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

摘要 与非西班牙裔白色美国人相比，癌症是西班牙裔美国人的主要死因， 这代表了一个显著的癌症健康差异。在20岁以下的拉美裔人中，白血病引起的 儿童和年轻人中死亡人数超过任何其他癌症，而急性淋巴细胞白血病（ALL） 显示了任何群体中最高的发病率。为了解决这些健康不平等问题，我们制作了一部小说， ALL生物储存库从我们的当地人口，并进行了全外显子组测序，这导致了一个 许多新的SNPs的几个假定的癌基因，包括Janus酪氨酸激酶3（Jak 3）。Jak 3没有 被大量研究或认为在这些类型的癌症中起主要作用。为了更好地 为了了解这种蛋白质，我们进行了自激酶反应与质谱联用，以鉴定新的 磷酸调节位点，指示“活性”Jak 3蛋白。11种独特和/或未报告的磷酸酪氨酸 网站已确定。随后，我们产生了一组高度特异性的磷酸化Jak 3抗体， 表征这种蛋白质的活化状态。这些工具使我们能够测试我们的中心假设，即Jak 3 具有独特的磷酸调节结构域，其通过引起的构象变化而组成性激活 会导致癌症的体细胞突变此外，这些相同的域也可以代表 用于治疗白血病的治疗靶点。我们的新工具使我们能够1）描述功能性 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ALL肿瘤细胞的活性Jak 3，和4）监测ALL SNPs对细胞周期的影响。 Jak 3激活状态，并根据其能力确定此类突变是否具有致癌潜力 以赋予细胞因子非依赖性生长。由于可用于破坏的临床治疗选择有限， Jak 3功能，没有FDA批准的特定Jak 3抑制剂（它们针对整个家族），总体目标 这项工作的目的是确定Jak 3独特的新调控域，用于未来的药物开发。电流激酶 靶向药物通常通过干扰高度保守的ATP结合口袋而起作用， 特异性和许多副作用。此外，由于Jak 3几乎仅限于造血细胞， 它代表了“真正的可下药目标”的特征。为了识别Jak 3的独特功能， 为了开发这些抗白血病药物，我们将采用高分辨率冷冻电子显微镜来阐明 这些Jak 3单核苷酸多态性和磷酸调节位点调节其功能的结构关系。这 这种方法代表了一种创新的策略，以确定蛋白质的未探索区域，将是肥沃的 Jak 3药物开发的基础。总的来说，拟议的工作将大大提高我们对以下问题的认识： 正常的Jak 3生理学，并在未来开发用于治疗高危 白血病在我们地区和国家代表性不足的少数民族中。

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.