BET Inhibitors Research Articles

Physachenolide C is a Potent, Selective BET Inhibitor.

A pulldown using a biotinylated natural product of interest in the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.

Analysis of differentially expressed long non-coding RNAs in LPS-induced human HMC3 microglial cells

BackgroundLong non-coding RNAs (lncRNAs) are emerging as key modulators of inflammatory gene expression, but their roles in neuroinflammation are poorly understood. Here, we identified the inflammation-related lncRNAs and correlated mRNAs of the lipopolysaccharide (LPS)-treated human microglial cell line HMC3. We explored their potential roles and interactions using bioinformatics tools such as gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and weighted gene co-expression network analysis (WGCNA).ResultsWe identified 5 differentially expressed (DE) lncRNAs, 4 of which (AC083837.1, IRF1-AS1, LINC02605, and MIR3142HG) are novel for microglia. The DElncRNAs with their correlated DEmRNAs (99 total) fell into two network modules that both were enriched with inflammation-related RNAs. However, treatment with the anti-inflammatory agent JQ1, an inhibitor of the bromodomain and extra-terminal (BET) protein BRD4, neutralized the LPS effect in only one module, showing little or even enhancing effect on the other.ConclusionsThese results provide insight into, and a resource for studying, the regulation of microglia-mediated neuroinflammation and its potential therapy by small-molecule BET inhibitors.

The Role of Bromodomain and Extraterminal (BET) Proteins in Controlling the Phagocytic Activity of Microglia In Vitro: Relevance to Alzheimer’s Disease

The correct phagocytic activity of microglia is a prerequisite for maintaining homeostasis in the brain. In the analysis of mechanisms regulating microglial phagocytosis, we focused on the bromodomain and extraterminal domain (BET) proteins: Brd2, Brd3, and Brd4, the acetylation code readers that control gene expression in cooperation with transcription factors. We used pharmacological (JQ1) and genetic (siRNA) inhibition of BET proteins in murine microglial cell line BV2. Inhibition of BET proteins reduced the phagocytic activity of BV2, as determined by using a fluorescent microspheres-based assay and fluorescently labelled amyloid-beta peptides. Gene silencing experiments demonstrated that all brain-existing BET isoforms control phagocytosis in microglia. From a set of 84 phagocytosis-related genes, we have found the attenuation of the expression of 14: Siglec1, Sirpb1a, Cd36, Clec7a, Itgam, Tlr3, Fcgr1, Cd14, Marco, Pld1, Fcgr2b, Anxa1, Tnf, Nod1, upon BET inhibition. Further analysis of the mRNA level of other phagocytosis-related genes which were involved in the pathomechanism of Alzheimer's disease demonstrated that JQ1 significantly reduced the expression of Cd33, Trem2, and Zyx. Our results indicate the important role of BET proteins in controlling microglial phagocytosis; therefore, targeting BET may be the efficient method of modulating microglial activity.

BRD4 inhibitor suppresses melanoma metastasis via the SPINK6/EGFR-EphA2 pathway

BET inhibition or BRD4 depletion is a promising and attractive therapy for metastatic melanoma; however, the mechanism is still unclear. Here, we indicated that BET inhibition suppressed melanoma metastasis both in vitro and in vivo and identified a new mechanism by which BET inhibitors suppress melanoma metastasis by blocking the direct interaction of BRD4 and the SPINK6 enhancer. Moreover, we demonstrated that SPINK6 activated the EGFR/EphA2 complex in melanoma and the downstream ERK1/2 and AKT pathways. Thus, these results identified the SPINK6/EGFR-EphA2 axis as a new oncogenic pathway in melanoma metastasis and support the further development of BRD4 inhibitors for the treatment of metastatic melanoma in the clinic.

Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair.

BRD4 functions as an epigenetic reader and plays a crucial role in regulating transcription and genome stability. Dysregulation of BRD4 is frequently observed in various human cancers. However, the molecular details of BRD4 regulation remain largely unknown. Here, we report that PRMT2- and PRMT4-mediated arginine methylation is pivotal for BRD4 functions on transcription, DNA repair, and tumor growth. Specifically, PRMT2/4 interacts with and methylates BRD4 at R179, R181, and R183. This arginine methylation selectively controls a transcriptional program by promoting BRD4 recruitment to acetylated histones/chromatin. Moreover, BRD4 arginine methylation is induced by DNA damage and thereby promotes its binding to chromatin for DNA repair. Deficiency in BRD4 arginine methylation significantly suppresses tumor growth and sensitizes cells to BET inhibitors and DNA damaging agents. Therefore, our findings reveal an arginine methylation-dependent regulatory mechanism of BRD4 and highlight targeting PRMT2/4 for better antitumor effect of BET inhibitors and DNA damaging agents.

Regulation of Epigenetic BET Proteins on Alzheimer’s Disease in Preclinical Models

AbstractBackgroundEpigenetic regulation plays substantial roles in human pathophysiology, which provides opportunities for intervention in human disorders through targeting epigenetic pathways. Emerging evidence from both preclinical and clinical studies suggested the potential in developing therapeutics of Alzheimer’s disease (AD) by targeting epigenetic regulatory proteins. Recently there has been keen interest in the family of bromodomain (BD)‐containing BET (bromodomain and extra terminal domain) proteins, which consists of 4 protein members. All BET proteins have two BD domains, which confer significant and complex regulatory mechanisms for AD.MethodHere, we developed domain‐specific small molecule probe targeting BD1 and evaluated expression of BET in AD transgenic animals compared to wild type animals using positron emission tomography‐based molecular imaging. We also investigated the effects of pharmacological inhibition of BET on AD cell models.ResultWe found that 5xFAD animals were characterized by increased signal in the brain in association with our BD1‐specific probe. Using BET inhibitors, AD‐related proteins were significantly changed.ConclusionOur study shows that BET protein may associate with AD by a gain of function mechanism and that inhibitors for specific domains may be utilized for AD in the presence of BET increase.

