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

基本信息

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
项目类别：
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

项目摘要

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 或钙网蛋白基因的突变导致染色质/转录的其他突变。修饰剂（表突变）会导致高达 20% 的 MPN-MF 患者转化为 AML (sAML)。 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 基因改变和表观基因组失调产生转录组失调。导致 MPN 后 sAML 母细胞祖细胞的表型转变和治疗难治性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的癌基因的超级增强子和启动子中。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此外，患者来源的 (PD) sAML BPC 还具有 CDK9 或 BETP 耐药性的表观遗传机制。 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，这与抑制我们捕获了 sAML 相关癌基因，例如 c-MYC、STAT3/5、NFkB、Bcl-xL 和 MCL-1。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，这些研究的具体目的是：目的 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的培养细胞系和 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。