The role of AID/APOBEC3 proteins in genome instability in multiple myeloma

摘要 多发性骨髓瘤(MM)是一种骨髓浆细胞的恶性肿瘤，在此之前有一系列癌前病变 和过渡阶段。所有阶段的细胞都表现出显著的基因组异常，其来源是 不是很清楚。这在我们对驱动MM的机制的理解上留下了很大的空白 启动和进展，这一提议旨在填补这一空白。最近的证据表明 胞苷脱氨酶-DNA中将胞嘧啶转化为尿嘧啶的酶-是基因组中的重要罪魁祸首 MM的不稳定性一个重要的例子是激活诱导的脱氨酶，AID，它启动了体细胞 高突变和类转换重组，但也突变B细胞基因组的许多区域 引起与MM相似的易位。相关的胞苷APOBEC3家族 脱氨酶也可以使DNA发生突变，许多发现将AID和APOBEC3酶与基因组联系起来 我们的初步数据表明，MM细胞表达APOBEC3B，C， D、F和G，其中APOBEC3B的表达尤为强烈。有趣的是，APOBEC3B最近 与乳腺癌的基因组不稳定有关。我们进一步发现，AID和某些 APOBEC3酶可增加多发性骨髓瘤细胞DNA链断裂水平。在此基础上，我们假设 AID和APOBEC3家族酶是基因组异常和疾病进展的主要原因 在MM中，最近的发现提供了脂质失调、免疫激活和MM之间的强烈联系，与 在多发性骨髓瘤患者中发现的对脂质有反应的克隆性伽玛病多达三分之一。因此，一个重要的 我们工作的总体指导性假设是，慢性B细胞激活是由于血浆中 炎性脂类参与了AID/APOBEC3活性的增加和MM疾病的进展。我们 从三个方面系统地检验这些假说。在目标1中，我们确定了 AID/APOBEC3酶单独和协同作用有助于生物化学和分子测量 核脱氨酶活性与基因组损伤。在目标2中，我们确定这些脱氨酶是如何 调节，揭示MM基因组中那些容易发生突变、DNA断裂和 由于他们的行为而导致的移位。最后，在目标3中，我们使用已建立的和新的小鼠模型系统 和MM及其癌前阶段在人源化小鼠体内繁殖，以评估在体内的作用 炎性脂质对AID/APOBEC3因子表达/活性、DNA损伤及突变的影响 MM基因组和MM的克隆进化，拟议的实验将提供 多发性骨髓瘤中胞苷脱氨酶作用的活性、靶点和结果的全面图像， 对疾病发病机制的广泛影响。

SUMMARY Multiple Myeloma (MM) is a malignancy of bone marrow plasma cells preceded by a series of premalignant and transitional stages. Cells at all stages exhibit significant genomic aberrations, the sources of which are not well understood. This has left a major gap in our understanding of the mechanisms that drive MM initiation and progression, a gap that this proposal is designed to fill. Recent evidence suggests that cytidine deaminases—enzymes that convert cytosine to uracil in DNA—are important culprits in genomic instability in MM. One important example is the activation induced deaminase, AID, which initiates somatic hypermutation and class switch recombination but which also mutates many regions of the B cell genome and causes translocations similar to those found in MM. The related APOBEC3 family of cytidine deaminases can also mutate DNA, and numerous findings link AID and APOBEC3 enzymes to genome instability and mutations in MM. Our preliminary data demonstrate that MM cells express APOBEC3B, C, D, F and G, with APOBEC3B being expressed particularly strongly. Interestingly, APOBEC3B has recently been implicated in genome instability in breast cancer. We further find that expression of AID and certain APOBEC3 enzymes increases levels of DNA strand breaks in MM cells. Based on this, we hypothesize that AID and APOBEC3 family enzymes are a major cause of genomic aberrations and disease progression in MM. Recent findings provide a strong link between lipid disregulation, immune activation, and MM, with as much as a third of the clonal gammopathies found in MM patients reacting to lipids. Hence, an important overall guiding hypothesis of our work is that chronic B cell activation arising from elevated levels of inflammatory lipids contributes to increased activity of AID/APOBEC3 and MM disease progression. We systematically test these hypotheses in three Aims. In Aim 1, we determine the extent to which AID/APOBEC3 enzymes, alone and in concert, contribute to biochemical and molecular measures of nuclear deaminase activity and genomic damage. In Aim 2, we determine how these deaminases are regulated, revealing those regions of the MM genome that are susceptible to mutation, DNA breaks, and translocations due to their action. Finally, in Aim 3 we use established and novel mouse model systems and MM and its premalignant stage propagated in humanized mice to assess the in vivo effects of inflammatory lipids on the expression/activity of AID/APOBEC3 factors, DNA damage and mutation of the MM genome, and clonal evolution of MM. Together, the proposed experiments will provide a comprehensive picture of the activity, targeting, and outcome of cytidine deaminase action in MM, with broad implications for disease pathogenesis.