The traditional view is that cancer cells predominately produce ATP by glycolysis, rather than by oxidation of energy-providing substrates. Mitochondrial uncoupling -the continuing reduction of oxygen without ATP synthesis -has recently been shown in leukemia cells to circumvent the ability of oxygen to inhibit glycolysis, and may promote the metabolic preference for glycolysis by shifting from pyruvate oxidation to fatty acid oxidation (FAO). Here we have demonstrated that pharmacologic inhibition of FAO with etomoxir or ranolazine inhibited proliferation and sensitized human leukemia cells -cultured alone or on bone marrow stromal cells -to apoptosis induction by ABT-737, a molecule that releases proapoptotic Bcl-2 proteins such as Bak from antiapoptotic family members. Likewise, treatment with the fatty acid synthase/lipolysis inhibitor orlistat also sensitized leukemia cells to ABT-737, which supports the notion that fatty acids promote cell survival. Mechanistically, we generated evidence suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Importantly, etomoxir decreased the number of quiescent leukemia progenitor cells in approximately 50% of primary human acute myeloid leukemia samples and, when combined with either ABT-737 or cytosine arabinoside, provided substantial therapeutic benefit in a murine model of leukemia. The results support the concept of FAO inhibitors as a therapeutic strategy in hematological malignancies. IntroductionMore than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that circumvents the Pasteur effect, i.e., the inhibition of anaerobic fermentation by oxygen (1). However, we have recently demonstrated that in leukemia cells, mitochondrial uncoupling - the continuing reduction of oxygen without the synthesis of ATP - could mimic the Warburg effect in the absence of permanent, transmissible alterations to the oxidative capacity of cells (2). This metabolic pattern was observed when leukemia cells were cultured on feeder layers of bone marrow-derived mesenchymal stromal cells (MSCs). MSCs have previously been reported to support both normal and malignant hematopoiesis (reviewed in refs. 3-5) and have become an important component in the in vitro modeling of the bone marrow microenvironment. Leukemia cells cultured on MSC feeder layers demonstrated increased lactate generation, and, most curiously, decreased mitochondrial membrane potential in the presence of a transient (6-8 hour) increase in oxygen consumption. Additionally, this uncoupled phenotype appeared to be associated with the antiapoptotic effect of MSC feeder layers, and we hypothesized a shift away from the complete oxidation of glucose. This concept has already been alluded to by Lynen (6), and by Ronzoni and Ehrenfest in experiments using the prototypical protonophore 2,4-dinitrophenol, and suggests a metabolic shift to fatty acid oxidation (FAO) rather than pyruvate oxidation (2, 7). Although i...

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relationship studies on phosphopeptide mimics targeted to the SH2 domain of Stat3. Inclusion of a methyl group on the β-position of the pTyr mimic, 4-phosphocinfnamide, enhanced affinity 2–3 fold. Bis-pivaloyloxymethyl prodrugs containing β-methyl cinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent, completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5–1 µM in a variety of cancer cell lines. The inhibitors were selective for Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby showing clear distinction between cytotoxicity and effects downstream of activated Stat3.

Nucleotides U(-67) to C(-40) at the extreme 5' end of the gene 32 mRNA in bacteriophage T4 have been shown to fold into an RNA pseudoknot proposed to be important for translational autoregulation. The thermal denaturation of three in vitro transcribed RNAs corresponding to the pseudoknot region has been investigated as a function of Mg2+ concentration to begin to elucidate the determinants of the structure and stability of this conformation. T4-35 is a 35-nucleotide RNA containing a 5' G followed by the natural T4 sequence starting with the mature 5' end of the mRNA, nucleotides A(-71) to C(-38). A 32-nucleotide RNA, termed T4-32, contains the native sequence form U(-67) to C(40) with 5'GC and 5'CA single-stranded regions appended to the 5' and 3' ends of the core sequence, respectively. T4-28 contains only the 28 core nucleotides, and the predicted closing U(-67)-A(-52) base pair in stem 1 has been replaced with a phylogenetically allowed G(-67)-C(-52) base pair. Ribonuclease mapping of T4-32 and imino proton NMR experiments of T4-35 show that both sequences adopt a pseudoknotted conformation. At pH 6.9 and 50 mM NaCl, T4-35 and T4-32 RNAs are characterized by a single major melting transition over a wide range of [Mg2+] (0-6 mM). The delta H degree of unfolding for T4-35 and T4-32 shows a large dependence on Mg2+ concentration; the maximum delta H degree occurs at about 2.0 mM Mg2+ with further addition of Mg2+ simply increasing the tm. Investigation of the [Mg2+] dependence of the tm suggests that a net of one Mg2+ ion is released upon denaturation of T4-35 and T4-32 RNAs. Over the entire [Mg2+] range, the delta G degree (37 degrees C) for the folding of T4-35 is consistently 1-1.5 kcal mol(-1) more negative than T4-32 due to a higher stabilization enthalpy for the natural sequence molecule. In contrast to this behavior, T4-28 gives consistently higher tm's but less negative enthalpies and is destabilized (at 37 degrees C) by about 0.5-1.5 kcal mol(-1) relative to T4-32 and by about 2-3 kcal mol(-1) relative to T4-35, depending upon cation concentration. (1)H NMR experiments suggest that, even in the presence of 4.0 mM Mg2+, T4-28 RNA does not adopt a stable pseudoknotted conformation. These data show that the stability of the pseudoknot in the gene 32 mRNA encoded by the 28-nucleotide core sequence is significantly influenced by the number and nature of the immediately adjacent "single-stranded" 5' and/or 3' nucleotides appended to the core structure. These findings are discussed within the context of the structural model for the evolutionarily related phage T2 and T6 gene 32 mRNA pseudoknots presented in the following paper [Du, Z., Giedroc, D. P., & Hoffman, D. W. (1996) Biochemistry 35, 4187-4198].

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced l5N-'H. three-dimensional "N-IH, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 2 1-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 2 1-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the ps-ms time scale at 25 "C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66). comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and "N RI, R2, and {'H-I'NN) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Am75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs. Keywords:Mason-Pfizer monkey virus (MPMV) is the prototypical member of the primate D-type retroviruses that include simian AIDS retrovirus type 1 (SRV-I) and SRV-2, the causative agents of simian AIDS in captive macaque populations (Power et al., 1986). Mouse mammary tumor virus (MMTV) is a type B retrovirus most closely