Date of Award

2016-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Robert A. Kirken

Abstract

A medical need exists for successfully treating people afflicted with leukemia, especially those who develop drug resistant forms. Relapse leukemia cases are particularly high within Hispanic populations where this disease is among the most frequently occurring cancer. Fourteen somatic mutations have been reported in Janus tyrosine kinase 3 (Jak3), including M511I and A573V, from patients with various forms of leukemia. To monitor drug sensitivity, a model system was developed. Indeed, many of these mutations have been shown to possess transforming ability in cell lines such as the IL-3 dependent pro-B cell line Ba/F3. As such, Ba/F3 cells were transformed to cytokine independent growth using a retrovirus system expressing either the Jak3 mutation M511I or A573V. Here we demonstrate that Jak3 M511I and A573V possess this ability to induce cytokine independent Ba/F3 cell growth. Next, cells were used to test the sensitivity to pre-clinical and clinical Jak3 selective inhibitors on cellular viability. The level of sensitivity of transformed Ba/F3 cells varied with treatment to Jak3 inhibitors NC1153 and CP-690,550, while neither responded to the pre-clinical Jak3 inhibitor, EP-009. Screening of one hundred and three patient samples with various forms of leukemia and lymphoma for mutations M511I and A573V in exons 11 and 13 of Jak3 failed to demonstrate the presence of either mutation, respectively, confirming that these mutations are rare. However, this study provides a rational for the use of Jak3 inhibitors with distinct chemical composition for the treatment of Jak3 driven cancers, including patients with the transforming mutations M511I and A573V. This work has also identified a novel Jak3 tyrosine phosphorylation site, Y841, through the use of kinase assays coupled to mass spectrometry. Y841 is evolutionarily conserved across multiple species and Jak family members, and is positionally conserved in several tyrosine and serine/threonine kinases. Through the use of phospho-deletion mutants, it was determined that Y841 is necessary for full activation of Stat5B, most likely due to its position in the glycine lid of the catalytic domain of Jak3, adjacent to the ATP binding pocket. Phospho-specific antibodies were generated against this site and showed specificity for phospho-peptide over non-phospho peptide, and recognized phosphorylated Jak3 in T-cell cell lines. Interestingly, this work shows that Jak3 mutation A573V has higher phosphorylation at Y841 than Jak3 WT. Furthermore, Jak1, Jak2 and Tyk2 are recognized by the anti-Jak3 Y841 antibody, potentially broadening the scope of this tool to explore hematopoietic cancers driven by other Jak family members.

Language

en

Provenance

Received from ProQuest

File Size

114 pages

File Format

application/pdf

Rights Holder

George Steven Martinez

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