Role of Shp2 in FLT3-ITD induced leukemogenesis

项目总结/摘要 虽然大多数急性髓细胞白血病（AML）患者对一线治疗的反应短暂， 化疗，复发经常发生，是老年AML患者死亡的最常见原因， 总体治愈率仅为15%，占AML患者人群的约90%。最新全基因组 和外显子组测序研究表明，逐步积累的遗传和表观遗传变化， 造血干细胞（HSC）导致白血病前干细胞（pre-LSC）的形成， 不仅在疾病起源中起作用，而且在白血病复发中起作用。虽然前LSC的存在相当 在人类和小鼠AML模型中有充分的记录，负责生长/存活的机制 前LSC和导致这些前LSC进展为完全成熟的LSC和AML母细胞的信号是 不太了解。表观遗传修饰基因（如TET 2和DNMT 3A）的突变经常被发现， 在前LSC中，当与FLT 3-ITD等基因突变配对时，会导致完全成熟的AML。基于 在动物模型中进行的研究中，单独的Tet 2或Dnmt 3A突变不会导致AML，因此单独的Tet 2或Dnmt 3A突变不会导致AML。 这些基因中的突变重现了前LSC状态。然而，这些突变与FLT 3- ITD会导致AML的全面发展，并预示着人类的预后不良。这些AML鼠模型是 特征在于可定义的、功能改变的前LSC和LSC，与人类疾病非常相似， 考虑到关键的分子，细胞和表型特征，并允许前瞻性识别和功能 研究AML中驱动前LSC形成和向LSC进展的机制。鉴于FLT 3-ITD 通常在存在其他协同表观遗传突变（如TET 2和DNMT 3A）的情况下发生， 竞争性更新，我们已经集中在如何Shp 2整合信号从这些不同的基因（表观遗传 调节剂相对于受体酪氨酸激酶）来调节前LSC和LSC两者的生长和存活。本 最后，我们有新的初步数据表明，Shp 2调节Tet 2介导的克隆造血功能的丧失， 在前LSC中通过形成涉及炎性细胞因子（包括IL-6）的产生的前馈回路， 以及通过诱导一种新的lncRNA Morrbid的表达。MORBID在AML中显著上调 患者来源的细胞，包括在具有FLT 3-ITD以及携带TET 2突变的AML患者中， 与总体生存率低有关。我们发现，在缺乏Tet 2的前LSC或缺乏Tet 2的LSC中， Tet 2和表达FLT 3-ITD，使这些细胞对凋亡敏感，部分是通过上调促凋亡蛋白， 凋亡蛋白Bim.我们进一步证明，使用一种新的变构SHP 2抑制剂，目前在临床试验中， 有效抑制小鼠和人类白血病AML细胞的生长。重要的是，SHP 2抑制剂显示， 对正常细胞的毒性，但单独和与5- 氮杂胞苷。我们假设靶向SHP 2，靶向Tet 2丢失介导的信号，以及FLT 3-ITD 诱导的信号，其分别在pre-LSC和LSC中会聚于新的lncRNA，Morrbid。

PROJECT SUMMARY/ABSTRACT Although majority of patients with acute myeloid leukemia (AML) do respond transiently to frontline chemotherapy, relapse occurs frequently and is the most common cause of death in older AML patients who have an overall cure rate of only 15% and make up ~90% of the AML patient population. Recent whole genome and exome sequencing studies suggest that accumulation of stepwise genetic and epigenetic changes in hematopoietic stem cells (HSCs) results in the formation of pre-leukemia stem cells (pre-LSC) that play a crucial role not only in disease origination but also in leukemia relapse. While the presence of pre-LSCs has been fairly well documented in both humans and mouse models of AML, mechanisms responsible for the growth/survival of pre-LSCs and signals leading to the progression of these pre-LSCs into full-blown LSCs and AML blasts are poorly understood. Mutations in epigenetic-modifying genes, such as TET2 and DNMT3A, are frequently found in pre-LSCs and when paired with genetic mutations such as FLT3-ITD, result in full-blown AML. Based on studies performed in animal models, Tet2 or Dnmt3A mutations alone do not result in AML, and thus single mutations in these genes recapitulate a pre-LSC state. However, combinations of these mutations with FLT3- ITD lead to full-blown AML and portend a poor prognosis in humans. These AML murine models are characterized by definable, functionally altered pre-LSCs and LSCs, closely resembling human disease with regard to key molecular, cellular and phenotypic features, and allow for prospective identification and functional study of mechanisms driving the formation of pre-LSCs and progression to LSCs in AML. Given that FLT3-ITD often occurs in the presence of other cooperating epigenetic mutations such as TET2 and DNMT3A, in this competitive renewal, we have focused on how Shp2 integrates signals from these distinct genes (an epigenetic regulator vs. a receptor tyrosine kinase) to regulate the growth and survival of both pre-LSCs and LSCs. To this end, we have novel preliminary data to suggest that Shp2 regulates loss of Tet2 mediated clonal hematopoiesis in pre-LSCs by forming a feed-forward loop involving the production of inflammatory cytokines including IL-6 as well as by inducing the expression of a novel lncRNA, Morrbid. MORRBID is significantly upregulated in AML patient derived cells including in AML patients with FLT3-ITD as well as bearing TET2 mutations, where it is associated with poor overall survival. We show that loss of Morrbid in pre-LSCs lacking Tet2 or in LSCs lacking Tet2 and expressing FLT3-ITD, renders these cells susceptible to apoptosis, in part by upregulation of a pro- apoptotic protein Bim. We further demonstrate, using a novel allosteric SHP2 inhibitor, currently in clinical trials, to potently inhibit the growth of mouse and human leukemic AML cells. Importantly, SHP2 inhibitor, shows no toxicity against normal cells but uniquely impacts the growth of leukemic cells alone and in combination with 5- Azacytidine. We hypothesize that targeting SHP2, targets both Tet2 loss mediated signals, as well as FLT3-ITD induced signals, that converge on a novel lncRNA, Morrbid in pre-LSCs and LSCs, respectively.