Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Maize miRNAs and their putative target genes involved in chilling stress response in 5-day old seedlings.

BACKGROUND: In the context of early sowing of maize as a promising adaptation strategy that could significantly reduce the negative effects of climate change, an in-depth understanding of mechanisms underlying plant response to low-temperature stress is demanded. Although microRNAs (miRNAs) have been recognized as key regulators of plant stress response, research on their role in chilling tolerance of maize during early seedling stages is scarce. Therefore, it is of great significance to explore chilling-responsive miRNAs, reveal their expression patterns and associated target genes, as well as to examine the possible functions of the conserved and novel miRNAs. In this study, the role of miRNAs was examined in 5d-old maize seedlings of one tolerant and one sensitive inbred line exposed to chilling (10/8 °C) stress for 6 h and 24 h, by applying high throughput sequencing. RESULTS: A total of 145 annotated known miRNAs belonging to 30 families and 876 potentially novel miRNAs were identified. Differential expression (DE) analysis between control and stress conditions identified 98 common miRNAs for both genotypes at one time point and eight miRNAs at both time points. Target prediction and enrichment analysis showed that the DE zma-miR396, zma-miR156, zma-miR319, and zma-miR159 miRNAs modulate growth and development. Furthermore, it was found that several other DE miRNAs were involved in abiotic stress response: antioxidative mechanisms (zma-miR398), signal transduction (zma-miR156, zma-miR167, zma-miR169) and regulation of water content (zma-miR164, zma-miR394, zma-miR396). The results underline the zma-miRNAs involvement in the modulation of their target genes expression as an important aspect of the plant's survival strategy and acclimation to chilling stress conditions. CONCLUSIONS: To our understanding, this is the first study on miRNAs in 5-d old seedlings' response to chilling stress, providing data on the role of known and novel miRNAs post-transcriptional regulation of expressed genes and contributing a possible platform for further network and functional analysis.

Intronic elements associated with insomnia and restless legs syndrome exhibit cell-type-specific epigenetic features contributing to MEIS1 regulation.

A highly evolutionarily conserved myeloid ecotropic viral integration site 1 (MEIS1) intronic region is strongly associated with restless legs syndrome (RLS) and insomnia. To understand its regulatory function, we dissected the region by analyzing chromatin accessibility, enhancer-promoter contacts, DNA methylation and expression quantitative trait locus (eQTLs) in different human neural cell types and tissues. We observed specific activity with respect to cell type and developmental maturation, indicating a prominent role for distinct highly conserved intronic elements in forebrain inhibitory neuron differentiation. Two elements were hypomethylated in neural cells with higher MEIS1 expression, suggesting a role of enhancer demethylation in gene regulation. MEIS1 eQTLs showed a striking modular chromosomal distribution, with forebrain eQTLs clustering in intron 8/9. Clustered regularly interspersed short palindromic repeats interference targeting of individual elements in this region attenuated MEIS1 expression, revealing a complex regulatory interplay of distinct elements. In summary, we found that MEIS1 regulation is organized in a modular pattern. Disease-associated intronic regulatory elements control MEIS1 expression with cell type and maturation stage specificity, particularly in the inhibitory neuron lineage. The precise spatiotemporal activity of these elements likely contributes to the pathogenesis of insomnia and RLS.

First report of Aspergillus welwitschiae causing maize ear rot in Serbia.

In recent years, countries in Southeast Europe are facing climate changes characterized by extreme hot weather, which contribute to the increased frequency of Aspergillus species. Because of these changes, Aspergillus parasiticus was isolated, for the first time, from maize grain in Serbia (Nikolic et al, 2018). The presence of black powdery mycelia on maize ears indicated occurrence of species of the genus Aspergillus section Nigri, which led to the need for detailed identification of these fungi. Disease incidence ranged from 10 and 15% in August 2013. Maize ears with black powdery symptoms were collected from field in Zemun Polje, Serbia. Symptomatic kernels were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed three times with sterilized water, then incubated at 25°C in the dark for 7 days on potato dextrose agar (PDA). Twenty isolates were identified as genus Aspergillus section Nigri. Monospore cultures formed black cottony colonies with a yellowish border on PDA. The average colony diameter was 50 mm. In order to reliably identify, isolates were transferred to Malt Extract agar (MEA) and Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. On MEA fungal colonies were dense, black, with yellowish border. The reverse side was colorless to pale yellow, with a yellow ring in the middle. The average size of conidia was 4.3 µm. The conidia were globose to sub-globose, smooth to roughened, which coincides with previous research (Silva et al, 2020). Given that the fungi Aspergillus niger and Aspergillus welwitschiae are morphologically indistinguishable (Susca et al, 2016), species level identification was completed by analysis of a partial sequence of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers) and calmodulin gene (CMD5/CMD6 primers) (Samson et al., 2014). The sequences were compared with the sequences of A. welwitschiae strains registered in the GenBank database based on nucleotide similarity, and results showed 99,64 and 100% similarity with ITS (OL711714) and calmodulin (KX894585), respectively. The sequence was deposited in GenBank with accession numbers OQ456471 (ITS) and OQ426518 (calmodulin). We also confirmed the presence of this species with specific primers (AWEL1/AWEL2) designed by Susca et al. 2020. Pathogenicity test was performed in Zemun Polje on the same maize hybrid from which the fungal species was isolated. Using artificial inoculations by the injecting conidial suspension into the silk channel, three days after 50% of plants reached the silking stage. Twenty ears were inoculated with each isolate, in four replicates (Reid et al, 1996). Inoculum was prepared from 7-day-old colonies on PDA, and 2 ml of a conidial suspension (1×106 spores/ml) was used. Control plants were inoculated with sterile water. All inoculated ears showed symptoms, similar to those from field infections. Control ears were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. Based on molecular, morphological and pathogenic properties, the isolates were identified as A. welwitschiae. This is the first report of A. welwitschiae as the causal agent of black maize ear rot not only in Serbia, but also in the other countries of the Western Balkans. Given that the fungus A. welwitschiae synthesizes both ochratoxin A (OTA) (Battilani et al, 2006) and fumonisin (FB) (Frisvad et al, 2011), further studies should be focused on assessment its aggressiveness and toxicological profile.

