Studying Side Effects of Tyrosine Kinase Inhibitors in a Juvenile Rat Model with Focus on Skeletal Remodeling

The tyrosine kinase (TK) inhibitor (TKI) imatinib provides a highly effective treatment for chronic myeloid leukemia (CML) targeting at the causative oncogenic TK BCR-ABL1. However, imatinib exerts off-target effects by inhibiting other TKs that are involved, e.g., in bone metabolism. Clinically, CML patients on imatinib exhibit altered bone metabolism as a side effect, which translates into linear growth failure in pediatric patients. As TKI treatment might be necessary for the whole life, long-term side effects exerted on bone and other developing organs in children are of major concern and not yet studied systematically. Here, we describe a new juvenile rat model to face this challenge. The established model mimics perfectly long-term side effects of TKI exposure on the growing bone in a developmental stage-dependent fashion. Thus, longitudinal growth impairment observed clinically in children could be unequivocally modeled and confirmed. In a “bench-to-bedside” manner, we also demonstrate that this juvenile animal model predicts side effects of newer treatment strategies by second generation TKIs or modified treatment schedules (continuous vs. intermittent treatment) to minimize side effects. We conclude that the results generated by this juvenile animal model can be directly used in the clinic to optimize treatment algorithms in pediatric patients

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

The eukaryotic CO2-concentrating organelle is liquid-like and exhibits dynamic reorganization

Approximately 30%–40% of global CO 2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO 2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a “magic number” effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles

Gel or Die: Phase Separation as a Survival Strategy.

Stress conditions trigger protein assembly by demixing from the cytoplasm, but the biological significance is still unclear. In this issue of Cell, Riback et al. report that the yeast poly(A)-binding protein 1 (Pab1) is a phase-separating stress sensor that boosts organismal fitness under physiological stress conditions

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly

Imatinib mesylate and nilotinib decrease synthesis of bone matrix in vitro.

Tyrosine kinase inhibitors (TKIs), such as imatinib (IMA) and nilotinib (NIL), are the cornerstone of chronic myeloid leukemia (CML) treatment via the blockade of the oncogenic BCR-ABL1 fusion protein. However, skeletal side effects are commonly observed in pediatric patients receiving long-term treatment with IMA. Additionally, in vitro studies have shown that IMA and NIL alter vitamin D metabolism, which may further impair bone metabolism. To determine whether TKIs directly affect bone cell function, the present study treated the human osteoblastic cell line SaOS-2 with IMA or NIL and assessed effects on their mineralization capacity as well as mRNA expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), two cytokines that regulate osteoclastogenesis. Both TKIs significantly inhibited mineralization and downregulated osteoblast marker genes, including alkaline phosphatase, osteocalcin, osterix, as well as genes associated with the pro-osteogenic Wnt signaling pathway; NIL was more potent than IMA. In addition, both TKIs increased the RANKL/OPG ratio, which is known to stimulate osteoclastogenesis. The present results suggested that the TKIs IMA and NIL directly inhibited osteoblast differentiation and directly promoted a pro-osteoclastogenic environment through the RANKL-OPG signaling axis. Thus, we propose that future work is required to determine whether the bone health of CML patients undergoing TKI-treatment should be routinely monitored