Developing Novel Therapies for High Risk Pediatric Cancers

开发高危儿童癌症的新疗法

• 批准号: 10702412
• 负责人: Javed Khan
• 金额: $ 85.43万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702412
• 关键词: ALK gene; Affinity; Amino Acids; Animal Model; Antigens; Apoptosis; Automobile Driving; Biological Assay; Biological Products; Biology; Bromodomain; Burkitt Lymphoma; CD8B1 gene; CMV promoter; CRISPR screen; CRISPR/Cas technology; Cell Count; Cell Death; Cell Line; Cell Survival; Cell surface; Cells; Cetuximab; Childhood; Childhood Solid Neoplasm; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; DNA; Data; Detection; Development; Down-Regulation; Drug Compounding; Drug Screening; Enhancers; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor Receptor; Epigenetic Process; Extracellular Domain; FDA approved; FGFR4 gene; FOXO1A gene; Family member; Firefly Luciferases; Freezing; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Green Fluorescent Proteins; Growth; Guide RNA; Human; Immune Targeting; Immunohistochemistry; Immunotherapeutic agent; Immunotherapy; Injury; Investigational Drugs; Knock-in; Laboratories; Lead; Libraries; Luciferases; Malignant Childhood Neoplasm; Measurement; Mediating; Methods; Molecular Target; Monitor; Monoclonal Antibodies; Mus; Mutate; Mutation; Myoblasts; National Center for Advancing Translational Sciences; Neuroblastoma; Normal tissue morphology; Nucleotides; Oncogenes; Organ; PAX3 gene; PAX7 gene; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phase; Plasmids; Post-Translational Protein Processing; Principal Component Analysis; Prognostic Marker; Property; Protein Biosynthesis; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Renilla Luciferases; Reporter; Reporting; Rhabdomyosarcoma; Signal Transduction; Site-Directed Mutagenesis; System; T-Lymphocyte; Technology; Tertiary Protein Structure; Testing; Tissue Microarray; Toxic effect; Translating; Transmembrane Domain; Work; Xenograft procedure; cancer therapy; cell growth; chimeric antigen receptor; chimeric antigen receptor T cells; clinical development; design; diagnostic biomarker; differential expression; drug mechanism; experimental study; fusion gene; gene repression; genome-wide; genomic locus; high risk; high throughput screening; in vivo; inhibitor; kinase inhibitor; knockout gene; luminescence; muscle regeneration; nano; novel; novel therapeutics; overexpression; patient derived xenograft model; peripheral blood; preclinical development; protein protein interaction; proteostasis; receptor; response; skeletal muscle differentiation; small hairpin RNA; small molecule; stoichiometry; synergism; targeted treatment; therapeutic target; trafficking; transcriptome; transcriptome sequencing

Among other assays we will use the Incutyte and ACEA systems to monitor cell growth. For the CRISPR screens we are performing genome wide and epigenetic focused libraries. For the drug screen we will use single agent and combination responses of a panel of 2000 drugs (Mechanism Interrogation Plate (Libraries) developed by NCATS. The content of this library include FDA approved compounds, several of which are approved for cancer therapy, those in clinical trials (phase 1, 2 or 3), several