EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22.

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays.

Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA polymerase ß.

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid.

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF(2)-substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF(2)-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF(2) groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol beta) binding pocket by obtaining a 2.5 A resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg(2+) interaction over Watson-Crick hydrogen bonding was found and discussed.

Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

Catalytic asymmetric C-H insertions of rhodium(II) azavinyl carbenes.

A highly efficient enantioselective C-H insertion of azavinyl carbenes into unactivated alkanes has been developed. These transition metal carbenes are directly generated from readily available and stable 1-sulfonyl-1,2,3-triazoles in the presence of chiral Rh(II) carboxylates and are used for C-H functionalization of alkanes to access a variety of ß-chiral sulfonamides.

A new route to α-alkyl-α-fluoromethylenebisphosphonates.

A new route to α-alkyl-α-fluoromethylenebisphosphonates, 2 has been developed starting from commercially available tetraethyl fluoromethylenebisphosphonate (1), and alkyl halides using either caesium carbonate in DMF or sodium dimsyl. De-esterification of 2 provided biologically important α-alkyl-α-fluoromethylenebisphosphonic acid, 3, while alkoxide-induced carbon-phosphorus bond cleavage of 2 gave α-fluorophosphonates, 4, which are useful synthons in organic synthesis.

Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4.

BACKGROUND: Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). METHODS: We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. RESULTS: Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. CONCLUSION: Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. STATEMENT OF SIGNIFICANCE: CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as a Single Agent and in Combination with Checkpoint Inhibitors.

The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.

Discovery of a Potent and Selective CCR4 Antagonist That Inhibits Treg Trafficking into the Tumor Microenvironment.

Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Nucleophilic difluoromethylation and difluoromethylenation of aldehydes and ketones using diethyl difluoromethylphosphonate.

New methodology for difluoromethylation and difluoromethylenation of aldehydes and ketones based on nucleophilic fluorination using diethyl difluoromethylphosphonate (1) was developed.

A synthetic hydrogel for the high-throughput study of cell-ECM interactions.

It remains extremely challenging to dissect the cooperative influence of multiple extracellular matrix (ECM) parameters on cell behaviour. This stems in part from a lack of easily deployable strategies for the combinatorial variation of matrix biochemical and biophysical properties. Here we describe a simple, high-throughput platform based on light-modulated hyaluronic acid hydrogels that enables imposition of mutually independent and spatially continuous gradients of ligand density and substrate stiffness. We validate this system by showing that it can support mechanosensitive differentiation of mesenchymal stem cells. We also use it to show that the oncogenic microRNA, miR18a, is nonlinearly regulated by matrix stiffness and fibronectin density in glioma cells. The parallelization of experiments enabled by this platform allows condensation of studies that would normally require hundreds of independent hydrogels to a single substrate. This system is a highly accessible, high-throughput technique to study the combinatorial variation of biophysical and biochemical signals in a single experimental paradigm.

Stimuli-responsive electrodes detect oxidative stress and liver injury.

A digital point-of-care biosensor for measuring reactive oxygen species is presented based on novel reactive oxygen species responsive polymer-based electrodes. The biosensor is able to detect a drug-induced liver injury by monitoring the oxidative stress in the blood.

Reactivity of N-(1,2,4-triazolyl)-substituted 1,2,3-triazoles.

Synthetically useful rhodium(II) carbenes were obtained from N-(1,2,4-triazolyl)-substituted 1,2,3-triazoles and Rh(II) carboxylates. The electron-withdrawing 1,2,4-triazolyl group reveals the heretofore unknown reactivity of nonsulfonyl 1,2,3-triazoles, which exhibit the reactivity of diazo compounds. The resulting carbenes provide ready asymmetric access to secondary homoaminocyclopropanes (80-95% ee, dr >20:1) via reactions with olefins and also engage in efficient transannulation reactions with nitriles.