressive malignancy with a dismal prognosis and a median survival of 6–9 months only. Although very few cases of NC are reported each year, the true prevalence is estimated to be much higher, with NC potentially widely underdiagnosed due to the lack of awareness. NC primarily occurs in midline structures including thorax, head, and neck; however, other sites such as pancreas and kidney are also affected, albeit at lower frequencies. NC is characterized by a single translocation involving the NUTM1 (NUT midline carcinoma family member 1) gene and different partner genes. The resulting fusion proteins initiate tumorigenesis through a mechanism involving BET (bromo-domain and extra-terminal motif) proteins such as Bromodomain-containing protein 4 (BRD4) and inordinate acetylation of chromatin, leading to the dysregulation of growth and differentiation genes. While no clinical characteristics are specific for NC, some histologic features can be indicative; therefore, patients with these tumor characteristics should be routinely tested for NUTM1. The diagnosis of NC using immunohistochemistry with a highly specific antibody is straightforward. There are currently no standard-of-care treatment options for patients with NC. However, novel therapies specifically addressing the unique tumorigenic mechanism are under investigation, including BET inhibitors. This review aims to raise awareness of this underdiagnosed cancer entity and provide all patients the opportunity to be properly diagnosed and referred to a clinical study.

Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. It is classified into two main subtypes: embryonal (eRMS) and alveolar (aRMS). MYC family proteins are frequently highly expressed in RMS tumors, with the highest levels correlated with poor prognosis. A pharmacological approach to inhibit MYC in cancer cells is represented by Bromodomain and Extra-Terminal motif (BET) protein inhibitors. In this paper, we evaluated the effects of BET inhibitor (+)-JQ1 (JQ1) on the viability of aRMS and eRMS cells. Interestingly, we found that the drug sensitivity of RMS cell lines to JQ1 was directly proportional to the expression of MYC. JQ1 induces G1 arrest in cells with the highest steady-state levels of MYC, whereas apoptosis is associated with MYC downregulation. These findings suggest BET inhibition as an effective strategy for the treatment of RMS alone or in combination with other drugs.

Abstract PD14-07: Bromodomain and Extra-Terminal motif (BET) inhibitors are a rational therapeutic choice for treatment of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer accounting for approximately 10-15% of all breast tumours. ILC is characterized by inactivation of E-Cadherin and cancer cells that invade the stroma in a "single-file" pattern. Women with ILC are more likely to have hormone receptor-positive disease. ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries. ILC are currently treated in the same manner as all other ER+ breast cancers. Like invasive ductal carcinoma (IDC), anti-estrogen resistance has emerged as a significant problem in the management of ILC. Approximately half of the recurrences of ER+ breast tumors respond to anti-endocrine treatment, while the other half are resistant. Additionally, anti-estrogen-resistant breast tumor cells appear to have/acquire a more aggressive, invasive phenotype compared to their anti-estrogen-responsive counterparts. ILC is considered to be chemo-resistant and ILC patients receive no additional benefit from adjuvant chemotherapy. As such, there is a pressing need to develop tailored therapeutic options for endocrine-resistant ILC patients. The bromodomain and extra-terminal (BET) motif family are epigenetic readers that bind to acetylated histones, recruiting co-factors to regulate transcription. As part of the FP7 RATHER project aimed at identifying rational treatment options for ILC, we discovered that BRD3 (uniquely among the BET family members) is a marker of poor prognosis in ILC but not in ER+ breast cancers as a whole. We subsequently validated this in an independent cohort from the METABRIC study. These data suggest that Brd3 may play a significant role in tumour progression in ILC and may be a rational therapeutic target for lobular tumours. Using the two ILC cell lines SUM44PE (SUM44) and MDA MB 134 VI (MM134) previously shown to be anti-endocrine resistant we assessed the therapeutic potential of BET inhibition. We found that ILC cell lines that do not respond to anti-endocrine therapy are sensitive to a panel of BET inhibitors in both 2D and 3D assays. In particular the BET inhibitors JQ1 and Mivebresib had the highest potency, these BET inhibitors were selected for further research. Next, a multi-omics approach merging RNA-sequencing with mass spectrometry was utilised to dissect the transcriptional networks employed by BET inhibitors in this ILC setting. RNA-sequencing revealed dysregulated pathways in cell cycle division, DNA damage, apoptosis and MAPK signalling following treatment. Further, rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was carried out to find the binding partners of the BRD proteins. Integrated pathway analysis through Genome Enhancer was used to assess the master regulators (MTRs) which drive BET inhibitor function. There was 142 MTRs found across the two cell lines treated with both inhibitors. Of note the MTR, FGFR3, was shown to regulate the downstream targets of both BET inhibitors. Further analysis of BET inhibition combined with erdafitinib, an FGFR inhibitor, drives increased cell inhibition compared to either agent alone. Finally, the efficacy of JQ1 in targeting ILC was assessed in vivo. We utilised the ILC cell-derived xenograft (CDX) models established by mammary intraductal implantation. The efficacy of BET inhibition alone and in combination with anti-endocrine therapies, tamoxifen and fulvestrant were assessed in the MM134 cell line. We found that JQ1 works synergistically with tamoxifen to significantly decrease tumour burden and metastatic potential in this tamoxifen resistant ILC model. Taken together this data highlights the need for tailored therapeutics in ILC research and highlights the use of BET inhibition in the anti-endocrine resistant ILC setting. Citation Format: Elspeth Ward, Anna Blümel, Emer Conroy, Grainne Cremin, Binbin Gao, William Gallagher, Idalia Cruz, Leena Hilakivi-Clarke, George Sflomos, Cathrin Brisken, Darran O'Connor. Bromodomain and Extra-Terminal motif (BET) inhibitors are a rational therapeutic choice for treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-07.

