Beta-blockade enhances anthracycline control of metastasis in triple-negative breast cancer.

Beta-adrenergic blockade has been associated with improved cancer survival in patients with triple-negative breast cancer (TNBC), but the mechanisms of these effects remain unclear. In clinical epidemiological analyses, we identified a relationship between beta-blocker use and anthracycline chemotherapy in protecting against TNBC progression, disease recurrence, and mortality. We recapitulated the effect of beta-blockade on anthracycline efficacy in xenograft mouse models of TNBC. In metastatic 4T1.2 and MDA-MB-231 mouse models of TNBC, beta-blockade improved the efficacy of the anthracycline doxorubicin by reducing metastatic development. We found that anthracycline chemotherapy alone, in the absence of beta-blockade, increased sympathetic nerve fiber activity and norepinephrine concentration in mammary tumors through the induction of nerve growth factor (NGF) by tumor cells. Moreover, using preclinical models and clinical samples, we found that anthracycline chemotherapy up-regulated ß2-adrenoceptor expression and amplified receptor signaling in tumor cells. Neurotoxin inhibition of sympathetic neural signaling in mammary tumors using 6-hydroxydopamine or genetic deletion of NGF or ß2-adrenoceptor in tumor cells enhanced the therapeutic effect of anthracycline chemotherapy by reducing metastasis in xenograft mouse models. These findings reveal a neuromodulatory effect of anthracycline chemotherapy that undermines its potential therapeutic impact, which can be overcome by inhibiting ß2-adrenergic signaling in the tumor microenvironment. Supplementing anthracycline chemotherapy with adjunctive ß2-adrenergic antagonists represents a potential therapeutic strategy for enhancing the clinical management of TNBC.

Discovery of TAK-925 as a Potent, Selective, and Brain-Penetrant Orexin 2 Receptor Agonist.

TAK-925, a potent, selective, and brain-penetrant orexin 2 receptor (OX2R) agonist, [methyl (2R,3S)-3-((methylsulfonyl)amino)-2-(((cis-4-phenylcyclohexyl)oxy)methyl)piperidine-1-carboxylate, 16], was identified through the optimization of compound 2, which was discovered by a high throughput screening (HTS) campaign. Subcutaneous administration of compound 16 produced wake-promoting effects in mice during the sleep phase. Compound 16 (TAK-925) is being developed for the treatment of narcolepsy and other related disorders.

Dynamic alterations of circulating T lymphocytes and the clinical response in patients with head and neck squamous cell carcinoma treated with nivolumab.

Cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). However, only approximately 20-30% of patients with recurrent/metastatic (R/M) HNSCC benefit. Moreover, the mechanisms underlying the response to ICIs remain unclear. We investigated the proportion, activation status, and expression level of immune checkpoint molecules in circulating T cell subsets in R/M HNSCC patients treated with nivolumab using flow cytometry and mass cytometry, and then determined whether treatment response was associated with these values. We also assessed the changes in the frequency of tumor-associated antigens, MAGE-A4 and p53, -specific T cells prior to and after nivolumab treatment using the IFN-γ ELISPOT assay. The proportion of activated CD4+ and CD8+ TEMRA cells significantly increased in the disease-controlled patients but not in disease-progressed patients. As expected, the expression of PD-1 in T cells markedly decreased regardless of the therapeutic response. Meanwhile, T cell immunoglobulin mucin-3 expression on CD8+ T cells was significantly higher in patients with disease progression than in disease-controlled patients after treatment. The frequency of the tumor-associated antigens, MAGE-A4- and p53-specific T cells, was not correlated with clinical responses; however, in the disease-controlled patients, the frequency of MAGE-A4-specific T cells was significantly augmented. We concluded that in R/M HNSCC patients treated with nivolumab, circulating T cells show dynamic alterations depending on treatment efficacy. An analysis of the immunokinetics of circulating T cells could thus provide new insights into rational therapeutic strategies in cancer immunotherapy for HNSCC.

MYC Causes Multiple Myeloma Progression via Attenuating TP53-Induced MicroRNA-34 Expression.

