Fu-Yu Chueh

Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in a leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine-stimulated cells. Our novel findings of cytokine-induced STAT5 translocation into mitochondria and its link to oncogenesis provide important insights into the underlying mechanisms of this characteristic metabolic shift.

Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription

STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2.

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

Engagement of T-cell antigen receptor and CD4/CD8 co-receptors induces prolonged STAT activation through autocrine/paracrine stimulation in human primary T cells.

Signal transducer and activator of transcription (STAT) proteins are key signaling molecules in response to cytokines and in regulating T cell biology. However, there are contradicting reports on whether STAT is involved in T-cell antigen receptor (TCR) signaling. To better define the role of STAT in TCR signaling, we activated the CD4/CD8-associated Lck kinase by co-crosslinking TCR and CD4/CD8 co-receptors in human peripheral blood T cells. Sequential STAT1, STAT3 and STAT5 activation was observed 1 h after TCR stimulation suggesting that STAT proteins are not the immediate targets in the TCR complex. We further identified interferon-γ as the key cytokine in STAT1 activation upon TCR engagement. In contrast to transient STAT activation in cytokine response, this autocrine/paracrine-induced STAT activation was sustained. It correlated with the absence of two suppressors of cytokine signaling (SOCS) proteins, SOCS3 and cytokine-inducible SH2 containing protein that are negative feedback regulators of STAT signaling. Moreover, enforced expression of SOCS3 inhibited tyrosine phosphorylation of zeta-associated protein kinase of 70 kD in TCR-stimulated human Jurkat T cells. This is the first report demonstrating delayed and prolonged STAT activation coordinated with the loss of SOCS expression in human primary T cells after co-crosslinking of TCR and CD4/CD8 co-receptors.

Abstract 4008: Nuclear localization of Lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (LMO2) gene

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Lmo2 is a transcription factor that plays a critical role in both hematopoietic development and vascular remodeling. Lmo2 - associated chromosomal translocations are believed to play a major role in the development of T-cell acute lymphoblastic leukemia (T-ALL). Lmo2 is also shown to interact with the T-cell acute lymphocytic leukemia protein (TAL1) in leukemia. A previous report established a link between Lmo2 expression and the unexpected nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. However, it is not known whether other oncogenic tyrosine kinases regulate Lmo2 expression through a similar mechanism. We showed previously that oncogenic Lck kinase promotes cellular transformation through the activation of signal transducer and activator of transcription 5b (Mol Cancer Res 4:39). In this report, we describe novel nuclear localization of Lck and its role in regulating Lmo2 expression. Lck, like most Src family kinases, is primarily known as a cytosolic or membrane-associated kinase. Using subcellular fractionation and immunofluorescence microscopy, we identified nuclear localization of Lck in murine leukemia cells (LSTRA) that overexpress Lck kinase. Nuclear Lck was also detected in 293 cells expressing constitutively active Y505FLck. Additionally, phosphorylation of the positive-regulatory tyrosine 394 confirmed the active status of nuclear Lck. These data suggest that oncogenic Lck may have an uncharacterized function within the nucleus that supports cellular transformation. Lmo2 expression was significantly elevated both in LSTRA leukemia and in 293 cells expressing oncogenic Y505FLck kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Y41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. However, expression of oncogenic Lck kinase does not result in significant increase in H3Y41 phosphorylation. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction. The resulting increase in Lmo2 expression can potentially contribute to the transforming capacity of oncogenic Lck. All together, we report here a novel mechanism of Lmo2 gene regulation by the oncogenic Lck kinase. Previously, we have characterized the role of STAT5b in Lck-mediated transformation. The Lmo2 gene promoter lacks predicted STAT5 binding sites suggesting that Lck-mediated Lmo2 promoter activation is STAT5-independent. Our data suggest that oncogenic Lck can initiate both STAT5- dependent and independent signals that are likely to be controlled by the spatial localization of the oncoprotein. Subcellular compartmentalization of Lck therefore adds to the complexity that defines cellular transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4008. doi:1538-7445.AM2012-4008

Mouse LSTRA leukemia as a model of human natural killer T cell and highly aggressive lymphoid malignancies

Human large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic lymphocytes. Th ere are two major forms of human LGL leukemia: T-cell type (85%) and natural killer (NK)-cell type (15%). When NK-cell markers are coexpressed, T-LGL leukemia becomes very aggressive with a poor prognosis [1]. In patients with LGL leukemia, there is a high frequency of somatic activating mutations of signal transducer and activator of transcription 3 (STAT3) and STAT5 [2,3]. Consistent with the contribution of STAT3 to the malignant transformation of LGL, STAT3 inhibition has been shown to induce apoptosis of leukemic LGL cells from patients [4]. Th ese fi ndings strongly support the critical role

Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells

Overexpression and hyperactivation of lymphocyte-specific protein tyrosine kinase (Lck) have been associated with leukemia development. We previously showed that, other than its known function as a cytoplasmic signal transducer, Lck also acts as a nuclear transcription factor in mouse leukemic cells. In the present study, we demonstrated the presence of nuclear Lck in human leukemic T cells and in primary cells. We further established a positive correlation between Lck nuclear localization and its kinase activity. Proteomic analysis identified CR6-interacting factor 1 (CRIF1) as one of the Lck-interacting proteins. CRIF1 and Lck association in the nucleus was confirmed both by immunofluorescence microscopy and co-immunoprecipitation in human leukemic T cells. Close-range interaction between Lck and CRIF1 was validated by in situ proximity ligation assay (PLA). Consistent with the role of nuclear CRIF1 as a tumor suppressor, CRIF1 silencing promotes leukemic T cell survival in the absence of growth factors. This protective effect can be recapitulated by endogenous Lck or reconstituted Lck in leukemic T cells. All together, our results support a novel function of nuclear Lck in promoting human leukemic T cell survival through interaction with a tumor suppressor. It has important implications in defining a paradigm shift of non-canonical protein tyrosine kinase signaling.

