Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction.

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.

Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.

Flualprazolam distribution in postmortem samples.

The constant emergence of novel psychoactive substances is troubling to both public health officials and legislators. Additionally, sufficient data collection for each new compound can take months up to years. Flualprazolam, a triazolobenzodiazepine, quickly garnered attention as a sedative drug that likely expresses adverse reactions similarly to alprazolam. This study focuses on the distribution of flualprazolam in multiple common postmortem matrices. Central blood, vitreous humor, liver homogenate, brain homogenate, gastric contents, and urine samples from death investigation cases were quantitated when available. Samples were screened with liquid chromatography quadrupole time-of-flight with limit of detection set at 4 ng/ml and quantitated on liquid chromatography tandem mass spectrometry, with concentration range from 4 to 256 ng/ml. From August 2018 to September 2020, 24 central blood samples were quantitated for flualprazolam. Central bloods of 22 cases had concentrations above the limit of quantitation. The average flualprazolam central blood concentration was 16.3 ng/ml with a median of 9.95 ng/ml (4.24-48.0). Additional analyses for unconjugated flualprazolam were performed on at a total of 15 urine samples ( x ¯  = 14.4, 4.07-36.1 ng/ml), 23 brain homogenates ( x ¯  = 23.2, 3.99-69.3 ng/g), 23 liver homogenates ( x ¯  = 50.7, 13.6-156 ng/g), five vitreous humor samples ( x ¯  = 7.70, 4.03-12 ng/ml), and 12 gastric contents samples ( x ¯  = 0.36, 0.02-2.51 mg). The cause of death for 13 of the 24 cases listed flualprazolam as a contributing factor of death.

Endogenous Gamma-Hydroxybutyrate in Postmortem Samples.

Gamma-hydroxybutyrate (GHB) is a naturally occurring molecule present in the human body as a catabolite of the neurotransmitter gamma-aminobutyrate (GABA). In the USA, GHB has a history of being manufactured illicitly and abused, with misguided proposed benefits for the body-building community and a persistent party drug with reported GHB overdoses occurring worldwide. The interpretation of GHB in postmortem biological fluids is complicated by the endogenous nature of the molecule. Analysis often requires more than one biological matrix to detect exogenous exposure, typically in urine. The analysis is further complicated by the endogenous de novo production of GHB in postmortem specimens. This work sought to examine the prevalence of endogenous GHB concentrations in postmortem toxicology samples from Orange County, CA, and to establish suitable in-house secondary matrices to confirm or rule out exogenous GHB exposure. A total of 348 postmortem heart blood samples were randomly selected and analyzed for GHB using liquid-liquid extraction followed by gas chromatography-mass spectrometry with selective ion monitoring and GHB-d6 as an internal standard. Of the 348 cases analyzed, 39 cases resulted in positive GHB detection with the median concentration of 22.45 mg/L (10.4-62.16 mg/L). None of the positive samples had suspected GHB ingestion or usage from the case report. GHB concentrations were then examined in secondary matrices collected at autopsy from the positive cases that included (when available) peripheral blood, urine, vitreous humor, liver homogenate and brain homogenate. Within the secondary matrices, GHB levels in peripheral blood compared to that of heart blood, while liver homogenate levels were variable. Quantifiable GHB levels were not identified in vitreous humor and brain homogenate samples. Our findings reaffirm the importance of multi-matrix analysis in postmortem toxicology and further confirm the utility of vitreous humor and brain tissue to distinguish exogenous GHB exposure from endogenous production.

Role of the CXCL8-CXCR1/2 Axis in Cancer and Inflammatory Diseases.

The chemokine receptors CXCR1/2 and their ligand CXCL8 are essential for the activation and trafficking of inflammatory mediators as well as tumor progression and metastasis. The CXCL8-CXCR1/2 signaling axis is involved in the pathogenesis of several diseases including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis and cancer. Interaction between CXCL8 secreted by select cancer cells and CXCR1/2 in the tumor microenvironment is critical for cancer progression and metastasis. The CXCL8-CXCR1/2 axis may play an important role in tumor progression and metastasis by regulating cancer stem cell (CSC) proliferation and self-renewal. During the past two decades, several small-molecule CXCR1/2 inhibitors, CXCL8 releasing inhibitors, and neutralizing antibodies against CXCL8 and CXCR1/2 have been reported. As single agents, such inhibitors are expected to be efficacious in various inflammatory diseases. Several preclinical studies suggest that combination of CXCR1/2 inhibitors along with other targeted therapies, chemotherapies, and immunotherapy may be effective in treating select cancers. Currently, several of these inhibitors are in advanced clinical trials for COPD, asthma, and metastatic breast cancer. In this review, we provide a comprehensive analysis of the role of the CXCL8-CXCR1/2 axis and select genes co-expressed in this pathway in disease progression. We also discuss the latest progress in developing small-molecule drugs targeting this pathway.

Discovery of Novel CXCR2 Inhibitors Using Ligand-Based Pharmacophore Models.

The chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. CXCR2 and its ligand, CXCL8, are implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and cancer. Though the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents has been pursued by pharmaceutical companies within the past decade, there are currently no clinically approved CXCR2 inhibitors. A pharmacophore model based on previously reported CXCR2 antagonists was developed to screen a database of commercially available compounds. Small-molecule compounds identified from the pharmacophore screening were selected for in vitro screening in a cell-based CXCR2-mediated ß-arrestin-2 recruitment assay and further characterized in several cell-based assays and lipopolysaccharide (LPS)-induced lung inflammation studies in mice. CX compounds identified from pharmacophore modeling inhibited cell migration, lung and colon cancer cell proliferation, and colony formation. Mechanistic studies of CX4152 showed that this compound inhibits CXCR2 signaling through downregulation of surface CXCR2. Additionally, CX4152 significantly inhibits CXCL8-mediated neutrophil migration and LPS-induced lung inflammation in mice. Using a CXCR2-inhibitor-based pharmacophore model, we identified a novel set of sulfonamides from a diverse library of small molecules. These compounds inhibit CXCR2/ß-arrestin-2 association, cell migration and proliferation, and acute inflammation in mouse models. CX compounds identified from our pharmacophore models are potential leads for further optimization and development as anti-inflammatory and anticancer agents.

Pyrimidine-based compounds modulate CXCR2-mediated signaling and receptor turnover.

Chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Because of its role in inflammation, it has been implicated in a number of immune-mediated inflammatory diseases such as psoriasis, arthritis, COPD, cystic fibrosis, asthma, and various types of cancer. CXCR2 and its ligands are up-regulated in cancer cells as well as the tumor microenvironment, promoting tumor growth, angiogenesis, and invasiveness. Although pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the last decades, there are currently no clinically approved CXCR2 inhibitors. Using a high-throughput, cell-based assay specific for CXCR2, we screened an in-house library of structurally diverse compounds and identified a class of pyrimidine-based compounds that alter CXCR2-mediated second messenger signaling. Our lead compound, CX797, inhibited IL8-mediated cAMP signaling and receptor degradation while specifically up-regulating IL8-mediated ß-arrestin-2 recruitment. CX797 also inhibited IL8-mediated cell migration. Mechanistic comparison of CX797 and a previously reported CXCR2 inhibitor, SB265610, show these two classes of compounds have a distinct mechanism of action on CXCR2.

A novel phenylcyclohex-1-enecarbothioamide derivative inhibits CXCL8-mediated chemotaxis through selective regulation of CXCR2-mediated signalling.

BACKGROUND AND PURPOSE: Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function. EXPERIMENTAL APPROACH: We used the CXCR2 Tango(TM) assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338. KEY RESULTS: CX4338 selectively inhibited CXCR2-mediated recruitment of ß-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice. CONCLUSIONS AND IMPLICATIONS: A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated ß-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.

Overcoming doxorubicin-resistance in the NCI/ADR-RES model cancer cell line by novel anthracene-9,10-dione derivatives.

Overcoming drug resistance with remarkable cytotoxic activity by anthracene-9,10-dione derivatives would offer a potential therapeutic strategy. In this study, we report the synthesis and the cytotoxicity of a novel set of anthraquninones. (4-(4-Aminobenzylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl-4-methylbenzenesulfonate) (3) has excellent in vitro cytotoxicity against doxorubicin-resistant cancer cell line (IC50=0.8 µM), 20-fold higher than doxorubicin. The cytotoxic effect via G2/M arrest does not appear to be ROS.

Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity.

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50≈20 nM) and an efficient HIV-1 cell-entry blocker (EC50≈2 nM). This cyclotide also showed high stability in human serum, thereby providing a promising lead compound for the design of a novel type of peptide-based anticancer and anti-HIV-1 therapeutics.

Screening and association testing of common coding variation in steroid hormone receptor co-activator and co-repressor genes in relation to breast cancer risk: the Multiethnic Cohort.

BACKGROUND: Only a limited number of studies have performed comprehensive investigations of coding variation in relation to breast cancer risk. Given the established role of estrogens in breast cancer, we hypothesized that coding variation in steroid receptor coactivator and corepressor genes may alter inter-individual response to estrogen and serve as markers of breast cancer risk. METHODS: We sequenced the coding exons of 17 genes (EP300, CCND1, NME1, NCOA1, NCOA2, NCOA3, SMARCA4, SMARCA2, CARM1, FOXA1, MPG, NCOR1, NCOR2, CALCOCO1, PRMT1, PPARBP and CREBBP) suggested to influence transcriptional activation by steroid hormone receptors in a multiethnic panel of women with advanced breast cancer (n = 95): African Americans, Latinos, Japanese, Native Hawaiians and European Americans. Association testing of validated coding variants was conducted in a breast cancer case-control study (1,612 invasive cases and 1,961 controls) nested in the Multiethnic Cohort. We used logistic regression to estimate odds ratios for allelic effects in ethnic-pooled analyses as well as in subgroups defined by disease stage and steroid hormone receptor status. We also investigated effect modification by established breast cancer risk factors that are associated with steroid hormone exposure. RESULTS: We identified 45 coding variants with frequencies > or = 1% in any one ethnic group (43 non-synonymous variants). We observed nominally significant positive associations with two coding variants in ethnic-pooled analyses (NCOR2: His52Arg, OR = 1.79; 95% CI, 1.05-3.05; CALCOCO1: Arg12His, OR = 2.29; 95% CI, 1.00-5.26). A small number of variants were associated with risk in disease subgroup analyses and we observed no strong evidence of effect modification by breast cancer risk factors. Based on the large number of statistical tests conducted in this study, the nominally significant associations that we observed may be due to chance, and will need to be confirmed in other studies. CONCLUSION: Our findings suggest that common coding variation in these candidate genes do not make a substantial contribution to breast cancer risk in the general population. Cataloging and testing of coding variants in coactivator and corepressor genes should continue and may serve as a valuable resource for investigations of other hormone-related phenotypes, such as inter-individual response to hormonal therapies used for cancer treatment and prevention.