MicroRNAs (miRNAs and miRs) are small (19-25 base pairs) non-coding RNAs with the ability to modulate gene expression. Previously, we showed that the miR-34 family is downregulated in multiple myeloma (MM) as the cancer progressed. In this study, we aimed to clarify the mechanism of miRNA dysregulation in MM. We focused particularly on the interaction between MYC and the TP53-miR34 axis because there is a discrepancy between increased TP53 and decreased miR-34 expressions in MM. Using the nutlin-3 or Tet-on systems, we caused wild-type (WT) p53 protein accumulation in human MM cell lines (HMCLs) and observed upregulated miR-34 expression. Next, we found that treatment with an Myc inhibitor alone did not affect miR-34 expression levels, but when it was coupled with p53 accumulation, miR-34 expression increased. In contrast, forced MYC activation by the MYC-ER system reduced nutlin-3-induced miR-34 expression. We also observed that TP53 and MYC were negatively correlated with mature miR-34 expressions in the plasma cells of patients with MM. Our results suggest that MYC participates in the suppression of p53-dependent miRNA expressions. Because miRNA expression suppresses tumors, its inhibition leads to MM development and malignant transformation.

Prognostic value of tumor-infiltrating lymphocytes in estrogen receptor-positive and human epidermal growth factor receptor 2-negative breast cancer.

Tumor-infiltrating lymphocytes (TILs) are a significant prognostic factor in triple-negative breast cancer. However, the clinicopathological significance of TILs in estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer remains unclear. The purpose of the present study was to evaluate the role of TILs in the prognosis of ER-positive and HER2-negative breast cancer. A total of 65 consecutive patients with ER-positive and HER2-negative breast cancer were examined. TILs in stromal tissue (str-TILs) were graded using the International TILs Working Group criteria. The association between several clinicopathological factors and TIL grade were investigated, and the prognostic impact of TILs was compared between luminal A-like and luminal B-like breast cancer. A total of 51 patients (78.5%) had low-grade (0-10%), 11 (16.9%) had intermediate (10-40%) and 3 (4.6%) had high-grade (40-90%) str-TIL levels. There was a significant association between high levels of Ki67 expression and a high str-TIL count. Relapse-free survival was significantly worse in patients with luminal B-like cancer compared with that in patients with luminal A-like cancer. Patients with an intermediate or high str-TIL count had a better prognosis compared with those with a low str-TIL count. All patients with luminal B-like cancer and intermediate or high str-TIL levels developed no recurrence during follow-up. In conclusion, there was a significant correlation between high-grade str-TIL levels and high tumor cell proliferation rate, as well as high levels of Ki67 expression.

In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms.

The evaluation of antitumor immune responses is essential for immune monitoring to predict clinical outcomes as well as treatment efficacies in cancer patients. In this study, we produced two tumor antigen (TA) proteins, melanoma antigen family A4 and wild type p53, using TG silkworm systems and evaluated anti-TA-specific immune responses by enzyme-linked immunosorbent spot assays in patients with head and neck cancer. Eleven (61.1%) of 18 patients showed significant IFN-γ production in response to at least one TA; however, the presence of TA-specific immune responses did not significantly contribute to better prognosis (overall survival, p = 0.1768; progression-free survival, p = 0.4507). Further studies will need to be performed on a larger scale to better assess the clinical significance of these systems. The production of multiple TA proteins may provide new avenues for the development of immunotherapeutic strategies to stimulate a potent and specific immune response against tumor cells as well as precise assessment of antitumor immune responses in cancer patients.

Discovery of a Novel and Brain-Penetrant O-GlcNAcase Inhibitor via Virtual Screening, Structure-Based Analysis, and Rational Lead Optimization.

O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of tauopathies.

Long Noncoding RNA PVT1 Is Regulated by Bromodomain Protein BRD4 in Multiple Myeloma and Is Associated with Disease Progression.

