Sodium channelopathies in neurodevelopmental disorders.

The voltage-gated sodium channel α-subunit genes comprise a highly conserved gene family. Mutations of three of these genes, SCN1A, SCN2A and SCN8A, are responsible for a significant burden of neurological disease. Recent progress in identification and functional characterization of patient variants is generating new insights and novel approaches to therapy for these devastating disorders. Here we review the basic elements of sodium channel function that are used to characterize patient variants. We summarize a large body of work using global and conditional mouse mutants to characterize the in vivo roles of these channels. We provide an overview of the neurological disorders associated with mutations of the human genes and examples of the effects of patient mutations on channel function. Finally, we highlight therapeutic interventions that are emerging from new insights into mechanisms of sodium channelopathies.

Long-Term Downregulation of the Sodium Channel Gene Scn8a Is Therapeutic in Mouse Models of SCN8A Epilepsy.

OBJECTIVE: De novo mutations of the voltage-gated sodium channel gene SCN8A cause developmental and epileptic encephalopathy (DEE). Most pathogenic variants result in gain-of-function changes in activity of the sodium channel Nav1.6, poorly controlled seizures, and significant comorbidities. In previous work, an antisense oligonucleotide (ASO) reduced Scn8a transcripts and increased lifespan after neonatal administration to a mouse model. Here, we tested long-term ASO treatment initiated after seizure onset, as required for clinical application. METHODS: ASO treatment was initiated after observation of a convulsive seizure and repeated at 4 to 6 week intervals for 1 year. We also tested the long-term efficacy of an AAV10-short hairpin RNA (shRNA) virus administered on P1. RESULTS: Repeated treatment with the Scn8a ASO initiated after seizure onset provided long-term survival and reduced seizure frequency during a 12 month observation period. A single treatment with viral shRNA was also protective during 12 months of observation. INTERPRETATION: Downregulation of Scn8a expression that is initiated after the onset of seizures is effective for long-term treatment in a model of SCN8A-DEE. Repeated ASO administration or a single dose of viral shRNA prevented seizures and extended survival through 12 months of observation. ANN NEUROL 2024;95:754-759.

Dentate gyrus granule cells are a locus of pathology in Scn8a developmental encephalopathy.

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.

Neoadjuvant chemotherapy-induced hemoglobin decline as a prognostic factor in osteosarcoma around the knee joint: a single-center retrospective analysis of 242 patients.

PURPOSE: Anemia is relatively common in cancer patients, and is associated with poor survival in patients with various malignancies. However, how anemia would affect prognosis and response to neoadjuvant chemotherapy (NAC) in osteosarcoma (OS) is still without substantial evidence. METHODS: We retrospectively analysed 242 patients with stage II OS around the knee joint in our institute. Changed hemoglobin (Hb) levels (before and after NAC) were recorded to assess the prognostic value in DFS (disease-free survival) and tumor response to NAC. Univariate and multivariate analyses were conducted to identify prognostic factors related with outcome in OS patients. RESULTS: The mean Hb level significantly decreased after NAC (134.5 ± 15.3 g/L vs. 117.4 ± 16.3 g/L). The percentage of mild (21%), moderate (4.2%) and severe (0%) anemia patients markedly increased after NAC: 41%, 24% and 4.1% respectively. There was higher percentage of ≥ 5% Hb decline in patients with tumor necrosis rate < 90% (141 out of 161), compared with those with tumor necrosis rate ≥ 90% (59 out of 81). Further univariate and survival analysis demonstrated that Hb decline had a significant role in prediction survival in OS patients. Patients with ≥ 5% Hb decline after NAC had an inferior DFS compared with those with < 5% Hb decline. CONCLUSION: In osteosarcoma, patients with greater Hb decrease during neoadjuvant treatment were shown to have worse DFS and a poorer response to NAC than those without. Attempts to correct anemia and their effects on outcomes for osteosarcoma patients should be explored in future studies.

Spontaneous seizures and elevated seizure susceptibility in response to somatic mutation of sodium channel Scn8a in the mouse.

