Bromodomain And Extra-terminal Research Articles

Abstract P61: Distinct Roles of BRD4 Isoforms in Tumor Progression and Metastasis in Embryonal Rhabdomyosarcoma

Abstract The deregulation of BRD4, a bromodomain and extraterminal (BET) protein, has been implicated in various cancers, positioning it as a promising target for drug development. Yet, the specific roles of the two primary BRD4 isoforms, BRD4-L and BRD4-S, remain poorly understood across most cancer types. This knowledge gap poses challenges in assessing the effectiveness of pan-BET inhibitors as potential therapies. This study seeks to address this gap by employing functional and transcriptomic analyses to elucidate the distinct contributions of BRD4 isoforms in embryonal rhabdomyosarcoma (ERMS). Through a comprehensive investigation, the research uncovers that BRD4-L and BRD4-S isoforms exert discrete functions in ERMS. Specifically, BRD4-L is found to exert an oncogenic role by inhibiting myogenic differentiation, partially through the activation of myostatin expression. Importantly, depletion of BRD4-L in vivo is shown to impede tumor progression without significantly affecting metastasis. In contrast, the absence of BRD4-S has an unexpected impact-while it has a minimal effect on tumor growth, it markedly enhances metastatic potential in vivo. Intriguingly, the depletion of BRD4-S leads to the heightened presence of BRD4-L and RNA Polymerase II at integrin gene promoters, consequently activating these genes. This novel insight underscores that BRD4-S acts as a gatekeeper, restraining the full scope of oncogenic activities driven by BRD4-L. Collectively, this study reveals distinctive roles for BRD4 isoforms and uncovers a delicate interplay in ERMS. Importantly, the findings indicate that pan-BRD4 inhibition might not be the optimal therapeutic strategy for ERMS due to the intricate interplay between isoforms. By illuminating the intricate functions of BRD4 isoforms, this research advances our understanding of the complex regulatory mechanisms underlying tumor progression and metastasis in ERMS. Citation Format: Dipanwita Das, Jia Yu Leung, Amos Hong Pheng Loh, Reshma Taneja. Distinct Roles of BRD4 Isoforms in Tumor Progression and Metastasis in Embryonal Rhabdomyosarcoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P61.

Targeting BRD4: Potential therapeutic strategy for head and neck squamous cell carcinoma (Review).

As a member of BET (bromodomain and extra-terminal) protein family, BRD4 (bromodomain‑containing protein 4) is a chromatin‑associated protein that interacts with acetylated histones and actively recruits regulatory proteins, leading to the modulation of gene expression and chromatin remodeling. The cellular and epigenetic functions of BRD4 implicate normal development, fibrosis and inflammation. BRD4 has been suggested as a potential therapeutic target as it is often overexpressed and plays a critical role in regulating gene expression programs that drive tumor cell proliferation, survival, migration and drug resistance. To address the roles of BRD4 in cancer, several drugs that specifically target BRD4 have been developed. Inhibition of BRD4 has shown promising results in preclinical models, with several BRD4 inhibitors undergoing clinical trials for the treatment of various cancers. Head and neck squamous cell carcinoma (HNSCC), a heterogeneous group of cancers, remains a health challenge with a high incidence rate and poor prognosis. Conventional therapies for HNSCC often cause adverse effects to the patients. Targeting BRD4, therefore, represents a promising strategy to sensitize HNSCC to chemo‑ and radiotherapy allowing de‑intensification of the current therapeutic regime and subsequent reduced side effects. However, further studies are required to fully understand the underlying mechanisms of action of BRD4 in HNSCC in order to determine the optimal dosing and administration of BRD4‑targeted drugs for the treatment of patients with HNSCC.

Abstract LB159: Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis

Abstract Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by defective clonal hematopoiesis, bone marrow fibrosis, splenomegaly, severe anemia, and the propensity for transformation to acute myeloid leukemia (AML). Inhibitors targeting JAKs (Janus kinases) have revolutionized the treatment of MF, but are limited by modest efficacy or acquired resistance after prolonged treatment. Here, we report a novel inhibitor, HP560 that targets BET (bromodomain and extra-terminal) proteins and shows promising therapeutic activities against MF and AML. Through engaging the bromodomain pocket of BET proteins in a competitive manner with acetylated histones, HP560 causes the disruption of BET proteins from chromatin. HP560 has high binding affinities for all BET proteins and strong anti-proliferative activity as well as synergistic anti-proliferative effect in combination with ruxolitinib, a JAK1/2 inhibitor. In an MF cell line derived tumor xenograft (CDX) mouse model, HP560 prolongs survival and improves splenomegaly and anemia, and these effects are enhanced when combined with ruxolitinib. HP560 also significantly and dose-dependently inhibits tumor growth in an AML CDX mouse model. Together, HP560 is a potent pan-BET inhibitor and can be developed as a monotherapy or in combination with a JAK inhibitor for the treatment of MF. HP560 structure will not be disclosed. Citation Format: Jing Li, Lei Fan, Hongwei Yuan, Yongxu Huo, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB159.

BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney—an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.

Abstract 7600: Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer related mortality in the United States, largely due to ineffective systemic therapy. While novel KRAS target strategies are in development, few agents have been investigated to target the transcriptional machinery for global inhibition of expression. Recent small molecule inhibitors targeting transcription (i.e., CDK7) have shown preliminary evidence of disease control; however, combination therapies have lagged, likely due to uncertainty in clinical toxicity. Text mining analysis suggests combination BET and CDK9 inhibition may be promising against PDAC. Here, we present preclinical evidence of synergistic activity for the combination of bromodomain and extraterminal (BET) inhibitor (ZEN3694) and CDK9 inhibitor (VIP152) using patient derived cancer organoids (PCOs). Methods: Dose response curves were generated for ZEN3694 and VIP152 to assess their single agent activity against PCOs using a low volume format. ZEN3694 was treated continuously while VIP152 was removed after 24h to mimic pharmacokinetics with assay endpoint at 144h. Synergy assays were designed using a combination dose titration grid. Viability was determined in all assays using 3D CellTiter Glo (CTG, 50% v/v). Synergy scores were produced using SynergyFinder 3.0 including Zero Interaction Potency (ZIP), Bliss Independence (Bliss), Highest Single Agent (HSA), and Loewe Additivity (Loewe). Western blot analysis was performed against global markers of active RNA polymerase II (phosphorylation at serine 2, 5, 7) and antiapoptotic protein, BCL xL, and MCL 1. Results: ZEN3694 was observed to have a single agent IC50 in the single uM range across 3 independent PDAC PCO lines (PDAC1 3.7uM, PDAC2 1.1uM, PDAC3 2.9uM). VIP152 showed significant potency in the same 3 PDAC PCO lines (PDAC1 140.2nM, PDAC2 30.5nM, PDAC3 43.4nM). The combination of ZEN3694 and VIP152 was found to have increased therapeutic activity with double digit synergy scores across all reference models: PDAC1 (Loewe 17.3, HSA 19.8, ZIP 20.2, and Bliss 17.2) and PDAC2 (Loewe 34.7, HSA 28.3, ZIP 19.7, and Bliss 19.6). Western blots of protein collected from organoids treated for 48h showed on target activity, including decrease in the phosphorylation of Rbp1 (Ser 2, Ser 5, and Ser 7) compared to both control and single agents and decreased expression in antiapoptotic proteins (MCL 1 and BCL xL). Conclusion: Here, we show the activity of dual transcription targeting with BET and CDK9 inhibition in patient derived pancreatic cancer organoid models. The combination of ZEN3694 and VIP152 has in vitro synergy in PDAC PCO models with on target activity. Ongoing work is validating this combination in animal models to assess toxicity, in vivo activity, and RNA expression profiling to understand transcriptional pathway dependency and therapeutic resistance. Citation Format: Lucas J. Koeppel, Austin Stram, Robert J. Millikin, Ellie Riedl, Md Shahadat Hossan, Ethan Samuel Lin, Ron Stewart, Jeremy D. Kratz. Synergy of ZEN3694 and VIP152 for dual transcriptional targeting of BET and CDK9 in patient derived organoid models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7600.

Delivery of a BET protein degrader via a CEACAM6-targeted antibody–drug conjugate inhibits tumour growth in pancreatic cancer models