Long noncoding RNAs (lncRNAs) are deregulated in human cancers and are associated with disease progression. Plasmacytoma Variant Translocation 1 (PVT1), a lncRNA, is located adjacent to the gene MYC, which has been linked to multiple myeloma (MM). PVT1 is expressed in MM and is associated with carcinogenesis. However, its role and regulation remain uncertain. We examined PVT1/MYC expression using real-time PCR in plasma cells purified from 59 monoclonal gammopathy of undetermined significance (MGUS) and 140 MM patients. The MM cell lines KMS11, KMS12PE, OPM2, and RPMI8226 were treated with JQ1, an MYC super-enhancer inhibitor, or MYC inhibitor 10058-F4. The expression levels of PVT1 and MYC were significantly higher in MM than in MGUS (p < 0.0001) and were positively correlated with disease progression (r = 0.394, p < 0.0001). JQ1 inhibited cell proliferation and decreased the expression levels of MYC and PVT1. However, 10054-F4 did not alter the expression level of PVT1. The positive correlation between MYC and PVT1 in patients, the synchronous downregulation of MYC and PVT1 by JQ1, and the lack of effect of the MYC inhibitor on PVT1 expression suggest that the expression of these two genes is co-regulated by a super-enhancer. Cooperative effects between these two genes may contribute to MM pathogenesis and progression.

Integrin α7 and Extracellular Matrix Laminin 211 Interaction Promotes Proliferation of Acute Myeloid Leukemia Cells and Is Associated with Granulocytic Sarcoma.

Acute myeloid leukemia (AML) with granulocytic sarcoma (GS) is characterized by poor prognosis; however, its underlying mechanism is unclear. Bone marrow samples from 64 AML patients (9 with GS and 55 without GS) together with AML cell lines PL21, THP1, HL60, Kasumi-1, and KG-1 were used to elucidate the pathology of AML with GS. RNA-Seq analyses were performed on samples from seven AML patients with or without GS. Gene set enrichment analyses revealed significantly upregulated candidates on the cell surface of the GS group. Expression of the adhesion integrin α7 (ITGA7) was significantly higher in the GS group, as seen by RT-qPCR (p = 0.00188) and immunohistochemistry of bone marrow formalin-fixed, paraffin-embedded (FFPE) specimens. Flow cytometry revealed enhanced proliferation of PL21 and THP1 cells containing surface ITGA7 in the presence of laminin 211 and stimulated ERK phosphorylation; this effect was abrogated following ITGA7 knockdown or ERK inhibition. Overall, high ITGA7 expression was associated with poor patient survival (p = 0.0477). In summary, ITGA7 is highly expressed in AML with GS, and its ligand (laminin 211) stimulates cell proliferation through ERK signaling. This is the first study demonstrating the role of integrin α7 and extracellular matrix interactions in AML cell proliferation and extramedullary disease development.

The Role and Function of microRNA in the Pathogenesis of Multiple Myeloma.

Recently, attention has been drawn to the role of non-coding regions of the genome in cancer pathogenesis. MicroRNAs (miRNAs) are small non-coding RNAs with 19-25 bases of length that control gene expression by destroying messenger RNA or inhibiting its translation. In multiple myeloma (MM), the expression of several miRNAs, such as miR-15a and miR-16, is markedly decreased and their target genes upregulated, suggesting their role as tumor-suppressing miRNAs. In contrast, miRNAs such as miR-21 and miR-221 are highly expressed and function as oncogenes (oncomiRs). In addition, several miRNAs, such as those belonging to the miR-34 family, are transcriptional targets of p53 and mediate its tumor-suppressive functions. Many miRNAs are associated with drug resistance, and the modulation of their expression or activity might be explored to reverse it. Moreover, miRNA expression patterns in either MM cells or serum exosomes have been shown to be good prognostic markers. miRNA regulation mechanisms have not been fully elucidated. Many miRNAs are epigenetically controlled by DNA methylation and histone modification, and others regulate the expression of epigenetic modifiers, indicating that miRNA and other epigenetic effectors are part of a network. In this review, we outlined the roles of miRNAs in MM and their potential to predict MM prognosis and develop novel therapies.

Clinical Features, Molecular Genetics, and Long-Term Outcome in Congenital Chloride Diarrhea: A Nationwide Study in Japan.

