Selective Targeting of Alternative Splicing Deregulation in Pediatric Acute Myeloid Stem and Progenitor Cells
Selective Targeting of Alternative Splicing Deregulation in Pediatric Acute Myeloid Stem and Progenitor Cells
复制标题
儿科急性髓系干细胞和祖细胞中选择性剪接失调的选择性靶向
DOI：
10.1182/blood-2020-136596
复制
发表时间：
2020
期刊：
影响因子：
20.3
通讯作者：
C. Jamieson
中科院分区：
文献类型：
--
作者：
I. V. D. Werf;Phoebe K. Mondala;Raymond H. Diep;L. Balaian;Cayla N. Mason;J. Cloos;G. Kaspers;J. L. Clair;P. Wentworth;L. Crews;T. Whisenant;K. Fisch;M. Burkart;C. Jamieson
介绍
由于急性髓性白血病（AML）是儿童白血病复发相关死亡的主要原因，因此迫切需要开发新的治疗方法并提供对复发的细胞类型和分子背景特异性原因的机制见解。骨髓恶性肿瘤，包括AML，具有通过白血病干细胞中剪接因子基因突变的获得或表观遗传剪接体破坏来破坏剪接体的倾向（综述于Chua，货车Der Werf，Jamieson，Signer. Cancer Cell 2020;26：138-159）。虽然白血病干细胞（LSC）由于其固有的休眠能力和抵抗靶向分裂细胞的治疗而促进成人AML的复发，但选择性剪接（AS）失调在LSC复发和治疗脆弱性中的作用尚未在儿科AML中得到彻底研究。
方法
最近，我们用选择性剪接调节剂17 S-FD-895完成了IND前使能研究。以前，我们表明17 S-FD-895选择性消除成人AML LSC（Crews...伯卡特，杰米森。Cell Stem Cell 2016;19：599-612）。作为一种稳定的普拉地烯醇衍生的小分子剪接调节剂，17 S-FD-895靶向剪接体的关键组分SF 3B 1，从而调节mRNA剪接。为了研究剪接失调在儿科AML LSC维持中的作用，我们开发了灵敏的体外和体内慢病毒剪接报告基因测定、全转录组RNA测序（RNA-seq）干细胞和祖细胞剪接分析管道、qRT-PCR剪接亚型特异性反应生物标志物以及基质共培养、造血祖细胞集落存活和再铺板测定，以测定LSC根除。
结果
由于剪接调控是细胞类型和环境依赖性的，我们生成了FACS纯化的造血干细胞（HSC; CD 34 + CD 38-Lin-）和造血祖细胞（HPC; CD 34 + CD 38 +Lin）的全面转录组表达图谱。通过利用剪接变体特异性比对算法，我们评估了全基因组可变剪接事件，并发现与非白血病供体相比，儿科AML中广泛存在外显子跳跃。在儿科AML HSC和HPC中鉴定了超过2000个外显子跳跃事件。此外，儿童AML HSC和HPC均显示剪接调节因子RNA结合Fox 2（RBFOX 2）下调，RBFOX 2与胚胎干细胞剪接变体特征相关，而胚胎干细胞剪接变体特征对白血病细胞存活至关重要（霍尔姆.杰米森PNAS 2015;112：15444-15449; Denichenko等人Nat Commun 2019;10：1590）。用17 S-FD-895处理诱导从儿科AML样品中分离的CD 34+细胞的克隆形成性（p=0.001; Student t检验）和自我更新（p=0.001; Student t检验）的剂量依赖性降低。儿童AML样本对剪接调节的敏感性显著高于成人新发或成人继发性AML。此外，正常脐带血HSC和HPC样品不受剪接调节剂处理的影响。我们的慢病毒剪接报告基因测定证明了在儿科白血病细胞系中MAPT内含子保留的剂量依赖性增加，如通过从GFP到RFP的转换所测量的。最后，剪接异构体特异性RT-PCR证明了处理后SF 3B 1内含子保留的剂量依赖性增加以及MCL 1外显子2跳跃，产生促凋亡MCL 1-S转录物。
结论
累积起来，我们的数据表明，通过17 S-FD-895介导的SF 3B 1靶向剪接体调节构成了儿童AML患者的一种新的潜在治疗策略。
卡斯珀：勃林格殷格翰：实体董事会或咨询委员会成员; AbbVie：过去24个月内离职; Janssen R&D：过去24个月内离职; Helsinn Healthcare：过去24个月内离职。工作人员：Ionis Pharmaceuticals：研究资金。杰米森：四十七公司：专利和版税;百时美施贵宝：其他。
INTRODUCTION
Because acute myeloid leukemia (AML) is the leading cause of pediatric leukemia relapse-related mortality, there is a desperate need for developing new therapeutics and providing mechanistic insights into the cell type and molecular context specific causes of relapse. Myeloid malignancies, including AML, have a propensity to disrupt the spliceosome either through acquisition of splicing factor gene mutations or epigenetic spliceosome disruption in leukemia stem cells (reviewed in Chua, Van Der Werf, Jamieson, Signer. Cancer Cell 2020;26:138-159). While leukemia stem cells (LSCs) promote relapse in adult AML as a result of their inherent capacity to become dormant and resist therapies that target dividing cells, the role of alternative splicing (AS) deregulation in relapse and therapeutic vulnerability of LSCs has not been thoroughly studied in pediatric AML.
METHODS