Triple combination of BET plus PI3K and NF-κB inhibitors exhibit synergistic activity in adult T-cell leukemia/lymphoma

AbstractAdult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Co-clinical Modeling of the Activity of the BET Inhibitor Mivebresib (ABBV-075) in AML.

The therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BETtargeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broadspectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML). Co-clinical modeling involves taking the patient's biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%). Results from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively). These findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.

CG223, a novel BET inhibitor, exerts TGF-β1-mediated antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-β1 (TGF-β1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-β1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin β3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-β1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-β1 autocrine/paracrine loop.

Abstract 2317: Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4

Abstract In prostate cancer (PCa) cells, “pioneer” factor FOXA1 resides at distal enhancers enriched with histone 3 lysine 4 methylation (H3K4me) and lysine 27 acetylation (H3K27ac). FOXA1 is capable of remodeling the compact chromatin to facilitate androgen receptor (AR) recruitment. Recent studies revealed overexpression of FOXA1 caused by frequent structural alterations in the lethal stage of PCa, namely castration-resistant PCa(CRPC), suggesting FOXA1 as a critical target in CRPC. However, a lack of targetable domain hinders the effective targeting of FOXA1. Our recent (epi)genomic profiling study revealed that lysine-specific demethylase (LSD1) and FOXA1 co-occupy at active enhancers in PCa cells and LSD1 demethylase activity is required for FOXA1 chromatin binding. LSD1 inhibition can quickly and drastically reduce FOXA1 global binding and concurrently impair the “openness” of enhancers, leading to disrupted AR cistrome. Mechanistically, we demonstrated that this activator function of LSD1 is mediated through directly demethylating K270 of FOXA1. By testing an array of adenocarcinoma CRPC in vivo models, we further showed that the efficacy of LSD1 inhibitors may be correlated with FOXA1 levels. Collectively, these results provide the molecular reasoning on co-targeting the epigenetic regulators that are important for promoting AR activity in treating CRPC. Furthermore, we have found that LSD1, FOXA1, and BRD4 (a Bromodomain and Extra-Terminal motif (BET) protein) co-occupy the super enhancers characterized by distinguishable high levels of H3K4me2 and H3K27ac in PCa cells. Super enhancers have been found to drive oncogenic transcription program in PCa. As an active enhancer marker and an important AR coactivator, BRD4 recognizes H3K27ac signal and promotes the initiation and elongation steps in gene transcription process. Herein, we focus on the potential synergism of combined targeting of LSD1 and BRD4. We found that FOXA1 interacted with BRD4 in different cell lines and patient-derived xenograft (PDX) tumor tissues. In line with this association, either LSD1 inhibitors or BET inhibitors could decrease FOXA1 and BRD4 chromatin binding. Characterization of the physical interaction is an ongoing study. We found that the combinational treatment lead to a more effective reduction of AR transcriptional activity than either single treatment. The PCa cell proliferation in vitro was also more profoundly inhibited by combined therapy. More importantly, we demonstrated this synergism in both 22RV1 xenograft and PDX LuCaP35CR in the castrated mice, with a lower dose of LSD1 inhibitor and BET inhibitor (both in clinical trials) which did not induce overt toxicity in the animals. Currently, we are performing RNA-seq studies to identify the convergent downstream pathways by this co-treatment. In summary, our study provides important pre-clinical insights for targeting the CRPC enhancers by combining inhibitors of LSD1 and BRD4. Citation Format: Shuai Gao, Muqing Li, Mingyu Liu, Dong Han, Zifeng Wang, Wanting Han, Changmeng Cai. Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2317.