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

The BET inhibitor GNE-987 effectively induces anti-cancer effects in T-cell acute lymphoblastic leukemia by targeting enhancer regulated genes.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy originating from T progenitor cells. It accounts for 15% of childhood and 25% of adult ALL cases. GNE-987 is a novel chimeric molecule developed using proteolysis-targeting chimeras (PROTAC) technology for targeted therapy. It consists of a potent inhibitor of the bromodomain and extraterminal (BET) protein, as well as the E3 ubiquitin ligase Von Hippel-Lindau (VHL), which enables the effective induction of proteasomal degradation of BRD4. Although GNE-987 has shown persistent inhibition of cell proliferation and apoptosis, its specific anti-tumor activity in T-ALL remains unclear. In this study, we aimed to investigate the molecular mechanisms underlying the anti-tumor effect of GNE-987 in T-ALL. To achieve this, we employed technologies including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and CUT&Tag. The degradation of BET proteins, specifically BRD4, by GNE-987 has a profound impact on T-ALL cell. In in vivo experiments, sh-BRD4 lentivirus reduced T-ALL cell proliferation and invasion, extending the survival time of mice. The RNA-seq and CUT&Tag analyses provided further insights into the mechanism of action of GNE-987 in T-ALL. These analyses revealed that GNE-987 possesses the ability to suppress the expression of various genes associated with superenhancers (SEs), including lymphoblastic leukemia 1 (LCK). By targeting these SE-associated genes, GNE-987 effectively inhibits the progression of T-ALL. Importantly, SE-related oncogenes like LCK were identified as critical targets of GNE-987. Based on these findings, GNE-987 holds promise as a potential novel candidate drug for the treatment of T-ALL.

BRD4 isoforms have distinct roles in tumour progression and metastasis in rhabdomyosarcoma.

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analysed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in fusion negative embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumour progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumour growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. In fusion positive alveolar rhabdomyosarcoma, BRD4-L is unrestricted in its oncogenic role, with no evident involvement of BRD4-S. Our work unveils isoform-specific functions of BRD4 in rhabdomyosarcoma.

Combined BET and MEK Inhibition synergistically suppresses melanoma by targeting YAP1.

Rationale: The response rate to the MEK inhibitor trametinib in BRAF-mutated melanoma patients is less than 30%, and drug resistance develops rapidly, but the mechanism is still unclear. Yes1-associated transcriptional regulator (YAP1) is highly expressed in melanoma and may be related to MEK inhibitor resistance. The purpose of this study was to investigate the mechanism of YAP1 in MEK inhibitor resistance in melanoma and to screen YAP1 inhibitors to further determine whether YAP1 inhibition reverses MEK inhibitor resistance. Methods: On the one hand, we analyzed paired melanoma and adjacent tissue samples using RNA-seq and found that the Hippo-YAP1 signaling pathway was the top upregulated pathway. On the other hand, we evaluated the transcriptomes of melanoma samples from patients before and after trametinib treatment and investigated the correlation between YAP1 expression and trametinib resistance. Then, we screened for inhibitors that repress YAP1 expression and investigated the mechanisms. Finally, we investigated the antitumor effect of YAP1 inhibition combined with MEK inhibition both in vitro and in vivo. Results: We found that YAP1 expression levels upon trametinib treatment in melanoma patients were correlated with resistance to trametinib. YAP1 was translocated into the nucleus after trametinib treatment in melanoma cells, which could render resistance to MEK inhibition. Thus, we screened for inhibitors that repress YAP1 expression and identified multiple bromodomain and extra-terminal (BET) inhibitors, including NHWD-870, as hits. BET inhibition repressed YAP1 expression by decreasing BRD4 binding to the YAP1 promoter. Consistently, YAP1 overexpression was sufficient to reverse the proliferation defect caused by BRD4 depletion. In addition, the BET inhibitor NHWD-870 acted synergistically with trametinib to suppress melanoma growth in vitro and in vivo. Conclusions: We identified a new vulnerability for MEK inhibitor-resistant melanomas, which activated Hippo pathway due to elevated YAP1 activity. Inhibition of BRD4 using BET inhibitors suppressed YAP1 expression and led to blunted melanoma growth when combined with treatment with the MEK inhibitor trametinib.

One-Pot Synthesis of Cereblon Proteolysis Targeting Chimeras via Photoinduced C(sp2)-C(sp3) Cross Coupling and Amide Formation for Proteolysis Targeting Chimera Library Synthesis.

In this study, a one-pot synthesis via photoinduced C(sp2)-C(sp3) coupling followed by amide formation to access proteolysis targeting chimeras (PROTACs) was developed. The described protocol was studied on cereblon (CRBN)-based E3-ligase binders and (+)-JQ-1, a bromodomain inhibitor, to generate BET (bromodomain and extra terminal domain) targeting protein degraders. The generated PROTACs were profiled in vitro and tested for their degradation ability with several potent candidates identified. Upfront, the individual reactions of the one-pot transformation were carefully optimized for CRBN binder functionalization and multiple heterobifunctional linker moieties were designed and synthesized. Separate scopes detailing the C(sp2)-C(sp3) coupling and one-pot PROTAC synthesis are described in this report as well as a library miniaturization study showing the high-throughput compatibility. Overall, the developed protocol provides rapid access to PROTACs in a single process, thereby allowing efficient generation of CRBN-based PROTAC libraries.

