KX2-361: a novel orally bioavailable small molecule dual Src/tubulin inhibitor that provides long term survival in a murine model of glioblastoma.

PURPOSE: A major challenge to developing new therapies for patients with malignant brain tumors is that relatively few small molecule anticancer drugs penetrate the blood-brain barrier (BBB) well enough to provide therapeutically effective concentrations in brain tissue before drug exposure in non-CNS tissues results in unacceptable toxicity. METHODS: KX2-361, a member of a novel family of compounds with Src-kinase and tubulin polymerization inhibitory activity, demonstrates good oral bioavailability and readily crosses the BBB in mice. The objective of this study was to investigate the activity of KX2-361 against human and murine glioma cells and assess its therapeutic effect in a syngeneic orthotopic model of glioblastoma. RESULTS: In addition to reducing the level of Src autophosphorylation in the GL261 murine glioblastoma cell line, KX2-361 binds directly to tubulin and disrupts microtubule architecture in glioma cells maintained in culture. CONCLUSIONS: The drug is active in vivo against orthotopic GL261 gliomas in syngeneic C57BL/6 mice. Long term survival is not observed in mice lacking an adaptive immune system, indicating that KX2-361 works in concert with the host immune system to control tumor growth and promote long-term survival in the GL261 glioma model.

Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.

Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.

Distinct molecular mechanisms responsible for bortezomib-induced death of therapy-resistant versus -sensitive B-NHL cells.

Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.

The immunogenicity of L1210 lymphoma clones correlates with their ability to function as antigen-presenting cells.

Major histocompatibility complex class II (MHCII) antigen expression is directly correlated with immunogenicity, and inversely correlated with tumorigenicity, in clones of the L1210 murine B lymphoma. Moreover, loss of MHCII expression on human diffuse large B-cell lymphoma is associated with dramatic decreases in patient survival. Thus, the role that MHCII antigens play in the progression of B-cell lymphomas is clinically important. In this study, we investigated the basis for the immunogenicity of MHCII(+) L1210 clones. Immunogenic, but not tumorigenic L1210 clones stimulated the proliferation of naïve T cells and their interleukin (IL)-2 production, which indicates that the immunogenic clones can function as antigen-presenting cells (APCs). However, subclonal variants of the immunogenic L1210 clones, which form tumours slowly in mice, could not activate T cells. The costimulatory molecules B7-1, B7-2 and CD40 were expressed on the immunogenic L1210 clones, but not the tumorigenic clones. Importantly, the tumour-forming subclonal variants expressed MHCII and B7-1, but lacked B7-2 and CD40. These results suggest that MHCII and B7-1 expression on L1210 cells is insufficient to activate naïve T cells, and, furthermore, loss of B7-2 and/or CD40 expression contributes to the decreased immunogenicity of L1210 subclones. Blocking B7-1 or B7-2 function on immunogenic L1210 cells reduced their capacity to activate naïve T cells. Furthermore, incubation of immunogenic L1210 cells with CD40 antibodies significantly enhanced APC function. Therefore, the immunogenicity of L1210 cells directly correlates (i) with their ability to stimulate naïve T cells, and (ii) with the concomitant expression of MHCII, B7-1, B7-2, and CD40.

Vav1 regulates the migration and adhesion of dendritic cells.

Dendritic cells (DCs) are the most potent APCs for activating naive T cells, a process facilitated by the ability of immature DCs to mature and home to lymph nodes after encountering an inflammatory stimulus. Proteins involved in cytoskeletal rearrangement play an important role in regulating the adherence and motility of DCs. Vav1, a guanine nucleotide exchange factor for Rho family GTPases, mediates cytoskeletal rearrangement in hematopoietic cells following integrin ligation. We show that Vav1 is not required for the normal maturation of DCs in vitro; however, it is critical for DC binding to fibronectin and regulates the distribution but not the formation of podosomes. We also found that DC Vav1 was an important component of a signaling pathway involving focal adhesion kinase, phospholipase C-gamma2, and ERK1/2 following integrin ligation. Surprisingly, Vav1(-/-) DCs had increased rates of migration in vivo compared with wild-type control DCs. In vitro findings show that the presence of adhesive substrates such as fibronectin resulted in inhibition of migration. However, there was less inhibition in the absence of Vav1. These findings suggest that DC migration is negatively regulated by adhesion and integrin-mediated signaling and that Vav1 has a central role in this process.

Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation.

Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.

Acquired resistance to rituximab is associated with chemotherapy resistance resulting from decreased Bax and Bak expression.

