Signaling in persisting leukemic stem cells in acute myeloid leukemia

化疗方案可诱导60-70%的急性髓性白血病（AML）完全缓解。然而，大约80%的AML患者最终会由于患者体内的持续性白血病细胞（PLC）而复发。PLC的表型目前尚不清楚。因此，在之前的资助期内，我们采用同步功能和描述性流式细胞术分析（SFDC）对AML患者在诊断、缓解和复发时的骨髓干细胞和祖细胞室进行了表征。我们证明，在我们的AML患者队列中，功能性干细胞参数（如高醛脱氢酶和药物转运蛋白功能）的高活性与难治性疾病或高复发率相关。为了追踪AML患者干细胞和祖细胞区室中的PLC，我们建立了基因组单细胞PCR分析，以检测不同细胞区室中的核磷蛋白1 （NPM1）突变。初步数据表明，与原始干细胞区室相比，在祖细胞区室中可以检测到更多的NPM1突变细胞。因此，PLC的免疫表型可能与正常造血干细胞的表型不同。在第二个资助期，我们现在将询问，AML缓解患者的哪个祖细胞室确实具有最高的NPM1突变率，以便SFDC可以选择这些细胞进行进一步的遗传分析，以揭示与体内AML患者PLC生存相关的基因信号级联反应。为了在体内分析白血病祖细胞，我们建立了人类AML的小鼠模型。为此，我们移植了原发人类AML患者的干细胞和祖细胞区室，并发现仅2500个人类AML患者细胞在免疫缺陷的Rag2-/-γ-/-小鼠体内进行了移植。使用白血病细胞（KG1a）后，Rag2-/-γ-/-小鼠在5-6周内发生了人类AML。然后对这些小鼠进行化疗。对小鼠体内化疗后残留的人PLC进行全基因组微阵列分析，揭示了一些已知参与细胞存活的基因，以及以前未知的有趣的新耐药候选基因。这些基因将在第二个资助期进一步验证和研究。我们的AML- Rag2-/-γ-/-模型为慢病毒转导的人类白血病细胞提供了一个很好的平台。AML细胞现在正在用慢病毒shRNA文库进行转导，并且可以应用于我们的Rag2-/-γ-/-小鼠，以便通过体内化疗通过条形码筛选选择耐药相关基因。这两种策略都采用小鼠AML体内模型或原发性患者骨髓，类似于真实生活场景，以表征PLC。揭示PLC中的生存信号级联是开发新型靶向治疗方法以克服AML中PLC耐药机制的先决条件。最终根除这些持续性白血病克隆将为许多AML患者带来长期治愈。

Chemotherapy regimens induce complete remission of acute myeloid leukemia (AML) in 60-70%. However, about 80% of these AML patients will ultimately relapse due to persistent leukemic cells (PLC) that remain in the patient. The phenotype of PLC is currently unknown. In the previous funding period, we have therefore characterized stem and progenitor compartments from bone marrow of AML patients at diagnosis, in remission and relapse employing simultaneous functional and descriptive flow cytometry analysis (SFDC). We demonstrate that high activity of functional stem cells parameters such as high aldehyde dehydrogenase and drug transporter function are associated with refractory disease or high relapse rates in our AML patient cohort. In order to track PLC in stem and progenitor cell compartments from AML patients we established a genomic single cell PCR analysis in order to detect nucleophosmin 1 (NPM1) mutations in different cell compartments. Preliminary data indicate, that more NPM1 mutated cells can be detected in progenitor cell compartments compared to the primitive stem cell compartment. Thus, the PLC immunophenotype may not resemble the phenotype of normal hematopoietic stems cells. In the second funding period we will now ask, which progenitor cell compartment from AML remission patients does have the highest NPM1 mutation rate, so that these cells can be selected by SFDC for further genetic analyses to reveal gene signaling cascades that are relevant for survival of PLC in AML patients in vivo.In order to analyze leukemic progenitor cells in vivo, we have established a murine model of human AML. To this end, we transplant stem and progenitor cell compartments of primary human AML patients and found engraftment within immunodeficient Rag2-/-γ-/- mice from only 2500 human sorted AML patient cells. After application of leukemia cells (KG1a) Rag2-/-γ-/- mice developed a human AML within 5-6 weeks. These mice were then treated by chemotherapy. Whole genome microarray analysis from human PLC that remained in the mice after in vivo chemotherapy revealed some genes that are known to be involved in cell survival in addition to previously unknown interesting new resistance candidate genes. These genes shall be further validated and studied in the second funding period. Our AML- Rag2-/-γ-/- model is a good platform for human leukemia cells that are lentivirally transduced. AML cells are now being transduced with a lentiviral shRNA library and can be applied to our Rag2-/-γ-/- mice in order to select for resistance-associated genes via bar code screen by in vivo chemotherapy.Both strategies employing either a murine AML in vivo model or primary patient bone marrow resemble real life scenarios in order to characterize PLC. Uncovering survival signaling cascades in PLC is a prerequisite for the development of novel targeted therapeutic approaches to overcome PLC resistance mechanisms in AML. Final eradication of these persistent leukemic clones would lead to long-lasting cure for many AML patients.