Abstract A164: Activity of dual BET and CDK9 inhibition in Pancreatic Ductal Adenocarcinoma informed from KinderMiner Prediction

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer mortality in the United States (US) with a 10% 5-year survival. Acquired resistance plays a critical role in progression, however targeted therapeutics have shown minimal clinical activity. There remains an unmet need to provide combination targeted therapies across the breadth of clinical development. KinderMiner is a highly efficient text mining algorithm developed to predict association with search terms across publicly available research. BMS986158 is a novel bromodomain and extraterminal (BET) inhibitor in early phase clinical trials, recently reported to have preclinical activity in combination with chemotherapy. Methods: Potential genes for targeting in combination with BMS986158 were queried using KinderMiner (KM) and Serial KinderMiner (SKiM). Search terms included synonymous terms of “drug resistance” and “BET”. BMS986158 was assayed for single agent activity at 72h and 144h. Lead agents in active clinical trials were screened for synergistic activity. All assays were performed with patient derived organoids (PCOs) in a low volume format with high content imaging, using dose titration in combination grid to assay synergy. Viability was assessed using 3D CellTiterGlo (CTG, Promega Inc) (50% v/v) with synergy scores determined by SynergyFinder 3.0 for Bliss Independence (Bliss), Highest Single Agent (HSA), and Loewe Additivity (Loewe). Results: A KM/SKiM query was performed against >30M primary literature reports resulting in 39 unique genes with significant relationship with BET. CDK9 was selected as a lead gene target based on CDK9 inhibitor, AZD4573, in active clinical trials with highly significant prediction from text mining screen (p<0.001). BMS986158 was found to have nanomolar single agent IC50 activity across multiple primary PDAC PCOs (n=5) that improved from 72h [43-411nM] in comparison to 144h [12-54nM]. AZD4573 was found to have single agent IC50 ranging from 18-246nM across 3 primary cultures. Therapeutically relevant dosing of 6.25nM BMS986158 and 12.5nM AZD4573 in combination was found to induce significant response including normalized change in diameter relative to control, single agent BMS986158, and AZD4573 (effect sizes=1.7, 1.4, 0.7) The combination of BMS986158 and AZD4573 was also found to have augmented activity with positive synergy scores across all reference models: Bliss=3.47, HSA=8.81, and Loewe=15.92. Conclusion: Here, we adapt a novel text-mining algorithm, KinderMiner, to screen agents for combination with BMS986158 to inform combination therapeutics. The combination of BMS986158 and AZD4573 was shown to have improved activity in a model of PDAC PCOs. Validation of the combination efficacy across different PDAC PCOs is ongoing to compare this activity across a diversity of mutational profiles. Future directions will assay the mechanistic modeling to characterize the interaction between transcriptional regulation and the induction of apoptosis. Citation Format: Austin Stram, Luke Koeppel, Rob Millikin, Eleanor Riedl, Md Shahadat Hossan, Ethan S Lin, Ron Stewart, Jeremy D Kratz. Activity of dual BET and CDK9 inhibition in Pancreatic Ductal Adenocarcinoma informed from KinderMiner Prediction [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 A164.

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS). BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcriptomes were analyzed by RNA sequencing (RNAseq), followed by pathway enrichment analysis. We confirmed results for selective target genes by real-time PCR, ELISA, and Western blotting. BRD3 regulates the expression of several cytokines and chemokines in FLS, and positively correlates with inflammatory scores in the RA synovium. In addition, RNAseq pointed to a profound role of BRD3 in regulating FLS proliferation, metabolic adaption, and response to stress, including oxidative stress, and autophagy. BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in FLS and executes many functions of BET proteins that have previously been identified using pan-BET inhibitors.

Promise: Investigation into the Combination of a Potent Beti with Ruxolitinib in Patients with High or Intermediate-2 Risk Myelofibrosis Not Receiving an Adequate Response with Ruxolitinib Alone

