Targeting dysregulated transcriptome as therapy for post-myeloproliferative neoplasm (MPN) sAML

靶向转录组失调作为骨髓增殖后肿瘤 (MPN) sAML 的治疗方法

• 批准号: 10595080
• 负责人: KAPIL BHALLA
• 金额: $ 46.63万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-13 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595080
• 关键词: ATAC-seq; Acetylation; Apoptotic; Attenuated; BCL2 gene; BCL2L1 gene; Binding; Binding Sites; Bromodomain; Bromodomains and extra-terminal domain inhibitor; C-terminal; CDK9 Protein Kinase; Catalytic Domain; Cell Culture Techniques; Cell Line; Cell Survival; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Clinical; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinases; DNA Polymerase II; DNA Sequence Alteration; DNMT3a; Dependence; Disease Progression; EZH2 gene; Elements; Enhancers; Enzymes; Epigenetic Process; Evaluation; Exhibits; Family member; Genes; Genetic Transcription; Growth; Hematopoietic stem cells; Histones; In Vitro; Induction of Apoptosis; JAK1 gene; JAK2 gene; Janus kinase; Lysine; MCL1 gene; Mediating; Mediator; Messenger RNA; Mutation; Myelofibrosis; Myeloproliferative disease; NF-kappa B; Neoplasm Metastasis; Oncogenes; Outcome; PIM1 gene; Patients; Phenotype; Phosphorylation; Positive Transcriptional Elongation Factor B; Predisposition; Productivity; Protac; Protein Family; Proteins; RELA gene; RNA; RUNX1 gene; Reader; Refractory; Repression; Resistance; SRSF2 gene; STAT3 gene; Secondary acute myeloid leukemia; Serine; Signal Transduction; TP53 gene; Techniques; Testing; Therapeutic; Time; Transcript; Xenograft Model; antagonist; c-myc Genes; calreticulin; cell growth; chemotherapy; cyclin T1; epigenetic regulation; epigenome; genomic profiles; improved; in vivo; inhibitor; mimetics; negative elongation factor; novel; pre-clinical; preclinical efficacy; promoter; protein expression; recruit; resistance mechanism; stem cells; transcription factor; transcriptome; transcriptome sequencing

Myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit constitutive activity of JAK-STAT signaling due to mutations in JAK2, c-MPL or calreticulin genes. Additional mutations in chromatin/transcriptional modifiers (epimutations) induce transformation to AML (sAML) in up to 20% of patients with MPN-MF. Lack of significant activity of the JAK1 & 2 inhibitor (JAKi) ruxolitinib and of AML chemotherapy highlights the need to develop and test novel agents and combinations that would improve clinical outcome in patients with post- MPN sAML. Genetic alterations and dysregulated epigenome produce the dysregulated transcriptome responsible for the transformed phenotype and therapy-refractoriness in post-MPN sAML blast progenitor cells (BPCs). This dysregulated transcriptome is dependent on ‘chromatin-reader’ BET (bromodomain and extra-terminal) proteins (BETPs), e.g., BRD4, and on its interactor pTEFb (positive transcription elongation factor b), both recruited to super-enhancers and promoters of actively-transcribed oncogenes. Cyclin dependent kinase 9 (CDK9), the catalytic subunit of pTEFb, phosphorylates RNA pol II (RNAP2), promoting RNAP2-mediated mRNA transcript elongation of oncogenes essential for growth and survival of post-MPN sAML BPCs. However, effects of BETP-CDK9 axis inhibition on active super-enhancers/enhancers and promoters with resulting impact on the dysregulated transcriptome and survival have not been elucidated in patient-derived (PD) sAML BPCs. Additionally, epigenetic mechanisms of resistance to CDK9 or BETP inhibitor (CDK9i or BETi) treatment and their therapeutic abrogation in post-MPN sAML BPCs need evaluation. Our preliminary studies demonstrate that CDK9i or BETP-antagonist (BETi and BETP- PROTACs) treatment induces apoptosis of post-MPN sAML BPCs, which is associated with repression of sAML-relevant oncogenes, e.g., c-MYC, STAT3/5, NFkB, Bcl-xL and MCL-1. We hypothesize that BETP- antagonist and CDK9i-based combinations will repress the dysregulated transcriptome and oncogenes, and with JAKi or BCL2/Bcl-xL inhibitor co-treatment, synergistically induce in vitro and in vivo lethality in PD, post- MPN sAML BPCs. Specific aims of these studies are: Aim 1: To elucidate the effects of BETP-PROTAC and CDK9i on active super-enhancers/enhancers (by ATAC-Seq and ChIP-Seq), mRNA transcriptome (by RNA-Seq) and on protein expressions (by CyTOF), as well as determine their pre-clinical efficacy against genetically-profiled, cultured cell lines and PD, post-MPN sAML BPCs. Aim 2: To determine lethal activity of BETP-PROTAC and CDK9i-based combinations against JAKi-sensitive and JAKi-persister/resistant sAML BPCs, utilizing in vitro cell cultures and in vivo xenograft models. Aim 3: To elucidate the dysregulated epigenome and transcriptome as well as susceptibility to BETP-PROTAC-based combinations in BETi- or CDK9i-persister/resistant post-MPN sAML BPCs.