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma.

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.

Identification of Restless Legs Syndrome Genes by Mutational Load Analysis.

OBJECTIVE: Restless legs syndrome is a frequent neurological disorder with substantial burden on individual well-being and public health. Genetic risk loci have been identified, but the causatives genes at these loci are largely unknown, so that functional investigation and clinical translation of molecular research data are still inhibited. To identify putatively causative genes, we searched for highly significant mutational burden in candidate genes. METHODS: We analyzed 84 candidate genes in 4,649 patients and 4,982 controls by next generation sequencing using molecular inversion probes that targeted mainly coding regions. The burden of low-frequency and rare variants was assessed, and in addition, an algorithm (binomial performance deviation analysis) was established to estimate independently the sequence variation in the probe binding regions from the variation in sequencing depth. RESULTS: Highly significant results (considering the number of genes in the genome) of the conventional burden test and the binomial performance deviation analysis overlapped significantly. Fourteen genes were highly significant by one method and confirmed with Bonferroni-corrected significance by the other to show a differential burden of low-frequency and rare variants in restless legs syndrome. Nine of them (AAGAB, ATP2C1, CNTN4, COL6A6, CRBN, GLO1, NTNG1, STEAP4, VAV3) resided in the vicinity of known restless legs syndrome loci, whereas 5 (BBS7, CADM1, CREB5, NRG3, SUN1) have not previously been associated with restless legs syndrome. Burden test and binomial performance deviation analysis also converged significantly in fine-mapping potentially causative domains within these genes. INTERPRETATION: Differential burden with intragenic low-frequency variants reveals putatively causative genes in restless legs syndrome. ANN NEUROL 2020;87:184-193.

A proposed guideline for vagus nerve stimulator handling in palliative care and after death.

Vagus nerve stimulation (VNS) is often used for patients with drug-resistant epilepsy. Although this intervention may improve seizure control and mood, a number of factors must be considered when patients with VNS near end of life. We reviewed relevant literature to create a proposed guideline for management of patients with VNS in palliative care and after death. VNS has multiple possible side effects, including cough and swallowing difficulties. For patients with neurologic disease in palliative care, such adverse effects can severely affect quality of life and increase the risk for complications such as aspiration pneumonia. Patients with VNS should be screened regularly for such side effects, and VNS parameters should be adjusted if they are identified. If a patient requires urgent cardiac resuscitation involving external defibrillation, the VNS should be interrogated immediately afterwards to evaluate its function. During defibrillation, paddles should be placed perpendicular to the VNS, and as far as possible away from it. The VNS can be acutely turned off by taping the magnet to the patient's chest, thereby preventing any possible interference with restoration of a normal heart rhythm. After death, any staff involved with handling the body should be notified that a VNS is in place. The device must be removed prior to cremation, as it can explode with high heat. If the cause of death is unclear, a full postmortem examination should be undertaken, per sudden unexpected death in epilepsy guidelines. If there is concern about device malfunction, the device should be returned to the manufacturer for evaluation.

Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Survival and mortality of adult-onset myasthenia gravis in the population of Belgrade, Serbia.

