ASP210: a potent oligonucleotide-based inhibitor effective against TKI-resistant CML cells.

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

Surface-modified magnetite nanoparticles act as aneugen-like spindle poison.

Iron oxide nanoparticles are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy; hence, great attention should be paid to their bio-safety. Here, we investigate the ability of surface-modified magnetite nanoparticles (MNPs) to produce chromosome damage in human alveolar A549 cells. Compared to control cells, all the applied MNPs increased the level of micronuclei moderately but did not cause structural chromosomal aberrations in exposed cells. A rise in endoreplication, polyploid and multinuclear cells along with disruption of tubulin filaments, downregulation of Aurora protein kinases and p53 protein activation indicated the capacity of these MNPs to impair the chromosomal passenger complex and/or centrosome maturation. We suppose that surface-modified MNPs may act as aneugen-like spindle poisons via interference with tubulin polymerization. Further studies on experimental animals revealing mechanisms of therapeutic-aimed MNPs are required to confirm their suitability as potential anti-cancer drugs.

No clinical evidence for performing trough plasma and intracellular imatinib concentrations monitoring in patients with chronic myelogenous leukaemia.

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough ) and intracellular (IMA Cintrac ) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7-4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1-acid glycoprotein plasma concentrations (rS = 0.42; p < 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1-acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results.

Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys.

Introduction: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms. Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days. Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues. Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications.

Treatment of molecular relapse in patients with acute myeloid leukemia using clofarabine monotherapy.

Few studies have examined the treatment of molecular relapse in patients with acute myeloid leukemia (AML) using different treatment regimens. We describe for the first time in the literature experiences with administration of clofarabine monotherapy in the treatment of eight patients with AML with molecular relapse of the disease.

Dynamics of the base of ribosomal A-site finger revealed by molecular dynamics simulations and Cryo-EM.

Helix 38 (H38) of the large ribosomal subunit, with a length of 110 A, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 A from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.

Plate reader spectroscopy as an alternative to atomic absorption spectroscopy for the assessment of nanoparticle cellular uptake.

Fundamental studies investigating the biological effects induced by nanoparticles (NPs) explicitly require the correct assessment of their intracellular concentration. Ultrasensitive atomic absorption spectroscopy (AAS) is perceived as one of the gold standard methods for quantifying internalized NPs. Besides its limitation to metal-based NPs though, AAS also requires specific infrastructure and tedious sample preparation and handling, making it time-consuming and cost-intensive. Herein we report on a reliable, rapid, and affordable alternative to AAS - plate reader spectroscopy (PRS), which offers an accessible option for everyday laboratory use without sophisticated instrumentation. Our results demonstrate, that following a proper methodological approach, data on intracellular concentration of NPs obtained by PRS are fully comparable to AAS results. Specifically, the intracellular concentration of magnetite NPs coated with sodium oleate and bovine serum albumin in human alveolar A549 cells was assessed by PRS and AAS in parallel, with a remarkable correlation coefficient of R = 0.9914.

Effective Reduction of SARS-CoV-2 RNA Levels Using a Tailor-Made Oligonucleotide-Based RNA Inhibitor.

In only two years, the coronavirus disease 2019 (COVID-19) pandemic has had a devastating effect on public health all over the world and caused irreparable economic damage across all countries. Due to the limited therapeutic management of COVID-19 and the lack of tailor-made antiviral agents, finding new methods to combat this viral illness is now a priority. Herein, we report on a specific oligonucleotide-based RNA inhibitor targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It displayed remarkable spontaneous cellular uptake, >94% efficiency in reducing RNA-dependent RNA polymerase (RdRp) RNA levels in transfected lung cell lines, and >98% efficiency in reducing SARS-CoV-2 RNA levels in samples from patients hospitalized with COVID-19 following a single application.

Differences in surface chemistry of iron oxide nanoparticles result in different routes of internalization.

The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired to reach high therapeutic efficiency while minimizing the side effects. Despite intensive research, the impact of the surface coating on the mechanism of nanoparticle uptake is not sufficiently understood yet. Herein, we present a mechanistic study of cellular internalization pathways of two magnetic iron oxide nanoparticles (MNPs) differing in surface chemistry into A549 cells. The MNP uptake was investigated in the presence of different inhibitors of endocytosis and monitored by spectroscopic and imaging techniques. The results revealed that the route of MNP entry into cells strongly depends on the surface chemistry of the MNPs. While serum bovine albumin-coated MNPs entered the cells via clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CavME) or lipid rafts were preferentially involved in the internalization of polyethylene glycol-coated MNPs. Our data indicate that surface engineering can contribute to an enhanced delivery efficiency of nanoparticles.

Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient-specific NPM1 mutations.

Nucleophosmin (NPM1) mutations in exon 12 are the most common genetic alternation in cytogenetically normal AML (CN-AML). Although mutation types A, B, and D represent the majority of cases, rare mutation variants of the NPM1 gene in individual patients do occur. In this study, we have evaluated a novel, DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of three of the most commonly occurring mutations and for six rare patient-specific mutation types, which represent 28% of all of the NPM1 mutations in our group of 25 CN-AML patients. Furthermore, the prognostic relevance of NPM1-based monitoring of minimal residual disease (MRD) in peripheral blood (PB), bone marrow (BM), and in specific cell subsets (CD34(+), CD34(-), CD34(dim)) of BM were evaluated. In 80% of the evaluable patients, a molecular relapse preceded a hematological relapse. Moreover, in this subset of patients, the molecular relapse occurred at a median of 97 days before the hematological relapse. Our compartment analysis showed a strong correlation between BM and PB (r = 0.907, P < 0.001) as well as a high copy number of mutated NPM1 in CD34(+) BM cells. In conclusion, we have demonstrated applicability of our presented RQ-PCR method for a large percentage of mutated NPM1 patients with CN-AML as well as the usefulness for long-term follow-up monitoring of MRD and the prediction of hematological relapse.

Surface coating determines the inflammatory potential of magnetite nanoparticles in murine renal podocytes and mesangial cells.

Drug-induced nephrotoxicity is a frequent adverse event and a dose-limiting factor in patient treatment and is a leading cause of prospective drug attrition during pharmaceutical development. Despite the obvious benefits of nanotherapeutics in healthcare strategies, the clearance of imaging agents and nanocarriers from the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Considering the potency of nanoparticles and their massive utilization in biomedicine the impact of magnetic nanoparticles (MNPs) on cells forming the filtration apparatus of the kidney was studied. Using primary mouse renal glomerular podocytes and mesangial cells, we investigated their response to exposure to magnetic nanoparticles coated with polyethylene glycol and bovine serum albumin. Cultured podocytes were more sensitive to MNPs than mesangial cells displaying signs of cell damage and stronger inflammatory response. Both types of MNPs induced the remodeling of actin fibers, affected the cell shape and triggered expression of inflammatory cytokines TNFα and IL-6 in podocytes. On the other hand, iNOS was induced in both renal cell types but only by MNPs with a polyethylene glycol coating. Our results have revealed that the type of cell and the type of nanoparticle coating might be the strongest determinants of cellular response toward nanoparticle exposure. Differences in susceptibility of cells to MNPs might be evident also between neighboring renal cell subpopulations integrally forming functional sub-units of this organ.

Elastic properties of ribosomal RNA building blocks: molecular dynamics of the GTPase-associated center rRNA.

Explicit solvent molecular dynamics (MD) was used to describe the intrinsic flexibility of the helix 42-44 portion of the 23S rRNA (abbreviated as Kt-42+rGAC; kink-turn 42 and GTPase-associated center rRNA). The bottom part of this molecule consists of alternating rigid and flexible segments. The first flexible segment (Hinge1) is the highly anharmonic kink of Kt-42. The second one (Hinge2) is localized at the junction between helix 42 and helices 43/44. The rigid segments are the two arms of helix 42 flanking the kink. The whole molecule ends up with compact helices 43/44 (Head) which appear to be modestly compressed towards the subunit in the Haloarcula marismortui X-ray structure. Overall, the helix 42-44 rRNA is constructed as a sophisticated intrinsically flexible anisotropic molecular limb. The leading flexibility modes include bending at the hinges and twisting. The Head shows visible internal conformational plasticity, stemming from an intricate set of base pairing patterns including dynamical triads and tetrads. In summary, we demonstrate how rRNA building blocks with contrasting intrinsic flexibilities can form larger architectures with highly specific patterns of preferred low-energy motions and geometries.

Kidney nanotoxicity studied in human renal proximal tubule epithelial cell line TH1.

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe3O4NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (>700 µg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 µg/ml) concentration. Fe3O4NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO2NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake.

RNA kink-turns as molecular elbows: hydration, cation binding, and large-scale dynamics.