Targeting BET proteins inhibited the growth of non-small cell lung carcinoma through downregulation of Met expression.

Hepatocyte growth factor receptor (HGFR or Met) upregulation has been proven to play important roles in non-small cell lung carcinoma (NSCLC). Interestingly, chemoresistance against epidermal growth factor receptor (EGFR) inhibitors including erlotinib and gefitinib was also related to Met. Targeting bromodomain and extra terminal domain (BET) proteins, especially BRD4, has shown inhibitory effects on lung cancer, but the mechanism is unclear. Herein, we found that JQ1 (BET inhibitor) suppressed NSCLC cell growth, reduced the Met expression, and contributed to inactivation of PI3K/Akt and MAPK/ERK pathways. Moreover, another BET protein inhibitor I-BET151, or BRD4 depletion, also inhibited NSCLC cell growth and downregulated Met. JQ1 inhibited HGF-induced cell growth and Met/PI3K/Akt activation, also inhibited A549 tumor growth in xenograft mouse models, in parallel with Met downregulation. Moreover, JQ1 inhibited the growth of paired erlotinib-sensitive and resistant HCC827 cells in parallel with Met downregulation and PI3K/Akt signaling inactivation. JQ1 also exerted inhibitory influences on the growth of erlotinib-sensitive and resistant HCC827 tumors in xenograft mouse models. These results suggested that targeting BET proteins inhibited NSCLC via downregulating Met and inactivating PI3K/AKT pathway. Our findings reveal a novel mechanism of BET proteins implicated in NSCLC progression with Met taken into consideration.

Epigenetic BET reader inhibitor apabetalone (RVX-208) counters proinflammatory aortic gene expression in a diet induced obesity mouse model and in human endothelial cells

Background and aimsObese patients are at risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). A lipid-rich diet promotes arterial changes by inducing hypertension, oxidative stress, and inflammation. Bromodomain and extraterminal (BET) proteins contribute to endothelial and immune cell activation in vitro and in atherosclerosis mouse models. We aim to determine if BET inhibition can reduce lipid-rich diet-induced vascular inflammation in mice. MethodsBody weight, serum glucose and lipid levels were measured in mice fed a high-fat diet (HFD) or low-fat diet (LFD) for 6 weeks and at study termination. BET inhibitors apabetalone and JQ1 were co-administered with the HFD for additional 16 weeks. Aortic gene expression was analyzed post necropsy by PCR, Nanostring nCounter® Inflammation Panel and bioinformatics pathway analysis. Transcription changes and BRD4 chromatin occupancy were analyzed in primary human endothelial cells in response to TNFα and apabetalone. ResultsHFD induced weight gain, visceral obesity, high fasting blood glucose, glucose intolerance and insulin resistance compared to LFD controls. HFD upregulated the aortic expression of 47 genes involved in inflammation, innate immunity, cytoskeleton and complement pathways. Apabetalone and JQ1 treatment reduced HFD-induced aortic expression of proinflammatory genes. Congruently, bioinformatics predicted enhanced signaling by TNFα in the HFD versus LFD aorta, which was countered by BETi treatment. TNFα-stimulated human endothelial cells had increased expression of HFD-sensitive genes and higher BRD4 chromatin occupancy, which was countered by apabetalone treatment. ConclusionsHFD induces vascular inflammation in mice through TNFα signaling. Apabetalone treatment reduces this proinflammatory phenotype, providing mechanistic insight into how BET inhibitors may reduce CVD risk in obese patients.

Modulation of Virulence-Associated Traits in Aspergillus fumigatus by BET Inhibitor JQ1.

Aspergillus fumigatus is a disease-causing, opportunistic fungus that can establish infection due to its capacity to respond to a wide range of environmental conditions. Secreted proteins and metabolites, which play a critical role in fungal-host interactions and pathogenesis, are modulated by epigenetic players, such as bromodomain and extraterminal domain (BET) proteins. In this study, we evaluated the in vitro and in vivo capability of the BET inhibitor JQ1 to modulate the extracellular proteins and virulence of A. fumigatus. The abundance of 25 of the 76 extracellular proteins identified through LC-MS/MS proteomic analysis changed following JQ1 treatment. Among them, a ribonuclease, a chitinase, and a superoxide dismutase were dramatically downregulated. Moreover, the proteomic analysis of A. fumigatus intracellular proteins indicated that Abr2, an intracellular laccase involved in the last step of melanin synthesis, was absent in the JQ1-treated group. To investigate at which level this downregulation occurred and considering the ability of JQ1 to modulate gene expression we checked the level of ABR2, Chitinase, and Superoxide dismutase mRNA expression by qRT-PCR. Finally, the capacity of JQ1 to reduce the virulence of A. fumigatus has been proved using Galleria mellonella larvae, which are an in vivo model to evaluate fungal virulence. Overall, the promising activity exhibited by JQ1 suggests that A. fumigatus is sensitive to BET inhibition and BET proteins may be a viable target for developing antifungal agents.

