Exploring Spin-Orbit Effects in a [Cu6Tl]+ Nanocluster Featuring an Uncommon Tl-H Interaction.

Reaction of [CuH(PPh3)]6 with 1 equiv. of Tl(OTf) results in formation of [Cu6TlH6(PPh3)6][OTf] ([1]OTf]), which can be isolated in good yields. Variable-temperature 1H NMR spectroscopy, in combination with density functional theory (DFT) calculations, confirms the presence of a rare Tl-H orbital interaction. According to DFT, the 1H chemical shift of the Tl-adjacent hydride ligands of [1]+ includes 7.7 ppm of deshielding due to spin-orbit effects from the heavy Tl atom. This study provides valuable new insights into a rare class of metal hydrides, given that [1][OTf] is only the third isolable species reported to contain a Tl-H interaction.

LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

Tumor suppressor p53: from engaging DNA to target gene regulation.

The p53 transcription factor confers its potent tumor suppressor functions primarily through the regulation of a large network of target genes. The recent explosion of next generation sequencing protocols has enabled the study of the p53 gene regulatory network (GRN) and underlying mechanisms at an unprecedented depth and scale, helping us to understand precisely how p53 controls gene regulation. Here, we discuss our current understanding of where and how p53 binds to DNA and chromatin, its pioneer-like role, and how this affects gene regulation. We provide an overview of the p53 GRN and the direct and indirect mechanisms through which p53 affects gene regulation. In particular, we focus on delineating the ubiquitous and cell type-specific network of regulatory elements that p53 engages; reviewing our understanding of how, where, and when p53 binds to DNA and the mechanisms through which these events regulate transcription. Finally, we discuss the evolution of the p53 GRN and how recent work has revealed remarkable differences between vertebrates, which are of particular importance to cancer researchers using mouse models.

Correlating Mechanical Sensitivity with Spin Transition in the Explosive Spin Crossover Complex [Fe(Htrz)3]n[ClO4]2n.

Spin crossover complexes are known to undergo bond length, volume, and enthalpy changes during spin transition. In an explosive spin crossover complex, these changes could affect the mechanical and initiation sensitivity of the explosive and lead to the development of a new class of sensitivity switchable materials. To explore this relationship, the well-known spin crossover compound [Fe(Htrz)3]n[ClO4]2n (1) was re-evaluated for its explosive properties, and its mechanical impact sensitivity was correlated to spin transition. A variable temperature impact test was developed and used to evaluate the impact sensitivity of 1 in the low spin (LS, S = 0), thermally accessed high spin (HS, S = 2), and mixed LS and HS states. For comparison, the structurally similar Ni compound, [Ni(Htrz)3]n[ClO4]2n (2), which does not undergo a spin transition at accessible temperatures, was synthesized and characterized, and its explosive properties and variable temperature impact sensitivity measured. These results reveal a correlation between impact sensitivity and spin transition, where 1 exhibits lower impact sensitivity in the LS state and increases in sensitivity upon transition to the HS state. Density functional theory was used to predict structural changes that occur upon spin transition that correlate to the change in sensitivity. This demonstrates, for the first time, an explosive spin crossover compound (ExSCO) that exhibits switchable impact sensitivity with a fully reversible internal switching mechanism.

Lanthanide Complexes of Bis(tetrazolato)amine: A Route to Lanthanide Nitride Foams.

Metal nitrides are strong refractory ceramic materials known for applications in the coatings, catalysis, and semiconductor industries. Lanthanide nitrides are difficult to prepare in high purity and often require high temperatures and sophisticated equipment. In this work, we present an approach to the synthesis of high-purity f-element nitrides through the use of simple lanthanide salts and the nitrogen-rich ligand 5,5'-bis(1H-tetrazolyl)amine (H2BTA) to form lanthanide complexes of 5,5'-bis(tetrazolato)amine (BTA2-). We have demonstrated that, when dehydrated, these types of complexes undergo a self-sustained combustion reaction under an inert atmosphere to yield nanostructured f-element nitride foams for lanthanum and cerium. The synthesis, characterization, and single-crystal X-ray crystallography of the BTA2- complexes of lanthanum, cerium, praseodymium, neodymium, and europium are also discussed.