The presence of Kink-turns (Kt) at key functional sites in the ribosome (e.g., A-site finger and L7/L12 stalk) suggests that some Kink-turns can confer flexibility on RNA protuberances that regulate the traversal of tRNAs during translocation. Explicit solvent molecular dynamics demonstrates that Kink-turns can act as flexible molecular elbows. Kink-turns are associated with a unique network of long-residency static and dynamical hydration sites that is intimately involved in modulating their conformational dynamics. An implicit solvent conformational search confirms the flexibility of Kink-turns around their X-ray geometries and identifies a second low-energy region with open structures that could correspond to Kink-turn geometries seen in solution experiments. An extended simulation of Kt-42 with the factor binding site (helices 43 and 44) shows that the local Kt-42 elbow-like motion fully propagates beyond the Kink-turn, and that there is no other comparably flexible site in this rRNA region. Kink-turns could mediate large-scale adjustments of distant RNA segments.

Chronic myelogenous leukemia on target.

Chronic myelogenous leukemia (CML) is commonly treated with tyrosine kinase inhibitors (TKIs) that inhibit the pro-leukemic activity of the BCR-ABL1 oncoprotein. Despite the therapeutic progress mediated by TKI use, off-target effects, treatment-induced drug resistance, and the limited effect of these drugs on CML stem cells (SCs) are major drawbacks frequently resulting in insufficient or unsustainable treatment. Therefore, intense research efforts have focused on development of improved TKIs and alternative treatment strategies to eradicate CML SCs. Alongside efforts to design superior protein inhibitors, the need to overcome the poor therapeutic effect of TKIs on CML SCs has led to a renaissance of antisense strategies, as they are reported as effective in more primitive cell types. Despite the greater drug design flexibility offered by antisense sequence variability and remarkable chemical improvements, antisense drugs exhibit unacceptable levels of off-target effects, precluding them from large-scale clinical testing. Recent advances in antisense drug design have led to a pioneering mRNA recognition concept that may offer a helping hand in eliminating off-target effects, and has potential to bridge the gap between research and clinical practice.

Dissociation of chitosan/tripolyphosphate complexes into separate components upon pH elevation.

Ionically crosslinked chitosan/tripolyphosphate (Chit/TPP) particles have been widely tested in biomedical applications, particularly as potential carriers for controlled drug delivery. Since Chit/TPP particles are typically prepared under acidic conditions, their application in physiological environment and correct evaluation of biological data ultimately require knowledge on their physico-chemical properties and overall behaviour at physiological pH, as they may differ substantially from those exhibited after preparation. In this study, Chit/TPP complexes prepared at pH 4.43 were exposed to a physiological and slightly alkaline pH of 7.42 and 8.90, respectively, and analysed by inductively coupled plasma mass spectrometry and Fourier transform infrared spectroscopy with attenuated total reflectance for TPP content. In parallel, osmolarity measurements as well as theoretical calculations were used to interpret the composition and behaviour of Chit/TPP complexes upon pH elevation. Exposure of Chit/TPP complexes to a pH in the physiological range resulted in their practically complete dissociation into free chitosan chains. This leads to a significant consequence that Chit/TPP particles prepared at acidic pH do not exist under physiological conditions.

Structural changes in alginate-based microspheres exposed to in vivo environment as revealed by confocal Raman microscopy.

A next-generation cure for type 1 diabetes relies on immunoprotection of insulin-producing cells, which can be achieved by their encapsulation in microspheres made of non-covalently crosslinked hydrogels. Treatment success is directly related to the microsphere structure that is characterized by the localization of the polymers constituting the hydrogel material. However, due to the lack of a suitable analytical method, it is presently unknown how the microsphere structure changes in vivo, which complicates evaluation of different encapsulation approaches. Here, confocal Raman microscopy (CRM) imaging was tailored to serve as a powerful new tool for tracking structural changes in two major encapsulation designs, alginate-based microbeads and multi-component microcapsules. CRM analyses before implantation and after explantation from a mouse model revealed complete loss of the original heterogeneous structure in the alginate microbeads, making the intentionally high initial heterogeneity a questionable design choice. On the other hand, the structural heterogeneity was conserved in the microcapsules, which indicates that this design will better retain its immunoprotective properties in vivo. In another application, CRM was used for quantitative mapping of the alginate concentration throughout the microbead volume. Such data provide invaluable information about the microenvironment cells would encounter upon their encapsulation in alginate microbeads.