Structure-Function relationship of novel phosphoregulatory sites and effect of acute lymphoblastic leukemia SNPs on Jak3 activity: Implications for new cancer treatments

摘要 癌症是美国拉美裔美国人的主要死因，与非拉美裔美国白人相比， 代表着巨大的癌症健康差距。在20岁以下的拉美裔人中，白血病导致的 儿童和年轻人的死亡率比其他任何癌症都高，而急性淋巴细胞白血病(ALL) 显示任何组中发病率最高的组。为了解决这些健康不平等问题，我们产生了一种新的 所有来自我们当地人群的生物信息库，并进行了完整的外显子组测序，这导致了 多种可能癌基因的新SNPs，包括Janus酪氨酸激酶3(JAK3)。JAK3还没有 被显著研究或被认为在这些类型的癌症中起主要作用。因此，为了更好地 为了了解这种蛋白质，我们进行了自激酶反应和质谱分析来鉴定新的 表明JAK3蛋白具有“活性”的光调节位点。11个独特的和/或未报道的磷酸酪氨酸 确定了地点。随后，我们产生了一组高度特异的磷酸化JAK3抗体，以 描述这种蛋白质的激活状态。这些工具使我们能够测试我们的中心假设JAK3 含有独特的磷调节结构域，通过构象变化被结构性激活 通过可以促进癌症的体细胞突变。此外，这些相同的域也可能代表 白血病治疗的治疗靶点。我们的新工具使我们现在能够1)表征功能 这些位点在酶功能中的作用；2)确定这些残基对信号的下游影响 转导级联，3)筛选所有肿瘤细胞中活性JAK3，以及4)监测所有SNPs对 JAK3的激活状态，并根据其能力确定这些突变是否具有致癌潜力 赋予细胞因子独立生长的能力。由于可用于干扰的临床治疗选择有限 JAK3功能，FDA没有批准特定的JAK3抑制剂(它们针对整个家族)，这是首要目标 这项工作的目的是为未来的药物开发确定JAK3特有的新的调节域。电流激活酶 靶向药物通常通过干扰高度保守的ATP结合口袋起作用，导致低 特异性和许多副作用。此外，由于JAK3几乎仅限于造血细胞， 它代表了“真正的可用药靶子”的特征。为确定JAK3的独特功能， 这些抗白血病药物的开发，我们将使用高分辨率冷冻电子显微镜来阐明 这些JAK3 SNPs和光调节位点调控其功能的结构关系。这 该方法代表了一种创新的策略，以确定蛋白质中可能具有可育性的未探索区域 为JAK3药物开发奠定了基础。总的来说，拟议的工作将极大地增进我们对 JAK3的正常生理和未来开发用于治疗高危疾病的选择性抑制剂 在我们区域和国家内未被充分代表的少数民族中的白血病。

ABSTRACT Cancer is the leading cause of death among U.S. Hispanics, in contrast to non-Hispanic White Americans, representing a significant cancer health disparity. In Hispanics under the age of 20, leukemia causes more deaths than any other cancer among children and young adults while acute lymphoblastic leukemia (ALL) displays the highest incidence rates of any group. To address these health inequalities, we generated a novel ALL biorepository from our local population and performed whole exome sequencing which resulted in a multitude of novel SNPs for several putative oncogenes, including Janus Tyrosine Kinase 3 (Jak3). Jak3 has not been significantly studied or thought to play a major role in these types of cancers. Therefore, to better understand this protein, we performed autokinase reactions coupled with mass spectrometry to identify novel phosphoregulatory sites indicative of an “active” Jak3 protein. Eleven unique and/or unreported phospho-tyrosine sites were identified. Subsequently, we generated a battery of highly specific phospho-Jak3 antibodies to characterize the activated state of this protein. These tools position us to test our central hypothesis that Jak3 harbors unique phosphoregulatory domains that are constitutively activated via conformational change caused by somatic mutations which can promote cancer. Additionally, these same domains may also represent therapeutic targets for the treatment of leukemia. Our novel tools position us to now 1) characterize the functional role of these sites in enzyme function; 2) determine the downstream effects of these residues on signal transduction cascades, 3) screen ALL tumor cells for active Jak3, and 4) monitor the impact of ALL SNPs on Jak3 activation status and determine whether such mutations harbor oncogenic potential based on their ability to confer cytokine independent growth. Since there are limited clinical treatment options available for disrupting Jak3 function, and no FDA approved specific Jak3 inhibitors (they target the entire family), the overarching goal of this work is to identify novel regulatory domains unique to Jak3 for future drug development. Current kinase targeted drugs typically work by interfering with the highly conserved ATP binding pocket resulting in low specificity and numerous side effects. Additionally, since Jak3 is almost exclusively limited to hematopoietic cells, it represents characteristics of a “true druggable target”. To identify the unique features of Jak3 for the development of these anti-leukemic drugs, we will employ high resolution cryo-Electron Microscopy to elucidate the structural relationship by which these Jak3 SNPs and phosphoregulatory sites regulate its function. This approach represents an innovative strategy to identify unexplored regions of the protein that would be fertile ground for Jak3 drug development. Collectively, the proposed work will greatly enhance our understanding of normal Jak3 physiology and aide in the future development of selective inhibitors for the treatment of high-risk leukemia in underrepresented minorities within our region and nation.