Identifying novel drugs for the treatment of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Other than a need for alternative and more effective therapies there is also a growing appreciation for the need to understand the molecular underpinnings of RMS with the aim of identifying, in part, novel targets to develop highly specific and effective treatments with negligible adverse effects. The aim of this study was to identify novel drugs for the treatment of the two major RMS subtypes viz alveolar (ARMS) and embryonal (ERMS) RMS and to do so it adopted a two-pronged approach. Firstly, a novel binuclear palladacycle, AJ-5, was investigated for its anti-cancer activity and its mechanism(s) of action in RMS cells. The second approach involved a target-based drug repurposing strategy where a library of FDA-approved drugs was screened to identify leads that were able to negatively regulate the oncogenic TBX2 and TBX3 transcription factors that are known drivers of RMS. The binuclear palladacycle, AJ-5, was recently shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. To investigate the anti-cancer activity of AJ-5 in RMS cells, MTT assays were firstly performed in ERMS and ARMS as well as 'normal' cells. IC50 values determined from these experiments showed values of ≤ 0.2μM for the RMS cells and a favourable selectivity index of > 2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptotic and necrotic cell death. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Increased levels of necroptotic markers p-RIP3 and p-MLKL and inhibition of necroptosis with necrostatin-1 with a corresponding significant increase in cell viability suggests that AJ-5 is also capable of triggering a form of programmed necrosis. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1, and a significant reduction in autophagosome flux J. Pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. With the intention of improving the drug-like properties of AJ-5, specifically its water solubility, a derivative of AJ-5, BTC2, was synthesised and identified to display comparable anti-cancer activity against ERMS and ARMS cells. Together these findings suggest that AJ-5 and BTC2 may be effective chemotherapeutics with a desirable and novel mechanism of action for treating drug resistant and advanced RMS. The highly homologous T-box transcription factors TBX2 and TBX3 have both been implicated as key drivers of RMS and they have been identified as novel therapeutic targets for the treatment of this sarcoma subtype. Indeed, TBX2 or TBX3 overexpression in normal myoblasts inhibits muscle differentiation and overexpression and knock-down cell culture and mouse models show that RMS cells are addicted to them for their cancer phenotype. However, targeting transcription factors is notoriously challenging because unlike enzymes they do not have catalytic activity and deep binding pockets to which small molecule inhibitors can be designed which is further exacerbated by the length of time and costs associated with de novo drug development. Therefore, this study adopted a novel strategy to circumvent these challenges by combining a drug repurposing with a targeted approach to TBX2/3. Briefly, a high throughput cell-based immunofluorescence screen was designed and conducted to identify FDA-approved drugs that could negatively regulate TBX2 and/or TBX3 protein levels or nuclear localisation. Cells were engineered to express induced exogenous FLAG-tagged TBX2 and TBX3 using a Tet-On system and they were screened with the Pharmakon 1600 drug library at a concentration of 10μM. 'Hits' were identified by z-scores and amongst these, niclosamide, piroctone olamine and pyrvinium pamoate were validated to be potent inhibitors of TBX2/3 and were shown to display anti-cancer activity in RMS. These drugs have the potential to be repurposed for the treatment of RMS and other TBX2/3 driven cancers either as single agents or in combination with currently used chemotherapeutics

Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface

We present “molecular threading”, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers

Structural and optical properties of high quality zinc-blende/wurtzite GaAs hetero-nanowires

The structural and optical properties of 3 different kinds of GaAs nanowires with 100% zinc-blende structure and with an average of 30% and 70% wurtzite are presented. A variety of shorter and longer segments of zinc-blende or wurtzite crystal phases are observed by transmission electron microscopy in the nanowires. Sharp photoluminescence lines are observed with emission energies tuned from 1.515 eV down to 1.43 eV when the percentage of wurtzite is increased. The downward shift of the emission peaks can be understood by carrier confinement at the interfaces, in quantum wells and in random short period superlattices existent in these nanowires, assuming a staggered band-offset between wurtzite and zinc-blende GaAs. The latter is confirmed also by time resolved measurements. The extremely local nature of these optical transitions is evidenced also by cathodoluminescence measurements. Raman spectroscopy on single wires shows different strain conditions, depending on the wurtzite content which affects also the band alignments. Finally, the occurrence of the two crystallographic phases is discussed in thermodynamic terms.Comment: 24 page

Cluster scale composition determination in a boron-rich compound

Composition metrology is an important issue for compound nanostructure and devices. By imaging the internal structure of boron clusters in a boron-rich material using the high angle annular dark field technique in an aberration-corrected scanning transmission electron microscope, the authors have been able to determine quantitatively the local composition to be B6O1−x (x=0.30±0.04) through a relative image contrast analysis with the aid of a dynamical electron scattering simulation. Fluctuation of the oxygen occupancy is spatially resolved. This high precision and efficient measurement allows the study of the nonstoichiometry effect on an atomic scale for boron-rich materials