Functional Consequences of Ubiquitin Depletion During B Lymphocyte Differentiation

项目摘要/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， 以合成丰富的免疫球蛋白(Ig)为特征。大多数PC的寿命都很短，只有几台PC会死 分化后天数，分子机制不明。多发性骨髓瘤的发病机制也仍然难以捉摸 没有单一的基因驱动突变，但普遍存在DNA损伤。我们之前已经展示了 多泛素化(PolyUb)蛋白运载量增加，蛋白酶体活性下降 B淋巴细胞的分化和我们假设蛋白毒性是PC死亡的驱动因素。通过使用 在相同的B细胞分化模型中，用绝对质谱仪(Aqua)对泛素(Ub)进行定量， 我们的初步数据显示，与休息的B细胞相比，PC中60%的游离Ub耗尽， 同时伴有最大的免疫球蛋白分泌和细胞凋亡。第二，我们发现存活时间较长的细胞 可以识别术语后分化，并显示正在进行的DNA损伤的标记。作为Ub是 正确的DNA损伤反应(DDR)所必需的，PolyUb蛋白降解和DDR竞争 相同的Ub池。基于我们的工作，我们提出了一个短寿命PC死亡和MM的统一模型 发病机制以危重的Ub耗竭为中心。我们的核心假设是，在PC中，蛋白毒性应激 继发于持续的免疫球蛋白合成，导致游离Ub耗尽，从而导致DDR受损。大多数PC 将无法从这场危机中恢复，但一小部分基因不稳定的PC可能会 存活，可能是由于积累了促进生存的突变，构成了癌前病变 我们进一步推测Ub基因Ubc可能是一个单倍体不足的肿瘤抑制因子 B细胞恶性肿瘤中的基因。在此，申请者Giada Bianci博士提出了一项全面的计划 为了检验在两个具体目标中阐明的这些假设：(1)探索免疫球蛋白合成之间的因果联系， Ub耗竭与PC细胞凋亡；以及(2)评估Ub耗竭是否足以导致 基因组不稳定性与多发性骨髓瘤的发病机制密切相关。在我们的调查过程中收集的数据 研究将提供有关正常和恶性PC生物学的新数据，并可能 揭示一种新的致癌机制--Ub功能耗竭--适用于其他 恶性肿瘤和创新的分子靶向治疗的机会。为此，比安奇博士 精心挑选了一个指导委员会和合作者，他们都是世界知名的Ub专家， DDR、蛋白质组学和B细胞生物学。这些团队将提供知识、模型和技术 使比安奇博士的假设在拟议的5年框架内得到检验是可行的。这个 申请人的指导委员会进一步设计了详细的职业计划，基于定期 会议、参加讲习班、培训班和国际会议，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员的轨迹。

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.