Role of Fc gamma RIIb signaling in CML stem cell biology and therapy resistance

我们和其他人之前已经证明，尽管肿瘤驱动因子Bcr-Abl完全被抑制，CML干细胞仍能存活（Schemionek等人，BLOOD 2010; Hamilton*, Helgason*， Schemionek*等人，BLOOD 2012）。因此，我们现在的目标是确定新的和潜在的药物靶点，诱导白血病干细胞（LSC）的持久性。我们之前对CML干细胞进行了表达谱分析，发现ITIM（免疫受体酪氨酸基抑制基序）受体FcgRIIB （Fc γ受体2b）显著上调。这种上调并没有被酪氨酸激酶抑制剂（TKIs）的最新治疗所消除，这表明该受体可能有助于Bcr-Abl的非依赖性耐药。ITIM受体最近也被描述为支持Bcr-Abl阳性ALL和AML的白血病发生，这表明它在恶性细胞生物学中起着关键作用。我们的初步结果表明，FcgRIIB失活影响Bcr-Abl转导谱系阴性的骨髓细胞在体外和体内的白血病发生。我们的第一个目标是确定FcgRIIB缺失对CML干细胞功能的影响。为了实现这一目标，我们想用Bcr-Abl逆转录病毒感染原代FcgRIIB-/-或wt型HSC，并分析对CML效价、自我更新、生长、归巢、ROS（活性氧）水平、凋亡和存活的影响。我们的第二个目标是验证FcgRIIB作为治疗靶点的潜力。因此，我们希望建立一个小鼠模型，我们可以用它来区分CML和正常干细胞，在同一动物体内表达或不表达受体。然后，我们将使用第一代和第二代TKIs治疗这些小鼠，TKIs被批准用于CML的一线治疗，并监测体内LSC的持久性。最后，我们希望利用先前建立的过表达FcgRIIB受体的白血病细胞系、敲除小鼠模型和原代人CML干细胞来分析FcgRIIB介导的致癌信号传导机制。在此，我们希望研究Bcr-Abl诱导FcgRIIB激活的机制，并随后研究下游信号级联反应。然后，我们希望通过肽结合测试治疗性FcgRIIB抑制的效果。这些数据将显示FcgRIIB激活是否允许LSC除当前治疗外持续存在，并且可以在体内特异性靶向根除疾病驱动细胞群。

We and others have previously demonstrated that CML stem cells survive despite complete inhibition of the oncogenic driver Bcr-Abl (Schemionek et al., BLOOD 2010; Hamilton*, Helgason*, Schemionek* et al., BLOOD 2012). Therefore we are now aiming to identify new and potentially druggable targets that induce leukemic stem cell (LSC) persistence. We previously performed expression profiling of CML stem cells and identified significant upregulation of the ITIM (immunoreceptor tyrosine-based inhibition motif) receptor FcgRIIB (Fc gamma receptor 2b). This upregulation was not abrogated by state of the art therapy with tyrosine kinase inhibitors (TKIs) suggesting that the receptor may contribute to Bcr-Abl independent resistance. ITIM receptors have recently been described to support leukemogenesis also in Bcr-Abl positive ALL as well as AML suggesting a key role in malignant cell biology. Our preliminary results show that FcgRIIB inactivation affects leukemogenesis of Bcr-Abl transduced lineage negative bone marrow cells in vitro as well as in vivo. Our first aim is to determine the impact of FcgRIIB depletion on CML stem cell function. To achieve this we want to infect primary FcgRIIB-/- or wt HSC using Bcr-Abl retrovirus and analyze the effect on CML potency, self-renewal, growth, homing, ROS (reactive oxygen species) levels, apoptosis and survival. Our second aim is to validate the potential of FcgRIIB to act as a therapeutic target. Therefore we want to generate a mouse model that we can use to distinguish CML and normal stem cells that do express or do not-express the receptor within the same animal. We will then treat these mice using first and second generation TKIs, approved for first-line therapy in CML and monitor LSC persistence in vivo. Finally, we want to analyze the mechanism of FcgRIIB mediated oncogenic signaling using previously established leukemic cell lines overexpressing the receptor as well as knock-out mouse models and primary human CML stem cells. Here we want to study the mechanism of Bcr-Abl induced FcgRIIB activation and subsequently study the downstream signaling cascades. We then want test for effects of therapeutic FcgRIIB inhibition by peptide-binding. These data will show if FcgRIIB activation allows for LSC persistence besides current therapy and can be specifically targeted in vivo to eradicate the disease driving cell population.