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 虽然在肿瘤免疫学和免疫调节的临床应用中取得了令人印象深刻的进展 实体瘤的特工，我们不完全了解如何有效地纳入免疫治疗策略 急性髓样白血病（AML）（Perna F等，癌症治疗RES 2022）。基因组和表观基因组的进步 AML的表征促进了对白血病的更好理解。平均AML基因组可能 只有13-16个编码突变，其中大约五个是可疑驱动基因的复发突变。 尽管批准了新型靶向疗法，但AML患者的临床结果仍然令人沮丧，5年 总体生存率约为27％，促使人们寻找额外的和协同的治疗原理。 缺乏表面靶标，为AML开发基于免疫的疗法受到了挑战。我以前 制定了目标发现策略，以鉴定AML中的嵌合抗原受体（CAR）靶标（Perna F. Al。，癌细胞2017）。由于我们无法识别具有有利表达曲线的单个目标，因此我们表明 组合配对对AML的汽车T细胞疗法有希望。 在过去的两年中，我的实验室调查了癌症特异性机制（例如替代机制）是否 由创始人突变驱动的mRNA剪接（即影响剪接因子的基因突变）可能会塑造 癌表面蛋白质组，提供促进疾病起步和进展的靶标 免疫疗法（Perna F. Molecular Therapy 2021和Dong等，Oncogene 2021）。我们现在已经开发了 我们根据5个步骤识别细胞变体的5个步骤，称为剪接 - 免疫捕获剂的新型管道 表面分子：1）正常脐带血CD34+ HSPC的RNA-seq分析病毒表达剪接因子 诸如SRSF2P95H突变之类的突变2）同工型量化的自定义转录组分析3）同工型 基于同工序列（例如编码势）的开关分析和功能后果预测 域获得4）基于在我们实验室中开发的集成数据集的细胞表面分子注释， 和5）候选蛋白质的蛋白质在SRSF2突变患者之外的原始AML患者样品中的验证。鉴于， 我们确定了六个顶级候选目标，a）至少参与一个开关同工型b）在srsf2中更新 与对照组相比，突变细胞源于编码细胞表面蛋白的基因。我们验证了他们 通过开发自定义 抗体专门识别从剪接事件中得出的同工型独特域。 这是我们建议研究这些承诺变体的功能作用和靶向的路径 具有免疫治疗干预措施的细胞外同工型特异性结构域。此方法 非常规靶抗原发现也可以推广到其他癌症，并将提供有用的 信息为AML患者的I期临床试验做准备。这些有吸引力的抗原的识别可能 还开放了新的途径，以了解剪接同工型在白血病中的作用。