Revealing a human p53 universe.

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

Lithium Enolates in the Enantioselective Construction of Tetrasubstituted Carbon Centers with Chiral Lithium Amides as Noncovalent Stereodirecting Auxiliaries.

Lithium enolates derived from carboxylic acids are ubiquitous intermediates in organic synthesis. Asymmetric transformations with these intermediates, a central goal of organic synthesis, are typically carried out with covalently attached chiral auxiliaries. An alternative approach is to utilize chiral reagents that form discrete, well-defined aggregates with lithium enolates, providing a chiral environment conducive of asymmetric bond formation. These reagents effectively act as noncovalent, or traceless, chiral auxiliaries. Lithium amides are an obvious choice for such reagents as they are known to form mixed aggregates with lithium enolates. We demonstrate here that mixed aggregates can effect highly enantioselective transformations of lithium enolates in several classes of reactions, most notably in transformations forming tetrasubstituted and quaternary carbon centers. Easy recovery of the chiral reagent by aqueous extraction is another practical advantage of this one-step protocol. Crystallographic, spectroscopic, and computational studies of the central reactive aggregate, which provide insight into the origins of selectivity, are also reported.

Subnanometer-Sized Copper Clusters: A Critical Re-evaluation of the Synthesis and Characterization of Cu8(MPP)4 (HMPP = 2-Mercapto-5-n-propylpyrimidine).

We report a critical re-evaluation of the synthesis and characterization of Cu8(MPP)4. This product was reportedly formed by the reaction of Cu(NO3)2 with 2-mercapto-5-n-propylpyrimidine (HMPP) and NaBH4, in ethanol, in the presence of [N(C8H17)4][Br]. In our hands, we found no experimental evidence to support the existence of Cu8(MPP)4 in the reaction mixture. Instead, we demonstrate that the material isolated from this reaction is a complex mixture containing [N(C8H17)4]+, Br-, NO3-, and 2-mercapto-5-n-propyl-1,6-dihydropyrimidine (H2MPP*), along with the Cu(I) coordination polymer, [Cu(MPP)]n. To support our conclusions, we have independently synthesized H2MPP* and [Cu(MPP)]n, as well as the related Cu(I) coordination complexes, [Cu(HMPP*)]n and [Cu2(MPP*)]n. All new materials were characterized by NMR spectroscopy and mass spectrometry, while H2MPP*, [Cu(HMPP*)]n (n = 4), and [Cu(MPP)]n (n = 6) were also characterized by X-ray crystallography.

Synthesis, Characterization, and Reactivity of the Group 11 Hydrido Clusters [Ag6H4(dppm)4(OAc)2] and [Cu3H(dppm)3(OAc)2].

The group 11 hydride clusters [Ag6H4(dppm)4(OAc)2] (1) and [Cu3H(dppm)3(OAc)2] (2) (dppm = 1,1-bis(diphenylphosphino)methane) were synthesized in moderate yields from the reaction of M(OAc) (M = Ag, Cu) with Ph2SiH2, in the presence of dppm. Complex 1 is the first structurally characterized homometallic polyhydrido silver cluster to be isolated. Both 1 and 2 catalyze the hydrosilylation of (α,ß-unsaturated) ketones. Notably, this represents the first example of hydrosilylation with an authentic silver hydride complex.

A Cu25 Nanocluster with Partial Cu(0) Character.

Atomically precise copper nanoclusters (NCs) are of immense interest for a variety of applications, but have remained elusive. Herein, we report the isolation of a copper NC, [Cu25H22(PPh3)12]Cl (1), from the reaction of Cu(OAc) and CuCl with Ph2SiH2, in the presence of PPh3. Complex 1 has been fully characterized, including analysis by X-ray crystallography, XANES, and XPS. In the solid state, complex 1 is constructed around a Cu13 centered-icosahedron and formally features partial Cu(0) character. XANES of 1 reveals a Cu K-edge at 8979.6 eV, intermediate between the edge energies of Cu(0) and Cu(I), confirming our oxidation state assignment. This assignment is further corroborated by determination of the Auger parameter for 1, which also falls between those recorded for Cu(0) and Cu(I).

