1. Super-enhancer profiling identifies novel critical and targetable cancer survival gene LYL1 in pediatric acute myeloid leukemia
Xin-Ran Chu,Si-Qi Jia,Di Wu,Yi Xie,Juan-Juan Yu,Yan-Fang Tao,Yuan-Yuan Tian,Jian-Wei Wang,Xiao-Lu Li,Jun Lu,Jing Ling,Yong-Ping Zhang,Yan-Ling Chen,Gen Li,Fang Fang,Chen-Xi Feng,Xu Sang,Jian Pan,Shao-Yan Hu,Shui-Yan Wu,Zi-Mu Zhang,Zhi-Heng Li J Exp Clin Cancer Res . 2022 Jul 16;41(1):225. doi: 10.1186/s13046-022-02428-9.
Background:Acute myeloid leukemia (AML) is a myeloid neoplasm makes up 7.6% of hematopoietic malignancies. Super-enhancers (SEs) represent a special group of enhancers, which have been reported in multiple cell types. In this study, we explored super-enhancer profiling through ChIP-Seq analysis of AML samples and AML cell lines, followed by functional analysis.Methods:ChIP-seq analysis for H3K27ac was performed in 11 AML samples, 7 T-ALL samples, 8 B-ALL samples, and in NB4 cell line. Genes and pathways affected by GNE-987 treatment were identified by gene expression analysis using RNA-seq. One of the genes associated with super-enhancer and affected by GNE-987 treatment was LYL1 basic helix-loop-helix family member (LYL1). shRNA mediated gene interference was used to down-regulate the expression of LYL1 in AML cell lines, and knockdown efficiency was detected by RT-qPCR and western blotting. The effect of knockdown on the growth of AML cell lines was evaluated by CCK-8. Western blotting was used to detect PARP cleavage, and flow cytometry were used to determine the effect of knockdown on apoptosis of AML cells.Results:We identified a total of 200 genes which were commonly associated with super-enhancers in ≧10 AML samples, and were found enriched in regulation of transcription. Using the BRD4 inhibitor GNE-987, we assessed the dependence of AML cells on transcriptional activation for growth and found GNE-987 treatment predominantly inhibits cell growth in AML cells. Moreover, 20 candidate genes were selected by super-enhancer profile and gene expression profile and among which LYL1 was observed to promote cell growth and survival in human AML cells.Conclusions:In summary, we identified 200 common super-enhancer-associated genes in AML samples, and a series of those genes are cancer genes. We also found GNE-987 treatment downregulates the expression of super-enhancer-associated genes in AML cells, including the expression of LYL1. Further functional analysis indicated that LYL1 is required for AML cell growth and survival. These findings promote understanding of AML pathophysiology and elucidated an important role of LYL1 in AML progression.
2. BRD4 Inhibitor GNE-987 Exerts Anticancer Effects by Targeting Super-Enhancer-Related Gene LYL1 in Acute Myeloid Leukemia
Juanjuan Yu,Lihui Lu,Yumeng Wu,Yuanyuan Tian,Kunlong Zhang,Jianwei Wang,Li Ma,Shaoyan Hu,Ran Zhuo,Shuiyan Wu,Li Gao,Di Yao,Yanling Chen,Zhen Zhang,Zhiheng Li,Zimu Zhang,Fang Fang,Xu Sang,Yanfang Tao,Xinran Chu,Jian Pan,Xiaolu Li,Yongping Zhang J Immunol Res . 2022 Aug 1;2022:7912484. doi: 10.1155/2022/7912484.
Background:AML (acute myeloid leukemia) is a common hematological malignancy in children with poor treatment effects and poor prognosis. Recent studies have shown that as a novel BRD4 (bromodomain containing 4) PROTACs (proteolysis targeting chimeras) degrader, GNE-987 can slow down the growth of various tumors and increase apoptosis, with promising clinical prospects. However, the function and molecular mechanism of GNE-987 in AML remain unclear. This study is aimed at investigating the therapeutic effect of GNE-987 on AML and its underlying mechanism.Methods:The association between BRD4 and AML was assessed by studying public databases. After GNE-987 was added to AML cells, cell proliferation slowed down, the cycle was disturbed, and apoptosis increased. Western blotting was used to detect BRD2 (bromodomain containing 2), BRD3 (bromodomain containing 3), BRD4, and PARP (poly ADP-ribose polymerase) proteins. The effect of GNE-987 on AML cells was analyzed in vivo. RNA-seq (RNA sequencing) and ChIP-seq (chromatin immunoprecipitation sequencing) validated the function and molecular pathways of GNE-987 in processing AML.Results:BRD4 expression was significantly elevated in pediatric AML samples compared with healthy donors. GNE-987 inhibited AML cell proliferation by inhibiting the cell cycle and inducing apoptosis. BRD2, BRD3, and BRD4 were consistent with decreased VHL (Von Hippel Lindau) expression in AML cells. In an AML xenograft model, GNE-987 significantly reduced the hepatosplenic infiltration of leukemia cells and increased the mouse survival time. Based on analysis of RNA-seq and ChIP-seq analyses, GNE-987 could target multiple SE- (super-enhancer-) related genes, including LYL1 (lymphoblastic leukemia 1), to inhibit AML.Conclusions:GNE-987 had strong antitumor activity in AML. GNE-987 could effectively inhibit the expression of SE-related oncogenes including LYL1 in AML. Our results suggested that GNE-987 had broad prospects in the treatment of AML.
3. Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity
Peter S Dragovich,Geoffrey Del Rosario,Jack D Sadowsky,Xiaoyu Zhu,Katherine R Kozak,Karen E Gascoigne,Aimee O'Donohue,Douglas D Leipold,Jinhua Chen,Pragya Adhikari,Hui Yao,Shang-Fan Yu,Ruina Li,Donglu Zhang,Rebecca K Rowntree,Hongyan Zhang,Melinda M Mulvihill,John Wai,Thomas H Pillow,Brandon Latifi,Cong Wu,Richard Zang,Chun Sing Li,Zijin Xu,Tracy Kleinheinz,Robert A Blake,Susan Kaufman,Gauri Deshmukh,Hao Zhou,Xinxin Wang,Amrita V Kamath,Isabel Figueroa ChemMedChem . 2020 Jan 7;15(1):17-25. doi: 10.1002/cmdc.201900497.
The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.
4. Native Mass Spectrometry for the Study of PROTAC GNE-987-Containing Ternary Complexes
Louise M Sternicki,Melinda M Mulvihill,Ronald J Quinn,Jim Nonomiya,Miaomiao Liu ChemMedChem . 2021 Jul 20;16(14):2206-2210. doi: 10.1002/cmdc.202100113.
PROteolysis TArgeting Chimeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous binding of both the target protein and an E3-Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE-987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3-Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high-throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.