伴有骨髓纤维化的骨髓增生性肿瘤(MPN-MF)表现出JAK-STAT信号的结构性活性 由于JAK2、c-MPL或钙网织蛋白基因突变所致。染色质/转录中的其他突变 在高达20%的MPN-MF患者中，修饰物(表型突变)诱导向AML(SAML)转化。缺乏 JAK1&2抑制剂(Jaki)ruxolitinib和AML化疗的显著活性突显了需要 开发和测试新的药物和组合，以改善术后患者的临床结果 MPN SAML。基因改变和异常的表观基因组产生异常的转录组 MPN后SAML祖细胞转化表型和治疗耐受性的研究 细胞(BPC)。这种失调的转录组依赖于染色质阅读器BET(溴域和 末端外)蛋白(BETPs)，例如BRD4，及其相互作用的pTEFb(正转录延伸 因子b)，两者都被招募到活跃转录的癌基因的超级增强子和启动子。骑自行车 依赖激酶9(CDK9)是pTEFb的催化亚基，它磷酸化RNAPolII(RNAP2)，促进 RNAP2介导的对MPN后生长和存活至关重要的癌基因的转录延长 SAML BPC。然而，BETP-CDK9轴抑制对活性的超级增强子/增强子和 启动子对失调的转录组和生存的影响尚未被阐明 患者来源(PD)SAML BPC。此外，CDK9或BETP耐药的表观遗传机制 MPN后SAML BPC所需的抑制剂(CDK9i或Beti)治疗及其治疗废止 评估。我们的初步研究表明，CDK9i或BETP拮抗剂(Beti和BETP- PROTACs)治疗诱导MPN SAML后BPC的凋亡，这与抑制 与SAML相关的癌基因，如c-myc、STAT3/5、NFkB、bclxl和mcl1。我们假设BETP- 拮抗剂和基于CDK9i的组合将抑制失调的转录组和癌基因，以及 Jaki或bcl2/bcl2/bclxl抑制剂联合应用，可协同诱导帕金森病的体内外致死作用。 MPN SAML BPC。这些研究的具体目的是：目的1：阐明BETP-PROTAC的作用 和CDK9i在活性超级增强子/增强子上(通过ATAC-Seq和ChIP-Seq)，mRNA转录组(通过 RNA-Seq)和蛋白质表达(通过细胞周期分析)，以及确定其临床前疗效 基因图谱，培养的细胞系和PD，后MPN SAML BPC。目的2：测定其致死活性 基于BETP-PROTAC和CDK9i的联合抗Jaki敏感和Jaki持久性/抗性SAML BPC，利用体外细胞培养和体内异种移植模型。目标3：阐明失调的 甜菜表观基因组和转录组以及对基于BETP-PROTAC的组合的敏感性 CDK9i-MPN SAML后耐药BPC。