Graham Hamilton

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development

SIX5 belongs to a family of highly conserved homeodomain transcription factors implicated in development and disease. The mammalian SIX5/SIX4 gene pair is likely to be involved in the development of mesodermal structures. Moreover, a variety of data have implicated human SIX5 dysfunction as a contributor to myotonic dystrophy type 1 (DM1), a condition characterized by a number of pathologies including muscle defects and testicular atrophy. However, this link remains controversial. Here, we investigate the Drosophila gene, D-Six4, which is the closest homolog to SIX5 of the three Drosophila Six family members. We show by mutant analysis that D-Six4 is required for the normal development of muscle and the mesodermal component of the gonad. Moreover, adult males with defective D-Six4 genes exhibit testicular reduction. We propose that D-Six4 directly or indirectly regulates genes involved in the cell recognition events required for myoblast fusion and the germline:soma interaction. While the exact phenotypic relationship between D-Six4 and SIX4/5 remains to be elucidated, the defects in D-Six4 mutant flies suggest that human SIX5 should be more strongly considered as being responsible for the muscle wasting and testicular atrophy phenotypes in DM1.

A genomics approach in determining nanotopographical effects on MSC phenotype

Topography and its effects on cell adhesion, morphology, growth and differentiation are well documented. Thus, current advances with the use of nanotopographies offer promising results in the field of regenerative medicine. Studies have also shown nanotopographies to have strong effects on stem cell self-renewal and differentiation. What is less clear however is what mechanotransductive mechanisms are employed by the cells to facilitate such changes. In fastidious cell types, it has been suggested that direct mechanotransduction producing morphological changes in the nucleus, nucleoskeleton and chromosomes themselves may be central to cell responses to topography. In this report we move these studies into human skeletal or mesenchymal stem cells and propose that direct (mechanical) signalling is important in the early stages of tuning stem cell fate to nanotopography. Using fluorescence in situ hybridization (FISH) and Affymetrix arrays we have evidence that nanotopography stimulates changes in nuclear organisation that can be linked to spatially regulated genes expression with a particular focus on phenotypical genes. For example, chromosome 1 was seen to display the largest numbers of gene deregulations and also a concomitant change in nuclear positioning in response to nanotopography. Plotting of deregulated genes in reference to band positioning showed that topographically related changes tend to happen towards the telomeric ends of the chromosomes, where bone related genes are generally clustered. Such an approach offers a better understanding of cell–surface interaction and, critically, provides new insights of how to control stem cell differentiation with future applications in areas including regenerative medicine.

Smooth Muscle Enriched Long Noncoding RNA (SMILR) Regulates Cell Proliferation

Background— Phenotypic switching of vascular smooth muscle cells from a contractile to a synthetic state is implicated in diverse vascular pathologies, including atherogenesis, plaque stabilization, and neointimal hyperplasia. However, very little is known about the role of long noncoding RNA (lncRNA) during this process. Here, we investigated a role for lncRNAs in vascular smooth muscle cell biology and pathology. Methods and Results— Using RNA sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein vascular smooth muscle cells following stimulation with interleukin-1&agr; and platelet-derived growth factor. We focused on a novel lncRNA (Ensembl: RP11-94A24.1), which we termed smooth muscle–induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal to SMILR was also altered by interleukin-1&agr;/platelet-derived growth factor treatment, and HAS2 expression was reduced by SMILR knockdown. In human samples, we observed increased expression of SMILR in unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. Conclusions— These results identify SMILR as a driver of vascular smooth muscle cell proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies.

Structure, mapping and expression of the human gene encoding the homeodomain protein, SIX2 ☆

Vertebrate genes with sequence similarity to the Drosophila homeobox gene, sine oculis (so), constitute the SIX family. There is notable expression of members of this family in anterior neural structures, and several SIX genes have been shown to play roles in vertebrate and insect development, or have been implicated in maintenance of the differentiated state of tissues. Mutations in three of these genes in man (SIX5, SIX6 and SIX3) are associated with severe phenotypes, and therefore, the cloning of other human genes from this family is of interest. We have cloned and characterised the gene that encodes human SIX2, elucidated its gene structure and conducted expression studies in a range of tissues. SIX2 is widely expressed in the late first-trimester fetus, but has a limited range of expression sites in the adult. The expression pattern of SIX2 and its localisation to chromosome 2p15-p16 will be of use in assessing its candidacy in human developmental disorders.

