Chemical metabolic inhibitors for the treatment of blood-borne cancers.

Tumor cells, including leukemic cells, remodel their bioenergetic system in favor of aerobic glycolysis. This process is called "the Warburg effect" and offers an attractive pharmacological target to preferentially eliminate malignant cells. In addition, recent results show that metabolic changes can be linked to tumor immune evasion. Mouse models demonstrate the importance of this metabolic remodeling in leukemogenesis. Some leukemias, although treatable, remain incurable and resistance to chemotherapy produces an elevated percentage of relapse in most leukemia cases. Several groups have targeted the specific metabolism of leukemia cells in preclinical and clinical studies to improve the prognosis of these patients, i.e. using L-asparaginase to treat pediatric acute lymphocytic leukemia (ALL). Additional metabolic drugs that are currently being used to treat other diseases or tumors could also be exploited for leukemia, based on preclinical studies. Finally, we discuss the potential use of several metabolic drugs in combination therapies, including immunomodulatory drugs (IMiDs) or immune cell-based therapies, to increase their efficacy and reduce side effects in the treatment of hematological cancers.

The protooncogene Vav1 regulates murine leukemia virus-induced T-cell leukemogenesis.

Vav1 is expressed exclusively in hematopoietic cells and is required for T cell development and activation. Vav1-deficient mice show thymic hypocellularity due to a partial block during thymocyte development at the DN3 stage and between the double positive (DP) and single positive (SP) transition. Vav1 has been shown to play a significant role in several non-hematopoietic tumors but its role in leukemogenesis is unknown. To address this question, we investigated the role of Vav1 in retrovirus-induced T cell leukemogenesis. Infection of Vav1-deficient mice with the Moloney strain of murine leukemia virus (M-MuLV) significantly affected tumor phenotype without modulating tumor incidence or latency. M-MuLV-infected Vav1-deficient mice showed reduced splenomegaly, higher hematocrit levels and hypertrophic thymi. Notably, Vav1-deficient mice with M-MuLV leukemias presented with markedly lower TCRß/CD3 levels, indicating that transformation occurred at an earlier stage of T cell development than in WT mice. Thus, impaired T cell development modulates the outcome of retrovirus-induced T cell leukemias, demonstrating a link between T cell development and T cell leukemogenesis.

Cyclin A2: a genuine cell cycle regulator?

Abstract Cyclin A2 belongs to the core cell cycle regulators and participates in the control of both S phase and mitosis. However, several observations suggest that it is also endowed with other functions, and our recent data shed light on its involvement in cytoskeleton dynamic and cell motility. From the transcription of its gene to its posttranslational modifications, cyclin A2 regulation reveals the complexity of the regulatory network shaping cell cycle progression. We summarize our current knowledge on this cell cycle regulator and discuss recent findings raising the possibility that cyclin A2 might play a much broader role in epithelial tissues homeostasis.

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1.

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

Oxidative phosphorylation induces de novo expression of the MHC class I in tumor cells through the ERK5 pathway.

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.

Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes.

Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and release of basic fibroblast growth factor (bFGF) are principal aspects of the pathology of osteoarthritis (OA). ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO) serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation-dependent transactivation capacity of Elk-1. This attenuates transcription of its downstream target gene MMP-13 to maintain the integrity of cartilage ECM homeostasis.

TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin.

The contractile activity of striated muscle depends on myofibrils that are highly ordered macromolecular complexes. The protein components of myofibrils are well characterized, but it remains largely unclear how signaling at the molecular level within the sarcomere and the control of assembly are coordinated. We show that the Rho GTPase TC10 appears during differentiation of human primary skeletal myoblasts and it is active in differentiated myotubes. We identify obscurin, a sarcomere-associated protein, as a specific activator of TC10. Indeed, TC10 binds directly to obscurin via its predicted RhoGEF motif. Importantly, we demonstrate that obscurin is a specific activator of TC10 but not the Rho GTPases Rac and Cdc42. Finally, we show that inhibition of TC10 activity by expression of a dominant-negative mutant or its knockdown by expression of specific shRNA block myofibril assembly. Our findings reveal a novel signaling pathway in human skeletal muscle that involves obscurin and the Rho GTPase TC10 and implicate this pathway in new sarcomere formation.

