Lipid uptake in chronic lymphocytic leukemia.

Many cancers rely on glucose as an energy source, but it is becoming increasingly apparent that some cancers use alternate substrates to fuel their proliferation. Chronic lymphocytic leukaemia (CLL) is one such cancer. Through the use of flow cytometry and confocal microscopy, low levels of glucose uptake were observed in the OSU-CLL and HG3 CLL cell lines relative to highly glucose-avid Raji cells (Burkitt's lymphoma). Glucose uptake in CLL cells correlated with low expression of the GLUT1 and GLUT3 receptors. In contrast, both CLL cell lines and primary CLL cells, but not healthy B cells, were found to rapidly internalise medium- and long-chain, but not short-chain, fatty acids (FAs). Differential FA uptake was also observed in primary cells taken from patients with unmutated immunoglobulin heavy variable chain usage (IGHV) compared with patients with mutated IGHV. Delipidation of serum in the culture medium slowed the proliferation and significantly reduced the viability of OSU-CLL and HG3 cells, effects that were partially reversed by supplementation with a chemically defined lipid concentrate. These observations highlight the potential importance of FAs in the pathogenesis of CLL and raise the possibility that targeting FA utilisation may represent a novel therapeutic and prognostic approach in this disease.

Altered expression of metabolic pathways in CLL detected by unlabelled quantitative mass spectrometry analysis.

Chronic lymphocytic leukaemia (CLL) remains the most common incurable malignancy of B cells in the western world. Patient outcomes are heterogeneous and can be difficult to predict with current prognostic markers. Here, we used a quantitative label-free proteomic technique to ascertain differences in the B-cell proteome from healthy donors and CLL patients with either mutated (M-CLL) or unmutated (UM-CLL) IGHV to identify new prognostic markers. In peripheral B-CLL cells, 349 (22%) proteins were differentially expressed between normal B cells and B-CLL cells and 189 (12%) were differentially expressed between M-CLL and UM-CLL. We also examined the proteome of proliferating CLL cells in the lymph nodes, and identified 76 (~8%) differentially expressed proteins between healthy and CLL lymph nodes. B-CLL cells show over-expression of proteins involved in lipid and cholesterol metabolism. A comprehensive lipidomic analysis highlighted large differences in glycolipids and sphingolipids. A shift was observed from the pro-apoptotic lipid ceramide towards the anti-apoptotic/chemoresistant lipid, glucosylceramide, which was more evident in patients with aggressive disease (UM-CLL). This study details a novel quantitative proteomic technique applied for the first time to primary patient samples in CLL and highlights that primary CLL lymphocytes display markers of a metabolic shift towards lipid synthesis and breakdown.

The Combination of Metformin and Valproic Acid Has a Greater Anti-tumoral Effect on Prostate Cancer Growth In Vivo than Either Drug Alone.

BACKGROUND/AIM: The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model. MATERIALS AND METHODS: Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice. The mice were treated with 200 µl/ml MET and/or 0.4% (w/v) VPA diluted in drinking water, or with vehicle control, and were monitored until the tumor volume reached 2,000 mm3 Evaluation of toxicity of the drug combination was determined in liver and kidney by histology. RESULTS: In both LNCaP and PC-3 xenografts, MET combined with VPA significantly reduced tumor growth during the first 4 weeks following treatment, and delayed the time-to-tumor volume of 2,000 mm3 by 90 days, as compared to MET or to VPA alone, and to vehicle control. There was no significant difference in total mouse weight, liver or kidney morphology in response to combination treatment (MET+VPA) compared to MET or VPA alone and vehicle control. CONCLUSION: The combination treatment of MET with VPA is more effective at slowing prostate tumor growth in vivo compared to either drug alone, in mouse xenografts. These pre-clinical results support previous in vitro data and also demonstrate the low toxicity of the combination of these drugs, suggesting that this may be a potential new therapy to be investigated in clinical trials for prostate cancer.

Identification of novel mutations causing pediatric cataract in Bhutan, Cambodia, and Sri Lanka.

BACKGROUND: Pediatric cataract is an important cause of blindness and visual impairment in children. A large proportion of pediatric cataracts are inherited, and many genes have been described for this heterogeneous Mendelian disease. Surveys of schools for the blind in Bhutan, Cambodia, and Sri Lanka have identified many children with this condition and we aimed to identify the genetic causes of inherited cataract in these populations. METHODS: We screened, in parallel, 51 causative genes for inherited cataracts in 33 probands by Ampliseq enrichment and sequencing on an Ion Torrent PGM. Rare novel protein coding variants were assessed for segregation in family members, where possible, by Sanger sequencing. RESULTS: We identified 24 rare (frequency <1% in public databases) or novel protein coding variants in 12 probands and confirmed segregation of variants with disease in the extended family where possible. Of these, six are predicted to be the cause of disease in the patient, with four other variants also highly likely to be pathogenic. CONCLUSION: This study found that 20%-30% of patients in these countries have a mutation in a known cataract causing gene, which is considerably lower than the 60%-70% reported in Caucasian cohorts. This suggests that additional cataract genes remain to be discovered in this cohort of Asian pediatric cataract patients.