Synthesis and characterization of a Cu14 hydride cluster supported by neutral donor ligands.

The copper hydride clusters [Cu14 H12 (phen)6(PPh3)4][X]2 (X=Cl or OTf; OTf=trifluoromethanesulfonate, phen=1,10-phenanthroline) are obtained in good yields by the reaction of [(Ph3P)CuH]6 with phen, in the presence of a halide or pseudohalide source. The complex [Cu14H12 (phen)6(PPh3)4][Cl]2 reacts with CO2 in CH2Cl2 , in the presence of excess Ph3 P, to form the formate complex [(Ph3P)2Cu(κ(2)-O2CH)], along with [(phen)(Ph3 P)CuCl].

The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop.

The tumor suppressor p53 is a transcription factor that responds to cellular stresses by initiating cell cycle arrest or apoptosis. One transcriptional target of p53 is Mdm2, an E3 ubiquitin ligase that interacts with p53 to promote its proteasomal degradation in a negative feedback regulatory loop. Here we show that the wild-type p53-induced phosphatase 1 (Wip1), or PPM1D, downregulates p53 protein levels by stabilizing Mdm2 and facilitating its access to p53. Wip1 interacts with and dephosphorylates Mdm2 at serine 395, a site phosphorylated by the ATM kinase. Dephosphorylated Mdm2 has increased stability and affinity for p53, facilitating p53 ubiquitination and degradation. Thus, Wip1 acts as a gatekeeper in the Mdm2-p53 regulatory loop by stabilizing Mdm2 and promoting Mdm2-mediated proteolysis of p53.

Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells.

The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels, binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin (DXR) or Nutlin-3 (Nutlin) led to strikingly different p53 gene binding patterns based on chromatin immunoprecipitation with high-throughput sequencing experiments. Although two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was six times greater for Nutlin than DXR, expression changes induced by Nutlin were much less dramatic compared with DXR. Unexpectedly, the solvent dimethylsulphoxide (DMSO) alone induced p53 binding to many sites common to DXR; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification histone H3 lysine 4 trimethylation. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.

Mutant TP53 posttranslational modifications: challenges and opportunities.

The wild-type (WT) human p53 (TP53) tumor suppressor can be posttranslationally modified at over 60 of its 393 residues. These modifications contribute to changes in TP53 stability and in its activity as a transcription factor in response to a wide variety of intrinsic and extrinsic stresses in part through regulation of protein-protein and protein-DNA interactions. The TP53 gene frequently is mutated in cancers, and in contrast to most other tumor suppressors, the mutations are mostly missense often resulting in the accumulation of mutant (MUT) protein, which may have novel or altered functions. Most MUT TP53s can be posttranslationally modified at the same residues as in WT TP53. Strikingly, however, codons for modified residues are rarely mutated in human tumors, suggesting that TP53 modifications are not essential for tumor suppression activity. Nevertheless, these modifications might alter MUT TP53 activity and contribute to a gain-of-function leading to increased metastasis and tumor progression. Furthermore, many of the signal transduction pathways that result in TP53 modifications are altered or disrupted in cancers. Understanding the signaling pathways that result in TP53 modification and the functions of these modifications in both WT TP53 and its many MUT forms may contribute to more effective cancer therapies.

Oxidation of alcohols and activated alkanes with Lewis acid-activated TEMPO.

