Alternatively spliced cell surface proteins as drivers of leukemogenesis and targets for immunotherapy

选择性剪接的细胞表面蛋白作为白血病发生的驱动因素和免疫治疗的靶点

基本信息

批准号：
10648346
负责人：
Fabiana Perna
金额：
$ 27.08万
依托单位：
H. LEE MOFFITT CANCER CTR & RES INST
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10648346
关键词：
Acute Lymphocytic Leukemia Acute Myelocytic Leukemia Address Affect Alternative Splicing Antibodies Antibody-drug conjugates Antigen Targeting Antigens Apoptosis Area Biological Biological Assay Biology CAR T cell therapy CD34 gene Cell Fractionation Cell Line Cell Proliferation Cell Surface Proteins Cell membrane Cell surface Cells Clinical Code Custom DNA Sequence Alteration Data Set Development Digestion Disease Engineering Epigenetic Process Event Flow Cytometry Fostering Future Genes Genetic Transcription Genome Genomics Goals Hematopoietic Neoplasms Hematopoietic stem cells IL3RA gene Immunologic Deficiency Syndromes Immunotherapeutic agent Immunotherapy In Vitro Laboratories Lead Leukemic Cell Lymphoma Malignant Neoplasms Messenger RNA Methods Modality Molecular Multiple Myeloma Mus Mutate Mutation Myeloproliferative disease NK cell therapy Normal tissue morphology Oncogenes Outcome Pathogenesis Patients Peptide Hydrolases Phase I Clinical Trials Post-Translational Protein Processing Protein Isoforms Proteins Proteome RNA Splicing Recurrence Refractory Relapse Role SRSF2 gene Safety Sampling Shapes Solid Neoplasm Spliceosomes Stains Surface Surface Antigens Survival Rate Therapeutic Therapeutic Intervention Translations Tumor Burden Umbilical Cord Blood Variant Viral Work acute myeloid leukemia cell cancer cell candidate validation chimeric antigen receptor chimeric antigen receptor T cells clinical application clinical practice combinatorial comparison control data integration diagnostic tool efficacy evaluation epigenomics extracellular founder mutation immune modulating agents knock-down lead candidate leukemia leukemia treatment leukemogenesis loss of function mutant new therapeutic target novel patient prognosis protein expression selective expression small hairpin RNA solute transcriptome transcriptome sequencing tumor immunology

项目摘要

Project Summary While impressive progress has been made in tumor immunology and clinical application of immunomodulatory agents in solid tumors, we don’t fully understand how to effectively incorporate immunotherapeutic strategies in Acute Myeloid Leukemia (AML) (Perna F et al., Cancer Treat Res 2022). Advances in genomic and epigenomic characterization of AML have fostered better understanding of leukemogenesis. The average AML genome may have only 13-16 coding mutations, of which around five are recurrent mutations in suspected driver genes. Despite approval of novel targeted therapies, the clinical outcome of AML patients remains dismal, with a 5-year overall survival of approximately 27%, prompting the search for additional and synergistic therapeutic rationales. Developing immune-based therapies for AML has been challenged by the lack of surface targets. I previously developed a target discovery strategy to identify Chimeric Antigen Receptor (CAR) targets in AML (Perna F. et al., Cancer Cell 2017). As we could not identify single targets with favorable expression profiles, we showed that combinatorial pairings hold promise for CAR T-cell therapy of AML. Over the past two years, my lab investigated whether cancer-specific mechanisms such as alternative mRNA splicing driven by founder mutations (i.e. genetic mutations affecting splicing factors) may shape the cancer surface proteome, providing targets for promoting disease initiation and progression and use of immunotherapy (Perna F. Molecular Therapy 2021 and Dong et al., Oncogene 2021). We have now developed a novel pipeline that we called Spliced-ImmuneTargetFinder based on 5 steps to identify splice variants of cell surface molecules: 1) RNA-seq analysis of normal cord blood CD34+ HSPCs virally expressing splicing factor mutations such as SRSF2P95H mutation 2) custom transcriptome analysis for isoform quantification 3) isoform switch analysis and prediction of functional consequences based on isoform sequences such as coding potential and domain gain 4) cell surface molecule annotation based on an integrated dataset developed in our laboratory, and 5) protein candidates validation in primary AML patient samples beyond SRSF2 mutant patients. Given that, we identified six top candidate targets that are a) involved in at least one switch isoform b) upregulated in SRSF2 mutant cells compared to controls and c) derive from genes coding for cell surface proteins. We validated their aberrant protein expression in several AML cell lines and primary AML patient samples by developing custom antibodies specifically recognizing isoform unique domains derived from splicing events. Thus, we propose to investigate the functional roles of these promising variants and the path for targeting the extracellular isoform-specific domains with immune-therapeutic interventions. This method for unconventional target antigens discovery is generalizable to other cancers as well and will provide useful information to prepare for a phase-I clinical trial in AML patients. Identification of these attractive antigens may also open new avenues for understanding the role of splice isoforms in leukemogenesis.