ERK5 knockdown generates mouse leukemia cells with low MHC class I levels that activate NK cells and block tumorigenesis.

Tumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine.

Erk5 controls Slug expression and keratinocyte activation during wound healing.

Reepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell-cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF.

Impaired anti-leukemic immune response in PKCtheta-deficient mice.

The cancer immunosurveillance hypothesis has found strong experimental support in recent years. It is believed that cytotoxic lymphocytes are important effectors in this process. PKCtheta plays an essential role in proliferation, activation and survival of these cells, but also proliferation and survival of leukemic T cells. In light of this, we tested the role of PKCtheta in T cell leukemia progression by inducing this disease in wild-type (wt) and PKCtheta-deficient mice with moloney-murine leukemia virus (M-MuLV). Leukemic PKCtheta(-/-) and wild-type (wt) mice showed the same profile of leukemic cell types, similar spleen and thymus sizes and comparable hematocrits. In contrast, disease incidence was higher and disease onset more rapid in PKCtheta(-/-) mice. Transfer of leukemic T cells from wt donors into PKCtheta-deficient and wt recipients induced leukemia in 100% and 40% of the mice, respectively. Interestingly, leukemic cells from PKCtheta(-/-) donors induced the disease in only 50% of the PKCtheta-deficient and 10% of the wt recipients. Intravenous injection of low numbers of EL4 cells induced tumors earlier in PKCtheta(-/-) mice. Taken together, our results show that PKCtheta is essential for the immune response to leukemia in mice and raise questions about the chronic treatment of humans with PKCtheta inhibitors.

SUMOylation regulates the transcriptional activity of JunB in T lymphocytes.

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modifications that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modifier (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant-negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol-13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4(+) T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation.

Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer.

Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G(0)/G(1)-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.

ERK5 activates NF-kappaB in leukemic T cells and is essential for their growth in vivo.

MAPK cascades play a central role in the cellular response to the environment. The pathway involving the MAPK ERK5 mediates growth factor- and stress-induced intracellular signaling that controls proliferation or survival depending upon the cell context. In this study, we show that reducing ERK5 levels with a specific small hairpin RNA 5 (shERK5) reduced cell viability, sensitized cells to death receptor-induced apoptosis, and blocked the palliative effects of phorbol ester in anti-Fas Ab-treated cells. shERK5 decreased nuclear accumulation of the NF-kappaB p65 subunit, and conversely, ectopic activation of ERK5 led to constitutive nuclear localization of p65 and increased its ability to trans activate specific reporter genes. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferated in vitro, but failed to induce s.c. tumors in mice. Our results suggest that ERK5 is essential for survival of leukemic T cells in vivo, and thus represents a promising target for therapeutic intervention in this type of malignancy.

Identification of Rho GTPases implicated in terminal differentiation of muscle cells in ascidia.

BACKGROUND INFORMATION: Members of the Rho GTPase family mediate changes in the actin cytoskeleton and are also implicated in developmental processes, including myogenesis. Nevertheless, a comprehensive analysis of these proteins during myofibrillogenesis has never been performed in any organism. RESULTS: Using the ascidian model to identify the role of Rho GTPases on myofibrillogenesis, we show that transcripts for all Rho GTPases are detected in muscle cells of the embryo. We find that activation of RhoA, TC10 and Cdc42 (cell division cycle 42) disturbs the polarity of muscle cells, whereas that of other Rho GTPases induced cell positioning defects. Moreover, dominant negative version of five Rho GTPases, RhoA, Rac2, RCL2 (Rac- and Cdc42-like 2), TC10 and WRCH (Wnt-1 responsive Cdc42 homologue), impaired the formation of mature myofibrils. CONCLUSIONS: Taken together, our results show that several Rho GTPase-dependent pathways are required to control the spatial localization of muscle cells in the embryo and to coordinate myofibril assembly. This stresses the importance of analysing the entire Rho family when studying a new biological process.