Abstract B044: Bromodomain and Extra-Terminal Protein inhibition decreases pancreatic cancer proliferation via MYC-independent pathways

Abstract Introduction: The bromodomain and extra-terminal (BET) protein family contains proteins which have evolutionarily conserved bromodomains (BRDs) that specifically recognize acetylated lysine residues on the histone tails of chromatin and regulate gene transcription. Deregulation of these BRD-containing proteins has been seen in carcinogenesis as they are also known to play role in the regulation of the cell-cycle and MYC oncogenes. BMS-986158 is an oral BET inhibitor which has been used in the clinical trials for both hematologic and advanced solid tumor cancers. We hypothesized that BET inhibition (BETi) decreases pancreatic ductal adenocarcinoma (PDA) proliferation. Methods: Pancreatic cancer cell lines and patient-derived cell-lines (PDCLs) were treated with 2.5 to 100nM BMS-986158 for 72 hours to study dose-response effect with Incucyte. We performed RNA-seq, qPCR, protein analysis, and flow cytometry to evaluate comprehensive changes and underlying mechanisms/pathways including epithelial to mesenchymal transition (EMT) and the cancer stem cell (CSC) phenotype. Various doses of BMS-986158 were used in PDCL and PDA patient-derived organoids (PDO) in combination with chemotherapy drug to evaluate combinational effects. Results: We found that BETi induced dose-dependent decrease in growth of PDA and PDCLs and induced cancer cell death. BETi demonstrated PDO growth inhibition which was enhanced when combined with gemcitabine and paclitaxel chemotherapy. Next, we found that BETi did not affect MYC expression but decreased expression of the SNAI1 (a mesenchymal marker) protein and increased E-cadherin protein expression in PDA cell line and PDO consistent with decreased activation of an EMT program. BETi also decreased expression of CD133, a marker of CSCs in PDO. BETi also induced G1/S Phase cell cycle arrest in PDA PDCLs. Furthermore, RNA-seq studies revealed that BETi treatment inhibits KRAS pathway and upregulates pathways involved in metabolism of lipids and lysosomal degradation. RNA-seq and qPCR demonstrated that GPX3 (glutathione peroxidase 3, a key protein in reactive oxygen species response) gene expression is upregulated in BETi treated samples. Conclusions: Thus, our data demonstrate that BETi through BMS-986158 is an additional therapy for inhibiting PDA growth through mechanisms that are independent of MYC pathway, inhibition of abnormal lipid metabolism and scavenging of reactive oxygen species. Citation Format: Manjul Rana, Rita G. Kansal, Jie Fang, Benjamin T. Allen, Jun Yang, Evan S. Glazer. Bromodomain and Extra-Terminal Protein inhibition decreases pancreatic cancer proliferation via MYC-independent pathways [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B044.

Lactate Utilization Provides a Metabolic Escape to Resist the Antileukemic Activity of BET Inhibition in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly heterogeneous malignant disease of the bone marrow resulting from arrest of hematopoietic precursors and uncontrolled proliferation of myeloblasts. Inhibition of the BET-family co-activator BRD4 (BETi) shows encouraging pre-clinical activity but limited clinical activity as a single agent. We previously demonstrated that MYC suppression by BETi induces metabolic and mitochondrial changes in AML blasts leading to a collapse in mitochondrial respiration. AML blasts that are resistant to BETi undergo comparable mitochondrial respiration despite impaired glycolysis and glutaminolysis. The metabolic pathway allowing BETi-resistant AML blasts to maintain metabolic integrity is unclear. Therefore, to identify how AML blast cells metabolically bypass BET inhibition, we used two different cell lines: BETi-resistant MOLM-13 and BETi-susceptible MV-4-11 cells. AML cell lines were treated with BETi for 48 hr and global untargeted metabolic mass spectrometry revealed that glycolysis, glutaminolysis, and fatty acid metabolism were not leveraged to maintain tricarboxylic acid (TCA) cycle activity (Fig. 1A). Rather, an increased NADH/NAD+ ratio and intracellular lactate accumulation suggested that lactate was being utilized as an alternative carbon source to sustain mitochondrial respiration. In support, MOLM-13 cells treated with BETi accumulated higher levels of mitochondrial lactate dehydrogenase, which preferentially converts lactate into pyruvate to fuel the TCA cycle. The heightened capacity of MOLM-13 cells to utilize lactate better maintained cellular viability but failed to support cellular replication, suggesting that lactate utilization promotes cellular senescence during BET inhibition. Therefore, we employed three chemical inhibitors of lactate utilization to couple with BETi: AZD3965 to prevent extracellular lactate uptake, UK5099 to block lactate import into the mitochondria, and oxamate to broadly impair lactate dehydrogenase activity. All three chemical inhibitors of lactate utilization were sufficient to render MOLM-13 cells susceptible to BET inhibition, as mitochondrial respiration, ATP production, and cellular viability were decreased to levels comparable to MV-4-11 cells. A similar correlation was observed in patient samples where AML blasts with increased resistance to BETi were predisposed to utilize lactate as an alternative carbon source and rendered susceptible to BET inhibition with lactate utilization inhibitors. Furthermore, these results were translated in vivo as low-dose combination of BETi and oxamate significantly reduced burdens of BETi-resistant cells in murine models (Fig. 1B). These results demonstrate that some AML blasts metabolically adapt to overcome BET inhibition by consuming lactate but that combinations of BETi and lactate utilization inhibitors can stymie this resistance. Recognizing the metabolic switching to utilize lactate in AML therapy resistance, and studying lactate utilization inhibitors in clinical settings, especially in BETi-based therapies should be considered. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activating a Non-Genetic MYC-Regulating Pathway Sensitizes Acute Myeloid Leukemia to Bromodomain Inhibition