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

A quinazoline-based bromodomain inhibitor, CN210, ameliorates indomethacin-induced ileitis in mice by inhibiting inflammatory cytokine expression.

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

INTASYL self-delivering RNAi therapy specifically dual-targeting BRD4 and PD-1 to elicit complete tumor responses and evidence of synergy in a subcutaneous Hepa1-6 model of murine hepatoma in C57BL/6N mice.

e14537 Background: The therapeutic potential of targeting bromodomain and extra-terminal motif (BET) protein BRD4 is supported by promising preclinical data in multiple cancer contexts. However, clinical trials of BET inhibitors (BETi) have overall shown limited clinical efficacy and only passable safety profiles as monotherapy. BET proteins are now also understood to control a wide range of immunoregulatory pathways. Preclinical data suggests a synergistic impact with immune checkpoint blockade (ICB) targeting PD-1. Trials assessing the potential of BETi in rational combination with ICB are underway. The INTASYL platform is a self-delivering RNAi technology enabling efficient delivery into target cells without need for specialized drug delivery systems. INTASYL provides specific, robust, and durable on-target gene silencing. Furthermore, INTASYL moieties targeting multiple specific targets can be formulated together, providing a highly versatile platform for providing multi-targeted combination therapy in a single drug substance. Methods: Here we assessed the efficacy of mouse/human BRD4-targeting INTASYL PH-894 as monotherapy or co-formulated with mouse PD-1 targeting INTASYL mPH-762, in treating a subcutaneous Hepa1-6 model of murine hepatoma in C57BL/6N mice. On-target, specific silencing of BRD4 and PD-1 was demonstrated in vitro. In vivo, mice bearing tumors of ̃150 mm3 (N = 12/arm) were treated intratumorally on Days 1, 3, 7, 10 & 14 with of PH-894 or mPH-762 as single-agent, or PH-894/mPH-762 in combination at sub-optimal doses (0.1 mg/INTASYL/dose), or with PH-894/mPH-762 at half-suboptimal dose (0.05mg/INTASYL/dose) to assess potential synergistic impacts. Positive control groups received systemic (IP) (+)-JQ-1 (13.33mg/dose; qdx5), anti-mouse PD-1 antibody (75 µg; Days 1, 3, 7, 10, 14), or both treatments. Negative control animals received vehicle only. Tumor volumes and body weights were recorded longitudinally. Results: Compared to vehicle, suboptimal (+)-JQ-1, anti-PD-1, PH-894 or mPH-762 each elicited tumor control but not resolution as expected; while treatment with suboptimal PH-894/mPH-762 combo produced complete response (CR) in 75% (9/12) mice, outperforming combination treatment with (+)-JQ-1 and anti-PD-1 mAb [CR 58% (7/12) of mice]. Treatment with half-suboptimal doses of PH-894/mPH-762 resulted in CR for 42% (5/12) mice and tumor control exceeding that elicited by each monotherapy, suggesting potential synergy of the dual-targeting formulation. Tumors were isolated for mechanistic analyses of immune and tumor cells. Conclusions: These data demonstrate proof-of-concept of the therapeutic potential of rational dual-targeted INTASYL provided as single formulation in vivo, suggesting potential for meaningful clinical therapeutic impact.

