Inequalities in Childhood Immunisation in South Asia.

Identifying the inequalities associated with immunisation coverage among children is crucial. We investigated the factors associated with complete immunisation among 12- to 23-month-old children in five South Asian countries: Afghanistan, Bangladesh, India, Nepal, and Pakistan, using nationally representative data sets from the Demographic and Health Survey (DHS). Descriptive statistics, bivariate association, and logistic regression analyses were employed to identify the prevalence and the factors in each country that affect the likelihood of full childhood immunisation coverage. The complete childhood immunisation coverage varied significantly within each country in South Asia. Afghanistan had the lowest immunisation rates (42.6%), whereas Bangladesh ranked the highest in complete childhood immunisation rates, at 88.2%. Similarly, 77.1% of Indian children, 79.2% of Nepali children, and 62.2% of Pakistani children were completely immunised. Household wealth status strongly correlated with full childhood immunisation in Afghanistan, India, and Pakistan at the bivariate level. The results from the logistic regression showed that a higher maternal educational level had a statistically significant association with complete childhood immunisation in all countries compared to mothers who did not attend any school. In conclusion, the study revealed the inequalities of complete childhood immunisation within South Asia. Governments must be proactive in their endeavours to address universal and equitable vaccine coverage in collaboration with national and international stakeholders and in line with the relevant Sustainable Development Goals.

Development of potent dimeric inhibitors of GAS41 YEATS domain.

GAS41 is an emerging oncogene overexpressed and implicated in multiple cancers, including non-small cell lung cancer (NSCLC). GAS41 is a dimeric protein that contains the YEATS domain, which is involved in the recognition of lysine-acylated histones. Here, we report the development of GAS41 YEATS inhibitors by employing a fragment-based screening approach. These inhibitors bind to GAS41 YEATS domain in a channel constituting a recognition site for acylated lysine on histone proteins. To enhance inhibitory activity, we developed a dimeric analog with nanomolar activity that blocks interactions of GAS41 with acetylated histone H3. Our lead compound engages GAS41 in cells, blocks proliferation of NSCLC cells, and modulates expression of GAS41-dependent genes, validating on-target mechanism of action. This study demonstrates that disruption of GAS41 protein-protein interactions may represent an attractive approach to target lung cancer cells. This work exemplifies the use of bivalent inhibitors as a general strategy to block challenging protein-protein interactions.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Cellular Stress Responses in Radiotherapy.

Radiotherapy is one of the major cancer treatment strategies. Exposure to penetrating radiation causes cellular stress, directly or indirectly, due to the generation of reactive oxygen species, DNA damage, and subcellular organelle damage and autophagy. These radiation-induced damage responses cooperatively contribute to cancer cell death, but paradoxically, radiotherapy also causes the activation of damage-repair and survival signaling to alleviate radiation-induced cytotoxic effects in a small percentage of cancer cells, and these activations are responsible for tumor radio-resistance. The present study describes the molecular mechanisms responsible for radiation-induced cellular stress response and radioresistance, and the therapeutic approaches used to overcome radioresistance.

GAS41 Recognizes Diacetylated Histone H3 through a Bivalent Binding Mode.

GAS41 is a chromatin-associated protein that belongs to the YEATS family and is involved in the recognition of acetyl-lysine in histone proteins. A unique feature of GAS41 is the presence of a C-terminal coiled-coil domain, which is responsible for protein dimerization. Here, we characterized the specificity of the GAS41 YEATS domain and found that it preferentially binds to acetylated H3K18 and H3K27 peptides. Interestingly, we found that full-length, dimeric GAS41 binds to diacetylated H3 peptides with an enhanced affinity when compared to those for monoacetylated peptides, through a bivalent binding mode. We determined the crystal structure of the GAS41 YEATS domain with H3K23acK27ac to visualize the molecular basis of diacetylated histone binding. Our results suggest a unique binding mode in which full-length GAS41 is a reader of diacetylated histones.

Radiation-induced overexpression of transthyretin inhibits retinol-mediated hippocampal neurogenesis.

