STAT5 Structure-Function in Hematopoiesis

描述(申请人提供)：造血干细胞(HSCs)可以对调节细胞增殖、分化、存活、自我更新和迁移的微环境信号做出反应。这些反应对于HSC在稳态和应激状态下长期促进造血至关重要。这些反应的缺陷可能会导致血液病。对于许多疾病，最持久和最彻底的治疗方法是干细胞置换，但目前许多良性疾病还不能接受干细胞移植。了解造血干细胞的基本生物学可能会为干细胞移植和促进骨髓清除术后内源性造血恢复提供新的方法。这些目标可能具有显著的临床益处。此外，同样的HSC调控机制可能也参与了白血病干细胞生物学。我们在R01的第一个周期的研究重点是确定JAK/STAT信号在正常HSC生物学中的作用。这项工作揭示了STAT5激活在HSC生长、长期竞争性再生活动和自我更新中的作用。我们发现，HSCs的缺陷与c-MPL基因敲除小鼠几乎相同，但在多潜能祖细胞分化方面的缺陷要大得多，这表明在造血中对STAT5的广泛需求。我们惊讶地发现，包括Gab2基因的Balb/c衍生修饰基因位点调节了W/WV宿主的稳定植入能力。我们现在提供的初步证据表明，Gab2也是正常造血所必需的，它通过直接相互作用与STAT5合作。此外，我们发现Gab2和STAT5在正常的HSC植入中协同作用，并且由于STAT5的结构性激活而引起的骨髓增殖性疾病可以通过缺乏Gab2来减轻。为了进一步探讨STAT5激活在造血中的作用，我们提出了以下具体目标：1)利用STAT5/Gab2复合突变小鼠，探索基于STAT5/PI3-激酶抑制的新型非清髓性适应机制。2)以完全缺失的STAT5基因敲除小鼠为模板，建立正常造血状态下STAT5的结构与功能关系。我们的初步数据证实了这种方法的可行性，该方法使用胎肝逆转录病毒转导来重新添加缺乏特定结构域功能的STAT5突变体。3)确定生存信号是否是白血病造血中STAT5结构性激活的致命弱点。我们将确定STAT5获得生存信号(如Bcl-2)以促进白血病发生的机制。这项工作有望显著提高我们对STAT5信号在正常和白血病造血中的关键作用的理解，并有可能发现基于抑制特定STAT5结构域功能的副作用较少的新的治疗策略。现在明确的是，JAK/STAT信号通路在许多血液疾病中存在缺陷，包括免疫缺陷、自身免疫和癌症。我们认为，更好地了解STAT5信号的结构和功能机制可能会对开发副作用更少的新疗法具有广泛的应用价值。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) can respond to microenvironmental cues that modulate cell proliferation, differentiation, survival, self-renewal, and migration. These responses are critical for the HSC to contribute to hematopoiesis long-term during steady-state and during stress. Defects in these responses can lead to hematologic disease. The most durable and complete cure for many diseases would be stem cell replacement, however stem cell transplant is currently not warranted for many benign disorders. Understanding the basic biology of HSCs may lead to novel approaches for stem cell transplant and for promoting endogenous hematopoietic recovery following myeloablation. These goals may have significant clinical benefit. Furthermore, the same mechanisms of HSC regulation may be involved in leukemic stem cell biology. Our research during the first cycle of this R01 was focused on defining the role of JAK/STAT signaling in normal HSC biology. This work uncovered roles for STAT5 activation in growth, long-term competitive repopulating activity, and self-renewal of HSCs. We showed that deficiencies in HSCs were almost identical to c-Mpl knockout mice but deficiencies in multipotent progenitor differentiation were considerably greater, illustrating a broad requirement for STAT5 in hematopoiesis. We were surprised to find that a Balb/c derived modifier locus that included the Gab2 gene modulated the steady- state engraftment ability as measured in W/Wv hosts. We now provide preliminary evidence that Gab2 is also required for normal hematopoiesis and it cooperates with STAT5 through a direct interaction. Furthermore, we show that Gab2 and STAT5 cooperate in normal HSC engraftment and that myeloproliferative disease resulting from constitutive activation of STAT5 is attenuated by Gab2 deficiency. We propose the following specific aims to further explore the role of STAT5 activation in hematopoiesis: 1) Explore novel non-myeloablative conditioning based on STAT5/PI3-kinase inhibition using STAT5/Gab2 compound mutant mice. 2) Establish the structure-function relationship for STAT5 in normal hematopoiesis using the complete null STAT5 knockout mouse background as a template. Our preliminary data has established feasibility for this approach using fetal liver retroviral transduction to add back STAT5 mutants lacking specific domain functions. 3) Determine whether survival signaling is the Achilles' heel of constitutive STAT5 activation in leukemic hematopoiesis. We will define mechanisms by which STAT5 acquires survival signals such as Bcl-2 to promote leukemogenesis. This work is expected to significantly advance our understanding of the pivotal role of STAT5 signaling in normal and leukemic hematopoiesis and potentially uncover new therapeutic strategies with fewer side effects based on suppression of specific STAT5 domain functions. It is now unequivocal that the JAK/STAT signaling pathway is defective in many blood diseases including immunodeficiency, autoimmunity, and cancer. We believe that a better understanding of the structure-function mechanisms of STAT5 signaling may have broad application for development of novel therapeutics with fewer side effects.