kinase inhibitors. For all these compounds, the target or mechanism of action is known. The most promising targets will be further evaluated in patient derived xenograft animal models. In a complementary effort with NCATs, we have teamed up the Molecular Targets Laboratory to identify inhibitors of the PAX3-FOXO1 fusion gene. To do this we will utilize the PAX3-FOXO1 activity reporter cell line developed by our laboratory using the super-enhancer region within the ALK gene which was cloned upstream of a minimal CMV promoter driving a Green Fluorescent Protein (GFP)-Luciferase reporters. We confirmed that RH41 cells (FP-RMS) appeared green with the stably transduced reporter construct, whereas RD cells (FN-RMS) containing the reporter construct were negative. We also confirmed that the ALK enhancer luciferase activity in the RH41 cell line was suppressed rapidly upon induction of the shRNA against PAX3-FOXO1. Of note decrease in luciferase activity preceded reduction of cell number. This property allows for the cell line to be utilized in drug screening experiments. This is currently being performed in large scale. For epigenetic studies our group has recently showed that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain protein family member BRD4, freezing FP-RMS cells in a myoblast-like state. These studies proved the feasibility of using unbiased high-throughput screening approaches to identify small molecules that disrupt the PAX3-FOXO1 core regulatory circuitry. In particular, we demonstrated that PAX3-FOXO1 transcriptional activity depends on its physical interaction with BRD4. The BRD4 inhibitor JQ1 ablated this interaction resulting in decreased PAX3-FOXO1 protein levels that correlated with suppression of FP-RMS xenograft growth in mice. In addition to protein-protein interactions, protein homeostasis (encompassing protein synthesis, folding, trafficking and degradation) is influenced by many other factors including post-translational modifications. Therefore, selectively targeting modifications that will result in decreased stability or activity of PAX3-FOXO1 is an attractive approach toward development of novel therapies for FP-RMS. Our previous screens used indirect approaches as they assessed PAX3-FOXO1-driven target gene expression or FP-RMS cell viability, with PAX3-FOXO1 protein levels being characterized post-hoc. Assay readouts of tagged endogenous proteins obviate the limitations associated with exogenous reporters where overexpression may disrupt the natural stoichiometry of interacting proteins or result in aggregation or mislocalization. Further, it is critical that the functional activity of the protein not be impeded by the addition of the tag and thus smaller tags are desirable because of their presumably reduced impact. For these reasons, we chose to fuse the pro-luminescent HiBiT peptide to endogenous PAX3-FOXO1 using CRISPR/Cas9-mediated knockin. HiBiT is a small 11-amino acid peptide capable of producing a luminescence signal that is about 100-fold brighter than firefly or Renilla luciferases through high affinity complementation with LgBiT, an 18 kDa subunit derived from the NanoLuc luciferase, thereby allowing detection at high