Aberrant determination of phenotypic markers in chronic lymphocytic leukemia (CLL) lymphocytes after cryopreservation.

The cryopreservation of peripheral blood mononuclear cells (PBMCs) is a routine research laboratory process, enabling long-term storage of primary patient blood samples. Retrospective analysis of these samples has the potential to identify markers that may be associated with prognosis and response to treatment. To draw valid biological conclusions from this type of analysis, it is essential to ensure that any observed changes are directly related to the pathology of the disease rather than the preservation process itself. Therefore, we have investigated 15 cell surface markers that are relevant to chronic lymphocytic leukemia (CLL) on matched fresh and thawed samples to determine the effect of cryopreservation on their detection. We found that the number of CLL cells positive for the markers CD22, CD40, CD49d, CD54, CD69, and CXCR3 was decreased significantly after cryopreservation. In addition, the mean fluorescence intensity of 10 of the 15 markers changed significantly after cryopreservation. These findings demonstrate that care must be taken when interpreting this type of analysis on thawed samples.

Development of locus specific sub-clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia.

Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia.

Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded) using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency <1% in public databases. Confirmed mutations were assessed for segregation with the phenotype in all available family members. All identified novel or previously reported cataract-causing mutations were screened in 326 unrelated Australian controls. We detected 11 novel mutations in GJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP; and three previously reported cataract-causing mutations in GJA8, CRYAA, and CRYBB2 The most commonly mutated genes were those coding for gap junctions and crystallin proteins. Including previous reports of pediatric cataract-associated mutations in our Australian cohort, known genes account for >60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.

The Combination of Metformin and Valproic Acid Induces Synergistic Apoptosis in the Presence of p53 and Androgen Signaling in Prostate Cancer.

We investigated the potential of combining the hypoglycemic drug metformin (MET) and the antiepileptic drug valproic acid (VPA), which act via different biochemical pathways, to provide enhanced antitumor responses in prostate cancer. Prostate cancer cell lines (LNCaP and PC-3), normal prostate epithelial cells (PrEC), and patient-derived prostate tumor explants were treated with MET and/or VPA. Proliferation and apoptosis were assessed. The role of p53 in response to MET + VPA was assessed in cell lines using RNAi in LNCaP (p53+) and ectopic expression of p53 in PC-3 (p53-). The role of the androgen receptor (AR) was investigated using the AR antagonist enzalutamide. The combination of MET and VPA synergistically inhibited proliferation in LNCaP and PC-3, with no significant effect in PrEC. LNCaP, but not PC-3, demonstrated synergistic intrinsic apoptosis in response to MET + VPA. Knockdown of p53 in LNCaP (p53+, AR+) reduced the synergistic apoptotic response as did inhibition of AR. Ectopic expression of p53 in PC-3 (p53-, AR-) increased apoptosis in response to MET + VPA. In patient-derived prostate tumor explants, MET + VPA also induced a significant decrease in proliferation and an increase in apoptosis in tumor cells. In conclusion, we demonstrate that MET + VPA can synergistically kill more prostate cancer cells than either drug alone. The response is dependent on the presence of p53 and AR signaling, which have critical roles in prostate carcinogenesis. Further in vivo/ex vivo preclinical studies are required to determine the relative efficacy of MET + VPA as a potential treatment for prostate cancer. Mol Cancer Ther; 16(12); 2689-700. ©2017 AACR.

Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome).

BACKGROUND: Cataract is a major cause of severe visual impairment in childhood. The purpose of this study was to determine the genetic cause of syndromic congenital cataract in an Australian mother and son. METHOD: Fifty-one genes associated with congenital cataract were sequenced in the proband using a custom Ampliseq library on the Ion Torrent Personal Genome Machine (PGM). Reads were aligned against the human genome (hg19) and variants were annotated. Variants were prioritised for validation by Sanger sequencing if they were novel, rare or previously reported to be associated with paediatric cataract and were predicted to be protein changing. Variants were assessed for segregation with the phenotype in the affected mother. RESULT: A novel likely pathogenic variant was identified in the transactivation domain of the MAF gene (c.176C > G, p.(Pro59Arg)) in the proband and his affected mother., but was absent in 326 unrelated controls and absent from public variant databases. CONCLUSION: The MAF variant is the likely cause of the congenital cataract, Asperger syndrome, seizures, hearing loss and facial characteristics in the proband, providinga diagnosis of Aymé-Gripp syndrome for the family.