ISRCTN: 12451433 Background: Myelofibrosis (MF), a chronic myeloproliferative neoplasm (MPN), often results in progressive splenomegaly and cytopenia (1). Acute myeloid leukaemia develops in 10-20% of patients, and median survival with intermediate-2 disease is four years (2). Stem cell transplant can be curative, but is only suitable for younger, comorbidity-free patients (median MF diagnosis is 73.6 years) (3). Janus Kinase 2 (JAK2) mutation is common in MPN, and the JAK1/2 inhibitor ruxolitinib was approved in 2011 (4). However, patients still experience high disease burden, with a clear need for further treatments. Research has now focused on bromodomain and extra terminal (BET) inhibitors (5), in particular the role these have in disease associated inflammation (6). Preclinical data suggests BETi disrupts this inflammation leading to anti-fibrotic activity within MPN models. BETi with ruxolitinib eliminates fibrosis in MF mouse models thus overcoming a limitation of ruxolitinib monotherapy (7). OPN-2853 (previously PLX2853) is potent against isolated bromodomains from multiple BET proteins with rapid drug absorption, high Cmax, and short half-life differentiating it from other BETi. Preclinical efficacy has been demonstrated across a range of hematologic malignancies. PROMise is evaluating OPN-2853 in combination with ruxolitinib to identify the recommended phase 2 dose (RP2D). Methods: Primary Objectives: To assess the RP2D of OPN-2853 in combination with ruxolitinib that is safe and tolerableTo assess the efficacy of the combination of OPN-2853 and ruxolitinib for reduction in spleen size PROMise is a single country (UK) multicentre study, recruiting up to 60 patients with high or intermediate-2 risk MF who are not receiving an adequate response on ruxolitinib alone. Patients must have been treated with ruxolitinib for at least 24 weeks, on a stable dose for at least 4 weeks, and have ongoing splenomegaly. PROMise is split into three ruxolitinib dose categories based on platelet count which are assessed independently, Low Dose (5mg-20mg daily), Mid Dose (25mg-45mg daily) and High Dose (≥50mg daily). These are evaluated in combination with three OPN-2853 dose levels, Level 1 (20mg OD Decreased Dose), Level 2 (40mg OD Starting Dose) and Level 3 (80mg OD Increased Dose). Cohorts of patients (n=2-4) receive up to eight 21-day cycles of OPN-2853 and ruxolitinib. Patients are closely monitored for Dose Limiting Toxicities in cycle 1. The Trial Safety Committee (TSC) meets following cycle 1 to establish the dose recommendations of OPN-2853 for each dose category using the continual reassessment method (CRM). The CRM is utilised independently for each dose category, as such it is possible for the low, mid and high doses to be allocated to different OPN-2853 doses. Following cycle 8, patients may continue to receive OPN-2853 if experiencing clinical benefit and are followed up annually. Peripheral blood, bone marrow aspirate and bone marrow trephine samples are taken from all patients to measure the proportion who achieve a molecular response, and to determine the on target transcriptional response in myeloid cells. Additionally, pharmacokinetic samples are taken at specified timepoints pre and post dose to establish PK properties of OPN-2853 in combination with ruxolitinib. Current Status: To date PROMise has recruited 14 patients across all dose categories, and two TSC meetings have taken place.

Modulation of Biomarkers By BET Inhibitor, BMS-986158, Including JAK2 Variant Allele Frequency (VAF), Bone Marrow (BM) Fibrosis, and Reversal of Abnormal Cytokine Production in Intermediate- or High-Risk Myelofibrosis (MF)

