Accepted Manuscript

Inflammatory and neuropathic gene expression signatures of chemotherapy-induced neuropathy induced by vincristine, cisplatin and oxaliplatin in C57BL/6J mice

Hana Starobova , Alexander Mueller , Jennifer R. Deuis , David A. Carter , Irina Vetter

PII: S1526-5900(19)30751-5 DOI: https://doi.org/10.1016/j.jpain.2019.06.008 Reference: YJPAI 3764

To appear in: Journal of Pain

Received date: 19 March 2019 Revised date: 4 June 2019 Accepted date: 13 June 2019

Please cite this article as: Hana Starobova , Alexander Mueller , Jennifer R. Deuis , David A. Carter , Irina Vetter , Inflammatory and neuropathic gene expression signatures of chemotherapy-induced neuropathy induced by vincristine, cisplatin and oxaliplatin in C57BL/6J mice, Journal of Pain (2019), doi: https://doi.org/10.1016/j.jpain.2019.06.008

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Inflammatory and neuropathic gene expression signatures of chemotherapy-induced neuropathy induced by vincristine, cisplatin and oxaliplatin in C57BL/6J mice

Hana Starobova1, Alexander Mueller1, Jennifer R. Deuis1, David A. Carter1, Irina Vetter1,2

1Centre for Pain Research, Institute for Molecular Bioscience, University of Queensland, St

Lucia, QLD, Australia

2School of Pharmacy, The University of Queensland, Woolloongabba, QLD, Australia

Running title:

Gene expression signatures of CIPN in C57BL/6J mice

Corresponding Author:

A/Prof Irina Vetter [email protected]

Institute for Molecular Bioscience, 306 Carmody Road, St. Lucia, 4072, QLD, Australia

Disclosures: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. There are no conflicts of interest.

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Abstract

Vincristine, oxaliplatin, and cisplatin are commonly prescribed chemotherapeutic agents for the treatment of many tumors. However, a main side-effect is chemotherapy-induced peripheral neuropathy (CIPN), which may lead to changes in chemotherapeutic treatment. Although symptoms associated with CIPN are recapitulated by mouse models, there is limited knowledge of how these drugs affect the expression of genes in sensory neurons. The present study carried out a transcriptomic analysis of dorsal root ganglia (DRG) following vincristine, oxaliplatin, and cisplatin treatment with a view to gain insight into the comparative pathophysiological mechanisms of CIPN. RNA-Seq revealed 368, 295 and 256 differential expressed genes (DEGs) induced by treatment with vincristine, oxaliplatin and cisplatin, respectively and only five shared genes were dysregulated in all three groups. Cell type enrichment analysis and gene set enrichment analysis showed predominant effects on genes associated with the immune system after treatment with vincristine, while oxaliplatin treatment affected mainly neuronal genes.

Treatment with cisplatin resulted in a mixed gene expression signature.

Perspective

These results provide insight into the recruitment of immune responses to DRG and indicate enhanced neuro-inflammatory processes following administration of vincristine, oxaliplatin, and cisplatin. These gene expression signatures may provide insight into novel drug targets for treatment of CIPN.

Key words:ACCEPTED neuro-inflammation, pain, anti -MANUSCRIPTneoplastic agents, gene expression profile, transcriptome

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Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) is an unfortunate side-effect of many anti- cancer therapies Quasthoff and Hartung [62]. In particular, patients receiving vincristine, oxaliplatin or cisplatin develop numbness, tingling, loss of reflexes, ataxia, and pain [67]. These dose-and time-dependent side-effects are characteristic of CIPN and can be so problematic that treatment cessation is required [59]. Unfortunately, there are no effective strategies for the treatment or prevention of CIPN symptoms despite the continued use of these highly effective cancer medications. Although these anti-cancer medications have diverse mechanisms of action - vincristine inhibits the assembly of β-tubulin [21], while the platinum derivates oxaliplatin and cisplatin bind to DNA and interfere with replication, transcription and cell cycle [1; 12; 19; 87] - all agents are defined by a predominantly peripheral sensorimotor neuropathy that includes sensory, motoric and autonomic symptoms (Table 1).

Sensory symptoms – including hypoesthesia, paresthesia, burning sensation, neuralgia, myalgia and decreased vibration sense – occur in patients receiving vincristine, oxaliplatin or cisplatin.

Interestingly, although cisplatin and oxaliplatin are closely related structurally, the symptoms induced by these agents suggest divergent pathophysiological mechanisms, as oxaliplatin elicits characteristic cooling-induced dysesthesias that are not typically induced by cisplatin.

