Chemotherapy for pain: reversing inflammatory and neuropathic pain with the anticancer agent mithramycin A.

ABSTRACT: The persistence of inflammatory and neuropathic pain is poorly understood. We investigated a novel therapeutic paradigm by targeting gene networks that sustain or reverse persistent pain states. Our prior observations found that Sp1-like transcription factors drive the expression of TRPV1, a pain receptor, that is blocked in vitro by mithramycin A (MTM), an inhibitor of Sp1-like factors. Here, we investigate the ability of MTM to reverse in vivo models of inflammatory and chemotherapy-induced peripheral neuropathy (CIPN) pain and explore MTM's underlying mechanisms. Mithramycin reversed inflammatory heat hyperalgesia induced by complete Freund adjuvant and cisplatin-induced heat and mechanical hypersensitivity. In addition, MTM reversed both short-term and long-term (1 month) oxaliplatin-induced mechanical and cold hypersensitivity, without the rescue of intraepidermal nerve fiber loss. Mithramycin reversed oxaliplatin-induced cold hypersensitivity and oxaliplatin-induced TRPM8 overexpression in dorsal root ganglion (DRG). Evidence across multiple transcriptomic profiling approaches suggest that MTM reverses inflammatory and neuropathic pain through broad transcriptional and alternative splicing regulatory actions. Mithramycin-dependent changes in gene expression following oxaliplatin treatment were largely opposite to and rarely overlapped with changes in gene expression induced by oxaliplatin alone. Notably, RNAseq analysis revealed MTM rescue of oxaliplatin-induced dysregulation of mitochondrial electron transport chain genes that correlated with in vivo reversal of excess reactive oxygen species in DRG neurons. This finding suggests that the mechanism(s) driving persistent pain states such as CIPN are not fixed but are sustained by ongoing modifiable transcription-dependent processes.

Ancient segmentally duplicated LCORL retrocopies in equids.

LINE-1 is an active transposable element encoding proteins capable of inserting host gene retrocopies, resulting in retro-copy number variants (retroCNVs) between individuals. Here, we performed retroCNV discovery using 86 equids and identified 437 retrocopy insertions. Only 5 retroCNVs were shared between horses and other equids, indicating that the majority of retroCNVs inserted after the species diverged. A large number (17-35 copies) of segmentally duplicated Ligand Dependent Nuclear Receptor Corepressor Like (LCORL) retrocopies were present in all equids but absent from other extant perissodactyls. The majority of LCORL transcripts in horses and donkeys originate from the retrocopies. The initial LCORL retrotransposition occurred 18 million years ago (17-19 95% CI), which is coincident with the increase in body size, reduction in digit number, and changes in dentition that characterized equid evolution. Evolutionary conservation of the LCORL retrocopy segmental amplification in the Equidae family, high expression levels and the ancient timeline for LCORL retrotransposition support a functional role for this structural variant.

An SNN retrocopy insertion upstream of GPR22 is associated with dark red coat color in Poodles.

Pigment production and distribution is controlled through multiple genes, resulting in a wide range of coat color phenotypes in dogs. Dogs that produce only the pheomelanin pigment vary in intensity from white to deep red. The Poodle breed has a wide range of officially recognized coat colors, including the pheomelanin-based white, cream, apricot, and red coat colors, which are not fully explained by the previously identified genetic variants involved in pigment intensity. Here, a genome-wide association study for pheomelanin intensity was performed in Poodles which identified an association on canine chromosome 18. Whole-genome sequencing data revealed an SNN retrocopy insertion (SNNL1) in apricot and red Poodles within the associated region on chromosome 18. While equal numbers of melanocytes were observed in all Poodle skin hair bulbs, higher melanin content was observed in the darker Poodles. Several genes involved in melanogenesis were also identified as highly overexpressed in red Poodle skin. The most differentially expressed gene however was GPR22, which was highly expressed in red Poodle skin while unexpressed in white Poodle skin (log2 fold change in expression 6.1, P < 0.001). GPR22 is an orphan G-protein-coupled receptor normally expressed exclusively in the brain and heart. The SNNL1 retrocopy inserted 2.8 kb upstream of GPR22 and is likely disrupting regulation of the gene, resulting in atypical expression in the skin. Thus, we identify the SNNL1 insertion as a candidate variant for the CFA18 pheomelanin intensity locus in red Poodles.

Recent, full-length gene retrocopies are common in canids.

Gene retrocopies arise from the reverse transcription and insertion into the genome of processed mRNA transcripts. Although many retrocopies have acquired mutations that render them functionally inactive, most mammals retain active LINE-1 sequences capable of producing new retrocopies. New retrocopies, referred to as retro copy number variants (retroCNVs), may not be identified by standard variant calling techniques in high-throughput sequencing data. Although multiple functional FGF4 retroCNVs have been associated with skeletal dysplasias in dogs, the full landscape of canid retroCNVs has not been characterized. Here, retroCNV discovery was performed on a whole-genome sequencing data set of 293 canids from 76 breeds. We identified retroCNV parent genes via the presence of mRNA-specific 30-mers, and then identified retroCNV insertion sites through discordant read analysis. In total, we resolved insertion sites for 1911 retroCNVs from 1179 parent genes, 1236 of which appeared identical to their parent genes. Dogs had on average 54.1 total retroCNVs and 1.4 private retroCNVs. We found evidence of expression in testes for 12% (14/113) of the retroCNVs identified in six Golden Retrievers, including four chimeric transcripts, and 97 retroCNVs also had significantly elevated F ST across dog breeds, possibly indicating selection. We applied our approach to a subset of human genomes and detected an average of 4.2 retroCNVs per sample, highlighting a 13-fold relative increase of retroCNV frequency in dogs. Particularly in canids, retroCNVs are a largely unexplored source of genetic variation that can contribute to genome plasticity and that should be considered when investigating traits and diseases.