De novo mutations of neuronal sodium channels are responsible for ~5% of developmental and epileptic encephalopathies, but the role of somatic mutation of these genes in adult-onset epilepsy is not known. We evaluated the role of post-zygotic somatic mutation by adult activation of a conditional allele of the pathogenic variant Scn8aR1872W in the mouse. After activation of CAG-Cre-ER by tamoxifen, the mutant transcript was expressed throughout the brain at a level proportional to tamoxifen dose. The threshold for generation of spontaneous seizures was reached when the proportion of mutant transcript reached 8% of total Scn8a transcript, equivalent to expression of the epileptogenic variant in 16% of heterozygous neurons. Expression below this level did not result in spontaneous seizures, but did increase susceptibility to seizure induction by kainate or auditory stimulation. The relatively high threshold for spontaneous seizures indicates that somatic mutation of sodium channels is unlikely to contribute to the elevated incidence of epilepsy in the elderly population. However, somatic mutation could increase susceptibility to other seizure stimuli.

A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas.

BACKGROUND: Cell-based  immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells. METHODS: In this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18-27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily. RESULTS: From October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months. CONCLUSIONS: Overall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04074564.

Translating AI to Clinical Practice: Overcoming Data Shift with Explainability.

To translate artificial intelligence (AI) algorithms into clinical practice requires generalizability of models to real-world data. One of the main obstacles to generalizability is data shift, a data distribution mismatch between model training and real environments. Explainable AI techniques offer tools to detect and mitigate the data shift problem and develop reliable AI for clinical practice. Most medical AI is trained with datasets gathered from limited environments, such as restricted disease populations and center-dependent acquisition conditions. The data shift that commonly exists in the limited training set often causes a significant performance decrease in the deployment environment. To develop a medical application, it is important to detect potential data shift and its impact on clinical translation. During AI training stages, from premodel analysis to in-model and post hoc explanations, explainability can play a key role in detecting model susceptibility to data shift, which is otherwise hidden because the test data have the same biased distribution as the training data. Performance-based model assessments cannot effectively distinguish the model overfitting to training data bias without enriched test sets from external environments. In the absence of such external data, explainability techniques can aid in translating AI to clinical practice as a tool to detect and mitigate potential failures due to data shift. ©RSNA, 2023 Quiz questions for this article are available in the supplemental material.

Structure and Expression Analysis of PtrSUS, PtrINV, PtrHXK, PtrPGM, and PtrUGP Gene Families in Populus trichocarpa Torr. and Gray.

Exogenous nitrogen and carbon can affect plant cell walls, which are composed of structural carbon. Sucrose synthase (SUS), invertase (INV), hexokinase (HXK), phosphoglucomutase (PGM), and UDP-glucose pyrophosphorylase (UGP) are the key enzymes of sucrose metabolism involved in cell wall synthesis. To understand whether these genes are regulated by carbon and nitrogen to participate in structural carbon biosynthesis, we performed genome-wide identification, analyzed their expression patterns under different carbon and nitrogen treatments, and conducted preliminary functional verification. Different concentrations of nitrogen and carbon were applied to poplar (Populus trichocarpa Torr. and Gray), which caused changes in cellulose, lignin, and hemicellulose contents. In poplar, 6 SUSs, 20 INVs, 6 HXKs, 4 PGMs, and 2 UGPs were identified. Moreover, the physicochemical properties, collinearity, and tissue specificity were analyzed. The correlation analysis showed that the expression levels of PtrSUS3/5, PtrNINV1/2/3/5/12, PtrCWINV3, PtrVINV2, PtrHXK5/6, PtrPGM1/2, and PtrUGP1 were positively correlated with the cellulose content. Meanwhile, the knockout of PtrNINV12 significantly reduced the cellulose content. This study could lay the foundation for revealing the functions of SUSs, INVs, HXKs, PGMs, and UGPs, which affected structural carbon synthesis regulated by nitrogen and carbon, proving that PtrNINV12 is involved in cell wall synthesis.

Assembly of the complexes of oxidative phosphorylation triggers the remodeling of cardiolipin.

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

Impact of Secondary Aneurysmal Bone Cysts on Survival of Patients with Enneking Stage IIB Extremity Osteosarcoma: A Propensity Score Matching Analysis.

