Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Naked mole-rat acid-sensing ion channel 3 forms nonfunctional homomers, but functional heteromers.

Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.

Field template-based design and biological evaluation of new sphingosine kinase 1 inhibitors.

PURPOSE: Sphingosine kinase 1 (SK1) is a protooncogenic enzyme expressed in many human tumours and is associated with chemoresistance and poor prognosis. It is a potent therapy target and its inhibition chemosensitises solid tumours. Despite recent advances in SK1 inhibitors synthesis and validation, their clinical safety and chemosensitising options are not well described. In this study, we have designed, synthesised and tested a new specific SK1 inhibitor with a low toxicity profile. METHODS: Field template molecular modelling was used for compound design. Lead compounds were tested in cell and mouse cancer models. RESULTS: Field template analysis of three known SK1 inhibitors, SKI-178, 12aa and SK1-I, was performed and compound screening identified six potential new SK1 inhibitors. SK1 activity assays in both cell-free and in vitro settings showed that two compounds were effective SK1 inhibitors. Compound SK-F has potently decreased cancer cell viability in vitro and sensitised mouse breast tumours to docetaxel (DTX) in vivo, without significant whole-body toxicity. CONCLUSION: Through field template screening, we have identified a new SK1 inhibitor, SK-F, which demonstrated antitumour activity in vitro and in vivo without overt toxicity when combined with DTX.

New FTY720-docetaxel nanoparticle therapy overcomes FTY720-induced lymphopenia and inhibits metastatic breast tumour growth.

PURPOSE: Combining molecular therapies with chemotherapy may offer an improved clinical outcome for chemoresistant tumours. Sphingosine-1-phosphate (S1P) receptor antagonist and sphingosine kinase 1 (SK1) inhibitor FTY720 (FTY) has promising anticancer properties, however, it causes systemic lymphopenia which impairs its use in cancer patients. In this study, we developed a nanoparticle (NP) combining docetaxel (DTX) and FTY for enhanced anticancer effect, targeted tumour delivery and reduced systemic toxicity. METHODS: Docetaxel, FTY and glucosamine were covalently conjugated to poly(lactic-co-glycolic acid) (PLGA). NPs were characterised by dynamic light scattering and electron microscopy. The cellular uptake, cytotoxicity and in vivo antitumor efficacy of CNPs were evaluated. RESULTS: We show for the first time that in triple negative breast cancer cells FTY provides chemosensitisation to DTX, allowing a four-fold reduction in the effective dose. We have encapsulated both drugs in PLGA complex NPs (CNPs), with narrow size distribution of ~ 100 nm and excellent cancer cell uptake providing sequential, sustained release of FTY and DTX. In triple negative breast cancer cells and mouse breast cancer models, CNPs had similar efficacy to systemic free therapies, but allowed an effective drug dose reduction. Application of CNPs has significantly reversed chemotherapy side effects such as weight loss, liver toxicity and, most notably, lymphopenia. CONCLUSIONS: We show for the first time the DTX chemosensitising effects of FTY in triple negative breast cancer. We further demonstrate that encapsulation of free drugs in CNPs can improve targeting, provide low off-target toxicity and most importantly reduce FTY-induced lymphopenia, offering potential therapeutic use of FTY in clinical cancer treatment.

Structural domains underlying the activation of acid-sensing ion channel 2a.

The acid-sensing ion channels (ASICs) are a family of ion channels expressed throughout the mammalian nervous system. The principal activator of ASICs is extracellular protons, and ASICs have been demonstrated to play a significant role in many physiologic and pathophysiologic processes, including synaptic transmission, nociception, and fear. However, not all ASICs are proton-sensitive: ASIC2a is activated by acid, whereas its splice variant ASIC2b is not. We made a series of chimeric ASIC2 proteins, and using whole-cell electrophysiology we have identified the minimal region of the ASIC2a extracellular domain that is required for ASIC2 proton activation: the first 87 amino acids after transmembrane domain 1. We next examined the function of different domains within the ASIC2b N-terminus and identified a region proximal to the first transmembrane domain that confers tachyphylaxis upon ASIC2a. We have thus identified domains of ASIC2 that are crucial to channel function and may be important for the function of other members of the ASIC family.

Activation-induced structural change in the GluN1/GluN3A excitatory glycine receptor.

Unlike GluN2-containing N-methyl-d-aspartate (NMDA) receptors, which require both glycine and glutamate for activation, receptors composed of GluN1 and GluN3 subunits are activated by glycine alone. Here, we used atomic force microscopy (AFM) imaging to examine the response to activation of the GluN1/GluN3A excitatory glycine receptor. GluN1 and GluN3A subunits were shown to interact intimately within transfected tsA 201 cells. Isolated GluN1/GluN3A receptors integrated into lipid bilayers responded to addition of either glycine or d-serine, but not glutamate, with a ∼1 nm reduction in height of the extracellular domain. The height reduction in response to glycine was abolished by the glycine antagonist 5,7-dichlorokynurenic acid. Our results represent the first demonstration of the effect of activation on the conformation of this receptor.

An in vivo and in vitro analysis of free radical scavenging potential possessed by Desmodium gangeticum chloroform root extract: interpretation by gsms.