INTRODUCTION: The objective of this study was to estimate mortality and survival in a large cohort of myasthenia gravis (MG) patients from Belgrade, Serbia, during the period 1979-2008. METHODS: Data for all patients with MG were collected from hospital records and the Belgrade MG Registry. RESULTS: Within the 30-year study period, death occurred in 107 (20%) of 562 patients with MG, with MG-related fatality below 2%. The average MG mortality rate was 1.76 per 1,000,000 population (1.26/1,000,000 women, 2.45/1,000,000 men). A statistically significant increase was recorded for the average standardized mortality rate for all patients (P < 0.01). The mean survival from disease onset was 34.3 ± 2.0 years. Significantly shorter survival was observed in men compared with women and in patients older than 50 years compared with younger ones (P < 0.01). DISCUSSION: We observed long survival and low frequency of MG-related fatalities but increasing average standardized mortality rate, most notably in older men with MG. Muscle Nerve 58: 708-712, 2018.

Employment status of patients with chronic inflammatory demyelinating polyradiculoneuropathy.

It has been previously shown that patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who are unemployed or retired have worse quality of life. The aim of this study was to assess predictors of early retirement in CIDP. One hundred five patients with CIDP were included. Following measures were used: questionnaire on employment status, Medical Research Council Sum Score, INCAT disability score, Beck Depression Inventory, and Krupp's Fatigue Severity Scale. At the moment of testing, 2% of patients were students, 15% were employed, 9% were unemployed due to CIDP, 9% were unemployed but not due to CIDP, 28% were retired early due to disability caused by CIDP, and finally 37% were in old-age pension. Mean age when patients retired due to CIDP was 50 ± 8 years. Mean time from CIDP onset to retirement was 2.7 ± 2.3 years. Older age at onset, lower education, and more severe weakness at the time of diagnosis were significant predictors of early retirement due to CIDP. Retired patients were 12 times more likely to suffer from depression, compared to employed patients (OR = 12.2, 95% CI = 1.41-100, P < 0.01), and eight times more likely to have fatigue (OR = 8.2, 95% CI = 1.89-35.82, P < 0.01). Older patients with lower education and more severe weakness at the time of diagnosis were most likely retired due to CIDP. Early retirement was associated with depression and fatigue. Therefore, maintaining employment should be an important aim in the management of CIDP patients.

Neuromuscular disease-specific questionnaire to assess quality of life in patients with chronic inflammatory demyelinating polyradiculoneuropathy.

To date, generic questionnaires have been used to investigate quality of life (QoL) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients. Although these measures are very useful, they are not usually precise enough to measure all specific characteristics of the disease. Our aim was to investigate QoL using the neuromuscular disease-specific questionnaire (individualized neuromuscular quality of life, INQoL) in a large cohort of patients with CIDP. Our study comprised 106 patients diagnosed with CIDP. INQoL questionnaire, Medical Research Council (MRC) sum score, Inflammatory Neuropathy Cause and Treatment (INCAT) disability score, Visual Analogue Pain Scale, Beck Depression Inventory, and Krupp's Fatigue Severity Scale were used in our study. Physical domains of INQoL were more affected than mental, and the overall score was 57 ± 25. Significant predictors of higher INQoL score in our patients with CIDP were severe fatigue (ß = 0.35, p < 0.01), higher INCAT disability score at time of testing (ß = 0.29, p < 0.01), and being unemployed/retired (ß = 0.22, p < 0.05). QoL was reduced in our cohort of CIDP patients, which was more pronounced in physical segments. Patients with fatigue, more severe disability, and unemployed/retired need special attention of neurologists because they could be at greater risk to have worse QoL.

Resolution of Middle Fossa Arachnoid Cyst after Minor Head Trauma - Stages of Resolution on MRI: Case Report and Literature Review.

Arachnoid cysts (ACs) are frequently encountered in pediatric neurosurgical practice. Their natural history and optimum treatment are not well determined. The authors describe a resolution of asymptomatic middle fossa AC after trivial head trauma in a 7-year-old child. Intracystic bleeding was noticed on interval MRI. Serial MRI follow-up demonstrates stages of resolution of the AC that did not require surgical intervention. Few cases of nonsurgical AC disappearance have been reported, much fewer with trauma as a triggering factor. Here, we present the stages of resolution in images.

Large scale genotype-phenotype analyses indicate that novel prognostic tools are required for families with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (≤ 8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of second- through fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family's genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families.

Effects of Different Therapeutic Approaches on Redox Balance in Psoriatic Patients.

