Functional Consequences of Ubiquitin Depletion During B Lymphocyte Differentiation

B 淋巴细胞分化过程中泛素耗竭的功能后果

• 批准号: 10238150
• 负责人: Giada Bianchi
• 金额: $ 20.73万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-13 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10238150
• 关键词: Apoptosis; B cell differentiation; B lymphoid malignancy; B-Lymphocytes; Biological Markers; Biology; Bone Marrow; Cancer Biology; Cancerous; Cell Death; Cell Survival; Cells; Cellular biology; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Damage; DNA Repair; DNA Sequence Alteration; DNA analysis; Data; Development; Disease; Educational workshop; Generations; Genetic; Genomic Instability; Goals; Heavy-Chain Immunoglobulins; Hematologic Neoplasms; Immunoglobulins; Impairment; Innovative Therapy; International; Investigation; Knock-out; Knowledge; Life; Link; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Modeling; Molecular; Monoclonal gammopathy of uncertain significance; Multiple Myeloma; Mutation; Nuclear; Oncogenic; Oncology; Outcome; Pathogenesis; Pathologic; Physiological; Plant Roots; Plasma Cells; Precancerous Conditions; Proliferating; Proteins; Proteomics; Public Health; Publishing; Research; Research Personnel; Rest; Secondary to; Stress; Structure of germinal center of lymph node; Technology; Testing; Tetracyclines; Time; Transfection; Transgenic Mice; Tumor Suppressor Genes; UBC gene; Ubiquitin; Western Blotting; Western World; Work; base; career; design; driver mutation; experimental study; innovation; knock-down; meetings; molecular targeted therapies; mouse model; multicatalytic endopeptidase complex; novel; novel therapeutics; plasma cell differentiation; protein degradation; proteotoxicity; response; small hairpin RNA

Project Summary/Abstract Multiple myeloma (MM) is an incurable cancer of terminally differentiated plasma cells (PC), characterized by abundant immunoglobulin (Ig) synthesis. Most PC are short lived and die only a few days after differentiation through unclear molecular mechanisms. MM pathogenesis also remain elusive with no single genetic driver mutation, but pervasive DNA damage. We have previously shown increased cargo of polyubiquitinated (polyUb) proteins and decreased proteasome activity in differentiating B lymphocytes and we hypothesized proteotoxicity as driver of PC death. By using absolute mass spectrometry (AQUA) to quantify ubiquitin (Ub) in the same B cell differentiation model, our preliminary data show that 60% free Ub depletion occurs in PC compared to resting B cells, concomitantly with maximal Ig secretion and apoptosis. Second, we discovered that cells surviving long term post differentiation can be identified and display markers of ongoing DNA damage. As Ub is necessary for proper DNA damage response (DDR), polyUb protein degradation and DDR compete for the same Ub pool. Based on our work, we propose a unifying model for short-lived PC death and MM pathogenesis centered on critical Ub depletion. Our core hypothesis is that in PC, proteotoxic stress secondary to sustained Ig synthesis, leads to free Ub depletion thus causing impaired DDR. Most PC will be unable to recover from this crisis, however a small subset of genomically unstable PC may survive, presumably due to accumulation of survival promoting mutations, constituting a premalignant state for MM. We further hypothesize that Ub gene Ubc may be a haploinsufficient tumor suppressor genes in B cell malignancies. Herein, the applicant, Dr. Giada Bianchi, presents a comprehensive plan to test these hypotheses articulated in 2 specific aims: (1) to probe a causative link between Ig synthesis, Ub depletion and apoptosis in PC; and (2) to evaluate whether Ub depletion is sufficient to cause genomic instability in PC and drive MM pathogenesis. Data gathered during the course of our investigation will provide novel data regarding the biology of normal and malignant PC and potentially uncover a novel oncogenic mechanism - functional depletion of Ub - with applicability to other malignancies and opportunity for innovative, molecularly targeted therapies. To this end, Dr. Bianchi has carefully selected a mentoring committee and collaborators who are world-renowned experts in Ub, MM, DDR, proteomics and B cell biology. Such team will provide knowledge, models and technologies to render the testing of Dr. Bianchi’s hypotheses feasible within the proposed 5-year frame. The applicant’s mentoring committee has furthermore designed a detailed career plan based on regular meeting, attendance of workshops, classes and international meetings which will further accelerate the trajectory of Dr. Bianchi to become an independent investigator in cancer biology in the next 5 years.

项目总结/摘要 多发性骨髓瘤（MM）是一种无法治愈的终末分化浆细胞（PC）癌症， 其特征在于大量的免疫球蛋白（IG）合成。大多数PC都是短命的，只有少数会死亡。 通过不清楚的分子机制分化后的天。MM发病机制也仍然难以捉摸 没有单一的基因驱动突变，但普遍的DNA损伤。我们先前已经表明 多聚泛素化（polyUb）蛋白的货物增加和蛋白酶体活性降低， 区分B淋巴细胞，我们假设蛋白毒性是PC死亡的驱动因素。通过使用 绝对质谱法（AQUA）定量相同B细胞分化模型中的泛素（UB）， 我们的初步数据显示与静息B细胞相比在PC中发生60%的游离UB耗竭， 伴随着最大的IG分泌和凋亡。第二，我们发现存活时间长的细胞 可以鉴定分化后术语并显示进行中的DNA损伤的标记。因为Ub是 适当的DNA损伤反应（DDR）所必需的，polyUb蛋白降解和DDR竞争 同一个游泳池基于我们的工作，我们提出了一个统一的模型，为短暂的PC死亡和MM 发病机制以临界Ub耗竭为中心。我们的核心假设是，在PC中，蛋白毒性应激 继发于持续的IG合成，导致游离Ub耗竭，从而导致受损的DDR。大多数PC 将无法从这场危机中恢复过来，然而一小部分基因组不稳定的PC可能 生存，可能是由于生存促进突变的积累，构成癌前病变。 我们进一步假设Ub基因Ubc可能是一个单倍不足肿瘤抑制基因， B细胞恶性肿瘤中的基因。在此，申请人Giada比安奇博士提出了一个全面的计划， 为了检验这些假设，有两个具体的目的：（1）探索IG合成之间的因果联系， 在PC中Ub缺失和细胞凋亡;和（2）评估Ub缺失是否足以引起PC中的细胞凋亡。 PC中的基因组不稳定性和驱动MM发病机制。在我们的过程中收集的数据 研究将提供关于正常和恶性PC生物学的新数据， 揭示了一种新的致癌机制-Ub的功能性缺失-适用于其他 恶性肿瘤和创新的机会，分子靶向治疗。为此，比安奇博士 精心挑选了一个指导委员会和合作者谁是世界知名的专家在乌布， MM、DDR、蛋白质组学和B细胞生物学。该团队将提供知识、模型和技术 使比安奇博士的假设在拟议的5年框架内可行。的 申请人的指导委员会还根据定期的职业规划设计了详细的职业规划， 会议，参加讲习班，课程和国际会议，这将进一步加快 比安奇博士在未来5年内成为癌症生物学独立研究者的轨迹。