MYC (c-Myc) plays a central role in the oncogenic progression and is commonly overexpressed or amplified in more than 50% of human cancers, including acute myeloid leukemia (AML), which is an aggressive disease with over 70% of patients who cannot survive more than five years. Elevated MYC expression is associated with therapeutic resistance and predicts a poorer outcome in AML patients. While numerous lines of evidence have validated MYC as a therapeutic target of AML and many other cancer types, MYC protein itself has been a challenge for therapeutic targeting. Recently, MYC gene transcription has been linked to the bromodomain and extra-terminal domain (BET) family proteins. BET inhibitors (BETi) inhibit MYC expression and have shown promising antitumor activities in preclinical models and are being evaluated in clinical trials. Despite the therapeutic promise, BETi-associated drug resistance and dose-limiting side effects hinder the clinical applications. Therefore, there is a pressing need to understand the mechanisms controlling drug response and resistance to BET inhibitors. To understand the mechanisms that regulate BETi sensitivity, we leveraged a functional CRISPR screen focusing on about 1,700 human transcription factors. We discovered the aryl hydrocarbon receptor (AHR) as a novel regulator of BETi sensitivity in AML. Our results have further identified that a novel AHR-ELMSAN1 axis is critically involved in regulating MYC and BETi sensitivity. Specifically, we show that constitutive or ligand activation of AHR sensitizes leukemia cells to BETi. AHR signaling represses MYC and synergizes with BETi for maximum inhibition of MYC transcription. Ectopic expression of MYC could rescue the anti-leukemia effects induced by combined AHR activation and BET blockade. Mechanistically, our unbiased bioinformatic analysis unexpectedly identified that AHR functions through activating a non-canonical target, ELMSAN1, a less-studied epigenetic factor known as a member of the MiDAC histone deacetylase complex. Depletion of ELMSAN1 leads to upregulation of MYC and blunts AHR signaling-mediated MYC repression and BETi sensitization. Importantly, we demonstrate that AHR agonist and BETi synergistically inhibit MYC and leukemia cell growth in CD34+ primary AML patient samples in vitro and patient-derived xenografts in vivo. In summary, our data suggest that AHR-ELMSAN1 is a novel pathway for MYC repression, activation of which could sensitize AML to BET-targeting agents. As neither AHR nor ELMSAN1 is found mutated in AML, our research may open a new door to new MYC targeting paradigms by activating novel non-genetic MYC repressors.

Preclinical Efficacy of Novel Drug Combination Against AML with 3q26 Lesions and EVI1 Overexpression

The EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc finger transcription factor (TF). EVI1 protein is expressed in leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. As a result of the chromosome translocation t(3;3) or inv(3) at 3q26, the distal enhancer of the TF and tumor suppressor GATA2 is dislocated and repositioned to induce expression of EVI1, while concomitantly repressing GATA2. EVI1 overexpression results in therapy refractoriness and inferior relapse-free and overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor has activity against AML cells with inv(3)/t(3;3). Among the targets of EVI1 are the pro-growth and pro-survival proteins c-Myc and Bcl-xL, expressions of which are also regulated by enhancer activity that is undermined by BRD4 inhibition. We have recently reported that the nuclear TBL1/R1-β-catenin-TCF7L2 complex transcriptionally regulates c-Myc, cyclin D and Survivin and sustains AML stem cells, such that the undermining of this transcriptional axis by the TBL1/R1-β-catenin-binding disruptor tegavivint (BC-2059, Iterion) represses MYC, cyclin D1 and Survivin and inhibits growth and survival of AML stem-progenitor cells. Based on these observations, we interrogated the pre-clinical efficacy of pan-BET inhibitor OTX015 and/or tegavivint (TV) against AML cells with inv(3)/t(3;3) and overexpression of EVI1. Treatment with BET inhibitor OTX015 dose-dependently (100 to 1000 nM) induced apoptosis of AML cell lines (UCSD-AML1, OCI-AML20, MUTZ-3 and HNT-34) and patient-derived (PD) AML cells (AML191 and AML194) with inv(3)/t(3;3) and with or without monosomy 7. OTX015 treatment was associated with repression of c-Myc, c-Myb, CDK4, MECOM, and MCL1 as well as induction of HEXIM1 and cleaved PARP levels. We also determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions with/without monosomy 7. Furthermore, TV treatment resulted in attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, while inducing p21, CD11b, BIM and cleaved PARP levels. Next, utilizing H3K27Ac antibody Hi-ChIP (Arima Hi-ChIP), we interrogated the active gene regulatory interactions through a combined Hi-C and ChIP workflow, followed by Tn5 transposase-mediated on-bead library construction and NGS. We observed that in untreated AML191 cells numerous loop interactions were present between the super-enhancers and promoters respectively of MYC and BCL2. Following TV treatment (100 nM for 16 hours), differential loop calling revealed a loss of these interactions. RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of TGFβ, inflammatory response, TNFα and interferon signaling, and apoptosis signaling, but negative enrichment of gene sets of MYC, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in the phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99) harboring inv(3) and EVI1 overexpression. Co-treatment with TV and OTX015 or the BCL2 inhibitor venetoclax synergistically induced in vitro apoptosis (determined by SynergyFinder 2.0) in the AML cell lines and the PD AML cells. Notably, in the flank-implanted PD AML191 or tail vein AML242 models in NSG mice, treatment with TV and OTX015 for 2 to 5 weeks, compared to vehicle control or each drug alone, significantly reduced AML growth (p < 0.01). Furthermore, in the tail-vein infused and engrafted PD AML191 PDX model, treatment with TV and OTX015 or venetoclax for 6 weeks, as compared to vehicle control or each drug alone, significantly improved overall survival of the NSG mice (p < 0.01), without inducing any toxicity. These findings highlight the promising pre-clinical efficacy of novel combinations of TV and BET protein or venetoclax against AML models harboring 3q26 lesions and EVI1 overexpression.