BACKGROUND: The clinicopathology of aneurysmal bone cysts (ABCs) secondary to osteosarcoma has not yet been reported. We conduct a retrospective review of ABCs secondary to osteosarcoma to characterize clinicopathology and influence on the survival of patients with Enneking stage IIB extremity osteosarcoma. PATIENTS AND METHODS: A total of 300 patients with Enneking stage IIB extremity osteosarcoma were eligible for analysis. These cases were divided, according to the pathology of biopsy and magnetic resonance imaging (MRI), into ABCs group and no ABCs group. Patients (ABCs versus no ABCs) were compared using a 1:2 propensity score analysis to best match between groups. Clinicopathology and survival data were analyzed. RESULTS: The total occurrence rate of secondary ABCs was 10.3%. A higher prevalence of pathological fractures was observed in the ABCs group (22.6%) compared with the no ABCs group (8.6%) (p = 0.032). Patients with ABCs were more likely to undergo amputation compared with patients without ABCs (p = 0.007). Those with secondary ABCs had poorer response to chemotherapy before and after propensity score matching (p = 0.006 and p = 0.048, respectively). Kaplan-Meier survival analysis showed that EFS and OS distributions were not significantly different between the two patient groups. ABCs were not significantly different in terms of EFS or OS in the multivariate analysis model (p > 0.05). CONCLUSIONS: The presence of secondary ABCs was associated with increased occurrence rate of pathological fracture and high percentage of amputation. Moreover, patients with secondary ABCs had poorer response to chemotherapy. However, the presence of secondary ABCs did not influence survival of patients with Enneking stage IIB extremity osteosarcoma.

Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.

OBJECTIVE: SCN8A encephalopathy is a developmental and epileptic encephalopathy (DEE) caused by de novo gain-of-function mutations of sodium channel Nav 1.6 that result in neuronal hyperactivity. Affected individuals exhibit early onset drug-resistant seizures, developmental delay, and cognitive impairment. This study was carried out to determine whether reducing the abundance of the Scn8a transcript with an antisense oligonucleotide (ASO) would delay seizure onset and prolong survival in a mouse model of SCN8A encephalopathy. METHODS: ASO treatment was tested in a conditional mouse model with Cre-dependent expression of the pathogenic patient SCN8A mutation p.Arg1872Trp (R1872W). This model exhibits early onset of seizures, rapid progression, and 100% penetrance. An Scn1a +/- haploinsufficient mouse model of Dravet syndrome was also treated. ASO was administered by intracerebroventricular injection at postnatal day 2, followed in some cases by stereotactic injection at postnatal day 30. RESULTS: We observed a dose-dependent increase in length of survival from 15 to 65 days in the Scn8a-R1872W/+ mice treated with ASO. Electroencephalographic recordings were normal prior to seizure onset. Weight gain and activity in an open field were unaffected, but treated mice were less active in a wheel running assay. A single treatment with Scn8a ASO extended survival of Dravet syndrome mice from 3 weeks to >5 months. INTERPRETATION: Reduction of Scn8a transcript by 25 to 50% delayed seizure onset and lethality in mouse models of SCN8A encephalopathy and Dravet syndrome. Reduction of SCN8A transcript is a promising approach to treatment of intractable childhood epilepsies. Ann Neurol 2020;87:339-346.

Clinical significance of indeterminate pulmonary nodules on the survival of 364 patients with nonmetastatic, high-grade, localized osteosarcoma: A 12-year retrospective cohort study.

BACKGROUND AND OBJECTIVES: We investigated the clinical significance of indeterminate pulmonary nodules (IPNs) in patients diagnosed with nonmetastatic, high-grade localized osteosarcoma. METHODS: We retrospectively analyzed the clinical data of 364 patients with nonmetastatic, high-grade localized osteosarcoma. Based on pulmonary computed tomography findings at presentation, the patients were categorized into the no-nodules and the IPNs group and were further categorized into subgroups based on age (<18 and ≥18 years). We performed an intergroup comparison of event-free survival (EFS) and overall survival (OS). RESULTS: At presentation, 276 (75.8%) patients showed no nodules, and 88 (24.2%) patients showed IPNs. The EFS and OS were similar between adults with IPNs (n = 54 [30.5%]) and without nodules (n = 123 [69.5%]) (p = .200 and p = .609, respectively). No significant intergroup difference in OS was observed in pediatric patients (p = .093). However, pediatric patients with IPNs (n = 34 [18.2%]) had poorer EFS than those without nodules (n = 153 [81.8%]) (p = .016). Multivariate analyses confirmed that IPNs were independently associated with poorer EFS in pediatric patients (hazard ratio 1.788, 95% confidence interval 1.092-2.926, p = .021). CONCLUSIONS: This study showed that IPNs at presentation did not affect the survival of adults with nonmetastatic, high-grade localized osteosarcoma but were associated with poorer EFS in pediatric patients.

Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Gabra2 is a genetic modifier of Scn8a encephalopathy in the mouse.

OBJECTIVE: SCN8A encephalopathy is a developmental epileptic encephalopathy typically caused by de novo gain-of-function mutations in Nav 1.6. Severely affected individuals exhibit refractory seizures, developmental delay, cognitive disabilities, movement disorders, and elevated risk of sudden death. Patients with the identical SCN8A variant can differ in clinical course, suggesting a role for modifier genes in determining disease severity. The identification of genetic modifiers contributes to understanding disease pathogenesis and suggesting therapeutic interventions. METHODS: We generated F1 and F2 crosses between inbred mouse strains and mice carrying the human pathogenic variants SCN8A-R1872W and SCN8A-N1768D. Quantitative trait locus (QTL) analysis of seizure-related phenotypes was used for chromosomal mapping of modifier loci. RESULTS: In an F2 cross between strain SJL/J and C57BL/6J mice carrying the patient mutation R1872W, we identified a major QTL on chromosome 5 containing the Gabra2 gene. Strain C57BL/6J carries a splice site mutation that reduces expression of Gabra2, encoding the α2 subunit of the aminobutyric acid type A receptor. The protective wild-type allele of Gabra2 from strain SJL/J delays the age at seizure onset and extends life span of the Scn8a mutant mice. Additional Scn8a modifiers were observed in the F2 cross and in an F1 cross with strain C3HeB/FeJ. SIGNIFICANCE: These studies demonstrate that the SJL/J strain carries multiple modifiers with protective effects against seizures induced by gain-of-function mutations in Scn8a. Homozygosity for the hypomorphic variant of Gabra2 in strain C57BL/6J is associated with early seizure onset and short life span. GABRA2 is a potential therapeutic target for SCN8A encephalopathy.

Regulation of Inositol Biosynthesis: Balancing Health and Pathophysiology.

Inositol is the precursor for all inositol compounds and is essential for viability of eukaryotic cells. Numerous cellular processes and signaling functions are dependent on inositol compounds, and perturbation of their synthesis leads to a wide range of human diseases. Although considerable research has been directed at understanding the function of inositol compounds, especially phosphoinositides and inositol phosphates, a focus on regulatory and homeostatic mechanisms controlling inositol biosynthesis has been largely neglected. Consequently, little is known about how synthesis of inositol is regulated in human cells. Identifying physiological regulators of inositol synthesis and elucidating the molecular mechanisms that regulate inositol synthesis will contribute fundamental insight into cellular processes that are mediated by inositol compounds and will provide a foundation to understand numerous disease processes that result from perturbation of inositol homeostasis. In addition, elucidating the mechanisms of action of inositol-depleting drugs may suggest new strategies for the design of second-generation pharmaceuticals to treat psychiatric disorders and other illnesses.

Production of Phosphorylated Ric-8A proteins using protein kinase CK2.

Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.

Valproate inhibits glucose-stimulated insulin secretion in beta cells.

Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18 h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.

Inositol Hexakisphosphate Kinase 1 (IP6K1) Regulates Inositol Synthesis in Mammalian Cells.

myo-Inositol, the precursor of all inositol compounds, has pivotal roles in cell metabolism and signaling pathways. Although physiological studies indicate a strong correlation between abnormal intracellular inositol levels and neurological disorders, very little is known about the regulation of inositol synthesis in mammalian cells. In this study, we report that IP6K1, an inositol hexakisphosphate kinase that catalyzes the synthesis of inositol pyrophosphate, regulates inositol synthesis in mammalian cells. Ip6k1 ablation led to profound changes in DNA methylation and expression of Isyna1 (designated mIno1), which encodes the rate-limiting enzyme inositol-3-phosphate synthase. Interestingly, IP6K1 preferentially bound to the phospholipid phosphatidic acid, and this binding was required for IP6K1 nuclear localization and the regulation of mIno1 transcription. This is the first demonstration of IP6K1 as a novel negative regulator of inositol synthesis in mammalian cells.