sensitivity. Plasmids encoding Cas9 and single guide RNAs targeting the carboxy terminus of FOXO1 together with a DNA construct for homology-directed HiBiT addition were transfected into two FP-RMS cell lines, RH4 and SCMC. The DNA homology construct encoding HiBiT was followed by a P2A "self-cleaving" peptide for coexpression of an mCherry fluorescent protein as a FACS-selectable marker. Single mCherry-positive clones were sorted into 96-well plates and expanded. Six clones were obtained for each cell line in which the HiBiT tag was demonstrated to be successfully appended to the carboxy terminus of PAX3-FOXO1 using the Nano-Glo HiBiT Blotting System. Nucleotide sequencing of clones RH4.P3F-HmC 1A9 (RH4.PAX3-FOXO1-HiBiT-P2A-mCherry, clone 1A9) and SCMC.P3F-HmC 3C4 (SCMC.P3F-HiBiT-PAX3-FOXO1-P2A-mCherry, clone 3C4) confirmed accurate in-frame addition of the HiBiT tag to PAX3-FOXO1. Principal component analysis of RNA-seq data showed that the edited clones faithfully recapitulated the transcriptome landscape of the parental FP-RMS cell lines. HiBiT signal was readily detectable and HiBiT-tagged PAX3-FOXO1 was reduced at the protein level upon treatment with JQ1 and the clinical BRD4 inhibitor CPI-0610. RH4.P3F-HmC 1A9 and SCMC.P3F-HmC 3C4 are being subjected to quantitative high-throughput screening using single agent and combination responses of a panel of 4500 FDA-approved and advanced investigational drugs toward the identification, and preclinical and clinical development of new treatment options for FP-RMS. For Rhabdomyosarcoma (RMS), FGFR4 is a rational target given that it is a key regulator of myogenic differentiation and muscle regeneration after injury; it is expressed in myoblasts, but not in differentiated skeletal muscle. We and others have found that FGFR4 is highly expressed in all RMS, and high expression is a diagnostic and prognostic biomarker. It is a direct target and strongly induced by PAX3-FOXO1, PAX3, and PAX7 and we reported that PAX3-FOXO1 established a super-enhancer at the gene's locus. We have reported that approximately 10% of FN-RMS have activating mutations in FGFR4 and that cells harboring FGFR4 mutations are oncogene addicted and sensitive to pharmacological inhibition by small molecules. Therefore, FGFR4 is a key cell surface tyrosine kinase receptor for RMS biology, growth and survival. We are developing monoclonal antibodies and human scFv binders. The majority detect the human FGFR4 protein by both ELISA and by FACS analysis . To mitigate for potential organ toxicity, we are examining FGFR4 expression levels in normal human organs. We are currently performing extensive RNAseq and immunohistochemistry (IHC) analysis of normal organ and rhabdomyosarcoma tissue arrays. We are testing our scFv binders as potential FGFR4 chimeric antigen receptors (CARs) to generate a second-generation CAR receptor lentiviral construct that contains the CD8 transmembrane region, 41BB and CD3zeta intracellular domains and a human EGFR extracellular domain. This design was chosen because of its efficacy in clinical trials and CAR T cell persistence in patient's peripheral blood for several months after therapy. The truncated EGFR in the CAR construct allows for the measurement of transduced T cells as well as therapeutic targeting of CAR T cells with Cetuximab in clinical trials in case of uncontrolled toxicity. Anti-FGFR4 CART cells could lyse RH30 but not RAJI, a FGFR4 negative Burkitt's lymphoma cell line (data not shown). Work is currently underway to validate FGFR4 CAR T cells in-vivo. We are also developing novel TCRs as therapies for pediatric solid tumors. If successful we anticipate the development of potent immunotherapeutic biologics and cell-ba *TRUNCATED*

在其他检测中，我们将使用Incutyte和ACEA系统来监测细胞生长。对于CRISPR筛选，我们正在执行全基因组和表观遗传重点文库。对于药物筛选，我们将使用单一药物和2000种药物的联合反应（由NCATS开发的机制询问板（Libraries））。该文库的内容包括FDA批准的化合物，其中一些已被批准用于癌症治疗，那些正在临床试验（1、2或3期），几种激酶抑制剂。所有这些化合物的作用靶点或作用机制都是已知的。最有希望的靶点将在患者来源的异种移植动物模型中进一步评估。在与NCATs的互补努力中，我们与分子靶标实验室合作鉴定PAX3-FOXO1融合基因的抑制剂。为了做到这一点，我们将利用我们实验室开发的PAX3-FOXO1活性报告细胞系，该细胞系使用ALK基因中的超级增强子区域，该区域克隆在驱动绿色荧光蛋白(GFP)-荧光素酶报告的最小CMV启动子上游。我们证实，RH41细胞（FP-RMS）在稳定转导的报告基因构建下呈绿色，而含有报告基因构建的RD细胞（FN-RMS）呈阴性。我们还证实了ALK增强子荧光素酶活性在RH41细胞系中被诱导抗PAX3-FOXO1的shRNA迅速抑制。值得注意的是，荧光素酶活性的降低先于细胞数量的减少。这一特性使得该细胞系可用于药物筛选实验。目前正在大规模地进行这项工作。在表观遗传学研究中，我们的研究小组最近发现，pax3 - fox01通过与bromodomain和extra-terminal domain蛋白家族成员BRD4合作诱导新生超级增强子，将FP-RMS细胞冻结在成肌细胞样状态，从而对顺式调控表观遗传学景观进行了重编程。这些研究证明了使用无偏高通量筛选方法识别破坏PAX3-FOXO1核心调控电路的小分子的可行性。特别是，我们证明了pax3 - fox01的转录活性取决于它与BRD4的物理相互作用。BRD4抑制剂JQ1消除了这种相互作用，导致PAX3-FOXO1蛋白水平下降，这与抑制FP-RMS异种移植物生长有关。除了蛋白质之间的相互作用外，蛋白质稳态（包括蛋白质合成、折叠、运输和降解）还受到许多其他因素的影响，包括翻译后修饰。因此，选择性靶向修饰会导致PAX3-FOXO1的稳定性或活性降低，这是开发FP-RMS新疗法的一个有吸引力的方法。我们之前的筛选使用间接方法来评估pax3 - fox01驱动的靶基因表达或FP-RMS细胞活力，pax3 - fox01蛋白水平是事后表征的。标记内源性蛋白的分析读数消除了外源性报告蛋白的局限性，其中过表达可能破坏相互作用蛋白的自然化学计量，或导致聚集或错误定位。此外，至关重要的是，蛋白质的功能活性不受添加标签的阻碍，因此较小的标签是可取的，因为它们可能会减少影响。基于这些原因，我们选择使用CRISPR/ cas9介导的敲入蛋白将前发光HiBiT肽融合到内源性PAX3-FOXO1上。HiBiT是一种由11个氨基酸组成的小肽，能够通过与LgBiT（来自NanoLuc荧光素酶的18 kDa亚基）的高亲和力互补，产生比萤火虫或Renilla荧光素酶亮约100倍的发光信号，从而实现高灵敏度检测。将编码Cas9和靶向FOXO1羧基末端的单导rna的质粒以及用于同源定向添加HiBiT的DNA构建物转染到两种FP-RMS细胞系RH4和SCMC中。编码HiBiT的DNA同源构建体之后是P2A“自切割”肽，用于作为facs选择标记的mCherry荧光蛋白的共表达。将单个麦克樱桃阳性克隆分选到96孔板中进行扩增。使用Nano-Glo HiBiT印迹系统，每个细胞系获得6个克隆，证明HiBiT标签成功地附加到PAX3-FOXO1的羧基末端。克隆RH4的核苷酸测序。p3f - hmc1a9 (RH4；pax3 - fox01 - hibit - p2a - mcherry，克隆1A9)和SCMC。P3F-HmC 3C4 (SCMC；P3F-HiBiT-PAX3-FOXO1-P2A-mCherry（克隆3C4）证实在帧内准确添加了HiBiT标签到PAX3-FOXO1。RNA-seq数据的主成分分析表明，编辑的克隆忠实地再现了亲本FP-RMS细胞系的转录组景观。用JQ1和临床BRD4抑制剂CPI-0610治疗后，HiBiT信号很容易检测到，HiBiT标记的PAX3-FOXO1在蛋白水平上降低。RH4。p3f - hmc1a9和SCMC。P3F-HmC 3C4正在接受一组4500种fda批准的和正在进行研究的药物的单药和联合反应的定量高通量筛选，以确定、临床前和临床开发FP-RMS的新治疗方案。对于横纹肌肉瘤（Rhabdomyosarcoma， RMS）， FGFR4是一个合理的靶点，因为它是损伤后肌源性分化和肌肉再生的关键调节因子；它在成肌细胞中表达，但在分化的骨骼肌中不表达。我们和其他人发现FGFR4在所有RMS中都是高表达的，高表达是一种诊断和预后的生物标志物。它是PAX3- foxo1、PAX3和PAX7强烈诱导的直接靶点，我们报道了PAX3- foxo1在该基因的位点建立了一个超级增强子。我们已经报道了大约10%的FN-RMS在FGFR4中具有激活突变，并且携带FGFR4突变的细胞是癌基因成瘾的，并且对小分子的药物抑制敏感。因此，FGFR4是RMS生物学、生长和存活的关键细胞表面酪氨酸激酶受体。我们正在开发单克隆抗体和人scFv结合物。大多数人通过ELISA和FACS分析检测人FGFR4蛋白。为了减轻潜在的器官毒性，我们正在检测正常人体器官中FGFR4的表达水平。我们目前正在对正常器官和横纹肌肉瘤组织阵列进行广泛的RNAseq和免疫组织化学（IHC）分析。我们正在测试我们的scFv结合物作为潜在的FGFR4嵌合抗原受体（CARs），以生成第二代CAR受体慢病毒结构体，该结构体包含CD8跨膜区、41BB和CD3zeta细胞内结构域以及人类EGFR细胞外结构域。之所以选择这种设计，是因为它在临床试验中的有效性，以及治疗后CAR - T细胞在患者外周血中的持久性。CAR结构中截断的EGFR允许在临床试验中测量转导的T细胞，以及在毒性失控的情况下使用西妥昔单抗靶向CAR - T细胞。抗FGFR4 CART细胞可以裂解RH30，但不能裂解RAJI，一种FGFR4阴性的伯基特淋巴瘤细胞系（数据未显示）。目前正在进行体内验证FGFR4 CAR - T细胞的工作。我们也在开发新的tcr作为儿童实体瘤的治疗方法。如果成功，我们预计将开发出有效的免疫治疗生物制剂和细胞抗体