Clinical Benefit Associated with Biomarker Changes Indicative of Disease Modification in Patients with Myelofibrosis Treated with the BET Inhibitor Pelabresib As Monotherapy or in Combination with Ruxolitinib

Bromodomain and extraterminal domain (BET) proteins play a key role in the regulation of neoplastic myeloproliferation and proinflammatory cytokine production in myelofibrosis (MF). Pelabresib (CPI-0610; PELA) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB signaling and other relevant genes involved in MF disease pathways (Mascarenhas J, et al. HemaSphere 2022;6:99-100). In the ongoing Phase 2 MANIFEST study (NCT02158858) (Kremyanskaya M, et al. Blood 2021;138:141) PELA is being evaluated as monotherapy in ruxolitinib (RUX)-intolerant, ineligible or refractory patients (pts) with MF (Arm 1) and in combination with RUX as an 'add-on' in pts with MF with suboptimal response to RUX (Arm 2) or in JAK inhibitor (JAKi)-naïve pts with MF (Arm 3). Assessment of ≥35% spleen volume reduction (SVR35) and ≥50% total symptom score reduction (TSS50) are standard clinical trial endpoints to evaluate disease response in MF. However, SVR35 and TSS50 do not fully reflect underlying disease improvement, and additional approaches are needed to understand what constitutes disease modification in MF (Pemmaraju N, et al. Cancer 2022;128:2420-2432) and to provide better insight into the mechanisms of novel drugs in development, such as PELA. Here we report results in pts enrolled in MANIFEST from exploratory analyses of treatment-induced changes at Week 24 in a range of biomarkers that are suggestive of disease modification (≥1 grade improvements in bone marrow fibrosis [BMF], reduction of Janus Kinase 2 V617F [JAK2V617F] variant allele frequency [VAF] in blood, improvements in hemoglobin and increased distance of CD61+ megakaryocytic [MK] cells in bone marrow ['declustering']) with respect to the clinical outcomes of SVR35 and TSS50. We previously demonstrated (Mascarenhas J, et al. HemaSphere 2022;6:99-100) that the increase in distance in MK cells correlated with SVR35 responses. Table 1 shows a summary of SVR35 and TSS50 response rates and changes in biomarkers associated with potential disease modification observed in the MANIFEST study in each study arm. Of note, with PELA monotherapy, 30% (7/23) of pts in a RUX-resistant/intolerant population (Arm 1) showed ≥1 grade improvement in BMF. Additional changes indicating biologic effect of PELA included declustering of MK cells (increase in distance of ≥15%) observed in 46% (5/11) of pts. Similar or better results were observed in pts treated with PELA/RUX combination. Changes in these markers of potential disease modification were associated with SVR35 and TSS50 endpoint responses. In evaluable pts with available data, splenic responses at Week 24 across the three arms were associated with ≥1 grade reduced BMF (in 18/43 pts, 42%), increased MK distance (in 20/26 pts, 77%, median change +34%) and JAK2V617F VAF reductions (in 41/50 pts, 82%, median change -13%). Of TSS50 responders at Week 24 with available data, 36% (16/44) showed ≥1 grade improvements in BMF; 71% (20/28) had an increase in MK distance (median change +22.3%); and 64% achieved reductions in JAK2V617F VAF (30/47, median change -8%). In JAKi-naïve pts (Arm 3), 68% (57/84) achieved SVR35 at Week 24; among these patients with available data, 38% (12/32) improved BMF ≥1 grade; 74% (14/19, median change +19.7%) increased MK distance; and 83% (34/41, median change -18%) reduced JAK2V617F VAF (any level of improvement). TSS50 was observed in 56% (46/82) of JAKi-naïve pts; among these patients with available data, improvements in ≥1 grade BMF, MK distance and JAK2V617F VAF (any level of improvement) were observed in 46% (11/24), 75% (12/16, median change +24%) and 78% (18/23, median change -14.3%) of pts, respectively. In nonresponders, changes in biomarkers were consistently smaller. Our data indicate differences in the association of individual markers of potential disease modification with SVR35 and TSS50. While reduction in JAK2V617F VAF and increase in MK distance appear to be associated with SVR35, a high incidence of BMF improvements were also observed in Arm 3 TSS50 responders. Extended multivariable, longitudinal, biomarker analyses for association with time-to-event endpoints (eg duration of splenic response) will be presented with a new data cutoff with the objective to investigate potential disease-modifying activity of PELA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bruton's Tyrosine Kinase (BTK) Degrader Nx-2127 Exhibits Lethal Activity and Synergy with Venetoclax and BET Protein Inhibitor Against MCL Cells Sensitive or Resistant to Covalent BTK Inhibitors