Given that oxidative stress represents an important etiological factor in the pathogenesis of psoriasis, the aim of this study was to assess the effects of different therapeutic approaches, methotrexate, secukinumab, and ustekinumab on systemic oxidative stress biomarkers in psoriatic patients. This study involved 78 psoriatic patients, divided into the group treated with methotrexate (23 patients), secukinumab (28 patients), and ustekinumab (27 patients), and 15 healthy controls. Oxidative stress biomarkers (index of lipid peroxidation measured as TBARS, nitrites (NO2-), superoxide anion radical (O2-), and hydrogen peroxide (H2O2)) and antioxidative defense system (superoxide dismutase (SOD) activity, catalase (CAT) activity, and reduced glutathione (GSH)) were determined spectrophotometrically from the blood before the initiation of therapy in 16th, 28th, and 52nd week. O2- and SOD showed the most prominent changes comparing the psoriatic patients and healthy controls. CAT activity was significantly lower in psoriatic patients, and methotrexate induced a further decline in CAT activity. Ustekinumab induced a significant increase in GSH level after 52 weeks of treatment, while methotrexate reduced GSH. All applied therapeutic options induced a reduction in PASI, BSA, DLQI, and EARP. Biological drugs exert more pronounced antioxidant effects compared to methotrexate, which is most clearly observed in the values of O2- and SOD.

Single-Cell Spatial Analysis Identifies Regulators of Brain Tumor-Initiating Cells.

Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/ß-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth. SIGNIFICANCE: Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor-initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

The Influence of Treatment Modalities on the Quality of Life of Psoriasis Patients.

Background: We aimed to identify the quality of life (QoL) of patients with psoriasis, to determine the possible differences depending on the therapeutic modalities (biologic, conventional treatment and phototherapy), and to examine other variables that could affect the success of the treatment. Methods: This research was a non-experimental, quantitative, observational study that included 183 psoriasis patients. The study was conducted from November 2021 to December 2022 at the University Clinical Center Kragujevac, Serbia. The following instruments were used: Dermatology Life Quality Index (DLQI), Psoriasis Area and Severity Index (PASI), as well as a general questionnaire that contained a set of questions which referred to sociodemographic data. Results: There was a statistically significant difference in the average values of the DLQI score concerning the application of different therapeutic modalities (P<0.001). Biologic treatment was the modality with the lowest impairments in the QoL domain (average value of DLQI score 10.6±7.3), followed by patients on conventional treatment (average value of DLQI score 12.9±7.9), and the highest levels of impaired QoL were in patients who received phototherapy (average value of the DLQI score 13.7±9.3). Conclusion: Patients on biological therapy at all four time points individually (baseline, 4, 12 and 16 weeks) had the lowest average values of the DLQI score, i.e. the best QoL compared to subjects who received other therapy.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition.

Signaling by growth factor receptor tyrosine kinases is manifest through networks of proteins that are substrates and/or bind to the activated receptors. FGF receptor-3 (FGFR3) is a drug target in a subset of human multiple myelomas (MM) and is mutationally activated in some cervical and colon and many bladder cancers and in certain skeletal dysplasias. To define the FGFR3 network in multiple myeloma, mass spectrometry was used to identify and quantify phosphotyrosine (pY) sites modulated by FGFR3 activation and inhibition in myeloma-derived KMS11 cells. Label-free quantification of peptide ion currents indicated the activation of FGFR3 by phosphorylation of tandem tyrosines in the kinase domain activation loop when cellular pY phosphatases were inhibited by pervanadate. Among the 175 proteins that accumulated pY in response to pervanadate was a subset of 52 including FGFR3 that contained a total of 61 pY sites that were sensitive to inhibition by the FGFR3 inhibitor PD173074. The FGFR3 isoform containing the tandem pY motif in its activation loop was targeted by PD173074. Forty of the drug-sensitive pY sites, including two located within the 35-residue cytoplasmic domain of the transmembrane growth factor binding proteoglycan (and multiple myeloma biomarker) Syndecan-1/CD138, were also stimulated in cells treated with the ligand FGF1, providing additional validation of their link to FGFR3. The identification of these overlapping sets of co-modulated tyrosine phosphorylations presents an outline of an FGFR3 network in the MM model and demonstrates the potential for pharmacodynamic monitoring by label-free quantitative phospho-proteomics.

Roles of Chromatin Remodelling and Molecular Heterogeneity in Therapy Resistance in Glioblastoma.

Cancer stem cells (CSCs) represent a therapy-resistant reservoir in glioblastoma (GBM). It is now becoming clear that epigenetic and chromatin remodelling programs link the stemlike behaviour of CSCs to their treatment resistance. New evidence indicates that the epigenome of GBM cells is shaped by intrinsic and extrinsic factors, including their genetic makeup, their interactions and communication with other neoplastic and non-neoplastic cells, including immune cells, and their metabolic niche. In this review, we explore how all these factors contribute to epigenomic heterogeneity in a tumour and the selection of therapy-resistant cells. Lastly, we discuss current and emerging experimental platforms aimed at precisely understanding the epigenetic mechanisms of therapy resistance that ultimately lead to tumour relapse. Given the growing arsenal of drugs that target epigenetic enzymes, our review addresses promising preclinical and clinical applications of epidrugs to treat GBM, and possible mechanisms of resistance that need to be overcome.

Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in situ hybridization probes are used to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof of principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.