B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLC; 2 , signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70 + cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials. [Cancer Res 2009;69(13):5424-32]
The Na+/H+ exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were produced and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503–545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633–815). CTD was functional as shown by Ca2+-dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (KD) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (KD = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = −48.6 kJ/mol, ΔH = −75.3 kJ/mol, −TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins.
Abbreviations: CaM, calmodulin; CBD, CHP-binding region of sodium/proton exchanger 1 (residues 503 to 545); CHP, calcineurin B homologous protein; CV, column volumes; EFCaBPs, EF-hand Ca 2+ -binding proteins; FPH assay, fluorescent probe hydrophobicity assay; ITC, isothermal titration calorimetry; MBP, maltose-binding protein; MST, microscale thermophoresis; NHE1, sodium proton exchanger 1; PAGE, polyacrylamide gel electrophoresis. AbstractCalcineurin B homologous proteins (CHPs) belong to the EF-hand Ca 2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na + /H + exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca 2+ with high affinity.Biochemical data indicated that Ca 2+ can regulate their functions. Experimental evidence for Ca 2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca 2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca 2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca 2+ .Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (K D s) in the nanomolar range.The K D for CHP1:CBD was not affected by Ca 2+ , whereas Ca 2+ -depletion increased the K D 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1. K E Y W O R D Scalcineurin B homologous proteins, Ca 2+ signaling, EF-hand Ca 2+ -binding proteins, NHE1, proteinprotein interaction 3254 | LIANG et AL.
Purpose This study aims to develop a molecular imaging strategy for response assessment of arginine deiminase (ADI) treatment in melanoma xenografts using 3′-[18F]fluoro-3′-deoxythymidine ([18F]-FLT) positron emission tomography (PET). Procedures F-FLT response to ADI therapy was studied in preclinical models of melanoma in vitro and in vivo. The molecular mechanism of response to ADI therapy was investigated, with a particular emphasis on biological pathways known to regulate 18F-FLT metabolism. Results Proliferation of SK-MEL-28 melanoma tumors was potently inhibited by ADI treatment. However, no metabolic response was observed in FLT PET, presumably based on the known ADI-induced degradation of PTEN, followed by instability of the tumor suppressor p53 and a relative overexpression of thymidine kinase 1, the enzyme mainly responsible for intracellular FLT processing. Conclusion The specific pharmacological properties of ADI preclude using 18F-FLT to evaluate clinical response in melanoma and argue for further studies to explore the use of other clinically applicable PET tracers in ADI treatment.
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