BET inhibitors repress expression of interferon-stimulated genes and synergize with HDAC inhibitors in glioblastoma.

BackgroundThe development of rational combination therapies is key to overcome inherent treatment resistance of glioblastoma (GBM). We aim at identifying new druggable targets by disturbing GBM cells with inhibitors of bromodomain and extra-terminal motif (BET) proteins to reveal cancer-relevant vulnerabilities that may sensitize to a second drug. BET proteins are epigenetic modulators and have been associated with proto-oncogene overexpression in cancer.MethodsA GBM-derived sphere-line was treated with the BET inhibitor (BETi) JQ1 over a time-course of 48 hours, followed by RNA-sequencing. Four chromatin marks were investigated by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Signatures of interest were functionally validated in vitro and in orthotopic xenografts. Combination therapies were evaluated for synergistic effects.ResultsCancer-relevant pathways significantly modulated by JQ1 comprised interferon alpha (IFN-α) response genes and response signatures to histone deacetylase inhibitors (HDACi). The IFN-signature was reminiscent of a GBM-derived IFN-signature comprising CD274 (PD-L1). Functional pathway analysis suggested that JQ1 was acting directly on the transcriptional level of IFN-response genes and not via the canonical JAK/STAT pathway. This was in line with JQ1 modulated expression and BRD4 and Pol II occupancy at IFN-signature genes, supporting a direct mechanistic interaction. Finally, we showed that combining HDACi with JQ1 acts synergistically in reducing cell viability of GS-lines.ConclusionsOur approach identified BETi-induced vulnerabilities in cancer-relevant pathways, potentially amenable to synergistic combinatorial therapy, such as combination with HDACi. The direct inhibitory effect of BETi on IFN-responsive genes in GBM cells, including CD274, indicates modulation of the tumor immune landscape and warrants further studies.

OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence.

Simple SummaryThe outcome for women diagnosed with ovarian cancer (OC), the most aggressive gynecological tumor worldwide, remains very poor. Encouraging therapeutic impact of epigenetic drugs has been suggested in a wide range of human solid tumors, including OC. The present study assessed the in vitro cytostatic and cytotoxic effects of OTX015, a pan Bromodomain and Extra-Terminal motif inhibitor, in human OC cells, both as single treatment and in combination with radiotherapy. Cellular, molecular and computational network analyses indicated the centrality of GNL3 downregulation in mediating the OTX015-related antitumor efficacy that blocks disease progression/maintenance and radioresistance acquisition. Our preclinical results confirm that targeted and combinatorial treatments represent effective anticancer strategies to be translated in the clinical research for improving OC patient care.Ovarian cancer (OC) is the most aggressive gynecological tumor worldwide and, notwithstanding the increment in conventional treatments, many resistance mechanisms arise, this leading to cure failure and patient death. So, the use of novel adjuvant drugs able to counteract these pathways is urgently needed to improve patient overall survival. A growing interest is focused on epigenetic drugs for cancer therapy, such as Bromodomain and Extra-Terminal motif inhibitors (BETi). Here, we investigate the antitumor effects of OTX015, a novel BETi, as a single agent or in combination with ionizing radiation (IR) in OC cellular models. OTX015 treatment significantly reduced tumor cell proliferation by triggering cell cycle arrest and apoptosis that were linked to nucleolar stress and DNA damage. OTX015 impaired migration capacity and potentiated IR effects by reducing the expression of different drivers of cancer resistance mechanisms, including GNL3 gene, whose expression was found to be significantly higher in OC biopsies than in normal ovarian tissues. Gene specific knocking down and computational network analysis confirmed the centrality of GNL3 in OTX015-mediated OC antitumor effects. Altogether, our findings suggest OTX015 as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with OC.

