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

• 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
• 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

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.

项目摘要 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， 尽管我们在实体瘤中使用了免疫抑制剂，但我们还不完全了解如何有效地将免疫抑制策略纳入实体瘤中， 急性骨髓性白血病（AML）（佩尔纳F等，Cancer Treat Res 2022）。基因组与表观基因组研究进展 AML的表征促进了对白血病发生的更好理解。平均AML基因组可能 只有13-16个编码突变，其中大约5个是疑似驱动基因的复发突变。 尽管新的靶向治疗获得批准，但AML患者的临床结局仍然令人沮丧，5年 总生存率约为27%，这促使人们寻找额外的和协同的治疗原理。 开发基于免疫的AML疗法一直受到缺乏表面靶点的挑战。我以前 开发了一种靶点发现策略来鉴定AML中的嵌合抗原受体（CAR）靶点（佩尔纳F. et 例如，Cancer Cell 2017）。由于我们无法鉴定具有有利表达谱的单一靶标，我们表明， 组合配对有望用于AML的CAR T细胞疗法。 在过去的两年里，我的实验室调查了癌症特异性机制是否存在，例如替代疗法 由创始者突变（即影响剪接因子的遗传突变）驱动的mRNA剪接可能会影响基因的表达。 癌症表面蛋白质组，提供促进疾病发生和发展的靶点，以及它们的用途 免疫疗法（佩尔纳F.分子治疗2021和Dong等人，Oncogene 2021）。我们现在已经开发 一种新型管道，我们称为Spliced-ImmuneTargetFinder，基于5个步骤来识别细胞的剪接变体 表面分子：1）病毒表达剪接因子的正常脐带血CD 34 + HSPC的RNA-seq分析 突变，如SRSF 2 P95 H突变2）用于亚型定量的定制转录组分析3）亚型 基于同种型序列（如编码潜能）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综合数据集的细胞表面分子注释， 和5）SRSF 2突变患者以外的原发性AML患者样本中的蛋白候选物验证。鉴于此， 我们鉴定了6个最佳候选靶点，它们a）涉及至少一种开关亚型B）在SRSF 2中上调 与对照相比的突变细胞和c）源自编码细胞表面蛋白的基因。我们验证了他们的 在几种AML细胞系和原发性AML患者样品中的异常蛋白表达， 特异性识别源自剪接事件的同种型独特结构域的抗体。 因此，我们建议研究这些有前途的变体的功能作用和靶向的途径 细胞外同种型特异性结构域与免疫治疗干预。该方法对 非常规靶抗原的发现也可推广到其他癌症， 为AML患者的I期临床试验做准备。这些有吸引力的抗原的鉴定可 也为理解剪接异构体在白血病发生中的作用开辟了新的途径。