OBJECTIVE: To clarify clinical and genetic features of Japanese children with congenital chloride diarrhea (CCD). STUDY DESIGN: This was a multi-institutional, retrospective survey of 616 pediatric centers in Japan with identified patients with CCD between 2014 and 2018. Mutations involving SLC26A3 were detected by Sanger sequencing. RESULTS: Thirteen patients met all entry criteria including mutations in SLC26A3, and 14 patients satisfied clinical diagnostic criteria. Homozygous or compound heterozygous mutations in SLC26A3, including 6 novel mutations, were identified in 13 of these 14 patients (93%). The most common (detected in 7 of 13) was c.2063-1g>t. Median age at diagnosis was 1 day. Nine of the patients meeting all criteria were diagnosed as neonates (69%). Median follow-up duration was 10 years. When studied, 8 patients had <5 stools daily (62%), and all had fewer than in infancy. Only 1 patient had nephrocalcinosis, and 3 (23%) had mild chronic kidney disease. Neurodevelopment was generally good; only 1 patient required special education. Five patients (38%) received long-term sodium, potassium, and chloride supplementation. CONCLUSIONS: Early fetal ultrasound diagnosis and prompt long-term sodium, potassium, and chloride supplementation were common management features. Genetic analysis of SLC26A3 provided definitive diagnosis of CCD. In contrast with previously reported localities, c.2063-1g>t might be a founder mutation in East Asia.

Optimal step-down approach for pediatric asthma controlled by salmeterol/fluticasone: A randomized, controlled trial (OSCAR study).

BACKGROUND: Several guidelines, including the Japanese Pediatric Guideline for the Treatment and Management of Asthma (JPGL), recommend salmeterol/fluticasone combination therapy (SFC) as step 3 to 4 treatment for moderate to severe asthma. However, the optimal step-down approach to SFC remains unclear. In the current study, we examined step-down approaches in asthmatic children whose symptoms had been stabilized by SFC 100/200 µg/day. METHODS: This randomized, multicenter, open-label, parallel-group study was conducted over 12 weeks. For step-down therapy, subjects aged 5-15 years were randomly assigned to an SFC group (25/50 µg b.i.d.) or an FP group (100 µg b.i.d.), and treated for 12 weeks. Childhood Asthma Control Test (C-ACT) scores, lung function, and exhaled nitric oxide (FeNO) levels were monitored. RESULTS: Of 131 enrolled subjects, 128 completed the study and were included in the analysis. Decreases in % peak expiratory flow rate and % forced expiratory flow at 50% of vital capacity (V50) were observed in the FP group at each time point. There was a significant difference between the two groups for the change in %V50 from its previous value at each time point. There were no significant changes in FeNO levels (range 15-20 ppb) or C-ACT scores (∼26 points) within or between groups. CONCLUSIONS: A high level of asthma control was maintained with both approaches. The use of SFC step-down resulted in somewhat better respiratory function, with no worsening of airway inflammation. However, halving the dose of SFC and switching to FP alone are both optimal step-down approaches.

Effects of transdermal tulobuterol in pediatric asthma patients on long-term leukotriene receptor antagonist therapy: results of a randomized, open-label, multicenter clinical trial in Japanese children aged 4-12 years.

BACKGROUND: Few studies have examined the efficacy or safety of a transdermal ß(2) agonist as add-on medication to long-term leukotriene receptor antagonist (LTRA) therapy in pediatric asthma patients. METHODS: In this randomized, open-label, multicenter clinical trial, children aged 4-12 years on long-term LTRA therapy were treated with tulobuterol patches (1-2mg daily) or oral sustained-release theophylline (usual dose, 4-5mg/kg daily) for 4 weeks. LTRAs were continued throughout the trial. Outcomes included volume of peak expiratory flow (% PEF), fractional exhaled nitric oxide (FeNO), clinical symptoms and adverse events. RESULTS: Thirty-three and 31 patients were treated with tulobuterol patches and theophylline, respectively. % PEF measured in the morning and before bedtime was significantly higher at all times in the treatment period compared with baseline in the tulobuterol patch group (p < 0.001), and was significantly higher in the tulobuterol patch group compared with the theophylline group. FeNO was similar and unchanged from baseline in both groups. There were no drug-related adverse events in either group. CONCLUSIONS: These results suggest that short-term use of a transdermal ß(2) agonist is an effective therapy for pediatric asthma without inducing airway inflammation in children on long-term LTRA therapy.

Effects of Transdermal Tulobuterol in Pediatric Asthma Patients on Long-Term Leukotriene Receptor Antagonist Therapy: Results of a Randomized, Open-Label, Multicenter Clinical Trial in Japanese Children Aged 4-12 Years.