Bruton's tyrosine kinase (BTK) is a Tec family member, non-receptor, tyrosine kinase, which is constitutively active in Mantle Cell Lymphoma (MCL) cells, downstream of B cell receptor (BCR), Toll like receptors (TLR) and Fc receptors. BTK plays a critical role in cell cycle progression and B cell activation. In murine models, loss of function and kinase-dead mutants of BTK show impaired B cell maturation. BTK activates multiple pro-growth and pro-survival mechanisms, involving PLCγ2 activity, RAS-RAF-MEK-ERK pathway and NFκB transcriptional activity. Covalent BTK inhibitors (BTKis), including ibrutinib and acalabrutinib, irreversibly bind to cysteine 481 in the kinase domain of BTK, inhibit growth, and induce loss of viability of MCL cells. Although treatment with covalent BTKi induces dramatic clinical remissions, intrinsic and acquired resistance to BTKis is observed in majority of patients. BTKi-resistance in MCL cells has been attributed to activation of the alternative NFκB signaling due to TRAF2, BIRC3 or MAP3K14 mutation, gain-of-function CARD11 mutation, CCND1 mutation and MYC activation. Emergence of C481S BTK mutation causes steric hindrance to the covalent BTKi to C481, leading to acquired resistance, followed by clinical relapse in CLL, and less commonly in MCL. However, this situation still underscores dependence on BTK signaling for growth and survival of MCL cells. It also supports the strategy of using a BTK degrader for degrading and depleting BTK, including C481S-BTK, in MCL cells. NX-2127 and NX-5948 are promising, cereblon-based, orally bioavailable, clinical grade BTK degraders, which degrade and deplete BTK levels. In the present studies, we determined that in vitro exposure of human MCL cells, including MCL cell lines: REC1, Mino and JeKo-1, as well as patient-derived (PD) MCL cells, to NX-2127 (10 to 250 nM) for 2 to 24 hours markedly depleted BTK levels via proteasomal degradation, since co-treatment with the proteasome inhibitor carfilzomib restored BTK levels. NX-2127 but not NX-5948 treatment also degraded and depleted IKZF1 and IKZF3 levels. However, both BTK degraders also depleted BTK levels in BTK1-resistant Mino-IR and Jeko1-IR cells (which display induced in vitro resistance after recovery from repeated exposures to high levels of ibrutinib, i.e., 30 μM for 48 hours). NX-2127 treatment reduced the mRNA levels of IL-10 and TNFα in the MCL cells. Additionally, NX-2127 mediated depletion of BTK was associated with downstream reduction in the protein levels of p-PLCγ2 (Y1217), p-AKT, pERK1/2 and SOX11 levels in Mino, Mino-IR as well as in PD MCL cells. Notably, NX-2127 treatment also markedly depleted the protein levels of C481S-BTK that confers clinical resistance to covalent BTKis in CLL cells. Exposure to NX-2127 dose-dependently inhibited growth and survival of REC1, Mino, JeKo-1, Mino-IR, JeKo-1-IR cells, as well as of PD MCL cells. We have previously reported that BET protein inhibitor and/or BCL2 inhibitor (venetoclax) induce in vitro lethality in cellular models of MCL (Blood, 126: 1565, 2015). Based on this, we determined the synergistic lethal activity of NX-2127 and venetoclax or the BD2-selective, BET protein inhibitor ABBV-744 in MCL cells. Co-treatment with NX-2127 (10 to 100 nM) and low nM levels of venetoclax or ABBV-744 was synergistically lethal against MCL cells (Delta synergy score above 1.0 by ZIP method). Collectively, these findings demonstrate that treatment with NX-2127 degrades and attenuates BTK and IKZF1/3 levels, as well as inhibits the downstream pro-growth and pro-survival signaling, resulting in loss of viability of MCL cells, including those resistant to covalent BTKi treatment. These findings also show promising anti-MCL activity of NX-2127-based combination with BCL2 or BET inhibitor which merits further in vivo validation.

INCB057643 Monotherapy in Patients with Relapsed or Refractory Myelofibrosis: A Phase 1 Study

Background: Bromodomain and extra-terminal (BET) proteins are a class of epigenetic readers that regulate expression of regulatory proteins, such as B-cell lymphoma-2, c-Myc, and nuclear factor kappa B, that are implicated in oncogenesis of hematologic malignancies, including myelofibrosis (MF). INCB057643 is a small-molecule inhibitor of BET that was evaluated as monotherapy and in combination with ruxolitinib in patients with advanced malignancies in 2 previous phase 1/2 clinical trials. Aim: This ongoing, phase 1, dose-confirmation/expansion study (NCT04279847) is evaluating the safety and tolerability of INCB057643 as monotherapy in patients with relapsed/refractory MF, myelodysplastic syndromes (MDS), or MDS/myeloproliferative neoplasm (MPN) overlap syndromes and in combination with ruxolitinib in patients with advanced MF and suboptimal response to ruxolitinib. Methods: The study includes a 3+3 design dose-escalation phase (part 1) followed by dose-expansion (part 2). Eligible patients in part 1 are aged ≥18 years with histologically confirmed MF and palpable spleen ≥5 cm below the left subcostal margin, MDS or MDS/MPN, Eastern Cooperative Oncology Group performance status of 0-2, and ≥1 line of prior therapy with no available subsequent therapy known to provide clinical benefit. Exclusion criteria include prior BET inhibitor treatment within 5 half-lives, platelet counts <50 × 109/L, absolute neutrophil count <0.75 × 109/L, and allogeneic transplant ≤6 months before enrollment. The initial part 1 dose is INCB057643 4 mg once daily (qd; escalation up to 12 mg qd), administered continuously in 28-day cycles. Doses deemed safe and tolerable in part 1 will be further evaluated in part 2 as monotherapy or in combination with ruxolitinib in patients with MF. The primary endpoint is safety and tolerability, including identification of dose-limiting toxicities (DLTs); pharmacokinetics (PK) are evaluated as a secondary endpoint. Additional secondary endpoints include overall response rate, symptom response, anemia response, red blood cell transfusion requirement, and change in spleen volume and length. PK data were analyzed using noncompartmental analysis. Results: Six patients have been evaluated in part 1 (age range, 59-77 years; men, n=4; study treatment duration range, 29-169 days); 4 had a diagnosis of MF, and 1 each had MDS/MPN unclassifiable and chronic myelomonocytic leukemia (CMML). All patients received INCB057643 4 mg qd. Four patients remain on treatment; treatment was discontinued in the remaining 2 patients for progressive disease. The most common treatment-emergent adverse events (TEAEs) occurring in >1 patient were thrombocytopenia (n=3), anemia (n=2), and hyperuricemia (n=2). Grade ≥3 TEAEs occurred in 1 patient each: acute myeloid leukemia (AML), anemia, chronic obstructive pulmonary disease, hypokalemia, leukocytosis, pancytopenia, and thrombocytopenia. There were 3 serious AEs (SAEs) across 2 patients (chronic obstructive pulmonary disease in 1 patient [dose was not changed; patient recovered with sequelae], pancytopenia and AML transformation from CMML in a second patient [study drug was interrupted; patient death followed related to disease progression]); none were considered related to study treatment. No DLTs were observed. The mean steady-state maximum plasma concentration (Cmax) and area under the curve (AUC) for a 4-mg qd dose were 92.4 nM and 1260 h·nM, respectively, indicating a dose proportionality from 4 mg to 12 mg. Conclusions: Treatment with a 4-mg qd dose of INCB057643 monotherapy in this patient population was generally well tolerated, with no treatment-related SAEs, no DLTs, and no TEAEs leading to treatment discontinuation. INCB057643 will be further evaluated at higher doses in dose-escalation and dose-expansion phases as monotherapy and in combination with ruxolitinib in patients with MF (preliminary combination data to be available for presentation).