The reactivity of MCl3(η(1)-TEMPO) (M = Fe, 1; Al, 2; TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl) with a variety of alcohols, including 3,4-dimethoxybenzyl alcohol, 1-phenyl-2-phenoxyethanol, and 1,2-diphenyl-2-methoxyethanol, was investigated using NMR spectroscopy and mass spectrometry. Complex 1 was effective in cleanly converting these substrates to the corresponding aldehyde or ketone. Complex 2 was also able to oxidize these substrates; however, in a few instances the products of overoxidation were also observed. Oxidation of activated alkanes, such as xanthene, by 1 or 2 suggests that the reactions proceed via an initial 1-electron concerted proton-electron transfer (CPET) event. Finally, reaction of TEMPO with FeBr3 in Et2O results in the formation of a mixture of FeBr3(η(1)-TEMPOH) (23) and [FeBr2(η(1)-TEMPOH)]2(µ-O) (24), via oxidation of the solvent, Et2O.

Lipid-Encapsulated mRNAs Encoding Complex Fusion Proteins Potentiate Antitumor Immune Responses.

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Wild-type p53-induced phosphatase 1 dephosphorylates histone variant gamma-H2AX and suppresses DNA double strand break repair.

In response to DNA double strand breaks, the histone variant H2AX at the break site is phosphorylated at serine 139 by DNA damage sensor kinases such as ataxia telangiectasia-mutated, forming gamma-H2AX. This phosphorylation event is critical for sustained recruitment of other proteins to repair the break. After repair, restoration of the cell to a prestress state is associated with gamma-H2AX dephosphorylation and dissolution of gamma-H2AX-associated damage foci. The phosphatases PP2A and PP4 have previously been shown to dephosphorylate gamma-H2AX. Here, we demonstrate that the wild-type p53-induced phosphatase 1 (WIP1) also dephosphorylates gamma-H2AX at serine 139 in vitro and in vivo. Overexpression of WIP1 reduces formation of gamma-H2AX foci in response to ionizing and ultraviolet radiation and blocks recruitment of MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1) to DNA damage foci. Finally, these inhibitory effects of WIP1 on gamma-H2AX are accompanied by WIP1 suppression of DNA double strand break repair. Thus, WIP1 has a homeostatic role in reversing the effects of ataxia telangiectasia-mutated phosphorylation of H2AX.

Functional conservation of an insect odorant receptor gene across 250 million years of evolution.

Pest insects have a profound negative impact on agriculture and human health. Significant global losses of crops, stored agricultural products, timber and livestock can be attributed to damage and destruction by insects . Blood-feeding insects such as mosquitoes, flies and ticks transmit many of humanity's most devastating infectious diseases. Insect-borne diseases account for more than one million annual fatalities, and insect-associated illnesses surpass 300 million annual reported cases . The medical and economic impact of these animals can be ascribed in part to the sensitivity and selectivity of their olfactory systems, essential for location of their preferred plant and animal hosts.

The Cytidine Deaminase APOBEC3 Family Is Subject to Transcriptional Regulation by p53.

The APOBEC3 (A3) family of proteins are DNA cytidine deaminases that act as sentinels in the innate immune response against retroviral infections and are responsive to IFN. Recently, a few A3 genes were identified as potent enzymatic sources of mutations in several human cancers. Using human cancer cells and lymphocytes, we show that under stress conditions and immune challenges, all A3 genes are direct transcriptional targets of the tumor suppressor p53. Although the expression of most A3 genes (including A3C and A3H) was stimulated by the activation of p53, treatment with the DNA-damaging agent doxorubicin or the p53 stabilizer Nutlin led to repression of the A3B gene. Furthermore, p53 could enhance IFN type-I induction of A3 genes. Interestingly, overexpression of a group of tumor-associated p53 mutants in TP53-null cancer cells promoted A3B expression. These findings establish that the "guardian of the genome" role ascribed to p53 also extends to a unique component of the immune system, the A3 genes, thereby integrating human immune and chromosomal stress responses into an A3/p53 immune axis.Implications: Activated p53 can integrate chromosomal stresses and immune responses through its influence on expression of APOBEC3 genes, which are key components of the innate immune system that also influence genomic stability. Mol Cancer Res; 15(6); 735-44. ©2017 AACR.

The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3-v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression.