HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human.

Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. Mitogen-activated protein kinases are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK. Currently, there have been 14 MKKK, 7 MKK, and 12 MAPK identified in mammalian cells. The mammalian MAPK can be subdivided into five families: MAPKerk1/2, MAPKp38, MAPKjnk, MAPKerk3/4, and MAPKerk5. Each MAPK family has distinct biological functions. In Saccharomyces cerevisiae, there are five MAPK pathways involved in mating, cell wall remodelling, nutrient deprivation, and responses to stress stimuli such as osmolarity changes. Component members of the yeast pathways have conserved counterparts in mammalian cells. The number of different MKKK in MAPK modules allows for the diversity of inputs capable of activating MAPK pathways. In this review, we define all known MAPK module kinases from yeast to humans, what is known about their regulation, defined MAPK substrates, and the function of MAPK in cell physiology.

Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death.

The activation of caspases is a final commitment step for apoptosis. It is now evident that signal transduction pathways involving specific protein kinases modulate the apoptotic response. Both pro-apoptotic and anti-apoptotic pathways integrate environmental cues that control the decision to undergo apoptosis. Pro- and anti-apoptotic signal pathways regulate the activation of the caspases. In this review we describe our current understanding of apoptotic signal transduction.

MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis.

MEK kinase 1 (MEKK1) is a 196-kDa protein that, in response to genotoxic agents, was found to undergo phosphorylation-dependent activation. The expression of kinase-inactive MEKK1 inhibited genotoxin-induced apoptosis. Following activation by genotoxins, MEKK1 was cleaved in a caspase-dependent manner into an active 91-kDa kinase fragment. Expression of MEKK1 stimulated DEVD-directed caspase activity and induced apoptosis. MEKK1 is itself a substrate for CPP32 (caspase-3). A mutant MEKK1 that is resistant to caspase cleavage was impaired in its ability to induce apoptosis. These findings demonstrate that MEKK1 contributes to the apoptotic response to genotoxins. The regulation of MEKK1 by genotoxins involves its activation, which may be part of survival pathways, followed by its cleavage, which generates a proapoptotic kinase fragment able to activate caspases. MEKK1 and caspases are predicted to be part of an amplification loop to increase caspase activity during apoptosis.

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P acts as a biased agonist toward neuropeptide and chemokine receptors.

Substance P derivatives are potential therapeutic compounds for the treatment of small cell lung cancer and can cause apoptosis in small cell lung cancer cells in culture. These peptides act as broad spectrum neuropeptide antagonists, blocking calcium mobilization induced by gastrin-releasing peptide, bradykinin, cholecystokinin, and other neuropeptides. We show that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P has unique agonist activities in addition to this described antagonist function. At doses that block calcium mobilization by neuropeptides, this peptide causes activation of c-Jun N-terminal kinase and cytoskeletal changes in Swiss 3T3 fibroblasts and stimulates migration and calcium flux in human neutrophils. Activation of c-Jun N-terminal kinase is dependent on the expression of the gastrin-releasing peptide receptor in rat 1A fibroblasts, demonstrating that the responses to the peptide are receptor-mediated. We hypothesize that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P acts as a biased agonist on neuropeptide and related receptors, activating certain guanine nucleotide-binding proteins through the receptor, but not others.

Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophages by TNF alpha.

A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. In this study, we have investigated the specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages in response to stimulation with TNF alpha. Exposure of macrophages to TNF alpha stimulated a preferential increase in catalytic activity of the p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as determined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange liquid chromatography and (ii) selective immunodepletion of the p46 JNK/SAPK from macrophage lysates. To investigate the level of regulation of p46 JNK/SAPK activation, we determined the ability of MKK4/SEK1/JNKK, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 was able to transphosphorylate both isoforms. In addition, both the p46 and p54 JNK/SAPK isoforms were phosphorylated on their TPY motif in response to TNF alpha stimulation as reflected by immunoblotting with a phospho-specific antibody that recognizes both kinases. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNF alpha regulates macrophage phenotypic heterogeneity and differentiation.

MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-Jun amino-terminal kinases?

Regulation of the mitogen-activated protein kinase (MAPK) family members - which include the extracellular response kinases (ERKs), p38/HOG1, and the c-Jun amino-terminal kinases (JNKs) - plays a central role in mediating the effects of diverse stimuli encompassing cytokines, hormones, growth factors and stresses such as osmotic imbalance, heat shock, inhibition of protein synthesis and irradiation. A rapidly increasing number of kinases that activate the JNK pathways has been described recently, including the MAPK/ERK kinase kinases, p21-activated kinases, germinal center kinase, mixed lineage kinases, tumor progression locus 2, and TGF-beta-activated kinase. Thus, regulation of the JNK pathway provides an interesting example of how many different stimuli can converge into regulating pathways critical for the determination of cell fate.

Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction.

In this paper we demonstrate for the first time a mitogen-induced activation of a nuclear acting phosphatidylcholine-phospholipase D (PLD) which is mediated, at least in part, by the translocation of RhoA to the nucleus. Addition of alpha-thrombin to quiescent IIC9 cells results in an increase in PLD activity in IIC9 nuclei. This is indicated by an increase in the alpha-thrombin-induced production of nuclear phosphatidylethanol in quiescent cells incubated in the presence of ethanol as well as an increase in PLD activity in isolated nuclei. Consistent with our previous report (Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400), the presence of ethanol decreases the alpha-thrombin-induced production of phosphatidic acid without affecting the induced increase in nuclear diglyceride, indicating that the increase in nuclear PLD activity is responsible for the effect on phosphatidic acid, but not that on diglyceride. Our data further demonstrate that RhoA mediates the activation of nuclear PLD. RhoA translocates to the nucleus in response to alpha-thrombin. Additionally, PLD activity in nuclei isolated from alpha-thrombin-treated cells is reduced in a concentration-dependent fashion by incubation with RhoGDI and restored by the addition of prenylated RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Western blot analysis indicates that this RhoGDI treatment results in the extraction of RhoA from the nuclear envelope. These data support a role for a RhoA-mediated activation of PLD in our recently described hypothesis, which proposes that a signal transduction cascade exists in the nuclear envelope and represents a novel signal transduction cascade that we have termed NEST (nuclear envelope signal transduction).

Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death.

Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine-threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response.

Nuclear lipid metabolism in NEST: Nuclear Envelope Signal Transduction.

There is increasing evidence that nuclear lipid metabolism in NEST is an important new component in signal transducing networks and as a result, this metabolism is beginning to attract more attention. While agonist-induced nuclear lipid metabolism adds further complexity to the ever increasing array of signal transduction components, it also provides further avenues by which nuclear activities may be regulated. Identification of the coupling mechanisms, regulation, and physiological roles of nuclear lipid metabolism represents a new and exciting area of research which will have a broad impact in our understanding of signal transduction pathways.

A V lambda x-bearing monoclonal antibody with similar specificity and sequence to encephalitogenic T cell receptors.

The fine specificity of mAb F28C4 to myelin basic protein (MBP), acetyl residues 1-9, has been compared with the previously described specificity of an encephalitogenic T cell clone, PJR-25. F28C4 has been found to express a cross-reactive idiotope (CRI) that is shared with MBP acetyl peptide 1-9-specific TCR. The CRI seems to be located at or near the Ag-combining site of F28C4 and the TCR and, thus, might possibly result from overlapping epitope specificity. We tested the fine epitope specificity of F28C4 by using alanine-substituted peptide analogues and found that residues critical for TCR recognition, Cln3 and Pro6, are also necessary for F28C4 recognition. By using nuclear magnetic resonance, we found that the MBP acetyl peptide 1-9 binds F28C4 in an extended conformation and that the central residues are more tightly bound than the terminal residues, much like the MBP-TCR interaction. Furthermore, sequence homology (75% overall) was found between the regions that contained CDR3 of F28C4 VL and VH and the VDJ junction of the TCR V beta. This homology is not shared by other Ig CDR3 regions and arises, in part, because F28C4 uses an unusual V lambda light chain, V lambda x. Thus, F28C4 shares a CRI with the TCRs, possibly as a result of having similar fine epitope specificity and sequence homology. The anti-CRI mAb can down-modulate experimental allergic encephalomyelitis; thus, it is possible that Abs that are similar to F28C4 may play an important immunoregulatory role in experimental allergic encephalomyelitis in vivo.

Predicted structural motif of IFN tau.

Ovine interferon tau (IFN tau) is a type I interferon that was originally identified as ovine trophoblast protein and is associated with the maternal recognition of pregnancy in sheep. Additionally, IFN tau possesses potent antiviral and antiproliferative activity without the corresponding toxicity found in known IFN alpha s. Structure-function studies with synthetic peptides have identified three discontinuous functional sites on the protein that are involved in receptor interaction and biological activity. However, the structural relationship of these regions is unknown. Therefore, a model relationship of these regions is unknown. Therefore, a model of the 3-D structure of IFN tau would be useful in interpretation of existing data and the design of future structure-function studies. Combining information from circular dichroism (CD) of both the full length recombinant IFN tau and synthetic peptides representing regions of the IFN tau molecule, with sequence homology of IFN tau to IFN beta, a protein of known 3-D structure, we have constructed a model of IFN tau using distance geometry and energy minimization methods. The most striking feature of this model is that functionally active domains of IFN tau, discontinuous in the primary structure, are localized to one side of the molecule and found to be spatially contiguous. This observation is consistent with multiple binding sites on IFN tau interacting simultaneously with the IFN tau receptor.

Alpha-thrombin-induced nuclear sn-1,2-diacylglycerols are derived from phosphatidylcholine hydrolysis in cultured fibroblasts.

Diglycerides play an important role in a number of agonist-induced signal transduction pathways. We have recently demonstrated that alpha-thrombin induces a rapid increase in the level of diglyceride mass in the nucleus and a selective increase in nuclear PKC-alpha [Leach, K.L., Ruff, V.A., Jarpe, M.B., Fabbro, D., Adams, L.D., & Raben, D.M. (1992) J. Biol. Chem. 267, 21816-21822]. In the present report, we examined the potential source of the induced nuclear diglycerides by examining the molecular species profiles of both the induced diglycerides and nuclear phospholipids by capillary gas chromatography. The molecular species profiles of the nuclear diglycerides generated resemble the species profiles of PC, and not PI species, at all times. In addition, while our previous data indicated that the molecular species of whole-cell phospholipids did not change in response to alpha-thrombin, nuclear PE was altered in a dramatic and selective manner in response to this agonist. These results demonstrate that PC hydrolysis is the predominant, if not exclusive, source of the alpha-thrombin-induced nuclear diglycerides in these fibroblasts.

Binding site on the murine IFN-gamma receptor for IFN-gamma has been identified using the synthetic peptide approach.

We have studied the structural parameters involved in the binding of murine IFN-gamma (MuIFN-gamma) to its receptor. Ten synthetic overlapping peptides corresponding to the extracellular domain of the MuIFN-gamma receptor (MuIFN-gamma R) were synthesized. In direct binding studies, biotinylated MuIFN-gamma bound specifically to receptor peptide (95-120). Further, the NH2-terminal IFN-gamma peptide, MuIFN-gamma (1-39), also specifically bound to receptor peptide (95-120). Binding of both labeled MuIFN-gamma and MuIFN-gamma (1-39) to MuIFN-gamma R peptide (95-120) was inhibited by either unlabeled molecule. The COOH-terminal receptor binding peptide, MuIFN-gamma (95-133), neither bound to any receptor peptides nor blocked the binding of intact MuIFN-gamma or MuIFN-gamma (1-39) to receptor peptide (95-120). Polyclonal antibodies to each of the peptides were then produced. Each of the anti-peptide antisera recognized its corresponding peptide and bound denatured cloned soluble receptor by Western blotting. Furthermore, the antisera to peptides representing the inner region of the extracellular domain of the receptor bound to nondenatured soluble MuIFN-gamma R. Specifically, antisera to the receptor peptides (73-97), (95-120), (118-143), (142-163), and (161-182) bound to soluble MuIFN-gamma R, whereas antisera to peptides (1-21), (20-49), (46-74), (178-203), and (202-227) did not bind. Most important, antisera to peptides (95-120) and (118-143) competed with [125I]MuIFN-gamma for binding to soluble receptor. These results show that the region of the MuIFN-gamma R encompassing amino acid residues (95-120) is a binding site on the receptor for the NH2-terminal of MuIFN-gamma by direct binding, and that the larger region (95-143) on the receptor may play a role in binding of intact MuIFN-gamma based on blocking of binding by site-specific antibodies.

The structure of a myelin basic protein-associated idiotope.

A cross-reactive idiotope (CRI) has been previously described on monoclonal antibodies (mAbs) specific for encephalitogenic peptides from myelin basic protein (MBP). The anti-CRI mAb, F25F7, binds an idiotope (Id) localized to the light chains of an anti-MBP peptide 1-9 mAb, denoted F23C6, and an anti-MBP peptide 80-89 mAb, denoted 845D3. It is the purpose of this study to further delineate the CRI being recognized by F25F7. To this end, we have found a structural correlation between the CRI and the antigen, a small synthetic peptide, denoted PBM 9-1, used to elicit the anti-Id mAb. Sequence comparison between the light chain of F23C6 and PBM 9-1 reveals a region of homology in CDR 2/FWK 3. The configuration of this site in the VL, as determined by comparison with a mAb, HyHEL-10, whose structure has been determined and is 97% homologous to the light chain of F23C6, conforms to the rules used to define antigenic determinants or Ids. A synthetic peptide having the F23C6 VL CDR 2/FWK 3 sequence inhibited the binding of F25F7 to F23C6 and 845D3. Taken together, these data suggest the Id recognized by F25F7 is defined, in part, by the PBM 9-1-like sequence of F23C6.

Solution structure of trypsin modulating oostatic factor is a left-handed helix.