PURPOSE: Targeting malignant B cells using rituximab (anti-CD20) has improved the efficacy of chemotherapy regimens used to treat patients with non-Hodgkin's lymphoma. Despite the promising clinical results obtained using rituximab, many patients relapse with therapy-resistant disease following rituximab-based treatments. We have created a cell line model of rituximab resistance using three B-cell non-Hodgkin's lymphoma-derived cell lines (Raji, RL, and SUDHL-4). In an attempt to define strategies to overcome rituximab resistance, we sought to determine the chemotherapy sensitivity of our rituximab-resistant cell lines (RRCL). EXPERIMENTAL DESIGN: Parental, rituximab-sensitive cell lines (RSCL) Raji, RL, and SUDHL-4, along with RRCLs derived from them, were exposed to several chemotherapeutic agents with different mechanisms of action and the ability of these agents to induce apoptotic cell death was measured. Expression of multidomain Bcl-2 family proteins was studied as potential mediators of chemotherapy/rituximab resistance. RESULTS: We found that RRCLs are resistant to multiple chemotherapeutic agents and have significantly decreased expression of the Bcl-2 family proteins Bax, Bak, and Bcl-2. RRCLs do not undergo rituximab- or chemotherapy-induced apoptosis but die in a caspase-dependent manner when either wild-type Bax or Bak is exogenously expressed. Furthermore, forced expression of Bak sensitized RRCL to chemotherapy-induced apoptosis. CONCLUSIONS: Whereas a single or limited exposure of lymphoma cells to rituximab may lead to a favorable ratio of proapoptotic to antiapoptotic Bcl-2 family proteins, repeated exposure to rituximab is associated with a therapy-resistant phenotype via modulation of Bax and Bak expression.

Ets-1 regulates plasma cell differentiation by interfering with the activity of the transcription factor Blimp-1.

Development of immunoglobulin-secreting plasma cells from B cells is a tightly regulated process controlled by the action of a number of transcription factors. In particular, the transcription factor Blimp-1 is a key positive regulator of plasmacytic differentiation via its ability to suppress expression of genes involved in the mature B cell program. The transcription factor Ets-1 is a negative regulator of plasmacytic differentiation, as indicated by the development of increased numbers of IgM-secreting plasma cells in Ets-1 knock-out mice. We have previously shown that Ets-1-deficient B cells undergo enhanced differentiation into IgM-secreting plasma cells in response to Toll-like receptor 9 (TLR9) signaling. We now explore the mechanism by which Ets-1 limits differentiation downstream of TLR9. Our results indicate that Ets-1 physically interacts with Blimp-1, which leads to a block in Blimp-1 DNA binding activity and a reduction in the ability of Blimp-1 to repress target genes without interfering with Blimp-1 protein levels. In addition, we show that Ets-1 induces the expression of several target genes that are repressed by Blimp-1, including Pax-5. These results reveal a previously unknown mechanism for the control of Blimp-1 activity by Ets-1 and suggest that expression of Ets-1 must be down-regulated before plasmacytic differentiation can occur.

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells.

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+ B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.

Impaired generation of CD8+ thymocytes in Ets-1-deficient mice.

The Ets family of transcription factors function as key regulators of multiple aspects of immune cell development and function. To date, Ets-1 has been implicated in regulating early stages of thymic maturation and lymphocyte function and homeostasis. This report describes a novel role for Ets-1 in supporting later stages of thymic selection, in that positive selection of MHC class I-restricted CD4+CD8+ double-positive thymocytes is markedly inhibited in mice expressing a hypomorphic allele of Ets-1. This effect is thymocyte intrinsic, as Ets-1 mutant thymocytes fail to efficiently generate CD8+ single-positive thymocytes in mixed bone marrow chimeric backgrounds. Although peripheral CD8+ T cells are present in Ets-1 mutant mice, both CD4+ and CD8+ subsets contain an elevated proportion of cells with an effector memory (CD62L-CD44+) phenotype. In addition, while thymic expression of Thy1 is relatively normal, peripheral T cells isolated from Ets-1 mutant mice display a striking loss of Thy1 expression. These data identify Ets-1 as a key transcription factor regulating thymocyte positive selection and lineage commitment of MHC class I-restricted thymocytes.

Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint.

