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: 10364667
• 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/10364667
• 关键词: ATAC-seq; Apoptosis; Apoptotic; Attenuated; BCL2 gene; BCL2L1 gene; Binding; Binding Sites; Bromodomain; C-terminal; CDK9 Protein Kinase; Catalytic Domain; Cell Culture Techniques; Cell Line; Cell Survival; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Clinical; Cyclin-Dependent Kinases; DNA Polymerase II; Dependence; Disease Progression; EZH2 gene; Elements; Enhancers; Enzymes; Epigenetic Process; Evaluation; Exhibits; Family member; Genes; Genetic Transcription; Growth; Hematopoietic stem cells; Histones; In Vitro; JAK1 gene; JAK2 gene; Janus kinase; Lysine; MCL1 gene; Mediating; Mediator of activation protein; Messenger RNA; Mutation; Myelofibrosis; Myeloproliferative disease; Oncogenes; Outcome; PIM1 gene; Patients; Phenotype; Positive Transcriptional Elongation Factor B; Predisposition; 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; base; c-myc Genes; calreticulin; cell growth; chemotherapy; cyclin T1; epigenetic regulation; epigenome; improved; in vivo; inhibitor; kinase 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 信号传导的组成活性 由于 JAK2、c-MPL 或钙网蛋白基因突变。染色质/转录的额外突变 修饰剂（表突变）会导致高达 20% 的 MPN-MF 患者转化为 AML (sAML)。缺乏 JAK1 和 2 抑制剂 (JAKi) 鲁索替尼和 AML 化疗的显着活性凸显了以下必要性： 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。遗传改变和失调的表观基因组产生失调的转录组 导致 MPN 后 sAML 母细胞祖细胞表型转变和治疗无效 细胞（BPC）。这种失调的转录组依赖于“染色质阅读器”BET（溴结构域和 额外末端）蛋白（BETP），例如 BRD4 及其相互作用子 pTEFb（正转录延伸 因子b），两者都被招募到活跃转录癌基因的超级增强子和启动子中。细胞周期蛋白 依赖性激酶 9 (CDK9) 是 pTEFb 的催化亚基，磷酸化 RNA pol II (RNAP2)，促进 RNAP2介导的癌基因mRNA转录延长对于MPN后的生长和存活至关重要 sAML BPC。然而，BETP-CDK9 轴抑制对活性超级增强子/增强子的影响和 启动子对转录组失调和生存产生影响尚未得到阐明 患者来源的 (PD) sAML BPC。此外，CDK9 或 BETP 抗性的表观遗传机制 MPN 后 sAML BPC 需要抑制剂（CDK9i 或 BETi）治疗及其治疗取消 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 后 sAML BPC 的凋亡，这与抑制 sAML 相关癌基因，例如 c-MYC、STAT3/5、NFkB、Bcl-xL 和 MCL-1。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，并且 与 JAKi 或 BCL2/Bcl-xL 抑制剂共同治疗，协同诱导 PD 的体外和体内致死率， MPN sAML BPC。这些研究的具体目标是： 目标 1：阐明 BETP-PROTAC 的作用 和 CDK9i 活性超级增强子/增强子（通过 ATAC-Seq 和 ChIP-Seq）、mRNA 转录组（通过 RNA-Seq）和蛋白质表达（通过 CyTOF），并确定其临床前疗效 基因分析、培养细胞系和 PD、MPN 后 sAML BPC。目标 2：确定致死活性 基于 BETP-PROTAC 和 CDK9i 的组合对抗 JAKi 敏感和 JAKi 持久/耐药 sAML BPC，利用体外细胞培养物和体内异种移植模型。目标 3：阐明失调的情况 BETi-或中表观基因组和转录组以及对基于BETP-PROTAC的组合的敏感性 CDK9i 持久/耐药 MPN 后 sAML BPC。