Vincristine and oxaliplatin but not cisplatin additionally induce hyperesthesia, jaw pain and arthralgia, while gait abnormalities or difficulty walking are characteristic of motoric dysfunction and developACCEPTED after treatment with all three drugs. MANUSCRIPT However, muscle weakness seems to develop specifically after treatment with vincristine or oxaliplatin (Table 1). In addition, patients treated ACCEPTED MANUSCRIPT

with vincristine develop frequent autonomic symptoms, including paralytic ileus, postural hypotension and urogenital dysfunction [53].

Although the pathophysiological mechanisms leading to CIPN induced by vincristine, oxaliplatin and cisplatin have been studied extensively and appear to involve altered calcium homeostasis of neurons, mitochondrial dysfunction and inflammatory processes (for review see [71]), a comparative analysis of gene expression changes in dorsal root ganglion neurons induced by these agents is lacking.

In the past, transcriptomic analysis of neuronal tissue has greatly extended our understanding of pain mechanism and progression [66; 70]. These techniques allow an understanding of the genomic and molecular responses of cells to pathological conditions, including the response of neurons and immune cells to chronic painful stimuli [70]. Therefore, we sought to compare the gene expression profile of dorsal root ganglia after treatment with vincristine, oxaliplatin or cisplatin to gain insight into the mechanisms of CIPN development. Overall, vincristine-induced gene expression changes were consistent with predominantly inflammatory processes, while treatment with oxaliplatin led to altered expression of genes associated with neuronal damage.

Furthermore, cisplatin caused gene expression changes consistent with a mixed inflammatory and neuropathic phenotype.

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Methods

Animal ethics

Ethics approval for in vivo experiments was obtained from the University of Queensland Animal

Ethics Committee. All experiments were conducted in accordance with the Animal Care and

Protection Act Qld (2002), the Australian Code of Practice for the Care and Use of Animals for

Scientific Purposes, 7th edition (2004) and the International Association for the Study of Pain

Guidelines for the Use of Animals in Research.

Induction of chemotherapy-induced neuropathy and behavioural assessment

Vincristine (Sapphire Bioscience), oxaliplatin (Sapphire Bioscience) or cis-

Diammineplatinum(II) dichloride (cisplatin, Sigma Aldrich) were administered as described previously [13; 15]. Briefly, 8-10 week old C57BL/6J mice were injected i.pl. with vincristine, oxaliplatin or cisplatin or with 5% glucose (control group) under 3% isoflurane anaesthesia and allowed to recover in their home cages. The injection schedules and the time of tissue collection were chosen based on previous studies and reports of vincristine, oxaliplatin and cisplatin induced neuropathy symptoms [13; 15; 55]. Vincristine (10 µg; i.pl.) was administered once a day for four days and DRGs were collected on day 7 (cumulative dose 40 µg). Oxaliplatin and cisplatin were injected once (40 µg; i.pl.) and tissue was collected after 72 hours.

Mechanical, thermal and cold allodynia were quantified as previously described [13; 14]. In brief, mechanicalACCEPTED allodynia was assessed using MANUSCRIPT an electronic von Frey apparatus (MouseMet, TopCat Metrology), which automatically determines the paw withdrawal threshold (PWT) upon linear increase of force applied via a blunt-tipped monofilament. Mice were acclimatized to individual enclosures in which animals stand on parallel bars, making the plantar surface of the ACCEPTED MANUSCRIPT

hind paws freely accessible to the von Frey filament. Three readings, taken at least 5 min apart, were averaged to obtain the PWT for each animal.

Heat allodynia was quantified using the Hargreaves test, in which animals are acclimatized to plexiglass enclosures positioned on a pre-warmed glass plate. A radiant heat source (IITC Life

Science Plantar Test), directed at the plantar surface of the hind paws, was used to elicit paw withdrawal, with the pre-determined cut-off set to 20 sec to avoid tissue damage.

Cold allodynia was quantified by counting the number of paw lifts, licks and flinches over 5 min in animals allowed to freely move on a Peltier-cooled plate maintained at 10 °C. All behavioural quantifications were carried out by a blinded observer who was unaware of the treatments animals had received.

Sample preparation for RNA-Seq analysis

Mice were euthanized 48 hours after i.pl. injection of vincristine, oxaliplatin or cisplatin using carbon dioxide, and the lumbar dorsal root ganglia (DRGs) at L3, L4 and L5 were dissected as described previously [83]. Total RNA was isolated using the RNeasy Mini Kit (Qiagen,

Melbourne, Australia, including on-column DNase digestion). To obtain sufficient amount of

RNA, DRGs from four animals were collected and pooled to obtain one biological replicate.

Three biological control replicates and three vincristine, oxaliplatin or cisplatin treated replicates were used for RNA-Seq. RNA-SeqACCEPTED MANUSCRIPT RNA-Seq, including bioinformatics analysis, were performed by the Institute for Molecular

Bioscience Sequencing Facility (The University of Queensland, Brisbane, QLD, Australia).