Partial duplication of the CRYBB1-CRYBA4 locus is associated with autosomal dominant congenital cataract.

Congenital cataract is a rare but severe paediatric visual impediment, often caused by variants in one of several crystallin genes that produce the bulk of structural proteins in the lens. Here we describe a pedigree with autosomal dominant isolated congenital cataract and linkage to the crystallin gene cluster on chromosome 22. No rare single nucleotide variants or short indels were identified by exome sequencing, yet copy number variant analysis revealed a duplication spanning both CRYBB1 and CRYBA4. While the CRYBA4 duplication was complete, the CRYBB1 duplication was not, with the duplicated CRYBB1 product predicted to create a gain of function allele. This association suggests a new genetic mechanism for the development of isolated congenital cataract.

From genome to proteome: Looking beyond DNA and RNA in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains the most common leukemia in the Western world. Whilst its disease course is extremely heterogeneous (ranging from indolent to aggressive), current methods are unable to accurately predict the clinical journey of each patient. There is clearly a pressing need for both improved prognostication and treatment options for patients with this disease. Whilst molecular studies have analyzed both genetic mutations and gene expression profiles of these malignant B-cells, and as a result have shed light on the pathogenesis of CLL, proteomic studies have been largely overlooked to date. This review summarizes our current knowledge of the proteomics of CLL, and discusses some of the issues in CLL proteomic research, such as reproducibility and data interpretation. In addition, we look ahead to how proteomics may significantly help in the development of a successful treatment for this currently incurable disease.

Recurrent mutation in the crystallin alpha A gene associated with inherited paediatric cataract.

BACKGROUND: Cataract is a major cause of childhood blindness worldwide. The purpose of this study was to determine the genetic cause of paediatric cataract in a South Australian family with a bilateral lamellar paediatric cataract displaying variable phenotypes. CASE PRESENTATION: Fifty-one genes implicated in congenital cataract in human or mouse were sequenced in an affected individual from an Australian (Caucasian) family using a custom Ampliseq library on the Ion Torrent Personal Genome Machine. Reads were mapped against the human genome (hg19) and variants called with the Torrent Suite software. Variants were annotated to dbSNP 137 using Ion Reporter (IR 1.6.2) and were prioritised for validation if they were novel or rare and were predicted to be protein changing. We identified a previously reported oligomerization disrupting mutation, c.62G > A (p.R21Q), in the Crystallin alpha A (CRYAA) gene segregating in this three generation family. No other novel or rare coding mutations were detected in the known cataract genes sequenced. Microsatellite markers were used to compare the haplotypes between the family reported here and a previously published family with the same segregating mutation. Haplotype analysis indicated a potential common ancestry between the two South Australian families with this mutation. The work strengthens the genotype-phenotype correlations between this functional mutation in the crystallin alpha A (CRYAA) gene and paediatric cataract. CONCLUSION: The p.R21Q mutation is the most likely cause of paediatric cataract in this family. The recurrence of this mutation in paediatric cataract families is likely due to a familial relationship.

Differential Telomere Shortening in Blood versus Arteries in an Animal Model of Type 2 Diabetes.

Vascular dysfunction is an early feature of diabetic vascular disease, due to increased oxidative stress and reduced nitric oxide (NO) bioavailability. This can lead to endothelial cell senescence and clinical complications such as stroke. Cells can become senescent by shortened telomeres and oxidative stress is known to accelerate telomere attrition. Sirtuin 1 (SIRT1) has been linked to vascular health by upregulating endothelial nitric oxide synthase (eNOS), suppressing oxidative stress, and attenuating telomere shortening. Accelerated leukocyte telomere attrition appears to be a feature of clinical type 2 diabetes (T2D) and therefore the telomere system may be a potential therapeutic target in preventing vascular complications of T2D. However the effect of T2D on vascular telomere length is currently unknown. We hypothesized that T2D gives rise to shortened leukocyte and vascular telomeres alongside reduced vascular SIRT1 expression and increased oxidative stress. Accelerated telomere attrition was observed in circulating leukocytes, but not arteries, in T2D compared to control rats. T2D rats had blunted arterial SIRT1 and eNOS protein expression levels which were associated with reduced antioxidant defense capacity. Our findings suggest that hyperglycemia and a deficit in vascular SIRT1 per se are not sufficient to prematurely shorten vascular telomeres.

High-resolution analysis of cis-acting regulatory networks at the α-globin locus.

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.

Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.

Generation of bivalent chromatin domains during cell fate decisions.

BACKGROUND: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. RESULTS: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. CONCLUSIONS: While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.

ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner.

ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.

Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition.

It is well established that all of the cis-acting sequences required for fully regulated human alpha-globin expression are contained within a region of approximately 120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the alpha-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the alpha-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of alpha-globin genes, affects the pattern of NME4 expression by altering the competition for the shared alpha-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.