项目概要肿瘤免疫学和免疫调节剂的临床应用取得了令人瞩目的进展实体瘤药物，我们并不完全了解如何有效地将免疫治疗策略纳入其中急性髓系白血病 (AML)（Perna F 等人，癌症治疗研究 2022）。 AML 的特征有助于更好地了解白血病的发生。只有 13-16 个编码突变，其中大约 5 个是可疑驱动基因中的反复突变。尽管新的靶向治疗已获得批准，但 AML 患者的临床结果仍然令人沮丧，5 年的治疗效果仍然不佳。总生存率约为 27%，促使人们寻找额外的协同治疗原理。开发基于免疫的 AML 疗法曾因缺乏表面靶点而受到挑战。开发了一种靶点发现策略来识别 AML 中的嵌合抗原受体 (CAR) 靶点（Perna F. 等人，2017）。 al.，Cancer Cell 2017）。由于我们无法识别具有良好表达谱的单一靶标，因此我们证明了这一点。组合配对为 AML 的 CAR T 细胞治疗带来希望。在过去的两年里，我的实验室研究了癌症特异性机制（例如替代疗法）是否由创始人突变（即影响剪接因子的基因突变）驱动的 mRNA 剪接可能会塑造癌症表面蛋白质组，提供促进疾病发生和进展的靶标以及使用免疫疗法（Perna F. Molecular Therapy 2021 和 Dong et al., Oncogene 2021）。我们称之为 Spliced-ImmuneTargetFinder 的新颖管道基于 5 个步骤来识别细胞的剪接变体表面分子：1) 对病毒表达剪接因子的正常脐带血 CD34+ HSPC 进行 RNA-seq 分析 SRSF2P95H 突变等突变 2) 用于异构体定量的定制转录组分析 3) 异构体基于异构体序列（例如编码潜力）的开关分析和功能后果预测和域增益4）基于我们实验室开发的集成数据集的细胞表面分子注释， 5) 在 SRSF2 突变患者之外的原发性 AML 患者样本中验证候选蛋白。我们确定了六个顶级候选靶标，它们 a) 涉及至少一种开关亚型 b) 在 SRSF2 中上调与对照细胞相比，突变细胞和 c) 来自编码细胞表面蛋白的基因。通过开发定制的方法，在几种 AML 细胞系和原发性 AML 患者样本中发现异常蛋白表达特异性识别源自剪接事件的同工型独特结构域的抗体。因此，我们建议研究这些有希望的变体的功能作用以及靶向路径这种方法可用于免疫治疗干预的细胞外亚型特异性结构域。非常规靶抗原的发现也适用于其他癌症，并将提供有用的为 AML 患者进行 I 期临床试验做准备的信息可能会鉴定这些有吸引力的抗原。还为了解剪接亚型在白血病发生中的作用开辟了新途径。