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

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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.