Translational Control of Leukemia Stem Cells - Resubmission - 1

在美国，每年诊断出大约13，000例成人AML新病例。 患者，治疗选择30年来基本保持不变，临床结果保持不变， 扶贫此外，人们对调节白血病干细胞（LSC）的基因知之甚少，这些基因代表了白血病细胞。 对化疗耐药的原始细胞群，对维持疾病和重新启动 治疗后的疾病因此，根除LSC是治愈的先决条件。 我们最近发现了一种新的LSC抗原，CD 99，它在绝大多数（约85%）的人淋巴细胞中表达。 AML [3]。我们已经证明CD 99在白血病原始细胞上表达，并且可以用于前瞻性地分离 来自残余造血的白血病原始细胞，类似于LSC抗原如TIM 3和CD 47，但CD 99表现出 几个独特的特点：1）CD 99是最常见的表达LSC抗原; 2）CD 99不只是标记 3）CD 99允许从具有CD 99 hi母细胞的母细胞中分离LSC 白血病起始细胞活性比CD 99低细胞富集约10-100倍; 4）CD 99是唯一的LSC抗原 可以识别CD 34+和CD 34-AML中的LSC;和，5）针对 CD 99通过激活Src家族激酶（SFKs）直接诱导细胞死亡。 我们已经评估了LSC和造血干细胞（HSC）中CD 99丢失的后果。我们 研究表明，LSC和HSC中CD 99表达的降低导致LSC和HSC中CD 99表达的总体上调。 蛋白质合成和自我更新的丧失。此外，针对CD 99的细胞毒性mAb模拟了CD 99的作用， 缺失，激活蛋白质合成并诱导与CD 99 null中观察到的相似的基因表达变化。 在大多数检测的AML基因亚型中的HSC或CD 99低原始细胞。总的来说，这些数据支持一个模型， LSC需要高度调节的蛋白质合成水平，类似于HSC，并基于我们在 在正常HSC中，我们预期这些翻译的改变会导致mRNA的选择性募集， 翻译核糖体（多核糖体）。总的来说，我们假设CD 99抑制了特异性的 mRNA，从而促进LSC自我更新所需的翻译程序。 鉴于我们富集LSC的能力，我们的团队在研究mRNA的作用方面处于独特的地位。 LSC函数中的翻译。我们精心优化了多核糖体分析和RNA分析的方法， 从来自少量细胞的多核糖体组分测序，我们已经开发了一种计算的 这是一条鉴定优先在LSC中翻译的mRNA的管道。这些工具结合独特的 试剂，如我们的CD 99 KO小鼠和模拟CD 99丢失的细胞毒性CD 99 mAb，使我们处于一个极好的 研究CD 99如何调节LSC中的翻译。了解分子途径， 调节蛋白质合成有可能帮助更好地描述AML中一个知之甚少的过程 生物学和认证使用mAb的靶向翻译作为AML中的潜在治疗策略。

Approximately 13,000 new cases of adult AML are diagnosed each year in the U.S. Unfortunately for these patients, treatment options have remained essentially unchanged for 30 years, and clinical outcomes remain poor. Moreover, little is known about the genes that regulate leukemia stem cells (LSCs), which represent the population of blasts that is resistant to chemotherapy and are critical for maintaining disease and re-initiating disease after therapy. Thus, eradication of the LSC is a prerequisite for cure. We recently identified a novel LSC antigen, CD99, that is expressed in the vast majority (~85%) of human AMLs [3]. We have shown that CD99 is expressed on leukemic blasts and can be used to prospectively separate leukemic blasts from residual hematopoiesis, similar to LSC antigens such as TIM3 and CD47, but CD99 exhibits several unique features: 1) CD99 is the most commonly expressed LSC antigen; 2) CD99 does not just mark LSCs, but regulates blast growth and survival; 3) CD99 allows isolation of LSCs from blasts with CD99hi blasts enriched ~10-100 fold for leukemia initiating-cell activity over CD99low cells; 4) CD99 is the only LSC antigen that can identify LSCs in both CD34+ and CD34- AMLs; and, 5) Novel monoclonal antibodies (mAbs) against CD99 induce cell death directly by activating Src-family kinases (SFKs). We have evaluated the consequences of CD99 loss in both LSCs and hematopoietic stem cells (HSCs). Our studies indicate that decreased expression of CD99 in both LSCs and HSCs results in global upregulation of protein synthesis and loss of self-renewal. Moreover, cytotoxic mAbs against CD99 mimic the effects of CD99 loss, activating protein synthesis and inducing similar gene expression changes to those observed in CD99 null HSCs or CD99low blasts in most genetic subtypes of AML tested. Collectively, these data support a model in which LSCs require highly regulated levels of protein synthesis, similar to HSCs, and based on our work in normal HSCs, we expect these alterations in translation to result in selective recruitment of mRNA’s to active translating ribosomes (polysomes). Overall, we hypothesize that CD99 constrains the translation of specific mRNA’s, thereby promoting a translational program required for LSC self-renewal. Given our ability to enrich for LSCs, our group is in a unique position to investigate the role of mRNA translation in LSC function. We have painstakingly optimized methods to perform polysome profiling and RNA- sequencing from polysome fractions from small numbers of cells, and we have developed a computational pipeline to identify mRNA’s that are preferentially translated in LSCs. These tools, in combination with unique reagents such as our CD99 KO mice and cytotoxic CD99 mAbs that mimic CD99 loss, place us in an excellent position to investigate how CD99 regulates translation in LSCs. Understanding the molecular pathways that regulate protein synthesis has the potential to help better characterize a poorly understood process in AML biology and to credential targeting translation using mAbs as a potential therapeutic strategy in AML.