Dynamic contrast-enhanced MRI to predict response to vinorelbine-cisplatin alone or with rh-endostatin in patients with non-small cell lung cancer and bone metastases: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Metastatic bone disease is a frequent complication of advanced non-small-cell lung cancer and causes skeletal-related events which result in a poor prognosis. A standard method to assess the therapeutic response of bone metastases does not currently exist. We used dynamic contrast-enhanced MRI to obtain quantitative measures to assess the suitability of this technique to gauge therapeutic response to vinorelbine-cisplatin plus rh-endostatinfor previously untreated non-small cell lung cancer with bone metastases. METHODS: We did a phase 4, randomised, prospective, double-blind, placebo-controlled clinical trial in Shanghai Sixth People's Hospital, Shanghai, China. Inclusion criteria were non-small-cell lung cancer patients with bone metastases confirmed by pathology or cytology; available imaging data of pelvic metastatic lesions; aged 18 to 75 years old; expected survival at least 3 months; not receiving taxane, bevacizumab, thalidomide, rh-endostatin, or bisphosphonate; not having radiation therapy within 3 months of enrollment into study; normal results of routine blood tests, liver and kidney function, and electrocardiogram; absence of cardiovascular disease, autoimmune disease, vasculitis, severe infection, diabetes, and other concomitant disease; and signed informed consent. Exclusion criteria were receiving granulocyte colony stimulating factor or granulocyte-macrophage colony stimulating factor during chemotherapy, intolerance to adverse reaction, and allergy to contrast agents. Patients were randomly assigned to treatment group and control group at a ratio of 2:1 by random code generation by an independent biostatistician in a double-blind fashion. Participants received either vinorelbine-cisplatin plus rh-endostatin or vinorelbine-cisplatin plus placebo. Vinorelbine (25 mg/m2) and cisplatin (75 mg/m2) were administered intravenously on the first day of a 21 day cycle. Patients received rh-endostatin (7·5 mg/m2) or placebo on days 1-14 of a cycle. The primary end points were objective response rate (complete remission+partial remission)/total × 100) and disease control rate (complete remission+partial remission+stable disease)/total × 100). Measurements including Ktrans, Kep, and Ve were evaluated by dynamic contrast-enhanced MRI before treatment and after completion of 2 treatment cycles. Blood concentrations of bone metabolites, tumour markers, and tumour vascular growth related factors were measured before and after treatment. Comparisons were made using paired t-test. Kaplan-Meier survival analysis was used to indicate the correlation between some measurements and progression-free survival or overall survival. The difference in Ktrans between patients who had partial remission or stable disease group and those who had disease progression was tested using the Chi-square test. All statistical analyses were performed with SPSS version 21.0. This trial was approved by the State Food and Drug Administration (No: S20050088) and China State Food and Drug Administration. The trial is registered with China Clinical Trials Registry, number chictr-ctr-09000569. Written informed consent and ethical approval was obtained. FINDINGS: We enrolled 33 patients (aged 52-70, 15 men and 18 women) of whom 28 were evaluable (20 in treatment group and 8 in control group). Five patients were excluded: 2 patients in treatment group and 1 patient in control group used granulocyte-macrophage colony stimulating factor, and 2 patients in the control group refused treatment. Objective response rate was higher (30% vs 0%; p<0·00001), mean overall survival was longer (21·44 [SD 17·28] vs 7·71 [4·68] months, p=0·008), and reduction in capillary permeability (measured by Ktrans) was greater (60·0% vs 4·4%; p=0·026) in the group given rh-endostatin than in the control group. Disease control rate was 80% in the treatment group and 75% in the control group (p=0·07). Overall survival was longer in patients with a greater than 50% reduction in Ktrans than in patients with a decrease of up to 50% (13·2 [1·8] vs 9·8 [0·2] months, p=0·026). INTERPRETATION: Addition of rh-endostatin to treatment with vinorelbine-cisplatin increased the treatment response in patients with non-small cell lung cancer and bone metastases. Quantitative analysis using dynamic contrast-enhanced MRI can be used to evaluate therapeutic response and to predict survival of bone metastases after anti-angiogenesis therapy. Limitations of this study include the small number of patients and the single-centre design. FUNDING: National Natural Science Foundation of China [grant number 81201628].