Abstract 2051: LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening

Large granular lymphocytic (LGL) leukemia is a rare form of leukemia that is caused by the increase in cytotoxic T-lymphocytes or natural killer (NK) cells. A subcategory of T-LGL leukemia that co-expresses T and NK cell surface markers is very aggressive and has a very poor prognosis. Current treatment of aggressive T-LGL is based on non-specific immunosuppressive therapy. Lack of model cell lines contributes to slow progress in designing targeted therapy and clinical trials. Our recent studies demonstrate that the mouse T-leukemic cell line LSTRA reproduces some important characteristics of aggressive T-LGL leukemia. This is the first report of a cell line model mimicking the deadly human leukemia and has great potentials for drug screening in treating T-LGL leukemia. Bortezomib is one of the drugs that show promising results in treating LGL leukemia patients in clinical trials. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. Therefore, it is important to develop novel strategies in overcoming LGL leukemic cells’ resistance to bortezomib and its derivatives. In our current study, we showed that bortezomib inhibited cell proliferation and induced apoptosis in LSTRA leukemic cells. Partial reversion after 48-72 hr of bortezomib treatment suggested development of resistance to bortezomib. We further established bortezomib-resistant LSTRA cells that tolerated significantly higher levels of bortezomib. Both carfilzomib (FDA-approved second generation irreversible proteasome inhibitor) and MLN 4924 (proteasome pathway upstream inhibitor) failed to overcome bortezomib resistance. Only luteolin, a common flavonoid found in plants, can effectively overcome bortezomib resistance. Metabolic reprogramming has been linked to drug resistance in cancer cells. Indeed, We found that bortezomib-resistant LSTRA cells had smaller cell size as well as reduced rate of glucose uptake, lactate secretion, and mitochondrial respiration. These results suggest that reduced mitochondrial activity may be an important molecular marker for LSTRA cell’s resistance to bortezomib. All together, we identified luteolin as a potential small molecule in overcoming LGL leukemia’s resistance to proteasome inhibitor therapy. Altered mitochondrial activity and cell metabolism in bortezomib-resistant cells provide additional targets in developing novel combined chemotherapy for cancer patients. Citation Format: Fu-Shin Chueh, Fu-Yu Chueh, Chao-Lan Yu. LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2017-2051

Abstract 2702: Therapeutic potentials of STAT5 inhibitors in overcoming bortezomib resistance in human T-cell leukemia

The purpose of our study is to define the mechanism of bortezomib resistance in cancer therapy and to identify novel approaches in overcoming the resistance. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. Numerous clinical trials are ongoing in testing the efficacy of bortezomib and its derivatives in treating human cancers. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. To explore the underlying mechanisms, we recently established a bortezomib-resistant human acute T lymphoblastic cell line Jurkat-BR. Jurkat-BR remains sensitive to carfilzomib, a FDA-approved second-generation irreversible proteasome inhibitor. An inhibitor targeting upstream proteasome pathway, MLN 4924, also exhibits cytotoxic effects toward Jurkat-BR. Overexpression and hyperactivation of signal transducer and activator of transcription 5 (STAT5) is associated with many forms of blood malignancies. Compared with parental Jurkat, Jurkat-BR has significant elevation of STAT5b, but not STAT5a, at both mRNA and protein levels. Pimozide is an antipsychotic drug that recently found to specifically inhibit STAT5 activity. Consistent with STAT5b overexpression, Jurkat-BR is more sensitive than parental Jurkat to cytotoxicity induced by pimozide. Another inhibitor targeting STAT5 SH2 domain, the nonpeptidic nicotinoyl hydrazone (CAS# 285968-31-4, STAT5I) also exhibits similar effects. Both pimozide and STAT5I synergize with bortezomib in killing Jurkat-BR cells. These results support the importance of STAT5b upregulation in acquired bortezomib resistance. We reported previously that STAT5 translocates to mitochondria (BBRC 402:778) and represses mitochondrial gene expression (manuscript submitted). In Jurkat-BR cells, we also observed STAT5b mitochondrial translocation, reduced mitochondrial gene expression, and repression of mitochondrial respiration. These results suggest that both STAT5b overexpression and reduced mitochondrial activity may be important molecular markers for cancer cell9s resistance to bortezomib. Our data further support the clinical benefit of STAT5 inhibitors in complementing proteasome inhibitors and in overcoming its resistance in patients who acquire resistance through upregulating STAT5. More importantly, the link between reduced mitochondrial respiration and bortezomib resistance highlights the potentials of targeting mitochondria in developing novel combined chemotherapy for cancer patients. Citation Format: Fu-Yu Chueh, Shahrooz Vahedi, Fu-Shin Chueh, Chao-Lan Yu. Therapeutic potentials of STAT5 inhibitors in overcoming bortezomib resistance in human T-cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2015-2702