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1.

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling.

Positive feedback regulation of PLCgamma1/Ca(2+) signaling by PKCtheta in restimulated T cells via a Tec kinase-dependent pathway.

PKCtheta plays an essential role in activation of mature T cells. Here, we report that the TCR/CD28-induced tyrosine phosphorylation and activation of PLCgamma1 was significantly impaired in PKCtheta (-/-) primary, restimulated T cells. Consistent with this finding, receptor-induced Ca(2+) mobilization, NF-AT DNA-binding activity and the membrane translocation of PKCalpha, a PLCgamma1-dependent conventional PKC, were also markedly reduced in the same cells. Moreover, a dominant-negative PLCgamma1 mutant blocked the PKCtheta-induced activation of an AP-1 reporter gene in Jurkat and primary cells. Regulation of PLCgamma1 signaling by PKCtheta required the tyrosine kinase Tec since a dominant-negative Tec mutant blocked PKCtheta-induced AP-1 (but not NF-kappaB) activation. In addition, wild-type Tec, but not Itk or Rlk, potently activated AP-1. Furthermore, Tec was found to constitutively associate with PKCtheta, an interaction that like AP-1 activation required the pleckstrin-homology domain of Tec. These findings define a novel PKCtheta-initiated pathway that regulates Ca(2+) signaling and AP-1 activation via Tec and PLCgamma1. Moreover, they identify Tec as a key point downstream of PKCtheta, where TCR- and PKCtheta-induced signaling pathways, leading to AP-1 versus NF-kappaB activation, diverge in T cells.

P2 receptors in bone--modulation of osteoclast formation and activity via P2X7 activation.

The concept that adenosine triphosphate (ATP) can act as an extracellular signaling molecule via interactions with specific purinergic receptors to mediate a wide variety of processes as diverse as neurotransmission (Edwards et al., 1992), inflammation (Perregaux et al., 1994), apoptosis (Chow et al., 1997), and bone remodelling (Jones et al., 1997; Morrison et al., 1998) is now widely accepted. Since the early work of Burnstock (Burnstock, 1972), the number of characterized P2 receptors responsive to extracellular nucleotides has increased dramatically. It is now known that both osteoblasts and osteoclasts express multiple P2 receptor subtypes, and the increasing number of nucleotide-induced effects reported to occur in bone serves to highlight the importance of these receptors in the bone microenvironment and the bone remodeling processes. In this article we will review work from our laboratory, and others, that has established nucleotides and P2 receptors as important signaling molecules in bone. In particular, we will focus on the expression of P2 receptors by osteoclasts and, more specifically, the P2X7 receptor and its paradoxical role in osteoclast function.

Activation of cyclin D1 expression by the ERK5 cascade.

Transcriptional activation of the cyclin D1 gene is a key step in cell proliferation. Accordingly, cyclin D1 overexpression is frequently an early step in neoplastic transformation, particularly in mammary epithelium. Numerous studies have linked elevated cyclin D1 promoter activity to a sustained activation of the ERK1/2 cascade. Here we show that the ERK5 cascade, a distinct mitogen-induced MAPK pathway, can also drive cyclin D1 expression. In CCL39 cells, serum induces a strong, prolonged peak of ERK1/2 and ERK5 phosphorylation, and subsequently elevates cyclin D1 mRNA and protein levels. Overexpression of constitutively active MEK5 and wt ERK5 induces a cyclin D1 reporter gene (D1 -973-luciferase) at least as well as constitutively active MEK1. Activation is blocked by kinase-dead mutants of ERK5 and ERK2, respectively. Mutation of the CRE at -50 in the cyclin D1 promoter decreases activation by the ERK5 but not the ERK1/2 cascade. Importantly, expression of kinase-dead ERK5 diminishes endogenous cyclin D1 protein induction by serum in CCL39 cells and the breast cancer cell lines MCF-7 and HS579. These data identify the cyclin D1 gene as a novel target of the ERK5 cascade, an observation with important implications in cancers involving cyclin D1 deregulation.