Date of Award

2025-05-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Robert A. Kirken

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and complex malignancy that accounts for 15% of pediatric acute lymphoblastic leukemia (ALL) cases in the United States. Tyrosine kinases control critical cellular processes, including the development, survival, and activation of T cells, and are established drivers of leukemia.Tyrosine kinases involved in hematological disorders include receptor tyrosine kinases and non-receptor tyrosine kinases family members such as SRC, ABL, FAK, and Janus kinases (JAKs). This study primarily focused on JAK1 gain-of-function (GoF) mutations. These mutations are recognized as hyperactivating mutations, maintaining kinase activity of JAKs on and essentially keeping downstream signaling effectors, like STATs (Signal Transducers and Activators of Transcription) phosphorylated. Employing in silico analysis, various GoF mutations associated with proliferative hematological diseases were identified in the structure of JAK proteins recognizing the JH2 pseudokinase domain as a "hot spot", in specific the region of this domain crucial for dimerization and transactivation of JAKs. It was observed a change in amino acid polarity in most of the GoF mutations identified, indicating a structural-functional impact. In silico analysis demonstrated the heterogeneity of these mutations and the urge to characterize and understand the functional impact of these mutations, thus allowing for personalized treatments. One of the most common JAK1 GoF mutation in refractory cases of T-cell acute lymphoblastic/lymphocytic leukemia (T-ALL) is the JAK1 V658F. Further analysis was made focused on this mutation because its functional role and how it drives oncogenesis have been unclear. A mass spectrometry analysis of an auto-kinase of JAK1 V658F vs JAK1 WT revealed a higher phosphorylation of Y463, Y598, and Y967 in JAK1 V658F compared to JAK1 WT. When auto-kinase assays were used to assess activity, the phosphodeletion of Y598(F) in JAK1 V658F, a reduced auto-activation response was observed. In silico analysis of this site indicated its close proximity between the JAK1 V658 site and Y598 within the JAK1 dimerization region. A decrease in the binding free energy was noted when the non-phosphorylated JAK1 V658F was compared to its phosphorylated form (JAK1 V658F pY598) during dimerization with either JAK3 or JAK1 itself for transactivation process. Nevertheless, the JAK1 V658F homodimer, phosphorylated at Y598, showed increased hydrogen bonds and salt bridges. This finding suggests that due to the increment of energy needs in cancerous cells, JAK1 V658F homodimer could be more stable in activating the JAK-STAT proliferative pathway. To elucidate the role of JAK1 V658F Y598 on the IL-2 JAK-STAT pathway, HEKblue IL-2 cells overexpressing JAK1 V658F, pY598, or F598 variants were examined to determine how the downstream transcription factor STAT5 (Signal Transducer and Activator of Transcription 5) is activated. Interestingly, no significant difference in STAT5 activation was found between JAK1 V658F and JAK1 V658F Y598F. Furthermore, the proliferation rate was not diminished, neither was the case in the absence of IL-2 when JAK1 V658F Y598F was expressed in Kit225 cells. Since there was no difference in activation of JAK1 V658F pY598 or F598 in a complete cell system, nor the proliferation of cells, further analysis needs to be performed to understand possible mechanisms that drive leukemogenesis in T cells. RNA sequencing analysis was made to evaluate how JAK1 V658F impacts gene expression in Kit225 cells. MYC and BCL-2 were found to be upregulated when compared to overexpression of JAK WT, as well as JAK1 V658F. CD8 upregulation, as changes in morphology passing from L1 to L3, suggests that oncogenesis by this mutation goes beyond an impact on the JAK-STAT signaling pathway. The results indicate that the JAK1 V658F mutation affects gene expression, suggesting that its phosphorylation and the JAK-STAT pathway may not completely regulate the disease process in T-ALL cases.

Language

en

Provenance

Received from ProQuest

File Size

111 p.

File Format

application/pdf

Rights Holder

Omar Javier Rodriguez Moncivais

Download

Share

COinS