Characterization of Nuclear and Mitochondrial Lck Oncoprotein in Leukemia Cell Lines and in Primary Human Leukemia (2)

Background: Protein tyrosine kinase (PTK) plays a key role in gene regulation by transmitting signals from cell surface to the nucleus. Uncontrolled PTK signaling can lead to malignant transformation of many cell types. In contrast to this paradigm, cytoplasmic PTK also exhibits additional functions by translocating to both nuclear and mitochondrial compartments. The detection of non-canonical nuclear and mitochondrial PTK in cancer cells strongly suggests a crosstalk of oncogenic PTK in different subcellular compartments. However, there is no systematic study in addressing nuclear and mitochondrial crosstalk of a distinct PTK in a defined cellular background. Our recent studies of human and mouse leukemic T cells demonstrated nuclear and mitochondrial localization of lymphocyte-specific protein tyrosine kinase (Lck). Nuclear and mitochondrial Lck functions as a transcription factor in activating other oncogenes and interferes with the intrinsic translational machinery in mitochondria, respectively. We hypothesize that nuclear and mitochondrial Lck coordinately contribute to different cancer hallmarks. Objective: As a continuation of the current 10-month grant, this three-year proposal extends on our cumulative findings of Lck’s distinct subcellular localization and will further focus on defining the mechanistic details and establishing clinical relevance of this unique spatial regulation. Methods: In the first and second years, we will define the molecular mechanisms of nuclear and mitochondrial translocation of Lck and characterize how nuclear and mitochondrial Lck regulates gene expression, mitochondrial respiration, and other cancer hallmarks. Lck domain mapping experiments will be performed to identify region(s) important for nuclear and mitochondrial import. Constitutively-active and kinase-dead Lck mutants will be cloned into targeting vectors specific for nuclear or mitochondrial localization. Constructs will be stably transfected into Lck-deficient human Jurkat and mouse LSTRA leukemic cell derivatives. We will use integrated next generation sequencing (NGS), proteomics, and metabolomics to identify novel Lck target genes, mitochondrial substrates, and related metabolic pathways, respectively. Oncogenic properties from organelle-specific expression of Lck mutants will be defined in culture, followed by xenograft/syngeneic mouse studies in the third year. Finally, paired samples at diagnosis before treatment and at complete hematologic remission from acute B and T lymphoblastic leukemia (ALL) patients at Chang Gung Memorial Hospital will be analyzed for organelle-specific localization of Lck and the pattern of Lck target gene expression to establish potential clinical correlation. Significance: The proposed research represents the first study to specifically define the functional importance of a cytoplasmic PTK in nuclear versus mitochondrial compartment. Results from this study will provide mechanistic insight of nuclear/mitochondrial crosstalk in oncogenesis. NGS, proteomic, and metabolomic studies will discover additional components in this network for future studies. Data from ALL patients will further provide the clinical relevance of non-canonical Lck signaling in leukemia progression and remission. It can potentially lead to the development of novel prognosis markers and/or new therapeutic approaches for leukemia patients as well as other Lck-associated hematologic malignancies and solid tumors.

Project ID:PC10703-0218
External Project ID:MOST106-2320-B182-030