Recently, we completed pre-IND enabling studies with a selective splicing modulator, 17S-FD-895. Previously, we showed that 17S-FD-895 selectively eliminated adult AML LSC (Crews...Burkart, Jamieson. Cell Stem Cell 2016;19:599-612). As a stable pladienolide-derived small molecule splicing modulator, 17S-FD-895 targets a key component of the spliceosome, SF3B1, thereby modulating mRNA splicing. To investigate the role of splicing deregulation in pediatric AML LSC maintenance, we developed a sensitive in vitro and in vivo lentiviral splicing reporter assay, whole transcriptome RNA sequencing (RNA-seq) stem and progenitor cell splicing analysis pipelines, qRT-PCR splice isoform specific biomarkers of response, as well as stromal co-culture, hematopoietic progenitor colony survival and replating assays to assay LSC eradication.
RESULTS
Because splicing regulation is cell type and context dependent, we generated a comprehensive transcriptome expression map of FACS-purified hematopoietic stem cells (HSCs; CD34+CD38-Lin-) and hematopoietic progenitor cells (HPCs;CD34+CD38+Lin). By utilizing a splice variant-specific alignment algorithm, we evaluated genome wide alternative splicing events and uncovered widespread exon skipping in pediatric AML compared to non-leukemic donors. More than 2000 exon skipping events were identified in pediatric AML HSCs and HPCs. In addition, both pediatric AML HSC and HPC demonstrated a downregulation of the splicing regulator RNA-Binding fox 2 (RBFOX2), which has been linked to embryonic stem cell splice variant signatures that are vital for leukemia cell survival (Holm...Jamieson. PNAS 2015;112:15444-15449; Denichenko et al Nat Commun 2019;10:1590). Treatment with 17S-FD-895 induced a dose-dependent reduction in clonogenicity (p=0.001; Student's t-test) and self-renewal of CD34+ cells (p=0.001; Student's t-test) isolated from pediatric AML samples. Pediatric AML samples were significantly more sensitive to splicing modulation than adult de novo or adult secondary AML. Moreover, normal cord blood HSC and HPC samples were unaffected by splicing modulator treatment. Our lentiviral splicing reporter assays demonstrated a dose dependent increase in MAPT intron retention in in pediatric leukemia cell lines as measured by a switch from GFP to RFP. Finally, splice isoform specific RT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment as well as MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts.
CONCLUSIONS
Cumulatively, our data indicate that spliceosome modulation via 17S-FD-895-mediated targeting of SF3B1 constitutes a novel potential therapeutic strategy for pediatric patients with AML.
Kaspers: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; AbbVie: Ended employment in the past 24 months; Janssen R&D: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months. Crews:Ionis Pharmaceuticals: Research Funding. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.