Background MF, driven by JAK/STAT pathway activation, results in an aberrant stem cell niche, leading to abnormal cytokine production, an inflammatory BM microenvironment, splenomegaly, and an array of other symptoms. Bromodomain and extra terminal (BET) proteins play a role in cancer cell proliferation, survival, and oncogenic progression. In combination with Janus kinase inhibitors (JAKi), ruxolitinib (RUX) or fedratinib (FED), BET inhibitors (BETi) have been shown to reduce inflammatory signals and disease burden in preclinical models of MF. BMS-986158 is an orally bioavailable, potent, and selective small-molecule BETi (Hilton et al. ESMO 2018. Abstr 1429). BMS-986158 in combination with RUX or FED is being evaluated in the phase 1b/2 CA011-023 (NCT04817007) study in patients (pts) with first-line (1L) and second-line (2L) MF, respectively. Clinical observations, including spleen volume reduction (SVR), were previously presented (Ayala et al. EHA 2023. Presentation S213). In this analysis, exploratory biomarkers were assessed for potential early disease-modifying activities of BMS-986158. These included: measurement of longitudinal JAK2 VAF, which may be indicative of mutation clonal burden; evaluation of the BM microenvironment, including fibrosis, megakaryocyte clusters, and other morphological features; and circulatory cytokines and other factors associated with MF (Hasselbalch. Cytokine Growth Factor Rev. 2013). Methods In dose escalation, RUX-naïve pts (1L MF) received BMS-986158 2.0, 3.0, or 3.75 mg QD 5d on/2d off over 28-day cycles and RUX 15 mg BID; pts relapsed, refractory or intolerant to prior RUX treatment (2L MF) received BMS-986158 0.5, 0.75, or 1.25 mg QD 5d on/2d off over 28-day cycles and FED 400 mg QD. Longitudinal assessment of JAK2 VAF was performed on multiple sample types, including isolated circulatory CD34+ cells, CD66b+ cells, and whole blood, using the MLL next-generation sequencing targeted exosome gene panel. Variants and indels were called using Pisces and Pindel, followed by annotation to COSMIC, ClinVar, gnomAd, and dbNSFP databases. BM assessments were performed on BM biopsies at baseline and post-treatment at the end of cycles 3 (optional), 6, and 12; fibrosis scores were determined by independent pathologist assessment (prognostic scoring system DIPSS PLUS-WHO 2017) and digital analysis of megakaryocyte clustering, reticulin pattern, and overall cellularity. The modulation of MF-associated circulatory cytokines and growth factors (79 soluble factors [SF] in total) was evaluated by multiple Rules-Based Medicine (RBM) custom MAP-HMP panels; aggregated data were analyzed. Results JAK2 VAF reduction was observed post-treatment with BMS-986158+RUX in 6/7 (86%) 1L MF pts with available data ( Figure A). The maximum VAF reduction from baseline was 52%, where > 25% VAF reduction looks to be associated with improved spleen response (6/6 pts [100%] achieved SVR35). In 2L MF pts treated with BMS-986158+FED, JAK2 VAF reduction was observed in 1/4 (25%) pts. Similar degree of changes in JAK2 VAFwere observed between CD34+ isolated cells and whole blood samples. In addition, BM fibrosis was either stabilized (3/9; 33%) or had ≥ 1 grade reduction (5/9; 56%) with BMS-986158+RUX at 24 weeks compared with baseline in 1L MF. Digital analysis further suggests potential normalized cellularity and decreased megakaryocyte clusters and reticulin levels in fibrosis-reduced samples. BM fibrosis reduction was also observed with BMS-986158+FED in 2L MF (3/8 [38%] stabilized and 2/8 [25%] reduced fibrosis). Lastly, sustained reduction of inflammatory cytokines downstream of NF-κB pathway was observed, including TNFα, IL-1ra, and VEGF ( Figure B). In addition, increased levels of metabolic adipokine/hormone SF (eg, leptin, erythropoietin) and reduction of SF specific to BETi+JAKi combination (eg, DKK1, CD27, TIMP3; Mascarenhas et al. J Clin Oncol. 2023) were observed as expected. Conclusions Preliminary data suggest combination treatment with BMS-986158 and JAKi in MF may modulate JAK2 VAF, BM microenvironment, circulatory cytokines, and other SF, providing evidence of early disease modifying potential of these drug combinations.

Bromodomain and Extra-Terminal Inhibitor INCB057643 (LIMBER-103) in Patients with Relapsed or Refractory Myelofibrosis and Other Advanced Myeloid Neoplasms: A Phase 1 Study