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

Significance Anti-trypanosomal drugs, used to tackle lethal human and animal diseases, target an unusual parasite DNA structure in a cellular compartment known as the mitochondrion. Using a high-throughput genetic approach to study drug resistance, we identified every component of a molecular rotor that couples ATP hydrolysis to proton transport across non-mitochondrial membranes. Surprisingly, this molecular machine was found to communicate with a related mitochondrial rotor and, when defective, rendered the mitochondrial DNA structure obsolete. Our findings reveal new potential mechanisms of multidrug resistance in trypanosomes. They also suggest that communication between these rotors in two separate cellular compartments could be conserved through evolution, reflecting an unanticipated and important aspect of environmental sensing and metabolic control in nucleated cells. Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM.

A2 Cryptic polyglutamine repeat sequence variation and somatic instability in huntington’s disease: drivers of pathology?

It is known that in addition to repeat length variation, the exact sequence of the polyglutamine repeat tract and the adjacent polyproline also vary. Likewise, it is known that the expanded CAG is somatically unstable in a process that is age-dependent, tissue-specific and expansion biassed. Notably, very large alleles exceeding greater than 1,000 repeats are observed in a subset of striatal neurons. These data strongly suggest that somatic instability contributes toward the tissue specificity and progressive nature of the symptoms. Indeed, it has been shown that the frequency of large expansions in cortical cells correlates with variation in age at onset not accounted for by inherited repeat length. Most recently, it has been demonstrated that mismatch repair genes lie under some of the association peaks for genome wide analysis of variants contributing to variation in age at onset. Thus, in order to further address these issues, we have developed a high-throughput sequencing pipeline that allows us to determine the precise sequence of the polyglutamine and polyproline tracts. These studies have revealed an unexpectedly high frequency of atypical non-pathogenic alleles. We have also detected novel CCG interruptions within the CAG array in a large ‘premutation’ length allele. The majority of expanded HD alleles retain the expected structure of CAG and CCG repeats, but a subset of atypical pathogenic alleles have been detected. Using this approach we can also estimate the degree of somatic mosaicism present in the blood DNA of each participant. These data confirm that as expected, somatic mosaicism in the blood DNA of HD individuals is age- and allele length-dependent. We are currently attempting to understand how atypical alleles and individual-specific mutational dynamics contribute toward phenotypic variability in HD. We are also investigating how variants in the mismatch repair genes contribute toward variation in somatic mosaicism.

Targeting BCR-ABL-Independent TKI resistance in chronic myeloid leukemia by mTOR and autophagy inhibition

Abstract Background Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4–6 mice per group). All statistical tests were two-sided. Results We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Genomic analysis of the role of transcription factor C/EBPδ in the regulation of cell behaviour on nanometric grooves

C/EBPδ is a tumour suppressor transcription factor that induces gene expression involved in suppressing cell migration. Here we investigate whether C/EBPδ-dependent gene expression also affects cell responses to nanometric topology. We found that ablation of the C/EBPδ gene in mouse embryonal fibroblasts (MEFs) decreased cell size, adhesion and cytoskeleton spreading on 240 nm and 540 nm nanometric grooves. ChIP-SEQ and cDNA microarray analyses demonstrated that many binding sites for C/EBPδ, and the closely related C/EBPβ, exist throughout the mouse genome and control the upregulation or downregulation of many adjacent genes. We also identified a group of C/EBPδ-dependent, trans-regulated genes, whose promoters contained no C/EBPδ binding sites and yet their activity was regulated in a C/EBPδ-dependent manner. These genes include signalling molecules (e.g. SOCS3), cytoskeletal components (Tubb2, Krt16 and Krt20) and cytoskeletal regulators (ArhGEF33 and Rnd3) and are possibly regulated by cis-regulated diffusible mediators, such as IL6. Of particular note, SOCS3 was shown to be absolutely required for efficient cell spreading and contact guidance on 240 nm and 540 nm nanometric grooves. C/EBPδ is therefore involved in the complex regulation of multiple genes, including cytoskeletal components and signalling mediators, which influence the nature of cell interactions with nanometric topology.