Pelabresib (CPI-0610) As Add-on to Ruxolitinib in Myelofibrosis: Durability of Response and Safety Beyond Week 24 in the Phase 2 MANIFEST Study

Background: Myelofibrosis (MF) is largely characterized by bone marrow (BM) fibrosis, splenomegaly and cytopenias. Progressive BM fibrosis results from aberrant megakaryopoiesis and proinflammatory cytokine expression, two processes that are tightly regulated by bromodomain and extraterminal domain (BET) protein-mediated expression of genes (eg NF-κB target genes, MYC and BCL-2) and often lead to myeloproliferation and cytopenias. Although the current standard of care for MF is treatment with the Janus kinase inhibitor (JAKi) ruxolitinib (RUX), many patients (pts) experience a suboptimal response to RUX monotherapy or must discontinue treatment due to cytopenias and therefore there is a significant unmet need for new therapies in this population. Pelabresib (CPI-0610; PELA) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB signaling and other relevant genes involved in MF disease pathways (Mascarenhas J, et al. HemaSphere 2022;6:99-100). Aims: In the ongoing, open-label Phase 2 MANIFEST study (NCT02158858), PELA is under investigation as monotherapy and in combination with RUX in pts with MF. In Arm 2 of MANIFEST, pts with suboptimal/lost response to RUX are treated with PELA as 'add-on’ to RUX. In this abstract we will discuss results from the September 2021 data cut; updated data with longer follow-up on efficacy (≥35% reduction in spleen volume from baseline [BL; SVR35], ≥50% total symptom score reduction from BL [TSS50], duration of response and BM fibrosis improvement) and safety data from the planned July 2022 data cut will be presented. Methods: Pts in Arm 2 are stratified as transfusion dependent (TD; Arm 2A) and nontransfusion dependent (non-TD; Arm 2B). The primary endpoints are conversion from TD to transfusion independence (TI) in Arm 2A (TD cohort) and SVR35 at Week (Wk) 24 for Arm 2B (non-TD cohort). The secondary endpoints include TSS50 per MF Symptom Assessment Form v4.0 at Wk 24 and SVR35 at Wk 24 in Arm 2A (TD cohort). BM biopsies to assess BM fibrosis, durability of responses and safety data are evaluated. Data reported here are from the September 2021 data cut. Results: At Wk 24 in Arm 2 (N=86), 20% (16/81) of pts achieved SVR35 (Figures 1 and 2); 17% for Arm 2A (TD cohort) and 26% for Arm 2B (non-TD cohort). SVR35 at any time was achieved by 30% (24/81) of pts. At Wk 24, 27% (22/81) pts achieved ≥25% reduction in spleen volume from BL (SVR25); 26% (14/54) pts in Arm 2A (TD cohort) and 30% (8/27) pts in Arm 2B (non-TD cohort). Median spleen volume reduction for the full Arm 2 cohort was -18% at Wk 24. Durable (up to Wk 60) spleen volume reductions were observed as depicted in mean spleen volume change over time, and spleen volume reduction deepened over time (Figure 1). Twenty percent (16/80) pts had SVR35 response at Wk 48. In Arm 2A (TD cohort), the TD to TI rate was 16% (6/38). TSS50 at Wk 24 was 37% (30/81); 36% for Arm 2A (TD cohort) and 39% for Arm 2B (non-TD cohort). A total of 54% (46/86) of pts achieved TSS50 at any time. Median total symptom score reduction was -47% at Wk 24. As of data cut off, the median follow-up time was 24.4 (23.0-30.7) months and median treatment duration was 14.0 (8.4-20.6) months. After 24 wks of treatment, BM fibrosis improvement by ≥1 grade was achieved in 26% of evaluable pts by central pathology review; 50% of pts maintained the improvement at the next available assessment or longer. A total of 39% of pts achieved ≥1 grade improvement at any time (best response). The two most common hematologic treatment-emergent adverse events (TEAEs) of any grade were thrombocytopenia, reported in 52% (Grade ≥ 3: 33%) pts, and anemia, reported in 27% (Grade ≥ 3: 19%) pts. Low-grade gastrointestinal TEAEs and respiratory infections were observed but rarely a reason for treatment discontinuation. A total of 20 pts (23%) reported TEAEs that led to PELA discontinuation. Serious adverse events reported in ≥3 pts were anemia (6 pts), respiratory tract infections (4 pts) and urinary tract infections (3 pts). Conclusions: Based on these preliminary Phase 2 data, PELA as 'add-on’ to RUX in pts with a suboptimal/lost response to RUX monotherapy resulted in durable splenic and symptom responses, deepening of spleen responses over time, and was generally well tolerated. Additionally, improvement in BM fibrosis, as a marker of disease modification, was observed in 39% of treated pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pelabresib (CPI-0610) Combined with Ruxolitinib for JAK Inhibitor Treatment-Naïve Patients with Myelofibrosis: Durability of Response and Safety Beyond Week 24