In the present study, the effect of DG chloroform root extract was assessed on isolated rat heart and in-vitro antioxidant models. Ischemia reperfusion injury was experimentally induced by using Langendroff apparatus. The free radical scavenging potential was studied in vitro by using different antioxidant models such as DPPH, super oxide scavenging activity, hydroxide scavenging activity and nitric oxide scavenging activity. Both experimental approaches not only substantiate its antioxidant potential but also the cardio-protection imparted by the extract. The cardio-stimulatory effects were investigated for the extract by treating it as a pre-conditioning agent against myocardial ischemia reperfusion injury. The improved antioxidant status of the myocardium indirectly predicts reduced oxidative stress mediated by ischemic reperfusion with evident reduction of infarct size determined by cardiac marker protein. These findings indicate that DG chloroform root extract may possess therapeutic potential against ischemia reperfusion injury.

Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer.

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Rationally engineered polymeric cisplatin nanoparticles for improved antitumor efficacy.

The use of cisplatin, a first line chemotherapy for most cancers, is dose-limited due to nephrotoxicity. While this toxicity can be addressed through nanotechnology, previous attempts at engineering cisplatin nanoparticles have been limited by the impact on the potency of cisplatin. Here we report the rational engineering of a novel cisplatin nanoparticle by harnessing a novel polyethylene glycol-functionalized poly-isobutylene-maleic acid (PEG-PIMA) copolymer, which can complex with cis-platinum (II) through a monocarboxylato and a coordinate bond. We show that this complex self-assembles into a nanoparticle, and exhibits an IC(50) = 0.77 ± 0.11 µM comparable to that of free cisplatin (IC(50) = 0.44 ± 0.09 µM). The nanoparticles are internalized into the endolysosomal compartment of cancer cells, and release cisplatin in a pH-dependent manner. Furthermore, the nanoparticles exhibit significantly improved antitumor efficacy in a 4T1 breast cancer model in vivo, with limited nephrotoxicity, which can be explained by preferential biodistribution in the tumor with reduced kidney concentrations. Our results suggest that the PEG-PIMA-cisplatin nanoparticle can emerge as an attractive solution to the challenges in cisplatin chemotherapy.

Core shell lipid-polymer hybrid nanoparticles with combined docetaxel and molecular targeted therapy for the treatment of metastatic prostate cancer.

Many prostate cancers relapse after initial chemotherapy treatment. Combining molecular and chemotherapy together with encapsulation of drugs in nanocarriers provides effective drug delivery and toxicity reduction. We developed core shell lipid-polymer hybrid nanoparticles (CSLPHNPs) with poly (lactic-co-glycolic acid) (PLGA) core and lipid layer containing docetaxel and clinically used inhibitor of sphingosine kinase 1 (SK1) FTY720 (fingolimod). We show for the first time that FTY720 (both free and in CSLPHNPs) re-sensitizes castrate resistant prostate cancer cells and tumors to docetaxel, allowing a four-fold reduction in effective dose. Our CSLPHNPs showed high serum stability and a long shelf life. CSLPHNPs demonstrated a steady uptake by tumor cells, sustained intracellular drug release and in vitro efficacy superior to free therapies. In a mouse model of human prostate cancer, CSLPHNPs showed excellent tumor targeting and significantly lower side effects compared to free drugs, importantly, reversing lymphopenia induced by FTY720. Overall, we demonstrate that nanoparticle encapsulation can improve targeting, provide low off-target toxicity and most importantly reduce FTY720-induced lymphopenia, suggesting its potential use in clinical cancer treatment.

Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1.

Sigma1 receptors (σ1Rs) are expressed widely; they bind diverse ligands, including psychotropic drugs and steroids, regulate many ion channels, and are implicated in cancer and addiction. It is not known how σ1Rs exert such varied effects. We demonstrate that σ1Rs inhibit store-operated Ca(2+)entry (SOCE), a major Ca(2+)influx pathway, and reduce the Ca(2+)content of the intracellular stores. SOCE was inhibited by expression of σ1R or an agonist of σ1R and enhanced by loss of σ1R or an antagonist. Within the endoplasmic reticulum (ER), σ1R associated with STIM1, the ER Ca(2+)sensor that regulates SOCE. This interaction was modulated by σ1R ligands. After depletion of Ca(2+)stores, σ1R accompanied STIM1 to ER-plasma membrane (PM) junctions where STIM1 stimulated opening of the Ca(2+)channel, Orai1. The association of STIM1 with σ1R slowed the recruitment of STIM1 to ER-PM junctions and reduced binding of STIM1 to PM Orai1. We conclude that σ1R attenuates STIM1 coupling to Orai1 and thereby inhibits SOCE.

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry.

Depletion of Ca(2+) from the endoplasmic reticulum (ER) lumen triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca(2+) store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.

Therapeutic potential of targeting SK1 in human cancers.

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipids ceramide and sphingosine into the antiapoptotic lipid sphingosine-1-phosphate and activates the signal transduction pathways that lead to cell proliferation, migration, the activation of the inflammatory response, and the impairment of apoptosis. There is compelling evidence that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization, and metastatic spread. High levels of SK1 expression or activity have been associated with a poor prognosis in several human cancers. Recent studies using cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of chemotherapy and radiotherapy; however, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery of SK1 inhibiting properties of a clinically approved drug FTY720 (Fingolimod), SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors may follow soon. This review provides an overview of the SK1 signaling, its relevance to cancer progression, and the potential clinical significance of targeting SK1 for improved local or systemic control of human cancers.