Developing Novel Therapies for Neuroblastoma and Rhabdomyosarcoma

对于神经母细胞瘤，我们使用四种细胞系，两种MYCN未扩增的细胞系和两种MYCN扩增的细胞系，可以在异种移植物模型中生长。对于RMS，我们使用了10个细胞系，其中两个带有PAX3-FOXO1融合基因，两个带有RAS通路突变基因。在其他分析中，我们将使用Incutyte系统。对于siRNA筛选，我们将使用由国家推进转化科学中心（NCATS）开发的超过6000个基因的可药物基因组文库。对于药物筛选，我们将使用1916种药物的单药和联合反应（机制询问板（MIPE-v4）库），也由NCATS开发。该文库的内容包括765种FDA批准的化合物，其中49种被批准用于癌症治疗，460种处于临床试验（1、2或3期），149种激酶抑制剂。其中1915种化合物的作用靶点或作用机制是已知的。如前所述，最有希望的靶点和合适的siRNA或药物组合将在异种移植动物模型中进一步评估。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。如果成功，我们期望开发有效的免疫治疗生物制剂和基于细胞的治疗侵袭性RMS患者。所有阳性结果将在更广泛的NB和RMS异种移植物和pdx中进一步筛选。

For neuroblastoma are using four cell lines, two MYCN not-amplified and two MYCN amplified cell line that can be grown in a xenograft model. For RMS we are using ten cell lines, tow with PAX3-FOXO1 fusion genes and two with RAS pathway mutated genes. Among other assays we will use the Incutyte system. For the siRNA screen we will use a druggable genome library of over 6000 genes developed by National Center for Advancing Translational Sciences (NCATS). For the drug screen we will use single agent and combination responses of a panel of 1916 drugs (Mechanism Interrogation Plate (MIPE-v4) Library) also developed by NCATS. The content of this library include 765 FDA approved compounds, 49 of which are approved for cancer therapy, 460 in clinical trials (phase 1, 2 or 3), 149 kinase inhibitors. For 1915 of these compounds, the target or mechanism of action is known. The most promising targets and the appropriate siRNA or drug combination will be further evaluated in the xenograft animal models as outlined above. 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. If successful we anticipate the development of potent immunotherapeutic biologics and cell based therapies for patients with aggressive RMS. All positive hits will be further screened in a wider panel of NB and RMS Xenografts and PDxs.