The solution structure of trypsin modulating oostatic factor (TMOF), a decapeptide (H-YDPAPPPPPP-OH) hormone that signals the termination of trypsin-like biosynthesis in mosquito midgut epithelial cells, was determined by 2-D 1H nuclear magnetic resonance spectroscopy and molecular modeling. The peptide forms a rod-shaped left-handed helix about 30 A long. No evidence was found to support a poly-L-proline beta-turn model. Hydrophobic contacts between the rings of tyrosine 1 and proline 3 may enhance the stability of the N-terminal segment. This peptide provides an interesting exception to the normal chemical shift index (csi) rules. Our results suggest that a sequence of positive csi indices, normally expected for a beta-strand structure, could also describe a left-handed poly-L-proline-like helix.

Proton NMR sequence-specific assignments and secondary structure of a receptor binding domain of mouse gamma-interferon.

Previous studies using synthetic peptides and monoclonal antibodies have implicated the N-terminal 39-residue segment as a receptor binding region of mouse gamma-interferon (MuIFN gamma). In this work, we report the solution structure of this fragment (dissolved in water with 40% trifluoroethanol) as determined by proton NMR spectroscopy. The proton sequence-specific assignments were determined from TOCSY and NOESY spectra using established procedures. The secondary structure is characterized by two well-defined alpha-helical regions composed of residues 5-16 and 22-37. These two helices are joined by a loop. No NOESY contacts between the two helical regions were detected. Molecular models consistent with the NMR data were generated for MuIFN gamma (1-39) using distance geometry and restrained molecular dynamics/energy minimization calculations. Comparison with similar N-terminal domains in the published NMR and crystallographic studies on the dimeric human and rabbit IFN gamma suggests some similarities in the structures except that the helical regions in the fragment are longer, and considerable variation may exist in the relative orientation of the two helices in the solution phase. The presence of stronger alpha N sequential NOE's suggests that this peptide is flexible. The absence of NOESY contacts involving the N-terminal tripeptide suggests that this region undergoes rapid segmental motion. The data presented here on MuIFN gamma (1-39), combined with the studies on human and rabbit IFN gamma, suggest that the N-terminal receptor binding domain of the protein can undergo structural changes, the understanding of which may provide insight into the basis for receptor interaction by this lymphokine.

Stable conformation of an interferon-gamma receptor binding peptide in aqueous solution is required for interferon-gamma antagonist activity.

A peptide antagonist for mouse interferon-gamma (IFN-gamma) activity corresponding to the amino-terminal 39 amino acids of mouse IFN-gamma [MuIFN-gamma (1-39)] has been identified previously. In an analogous manner, we have assessed the ability of the corresponding peptide of human IFN-gamma [HuIFN-gamma (1-39)] to antagonize human IFN-gamma. HuIFN-gamma (1-39) has the ability to block the antiviral activity of human IFN-gamma in a functional assay. In receptor competition, the peptide can also block human IFN-gamma receptor binding. Surprisingly, the murine analog, MuIFN-gamma (1-39), possesses a 10-fold greater ability to block human IFN-gamma antiviral activity and receptor binding than HuIFN-gamma (1-39). Both peptides showed alpha-helical structure in water by circular dichroism, however MuIFN-gamma (1-39) possessed a greater amount of alpha-helix compared to HuIFN-gamma (1-39), suggesting a requirement for a stable secondary structure for optimal antagonist activity. In trifluoroethanol, a helix-stabilizing agent, both peptides showed relatively equal alpha-helices, suggesting that both sequences have an equal potential for helical structure. As IFN-gamma is species specific, the observation that MuIFN-gamma (1-39) can antagonize human IFN-gamma raises questions about the role of this region in species specificity. These studies provide insight into the structural requirements, both primary and secondary, for IFN-gamma receptor binding. In the future, this information, along with more detailed three-dimensional structural analyses of the peptides, could prove useful in the rational design of IFN-gamma agonists and antagonists.

Identification of binding domains on the superantigen, toxic shock syndrome-1, for class II MHC molecules.

Toxic shock syndrome toxin-1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. In order to determine the regions on the TSST-1 molecule involved in binding to class II MHC, seven overlapping peptides of the entire TSST-1 molecule were synthesized and tested for their ability to compete with 125I-TSST-1 for binding to class II MHC on murine A20 cells and HLA on Raji cells. Peptides corresponding to N-terminal amino acid residues 39 through 68 and C-terminal residues 155 through 194 competed with 125I-TSST-1 for binding to class II MHC. Also, binding studies with class II MHC beta-chain peptides indicate that regions encompassed by I-A beta b(30-60) and I-A beta b(60-90) are binding regions for TSST-1. Thus, we have identified binding domains on the TSST-1 molecule for class II MHC molecule receptors on antigen presenting cells.