Introduction: Bromodomain and extra-terminal (BET) proteins are epigenetic readers that regulate expression of critical oncoproteins involved in the pathophysiology of myelofibrosis (MF) and other hematologic malignancies including B-lymphoma-2, nuclear factor kappa, and c-Myc. In a previous phase 1/2 clinical trial, the small-molecule oral BET inhibitor INCB057643, evaluated as monotherapy and in combination with ruxolitinib, was associated with favorable tolerability and encouraging clinical activity in patients with advanced malignancies. Methods: This ongoing phase 1, 3+3 dose-escalation/expansion study (NCT04279847) is evaluating the safety and tolerability of INCB057643 (4 mg once daily [qd] with escalation up to 12 mg qd) in patients aged ≥18 years as (1) monotherapy (part 1) in relapsed or refractory (R/R) MF, myelodysplastic syndromes (MDS), or MDS/myeloproliferative neoplasm (MPN) overlap syndromes (MDS/MPN) or (2) added to ruxolitinib (part 2) in patients with MF and suboptimal response to ruxolitinib. The primary endpoints are safety and tolerability, including identification of dose-limiting toxicities (DLTs). Secondary endpoints in patients with MF include spleen volume response (≥35% reduction from baseline at Week 24 per magnetic resonance imaging/computed tomography scan) and symptom response (≥50% reduction from baseline at Week 24 in MPN-Symptom Assessment Form Total Symptom Score [MPN-SAF TSS]). Results: As of data cutoff on June 1, 2023, 14 patients were treated in part 1 (4 mg, n=6; 8 mg, n=4; 10 mg, n=2; 12 mg, n=2), and 6 were treated in part 2 (4 mg, n=3, 6 mg, n=3). In part 1, patients had a median (range) age of 69.5 (50.0-79.0) years and a median (range) study treatment duration of 177 (9-375) days; 8 were men. In part 2, patients had a median (range) age of 71 (64-76) years; median (range) study treatment duration of 92.5 (46-274) days; 3 were men. 16 patients had MF; 4 had MDS/MPN, including 1 with chronic myelomonocytic leukemia. A total of 10 patients discontinued treatment, 4 for lack of efficacy or disease progression and 4 due to adverse events (AEs; 3 in the monotherapy group, 1 in the combination group). Thrombocytopenia (n=3) and anemia/thrombocytopenia (n=1) were the only treatment-emergent AEs (TEAEs) leading to discontinuation, and thrombocytopenia was the most common TEAE (n=11). Grade ≥3 TEAEs occurring in ≥1 patient were thrombocytopenia (n=5), anemia (n=4), and hypokalemia (n=2). 12 serious AEs occurred across 5 patients (only COVID-19 occurred in >1 patient [n=2]); all but one serious AE (pneumonia) was considered unrelated to study treatment. There were 2 DLTs: thrombocytopenia in 1 patient with MDS/MPN in the 12 mg cohort and hyperbilirubinemia in 1 patient with MF in the 12 mg cohort. 2 deaths occurred on study, both in the 4 mg monotherapy cohort due to disease progression (MF, n=1; MDS/MPN, n=1). Overall, 5 patients on monotherapy and 2 on combination therapy demonstrated a reduction in spleen volume at Week 12 (≥35% reduction in 2 patients and 1 patient, respectively); at Week 24, 3 patients on monotherapy and 2 on combination therapy had a reduction in spleen volume (≥35%: 1 patient and 1 patient, respectively; Figure 1). Percentage change from baseline in MPN-SAF TSS in individual patients is reported in Figure 2; at Week 12, ≥50% improvement from baseline occurred in 3 patients on monotherapy and 1 on combination therapy (≥50% improvement at Week 24: 2 patients and 1 patient). Conclusions: INCB057643 monotherapy (4 and 8 mg qd) and combined (4 and 6 mg qd) with ruxolitinib was generally well tolerated, whereas the 12 mg qd monotherapy dose caused 2 DLTs. There were no treatment-related fatal events. Improvements in spleen size and symptom burden were observed in patients receiving ≥8 mg in the monotherapy group and 4 mg in the combination therapy group. Dose finding (part 1) is ongoing with 10 mg qd, after which the recommended dose(s) for expansion will be declared. Dose escalation is also ongoing in the combination therapy group.

Structure-Guided Design of a Domain-Selective Bromodomain and Extra Terminal N-Terminal Bromodomain Chemical Probe.

Small-molecule-mediated disruption of the protein-protein interactions between acetylated histone tails and the tandem bromodomains of the bromodomain and extra-terminal (BET) family of proteins is an important mechanism of action for the potential modulation of immuno-inflammatory and oncology disease. High-quality chemical probes have proven invaluable in elucidating profound BET bromodomain biology, with seminal publications of both pan- and domain-selective BET family bromodomain inhibitors enabling academic and industrial research. To enrich the toolbox of structurally differentiated N-terminal bromodomain (BD1) BET family chemical probes, this work describes an analysis of the GSK BRD4 bromodomain data set through a lipophilic efficiency lens, which enabled identification of a BD1 domain-biased benzimidazole series. Structure-guided growth targeting a key Asp/His BD1/BD2 switch enabled delivery of GSK023, a high-quality chemical probe with 300-1000-fold BET BD1 domain selectivity and a phenotypic cellular fingerprint consistent with BET bromodomain inhibition.