BACKGROUND: Few studies have examined the efficacy or safety of a transdermal ß2 agonist as add-on medicationto long-term leukotriene receptor antagonist (LTRA) therapy in pediatric asthma patients. METHODS: In this randomized, open-label, multicenter clinical trial, children aged 4-12 years on long-term LTRA therapy were treated with tulobuterol patches (1-2 mg daily) or oral sustained-release theophylline (usual dose, 4-5 mg_kg daily) for 4 weeks. LTRAs were continued throughout the trial. Outcomes included volume peak expiratory flow (% PEF), fractional exhaled nitric oxide (FeNO), clinical symptoms and adverse events. RESULTS: Thirty-three and 31 patients were treated with tulobuterol patches and theophylline, respectively. % PEF measured in the morning and before bedtime was significantly higher at all times in the treatment period compared with baseline in the tulobuterol patch group (p < 0.001), and was significantly higher in the tulobuterol patch group compared with the theophylline group. FeNO was similar and unchanged from baseline in both groups. There were no drug-related adverse events in either group. CONCLUSIONS: These results suggest that short-term use of a transdermal ß2 agonist is an effective therapy for pediatric asthma without inducing airway inflammation in children on long-term LTRA therapy.

Effects of the tulobuterol patch on the treatment of acute asthma exacerbations in young children.

The tulobuterol patch (TP) is a beta(2)-adrenergic agonist with a favorable pharmacokinetic profile used for asthma management in Japan. Because it contains tulobuterol in a molecular, crystallized form that is gradually absorbed percutaneously, TP exerts a prolonged bronchodilator effect exceeding 24 hours. Although it is a well-established treatment for asthma and wheezing, few studies have investigated whether it can reduce or prevent the symptoms associated with upper respiratory tract infections (URTIs) in young children. This study evaluated the effect of TP on the long-term management of asthma in young children. In this 1-year, randomized, multicenter, double-blind, placebo-controlled study, children aged 0.5-3 years old with mild-to-moderate persistent asthma were treated with either TP or placebo patch. The parents/guardians applied the TP or placebo patch to their children after URTI symptoms appeared. Respiratory symptoms were recorded daily during the 1-year observation period. Overall, 86 patients were enrolled and 80 were treated and analyzed in this study. All patients had been treated with anti-inflammatory drugs before enrollment. The time to symptom resolution was significantly shorter (p = 0.001) and the total respiratory symptom score (p = 0.0457) was significantly lower in the TP group than in the placebo group. In young children with mild-to-moderate asthma who had been treated with anti-inflammatory drugs, using the TP soon after the appearance of URTI symptoms led to quicker resolution of respiratory symptoms and lower respiratory symptom scores.

Effects of the tulobuterol patch on the treatment of acute asthma exacerbations in young children.

The tulobuterol patch (TP) is a beta2-adrenergic agonist with a favorable pharmacokinetic profile used for asthma management in Japan. Because it contains tulobuterol in a molecular, crystallized form that is gradually absorbed percutaneously, TP exerts a prolonged bronchodilator effect exceeding 24 hours. Although it is a well-established treatment for asthma and wheezing, few studies have investigated whether it can reduce or prevent the symptoms associated with upper respiratory tract infections (URTIs) in young children. This study evaluated the effect of TP on the long-term management of asthma in young children. In this 1-year, randomized, multicenter, double-blind, placebo-controlled study, children aged 0.5-3 years old with mild-to-moderate persistent asthma were treated with either TP or placebo patch. The parents/guardians applied the TP or placebo patch to their children after URTI symptoms appeared. Respiratory symptoms were recorded daily during the 1-year observation period. Overall, 86 patients were enrolled and 80 were treated and analyzed in this study. All patients had been treated with anti-inflammatory drugs before enrollment. The time to symptom resolution was significantly shorter (p = 0.001) and the total respiratory symptom score (p = 0.0457) was significantly lower in the TP group than in the placebo group. In young children with mild-to-moderate asthma who had been treated with anti-inflammatory drugs, using the TP soon after the appearance of URTI symptoms led to quicker resolution of respiratory symptoms and lower respiratory symptom scores.