Background: Myelofibrosis (MF) is characterized by progressive bone marrow (BM) fibrosis, splenomegaly and cytopenias resulting from aberrant megakaryopoiesis and proinflammatory cytokine expression. These processes are heavily regulated by bromodomain and extraterminal domain (BET) protein-mediated gene expression (eg NF-κB target genes, MYC and BCL-2) and lead to myeloproliferation and cytopenias. The current standard of care for patients (pts) with MF is the Janus kinase inhibitor (JAKi) ruxolitinib (RUX); however, the depth and durability of responses, along with the percentage of pts that achieve ≥35% spleen volume reduction from baseline (BL; SVR35) and ≥50% total symptom score reduction (TSS) from BL (TSS50) with RUX monotherapy are limited. Pelabresib (CPI-0610; PELA) is an oral, small-molecule, investigational BET inhibitor that downregulates NF-κB signaling and other relevant genes involved in MF disease pathways (Mascarenhas J, et al. HemaSphere 2022;6:99-100). In the ongoing Phase 2 MANIFEST study (NCT02158858), PELA is under investigation as monotherapy and in combination with RUX in pts with MF. Here, we present results from Arm 3 of MANIFEST (in which JAKi-naïve pts with MF receive PELA in combination with RUX). Aims: The primary endpoint of MANIFEST Arm 3 is SVR35 after 24 weeks (wks) of treatment. Secondary endpoints include TSS50, duration of splenic response, pharmacokinetics and safety. Exploratory endpoints include transfusion independence and changes from BL in BM fibrosis. In this abstract we will discuss results from the September 2021 data cut; longer-term data from the July 2022 data cut will be presented. Methods: Spleen volume was assessed by computed tomography or magnetic resonance imaging every 12 wks; TSS per Myelofibrosis Symptom Assessment Form v4.0; and BM fibrosis by biopsies at Wk 24 and Wk 48. Duration of splenic response and safety data are also evaluated. Data presented are from the September 2021 data cut; a more mature data set will be presented at the conference. Results: Eighty-four pts received ≥1 dose of PELA and RUX. Median age was 68 years (range 37-85); 24%, 61% and 16% had Dynamic International Prognostic Scoring System intermediate-1, intermediate-2 or high risk, respectively. Pts who did not reach Wk 24 were counted as nonresponders. At Wk 24 and Wk 48, 68% (57/84) and 60% (47/79) of pts, respectively, achieved SVR35. SVR35 at any time was achieved by 80% pts (67/84), and 87% (58/67) pts with SVR35 maintained response at the time of data cutoff. Median spleen volume change was -50% (range -84.4% to 27.9%) at Wk 24. Median time to SVR35 response was 12 wks (first on-treatment assessment of splenic response); durable spleen volume reductions were observed as depicted in mean spleen volume change over time in Figure 1. TSS50 was achieved by 56% (46/82) pts at Wk 24 and 43% (34/79) at Wk 48. TSS50 at any time was achieved by 81% pts (68/84). The median change in TSS was −59% (range −100% to 225%) at Wk 24. Mean TSS change over time is shown in Figure 2, indicating sustained reduction in symptom burden. Median follow up for Arm 3 was 21.8 (range 21.2-22.5) months; median treatment duration was not reached as of data cutoff. BM fibrosis improved by ≥1 grade in 28% (16/57) of evaluable pts at Wk 24 as assessed by central pathology review; 56% maintained the improvement at the next available assessment (Wk 48 or beyond) or longer; and 40% achieved ≥1 grade improvement at any time (best response). The most common hematologic treatment-emergent adverse event (TEAE) of any grade was thrombocytopenia, reported in 52% (Grade ≥3: 12%) of pts. Anemia was reported in 42% (Grade ≥3: 34%). Serious adverse events reported in ≥2 pts were respiratory tract infections (6 pts), pyrexia (3 pts) and anemia, multiorgan failure, fall and pelvic fracture (2 pts each). TEAEs that led to PELA discontinuation occurred in 7 pts (8%). Updated data with longer follow-up, including durability of splenic response and safety data from a more mature data set, will be available for presentation at the conference. Conclusions: The combination of PELA and RUX in JAKi-naïve pts with MF showed durable improvements in spleen volume, total symptom score and BM fibrosis, and was generally well tolerated. The randomized, double-blind, active-controlled Phase 3 MANIFEST-2 study is open for enrollment and evaluating the safety and efficacy of PELA in combination with RUX in JAKi treatment-naïve pts with MF (NCT04603495). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BET Inhibitors Target the SCLC-N Subtype of Small-Cell Lung Cancer by Blocking NEUROD1 Transactivation.

Our findings suggest that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.