Delay of cell cycle progression in response to double-strand DNA breaks (DSBs) is critical to allow time for DNA repair and prevent cellular transformation. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is activated in immature thymocytes along with TcRbeta gene V(D)J recombination. Active p38 MAP kinase promotes a G2/M cell cycle checkpoint through the phosphorylation and activation of p53 in these cells in vivo. Inactivation of p38 MAP kinase and p53 is required for DN3 thymocytes to exit the G2/M checkpoint, progress through mitosis and further differentiate. We propose that p38 MAP kinase is activated by V(D)J-mediated DSBs and induces a p53-mediated G2/M checkpoint to allow DNA repair and prevent cellular transformation.

Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection.

The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.

Ets-1 deficiency leads to altered B cell differentiation, hyperresponsiveness to TLR9 and autoimmune disease.

It has been shown that mice with a targeted mutation in the Ets-1 gene exhibit increased B cell terminal differentiation to IgM-secreting plasma cells. Here, we show that mice, formerly described to lack Ets-1 protein, actually express low levels of an internally deleted Ets-1 protein. Mice harboring this Ets-1 hypomorphic allele possess very few marginal zone B cells and have increased expression of activation markers on follicular B cells. Adoptive transfer experiments indicate that this activated phenotype can be reversed upon transfer of Ets-1-deficient B cells to a wild-type host, suggesting a role for B cell-extrinsic factors in regulating the activated state. Supporting this observation, the reverse transfer experiment of wild-type B cells into an Ets-1-deficient host resulted in increased expression of activation markers on the transferred B cells. However, there are also cell-intrinsic changes in Ets-1-deficient B cells as demonstrated by their increased differentiation to plasma cells in vitro in response to stimulation with cytosine-phosphate-guanine DNA sequence-containing oligodeoxynucleotide [CpG DNA, a Toll-like receptor (TLR) 9 ligand]. Consistent with the activated phenotype and increased terminal differentiation of Ets-1-deficient B cells, Ets-1 mutant mice develop autoimmune disease. Hence, our studies establish Ets-1 as an important regulator of peripheral B cell differentiation and B cell responses to TLR9 activation.

Signaling through the murine T cell receptor induces IL-17 production in the absence of costimulation, IL-23 or dendritic cells.

IL-17 (IL-17A or CTLA-8) is the founding member of a novel family of inflammatory cytokines, and emerging evidence indicates that it plays a central role in inflammation and autoimmunity. IL-17 is made primarily, if not exclusively by T cells, but relatively little is known about how its expression is regulated. In the present study, we examined the requirements and mechanisms for IL-17 expression in primary mouse lymphocytes. Like many cytokines, IL-17 is induced rapidly in primary T cells after stimulation of the T cell receptor (TCR) through CD3 crossinking. Surprisingly, however, the pattern of regulation of IL-17 is different in mice than in humans, because "costimulation" of T cells through CD28 only mildly enhanced IL-17 expression, whereas levels of IL-2 were dramatically enhanced. Similarly, several other costimulatory molecules such as ICOS, 4-1BB and CD40L exerted only very weak enhancing effects on IL-17 production. In agreement with other reports, IL-23 enhanced CD3-induced IL-17 expression. However, IL-17 production can occur autonomously in T cells, as neither dendritic cells nor IL-23 were necessary for promoting short-term production of IL-17. Finally, to begin to characterize the TCR-mediated signaling pathway(s) required for IL-17 production, we showed that IL-17 expression is sensitive to cyclosporin-A and MAPK inhibitors, suggesting the involvement of the calcineurin/NFAT and MAPK signaling pathways.

Known and potential functions for the SLP-76 adapter protein in regulating T-cell activation and development.

The hematopoietic adapter protein SLP-76 is a critical component of multiple biochemical signaling 'circuits' in T cells that integrate proximal signaling events initiated by ligation of the T-cell receptor (TCR) into more distal pathways. Given the important role ascribed to TCR signaling in directing the outcome of thymocyte selection, it seems likely that SLP-76 may also function in signaling pathways that ultimately impact the establishment of the peripheral T-cell repertoire. It is generally accepted that the peripheral T-cell repertoire is selected in large part during T-cell development in the thymus. Molecular interactions between the TCR and self-peptide/major histocompatibility complexes expressed on thymic stromal elements dictate the fate of developing thymocytes. Thymocyte survival and further maturation (positive selection) require an active signal delivered to the cell as a consequence of TCR ligation. This raises the intriguing question of how a thymocyte can, for a narrow window of developmental time, obtain responsiveness to self while maintaining tolerance to these same determinants upon export to the periphery. This article reviews the current literature describing SLP-76-dependent signaling pathways in mature T cells and developing thymocytes. A potential role for this critical signaling intermediate in integrating signals leading to positive and negative selection of the peripheral T-cell repertoire is also discussed.