A Phase 1 Study of INCB057643 Monotherapy in Patients with Relapsed or Refractory Myelofibrosis (INCB 57643-103)

Background: Activation of transcription factors that regulate oncogenic processes is frequently observed in cancers such as myelofibrosis (MF). In preclinical studies, tumors associated with dysregulation of transcription factors appear to be responsive to inhibition of bromodomain and extra-terminal motif (BET) protein [Delmore JE, et al. Cell 2011; Shimamura T, et al. Clin Cancer Res 2013]. In addition to exerting a direct effect on tumor cells, BET inhibitors may also modulate tumorigenic inflammation; in a mouse model of MF, BET inhibition in combination with the Janus kinase inhibitor ruxolitinib resulted in decreased inflammation and reduced disease burden [Kleppe M, et al. Cancer Cell 2018]. In the first-in-human study INCB 57643-101, the small-molecule BET inhibitor INCB057643, was safe and generally well tolerated as monotherapy, and demonstrated preliminary efficacy in 2 out of 3 patients with MF when administered alone or in combination with ruxolitinib [Falchook G, et al. Clin Cancer Res 2020]. The INCB 57643-103 trial is designed to further evaluate the safety and tolerability of INCB057643 monotherapy in patients with relapsed or refractory MF. Methods: INCB 57643-103 (NCT04279847) is a phase 1, single-arm, open-label, two-part dose confirmation and expansion study evaluating INCB057643 in patients with measurable histologically or cytologically confirmed (World Health Organization criteria 2016), relapsed or refractory primary MF or secondary MF (post-polycythemia vera, post-essential thrombocythemia). Patients must be ≥18 years of age, have received ≥1 line of prior therapy including ruxolitinib and have no option for therapy known to provide clinical benefit, have a risk category of intermediate-2 or high according to the Dynamic International Prognostic Scoring System, have an Eastern Cooperative Oncology Group performance status 0-2, have a life expectancy of 24 weeks or more, and be willing to provide a pretreatment bone marrow biopsy and/or aspirate at baseline (or an archival sample obtained after most recent therapy). Patients will be excluded if they have received prior treatment with a BET inhibitor; received anticancer treatment within specified intervals before the first administration of study drug or receiving concurrent anticancer therapy; received allogeneic hematopoietic stem cell transplant (6 months or less before enrollment) or have active graft versus host disease or received immunosuppressive therapy after allogeneic transplant 2 weeks or less before study drug treatment; have significant and uncontrolled medical events such as gastrointestinal or cardiovascular; have a history of bleeding disorder or at a high risk of bleeding; or have abnormal hematologic, hepatic, renal, coagulation, or metabolic laboratory values. Approximately 15 patients will be enrolled. Part 1 will evaluate initial safety and tolerability of INCB057643. Up to 6 patients will self-administer oral INCB057643 4 mg once daily (QD) continuously. Doses will be declared tolerable if dose-limiting toxicities (DLT) occur in ≤2 patients and discontinuation due to treatment-related adverse events (TRAEs) occurs in ≤2 patients during the DLT evaluation period. Part 2 will further characterize safety and tolerability as well as evaluate preliminary efficacy. Up to 9 patients will receive INCB057643 at 4 mg QD if the dose is deemed tolerable in Part 1; if not, the starting dose will be 2 mg QD. Treatment may continue as long as benefit is derived and discontinuation criteria are not met. The primary study objective is assessment of safety and tolerability of INCB057643 monotherapy by monitoring the frequency and severity of AEs, performing physical examinations, and collecting vital signs and laboratory values. Secondary objectives include evaluation of anemia response by International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet (ELN) Consensus Report, red blood cell transfusion dependence, spleen volume, rate of spleen response and duration of spleen response by IWG-MRT and ELN Consensus Report, and impact on quality of life. Patients will be assessed every 3 cycles and will receive follow-up for safety for 30-35 days after last dose of study drug. Disclosures Lihou: Incyte: Current Employment, Current equity holder in publicly-traded company. Zhou:Incyte: Current Employment, Current equity holder in publicly-traded company. Zheng:Incyte: Current Employment, Current equity holder in publicly-traded company.