Cranial irradiation is the main therapeutic strategy for treating primary and metastatic brain tumors. However, radiation is well-known to induce several unexpected side effects including emotional disorders. Although radiation-induced depression may cause decreased quality of life after radiotherapy, investigations of its molecular mechanism and therapeutic strategies are still insufficient. In this study, we found that behavioral symptoms of depression on mice models with the decrease of BrdU/NeuN- and Dcx-positive populations and MAP-2 expression in hippocampus were induced by cranial irradiation, and transthyretin (TTR) was highly expressed in hippocampus after irradiation. It was shown that overexpression of TTR resulted in the inhibition of retinol-mediated neuritogenesis. PAK1 phosphorylation and MAP-2 expression were significantly reduced by TTR overexpression following irradiation. Moreover, we observed that treatment of allantoin and neferine, the active components of Nelumbo nucifera, interrupted irradiation-induced TTR overexpression, consequently leading to the increase of PAK1 phosphorylation, neurite extension, BrdU/NeuN- and Dcx-positive populations, and MAP-2 expression. Behavioral symptoms of depression following cranial irradiation were also relieved by treatment of allantoin and neferine. These findings demonstrate that TTR plays a critical role in neurogenesis after irradiation, and allantoin and neferine could be potential drug candidates for recovering the effects of radiation on neurogenesis and depression.

RNF138-mediated ubiquitination of rpS3 is required for resistance of glioblastoma cells to radiation-induced apoptosis.

An interaction between ribosomal protein S3 (rpS3) and nuclear factor kappa B or macrophage migration inhibitory factor in non-small-cell lung cancer is responsible for radioresistance. However, the role of rpS3 in glioblastoma (GBM) has not been investigated to date. Here we found that in irradiated GBM cells, rpS3 translocated into the nucleus and was subsequently ubiquitinated by ring finger protein 138 (RNF138). Ubiquitin-dependent degradation of rpS3 consequently led to radioresistance in GBM cells. To elucidate the apoptotic role of rpS3, we analyzed the interactome of rpS3 in ΔRNF138 GBM cells. Nuclear rpS3 interacted with DNA damage inducible transcript 3 (DDIT3), leading to DDIT3-induced apoptosis in irradiated ΔRNF138 GBM cells. These results were confirmed using in vivo orthotopic xenograft models and GBM patient tissues. This study aims to clarify the role of RNF138 in GBM cells and demonstrate that rpS3 may be a promising substrate of RNF138 for the induction of GBM radioresistance, indicating RNF138 as a potential target for GBM therapy.

TRAF4 promotes lung cancer aggressiveness by modulating tumor microenvironment in normal fibroblasts.

Normal fibroblasts surrounding tumor cells play a crucial role in cancer progression through formation of the tumor microenvironment. Because factors secreted from normal fibroblasts can modulate the tumor microenvironment, it is necessary to identify key factors associated with regulation of secreted factors and to investigate the molecular mechanisms contributing to the tumor microenvironment formation process. In this study, we found that radiation induced the expression and K63-linkage poly-ubiquitination of TRAF4 in normal lung fibroblasts. The K63-linkage poly-ubiquitinated TRAF4 formed complexes with NOX2 or NOX4 by mediating phosphorylated p47-phox in normal lung fibroblasts. Moreover, we showed that TRAF4 stabilized NOX complexes by decreasing lysosomal degradation of NOX2 and NOX4 after irradiation. NOX complexes increased endosomal ROS levels that were permeable into cytoplasm, leading to NF-κB-mediated ICAM1 up-regulation. Soluble ICAM1 was subsequently secreted into conditioned media of radiation-activated normal lung fibroblasts. The conditioned media from irradiated normal fibroblasts enhanced proliferation and epithelial-mesenchymal transition of non-small cell lung cancer cells both in vitro and in vivo. These results demonstrate that TRAF4 in irradiated fibroblasts is positively associated with aggressiveness of adjacent cancer cells by altering the tumor microenvironment. Thus, we suggest that regulation of TRAF4 might be a promising strategy for cancer therapy.

Surfactant Protein B Suppresses Lung Cancer Progression by Inhibiting Secretory Phospholipase A2 Activity and Arachidonic Acid Production.

BACKGROUND/AIMS: Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B). METHODS: To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM. RESULTS: In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-ß signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA2, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model. CONCLUSION: Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

The role of tumor microenvironment in therapeutic resistance.

