The biological and clinical impact of deletions before and after large chromosomal gains in multiple myeloma.

ABSTRACT: Acquisition of a hyperdiploid (HY) karyotype or immunoglobulin heavy chain (IgH) translocations are considered key initiating events in multiple myeloma (MM). To explore if other genomic events can precede these events, we analyzed whole-genome sequencing data from 1173 MM samples. By integrating molecular time and structural variants within early chromosomal duplications, we indeed identified pregain deletions in 9.4% of patients with an HY karyotype without IgH translocations, challenging acquisition of an HY karyotype as the earliest somatic event. Remarkably, these deletions affected tumor suppressor genes (TSGs) and/or oncogenes in 2.4% of patients with an HY karyotype without IgH translocations, supporting their role in MM pathogenesis. Furthermore, our study points to postgain deletions as novel driver mechanisms in MM. Using multiomics approaches to investigate their biologic impact, we found associations with poor clinical outcome in newly diagnosed patients and profound effects on both the oncogene and TSG activity despite the diploid gene status. Overall, this study provides novel insights into the temporal dynamics of genomic alterations in MM.

Longitudinal RNA Sequencing of Skin and DRG Neurons in Mice with Paclitaxel-Induced Peripheral Neuropathy.

Paclitaxel-induced peripheral neuropathy is a condition of nerve degeneration induced by chemotherapy, which afflicts up to 70% of treated patients. Therapeutic interventions are unavailable due to an incomplete understanding of the underlying mechanisms. We previously discovered that major physiological changes in the skin underlie paclitaxel-induced peripheral neuropathy in zebrafish and rodents. The precise molecular mechanisms are only incompletely understood. For instance, paclitaxel induces the upregulation of MMP-13, which, when inhibited, prevents axon degeneration. To better understand other gene regulatory changes induced by paclitaxel, we induced peripheral neuropathy in mice following intraperitoneal injection either with vehicle or paclitaxel every other day four times total. Skin and dorsal root ganglion neurons were collected based on distinct behavioural responses categorised as "pain onset" (d4), "maximal pain" (d7), "beginning of pain resolution" (d11), and "recovery phase" (d23) for comparative longitudinal RNA sequencing. The generated datasets validate previous discoveries and reveal additional gene expression changes that warrant further validation with the goal to aid in the development of drugs that prevent or reverse paclitaxel-induced peripheral neuropathy.

Analyzing chemotherapy-induced peripheral neuropathy in vivo using non-mammalian animal models.

Non-mammalian models of CIPN remain relatively sparse, but the knowledge gained from the few published studies suggest that these species have great potential to serve as a discovery platform for new pathways and underlying genetic mechanisms of CIPN. These models permit large-scale genetic and pharmacological screening, and they are highly suitable for in vivo imaging. CIPN phenotypes described in rodents have been confirmed in those models, and conversely, genetic players leading to axon de- and regeneration under conditions of chemotherapy treatment identified in these non-mammalian species have been validated in rodents. Given the need for non-traditional approaches with which to identify new CIPN mechanisms, these models bear a strong potential due to the conservation of basic mechanisms by which chemotherapeutic agents induce neurotoxicity.

Paclitaxel-induced peripheral neuropathy is caused by epidermal ROS and mitochondrial damage through conserved MMP-13 activation.

Paclitaxel induces peripheral neuropathy as a side effect of cancer treatment. The underlying causes are unclear, but epidermal, unmyelinated axons have been shown to be the first to degenerate. We previously utilized an in vivo zebrafish model to show that the epidermal matrix-metalloproteinase 13 (MMP-13) induces degeneration of unmyelinated axons, whereas pharmacological inhibition of MMP-13 prevented axon degeneration. However, the precise functions by which MMP-13 is regulated and affects axons remained elusive. In this study, we assessed mitochondrial damage and reactive oxygen species (ROS) formation as possible inducers of MMP-13, and we analyzed MMP-13-dependent damage. We show that the small ROS, H2O2, is increased in basal keratinocytes following treatment with paclitaxel. Cytoplasmic H2O2 appears to derive, at least in part, from mitochondrial damage, leading to upregulation of MMP-13, which in turn underlies increased epidermal extracellular matrix degradation. Intriguingly, also axonal mitochondria show signs of damage, such as fusion/fission defects and vacuolation, but axons do not show increased levels of H2O2. Since MMP-13 inhibition prevents axon degeneration but does not prevent mitochondrial vacuolation, we suggest that vacuolization occurs independently of axonal damage. Finally, we show that MMP-13 dysregulation also underlies paclitaxel-induced peripheral neuropathy in mammals, indicating that epidermal mitochondrial H2O2 and its effectors could be targeted for therapeutic interventions.

Discovery of Inhibitors for Proliferating Cell Nuclear Antigen Using a Computational-Based Linked-Multiple-Fragment Screen.

Proliferating cell nuclear antigen (PCNA) is a central factor in DNA replication and repair pathways that plays an essential role in genome stability. The functional roles of PCNA are mediated through an extensive list of protein-protein interactions, each of which transmits specific information in protein assemblies. The flexibility at the PCNA-protein interaction interfaces offers opportunities for the discovery of functionally selective inhibitors of DNA repair pathways. Current fragment-based drug design methodologies can be limited by the flexibility of protein interfaces. These factors motivated an approach to defining compounds that could leverage previously identified subpockets on PCNA that are suitable for fragment-binding sites. Methodologies for screening multiple connected fragment-binding events in distinct subpockets are deployed to improve the selection of fragment combinations. A flexible backbone based on N-alkyl-glycine amides offers a scaffold to combinatorically link multiple fragments for in silico screening libraries that explore the diversity of subpockets at protein interfaces. This approach was applied to discover new potential inhibitors of DNA replication and repair that target PCNA in a multiprotein recognition site. The screens of the libraries were designed to computationally filter ligands based upon the fragments and positions to <1%, which were synthesized and tested for direct binding to PCNA. Molecular dynamics simulations also revealed distinct features of these novel molecules that block key PCNA-protein interactions. Furthermore, a Bayesian classifier predicted 15 of the 16 new inhibitors to be modulators of protein-protein interactions, demonstrating the method's utility as an effective screening filter. The cellular activities of example ligands with similar affinity for PCNA demonstrate unique properties for novel selective synergy with therapeutic DNA-damaging agents in drug-resistant contexts.