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

伴有骨髓纤维化的骨髓增生性肿瘤（MPN-MF）表现出JAK-STAT信号传导的组成性活性 由于JAK 2、c-MPL或钙网蛋白基因突变。染色质/转录中的其他突变 修饰物（表突变）在高达20%的MPN-MF患者中诱导向AML（sAML）的转化。缺乏 JAK 1和2抑制剂（JAKi）ruxolitinib和AML化疗的显著活性强调了需要 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。遗传改变和失调的表观基因组产生失调的转录组 负责MPN后sAML原始祖细胞的转化表型和治疗难治性 细胞（BPC）。这种失调的转录组依赖于“染色质阅读器”BET（布罗莫结构域和 末端外）蛋白（BETP），例如，BRD 4及其相互作用子pTEFb（正转录延伸 因子B），两者都被募集到活性转录的癌基因的超级增强子和启动子。Cyclin 依赖性激酶9（CDK 9），pTEFb的催化亚基，磷酸化RNA pol II（RNAP 2），促进 RNAP 2介导的MPN后生长和存活所必需的癌基因的mRNA转录延长 sAML BPC。然而，BETP-CDK 9轴抑制对活性超级增强子/增强子和 对失调的转录组和存活产生影响的启动子尚未阐明， 患者源性（PD）sAML BPC。此外，对CDK 9或BETP的抗性的表观遗传机制 抑制剂（CDK 9 i或BETi）治疗及其在需要MPN后sAML BPC中的治疗性废除 评价我们的初步研究表明，CDK 9 i或BETP-拮抗剂（BETi和BETP-拮抗剂）， PROTACs）处理诱导MPN后sAML BPC的凋亡，这与MPN后sAML BPC的抑制有关。 sAML相关癌基因，例如，c-MYC、STAT3/5、NF κ B、Bcl-xL和MCL-1。我们假设BETP- 拮抗剂和基于CDK 9 i的组合将抑制失调的转录组和癌基因，并且 与JAKi或BCL 2/Bcl-xL抑制剂共处理，协同诱导PD中的体外和体内致死性， MPN sAML BPC。这些研究的具体目的是：目的1：阐明BETP-PROTAC的作用 和CDK 9 i对活性超级增强子/增强子的作用（通过ATAC-Seq和ChIP-Seq），mRNA转录组（通过 RNA-Seq）和蛋白质表达（通过CyTOF），以及确定其针对以下疾病的临床前功效： 遗传学分析的培养细胞系和PD、MPN后sAML BPC。目的2：确定 基于BETP-PROTAC和CDK 9 i的组合针对JAKi敏感性和JAKi持久性/耐药性sAML BPC，利用体外细胞培养物和体内异种移植模型。目的3：阐明失调的 表观基因组和转录组以及对基于BETP-PROTAC的组合的易感性， CDK 9 i-持久/耐药的MPN后sAML BPC。

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.