Discovery of (R)-4-(8-methoxy-2-methyl-1-(1-phenylethy)-1H-imidazo[4,5-c]quinnolin-7-yl)-3,5-dimethylisoxazole as a potent and selective BET inhibitor for treatment of acute myeloid leukemia (AML) guided by FEP calculation

The functions of the bromodomain and extra terminal (BET) family of proteins have been proved to be involved in various diseases, particularly the acute myeloid leukemia (AML). In this work, guided by free energy perturbation (FEP) calculation, a methyl group was selected to be attached to the 1H-imidazo[4,5-c]quinoline skeleton, and a series of congeneric compounds were synthesized. Among them, compound 10 demonstrated outstanding activity against BRD4 BD1 with an IC50 value of 1.9 nM and exhibited remarkable antiproliferative effects against MV4-11 cells. The X-ray cocrystal structure proved that 10 occupied the acetylated lysine (KAc) binding cavity and the WPF shelf of BRD4 BD1. Additionally, 10 displayed high selectivity towards BET family members, effectively inhibiting the growth of AML cells, promoting apoptosis, and arresting the cell cycle at the G0/G1 phase. Further mechanistic studies demonstrated that compound 10 could suppress the expression of c-Myc and CDK6 while enhancing the expression of P21, PARP, and cleaved PARP. Moreover, 10 exhibited remarkable pharmacokinetic properties and significant antitumor efficacy in vivo. Therefore, compound 10 may represent a new, potent and selective BET bromodomain inhibitor for the development of therapeutics to treat AML.

A Combination of CPI-0610 and SAHA Induces Apoptosis through STAT3 and p38 Signalling Pathways in Diffuse Large B-cell Lymphoma Cells.

Although immunotherapies have greatly improved diffuse large B-cell lymphoma (DLBCL) prognosis, a proportion of patients remain to be relapsed or refractory. Therefore, the identification of novel therapeutic targets and drugs is urgently required. Inhibition of the bromodomain and extra-terminal (BET) proteins has been a promising therapeutic strategy for various haematologic cancers. CPI-0610 is a potent and selective BET inhibitor. The effects of CPI-0610 in DLBCL cells have not been reported yet. The aim of this study was to assess the effects of CPI-0610 in DLBCL and its underlying mechanisms. DLBCL cells were treated with CPI-0610, followed by measuring cell viability, cell cycle, apoptosis, autophagy, and specific cell signaling pathways. Moreover, immunodeficient mice were engrafted with SUDHL2 cells and then treated with CPI-0610 for analysis of tumor burden. We also analyzed the synergistic effect of CPI-0610 with histone deacetylase inhibitor suberoylanilide hydroxamic acid. The present study demonstrated that CPI-0610 displayed cell cytotoxicity by arresting the G1 cell cycle and inducing endogenous and exogenous apoptotic pathways. Additionally, CPI-0610 decreased BRD4 and c-Myc expressions and affected MAPK, JAK/STAT, and AKT signalling pathways in human DLBCL cells. An in vivo experiment exhibited that CPI-0610 decreased the primary tumour growth of the DLBCL xenograft model. Furthermore, the use of CPI-0610 in combination with suberoylanilide hydroxamic acid exhibited a specific synergistic effect in inducing apoptosis through the regulation of STAT3 and p38. Targeting BET may be an effective therapeutic strategy and potentiated by a combination with histone deacetylase inhibition in DLBCL.

Integration of signaling pathway and bromodomain and extra-terminal domain inhibition for the treatment of mutant Kirsten rat sarcoma viral oncogene homolog cancer.

Mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) is now a drugable oncogenic driver and the KRAS G12C variant responds clinically to sotorasib and adagrasib that covalently block the cysteine of the active center and inhibit downstream signaling and proliferation. Unfortunately, progression-free survival (PFS) of lung cancer patients is only 5-6 months and no survival advantage has been found for sotorasib in comparison to docetaxel chemotherapy. Increased responses to KRAS inhibitors are tested in combination with the son of sevenless 1 (SOS1) inhibitors, upstream and downstream signaling modulators as well as chemotherapeutics. Some of these approaches are limited by toxicity to normal tissues and by diverse mechanisms of resistance. In essence, most of these attempts are directed to the inhibition of proliferation by impairment of the signal transduction pathways. The final target of KRAS-mediated growth stimulation is MYC in the cell nucleus that stimulates transcription of a host of genes. In detail, MYC alters genomic enhancer and super-enhancers of transcription that are frequently deregulated in cancer. Such enhancers can be targeted by bromodomain and extra-terminal (BET) inhibitors (BETi) or degraders and this review discusses whether integrated SOS1 inhibition and BET targeting of MYC synergizes against mutant KRAS tumor growth. BET degraders in the form of proteolysis-targeting chimeras (PROTACs) combined with BAY-293-mediated SOS1 inhibition revealed marked cytotoxic synergy against mutant KRAS cancer cells and may constitute a promising option for clinical treatment.

Apabetalone, a Clinical-Stage, Selective BET Inhibitor, Opposes DUX4 Target Gene Expression in Primary Human FSHD Muscle Cells.

Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4's deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor-and DUX4 transcriptional repressor-losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.