Delineating roles for a novel tri-protein complex (TRIM44) in the multiple myeloma stem cell niche

摘要： 多发性骨髓瘤（MM）是一种无法治愈的B细胞恶性肿瘤，其特征是生长和存活的骨髓瘤。 骨髓微环境中的肿瘤性浆细胞。尽管治疗取得了进展， MM的治疗，包括使用大剂量化疗和自体干细胞移植， 大部分患者对这些疗法是难治的。这种反应的变异性可能与细胞 形态学、分子遗传学和MM微环境内的功能异质性。 我们先前使用PKH 67荧光示踪剂的研究表明MM异质性与以下因素相关： 存在静止的MM细胞。我们分离了接近纯度的静止期MM细胞，其基础是它们具有以下能力： 保留亲脂性染料PKH 67，这是由于它们的静止性质。我们还允许MM细胞 在小鼠中而不是在体外循环，这反映了它们在微环境中的性质。我们的研究首次 揭示了一个静止的MM细胞龛和MM干细胞样细胞与 微环境在体内循环后，罕见的静止PKH+细胞优先驻留在成骨细胞中， 壁龛，而不是在骨髓或脾脏的血管壁龛中。PKH+细胞的功能分析 通过增加集落形成单位，成骨细胞龛在体外显示出增强的干样特性。在 此外，与PKH-细胞相比，这些PKH+细胞是高度致瘤性的，并且对多种 临床相关的化疗药物。 cDNA微阵列分析显示，TRIM 44在PKH+中高度上调。 成骨细胞龛中的细胞TRIM家族蛋白参与了广泛的生物过程 并且表达和功能的改变与病理学如出生缺陷有关， 神经变性疾病、病毒感染和癌症。与其他TRIM家族成员蛋白不同，TRIM 44 在N-末端区域含有锌指泛素结合结构域（ZF-UBP）而不是RING结构域。 UBP结构域通常存在于含有泛素特异性肽酶的去泛素化酶中。 总的来说，这些数据表明TRIM 44可能作为泛素特异性肽酶样TRIM起作用，并在细胞内起作用。 作为调节癌基因的去泛素化和/或稳定化的癌症促进基因。TRIM 44是第一个 2001年克隆出来，但对其功能知之甚少。有趣的是，有几份报告显示， TRIM 44在癌症中的过度表达，包括16%的TRIM 44表达扩增的上皮癌。 因此，TRIM 44可能作为MM的潜在癌症生物标志物。 和分子方法来描绘TRIM 44在静止MM细胞中的功能。我们假设TRIM 44 对于维持PKH+ MM细胞在成骨细胞龛内的静止是必需的。此外，我们建议 TRIM 44赋予MM细胞干细胞样功能。在具体目标1中，我们将定义TRIM 44函数， 体内静止MM龛。在具体目标2中，我们将研究TRIM 44在自噬形成中的作用， MM存活率。综上所述，本课题的完成将加深我们对MM干细胞及其分化的认识。 微环境，并建立TRIM 44作为MM的潜在治疗靶点。

Abstract: Multiple myeloma (MM) is an incurable B cell malignancy that is characterized by growth and survival of neoplastic plasma cells within the bone marrow microenvironment. Despite progress achieved in the treatment of MM, including the use of high-dose chemotherapy and autologous stem cell transplantation, a considerable proportion of patients are refractory to the therapies. This variability in response is likely related cellular morphology, molecular genetics and functional heterogeneity within the MM microenvironment. Our previous studies using PKH67 fluorescent tracers showed that MM heterogeneity is correlated with the presence of quiescent MM cells. We isolated quiescent MM cells near purity on the basis of their ability to retain the lipophilic dye, PKH67, as a consequence of their quiescent nature. We also allowed MM cells to cycle in mice instead of in vitro, which reflects their nature within the microenvironment. Our study is the first to reveal a quiescent MM cell niche and the effects of functional interactions between MM stem-like cells and microenvironment. After cycling in vivo, rare quiescent PKH+ cells preferentially reside within osteoblastic niches, rather than in vascular niches of the bone marrow or spleen. Functional analyses of PKH+ cells from osteoblastic niches revealed enhanced stem-like properties in vitro by increasing colony forming units. In addition, these PKH+ cells were highly tumorigenic, as compared to PKH- cells, and were resistant to a variety of clinically relevant chemotherapeutic drugs. cDNA microarray analyses revealed that a tripartite motif protein, TRIM44, is highly upregulated in PKH+ cells from the osteoblastic niche. TRIM family proteins are involved in a broad range of biological processes and alterations in expression and function are associated with pathologies such as birth defects, neurodegenerative diseases, viral infections and cancer. Unlike other TRIM family member proteins, TRIM44 contains a zinc finger ubiquitin binding domain (ZF-UBP) in the N-terminal region instead of a RING domain. UBP domains are often found in the deubiquitinating enzymes containing ubiquitin specific peptidase. Collectively, these data suggest that TRIM44 may function as an ubiquitin specific peptidase-like TRIM and act as a cancer promoting gene regulating deubiquitination and/or stabilization of oncogenes. TRIM44 was first cloned in 2001 yet very little is known about its function. Interestingly, several reports have shown overexpression of TRIM44 in cancer, including 16% of epithelial cancers with amplified TRIM44 expression. Therefore, TRIM44 may serve as a potential cancer biomarker for MM. In this project, we will use biochemical and molecular approaches to delineate TRIM44 functions in quiescent MM cells. We hypothesize that TRIM44 is essential for maintenance of PKH+ MM cell quiescence within osteoblastic niches. Furthermore, we propose that TRIM44 confers stem-like functions to MM cells. In Specific Aim 1, we will define TRIM44 functions in quiescent MM niches in vivo. In Specific Aim 2, we will investigate roles of TRIM44 in autophagy formation and MM survival. In summary, completion of this project will deepen our knowledge of MM stem-like cells and their microenvironment and establish TRIM44 as a potential therapeutic target in MM.