Cancer cells undergo unlimited progression and survival owing to activation of oncogenes. However, support of the tumor microenvironment is essential to the formation of clinically relevant tumors. Recent evidence indicates that the tumor microenvironment is a critical regulator of immune escape, progression, and distant metastasis of cancer. Moreover, the tumor microenvironment is known to be involved in acquired resistance of tumors to various therapies. Despite significant advances in chemotherapy and radiotherapy, occurrence of therapeutic resistance leads to reduced efficacy. This review highlights myeloid cells, cancer-associated fibroblasts, and mesenchymal stem cells consisting of the tumor microenvironment, as well as the relevant signaling pathways that eventually render cancer cells to be therapeutically resistant.

TFAP2C-mediated upregulation of TGFBR1 promotes lung tumorigenesis and epithelial-mesenchymal transition.

TFAP2C (transcription factor-activating enhancer-binding protein 2C) expression has been positively correlated with poor prognosis in patients with certain types of cancer, but the mechanisms underlying TFAP2C-mediated tumorigenesis in non-small-cell lung cancer (NSCLC) are still unknown. We previously performed a microarray analysis to identify TFAP2C regulation genes, and TGFBR1 (transforming growth factor-ß receptor type 1) was found to be upregulated by TFAP2C. We observed that TFAP2C or TGFBR1 overexpression led to oncogenic properties, such as cell viability, proliferation and cell cycle progression. TGFBR1 upregulation induced by TFAP2C also promoted cell motility and migration, leading to malignant development. We also found that PAK1 (p21 protein (Cdc42/Rac)-activated kinase 1) signaling was involved in TFAP2C/TGFBR1-induced tumorigenesis. These results were confirmed by an in vivo xenograft model and patient tissue samples. This study shows that TFAP2C promoted tumor progression by upregulation of TGFBR1 and consequent activation of PAK1 signaling.

Inhibition of hedgehog signalling attenuates UVB-induced skin photoageing.

The hedgehog (Hh) signalling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis and progression of a variety of aggressive human cancers including skin cancer. Despite its importance, little is known about its role in photoageing, a type of UV-induced skin lesions. In this study, we investigated the involvement of Hh signalling in the photoageing process as well as the use of an Hh-regulating alkaloid compound as a novel therapeutic drug to regulate photoageing in keratinocytes. We found that UVB induced Hh signalling by the expression of Hh ligands and Hh-mediated transcription factors, respectively. Moreover, UVB-induced Hh activation relied on mitogen-activated protein kinase (p38, ERK and JNK) activity and inflammatory responses (upregulation of COX-2, IL-1ß, IL-6 and TNF-α), resulting in premature senescence and photoageing in vitro and in vivo. Notably, a selected Hh inhibitor, evodiamine, mediated photoageing blockade in a mouse skin model. Taken together, our findings demonstrated that Hh signalling is associated with UVB-induced photoageing, while pharmacological inhibition of Hh signalling significantly reduced experimental photoageing, indicating its potential for use as a therapeutic target for this disease.

PAK1 tyrosine phosphorylation is required to induce epithelial-mesenchymal transition and radioresistance in lung cancer cells.

The p21-activated Ser/Thr kinase 1 (PAK1) kinase has an essential role in tumorigenesis and cell survival in many cancers, but its regulation is not fully understood. In this study, we showed that in response to irradiation of lung cancer cells, PAK1 was upregulated, tyrosine phosphorylated, and translocated to the nucleus. Tyrosine phosphorylation relied upon JAK2 kinase activity and was essential for PAK1 protein stability and binding to Snail. This radiation-induced JAK2-PAK1-Snail signaling pathway increased epithelial-mesenchymal transition (EMT) by regulating epithelial and mesenchymal cell markers. Notably, JAK2 inhibitors mediated radiosensitization and EMT blockade in a mouse xenograft model of lung cancer. Taken together, our findings offered evidence that JAK2 phosphorylates and stabilizes functions of PAK1 that promote EMT and radioresistance in lung cancer cells, with additional implications for the use of JAK2 inhibitors as radiosensitizers in lung cancer treatment.

Rhamnetin and cirsiliol induce radiosensitization and inhibition of epithelial-mesenchymal transition (EMT) by miR-34a-mediated suppression of Notch-1 expression in non-small cell lung cancer cell lines.

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.

PIM1 kinase inhibitors induce radiosensitization in non-small cell lung cancer cells.

Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the potential involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Consequently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic proteins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradiation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance.

Identification of T-type alpha1H Ca2+ channels (Ca(v)3.2) in major pelvic ganglion neurons.

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.