RNA-Seq was performed on the Illumina NextSeq 500 platform using 75-nucleotide single-end ACCEPTED MANUSCRIPT

runs. Libraries were constructed using TruSeq stranded total RNA library preparation and the reads were mapped to Ensembl Mus musculus mm10 genome (version GRCm38) using STAR aligner (version star/2.3.0 e) [17]and samtools [38]. The counts were generated using the HTSeq package [3] and the list of differential expressed genes was created using the R (version 3.2.2) package DESeq2 [45].

Bioinformatics analysis

The volcano plots were generated using the R package ggplot2. The list of genes was ranked by adjusted p-value (Padj), and only genes with Padj < 0.05 and total log2-fold change of > 1 were considered to be differentially expressed and used for further analysis. Cell type enrichment analysis was performed using the CTen tool as described previously [68]. The score was generated using one-sided, Fisher's Exact test for enrichment and it is shown as the -log10 of the

Benjamini-Hochberg (BH) Padj. Scores > 20 optimally minimize the false positive rate (FPR).

Each list of differentially expressed genes (DEG) was analyzed separately. Gene set enrichment analysis (GSEA) was conducted using GSEA Java desktop application and Molecular Signature

Database (MSigDB) [72]. Overlaps between differentially expressed genes and C5 curated gene set (Biological process) were computed with FDR q value setting < 0.05 and top 20 or 60 gene set overlaps were shown. The functional protein association network analysis was performed using STRING database [73] and only interactions with medium or high confidence were shown; proteins were clustered using the Markov Cluster Algorithm with inflation parameter of 1.8 [7]. The geneACCEPTED identifiers were converted into gene MANUSCRIPT names using DAVID Bioinformatics Resources 6.8, NIAID/NIH the Gene Name Batch Viewer tool. Lists of differentially expressed genes or

GO biological terms were compared using the Molbiotools, multiple list comparator ACCEPTED MANUSCRIPT

(http://www.molbiotools.com/listcompare.html). Data is available at the Gene Expression

Omnibus (GEO submission number: GSE125003).

Results

Vincristine, oxaliplatin and cisplatin induce neuropathy symptoms in mice

To confirm development of peripheral neuropathy after administration of vincristine, oxaliplatin and cisplatin, and to directly compare the behavioural phenotypes induced by these agents, we first assessed mechanical, heat and cold allodynia. As previously described, all three agents elicited robust mechanical allodynia (Figure 1A), with paw withdrawal thresholds decreasing from 3.2 ± 0.1 g (control group) to 1.0 ± 0.3 g (oxaliplatin, P < 0.05), 1.1 ± 0.2 g (cisplatin, P <

0.05) and 1.6 ± 0.1 g (vincristine, P < 0.05) within 24 h of injection. In contrast, heat withdrawal thresholds were unchanged from control (10.2 ± 1.0 s) after treatment with oxaliplatin (9.0 ± 1.6 s); decreased after treatment with cisplatin (6.0 ± 0.2 s, P < 0.05) and increased after treatment with vincristine (16.5 ± 0.9 s, P < 0.05) (Figure 1B). Only oxaliplatin (90.7 ± 6.1 flinches) caused cold allodynia, while no cold-induced responses were observed in either vehicle-treated animals (0 ± 0 flinches), cisplatin-treated animals (0 ± 0 flinches) or vincristine-treated animals

(2.2 ± 1.0 flinches) (Figure 1C).

ComparisonACCEPTED of vincristine-, oxaliplatin- and MANUSCRIPT cisplatin-induced changes in gene expression

In order to gain more insights into mechanism underlying the development of vincristine-, oxaliplatin- or cisplatin-induced peripheral neuropathy, and to compare the substance-specific ACCEPTED MANUSCRIPT

effects on peripheral neurons, we performed a transcriptome of mouse lumbar dorsal root ganglia

(L3-L5) after administration of vincristine, oxaliplatin or cisplatin (GEO submission number:

GSE125003).

Treatment with vincristine caused a significant (Padj < 0.05) differential expression of 368 genes,

323 of which were up-regulated, with only 45 down-regulated (Figure 2A & 2B) (For full list of

DEGs following vincristine administration see Supplementary Table S1). Among the highly down-regulated genes (log2fold change < - 1) was Ms4a1, which encodes Membrane Spanning

4-Domains A1, also called CD20 antigen; a B-lymphocyte antigen. Highly upregulated DEGs

(log2fold change > 1) included Nts (Neurotensin), Atf3 (Activating Transcription Factor 3),

Sprr1a (Small Proline Rich Protein 1A), Ecel1 (Endothelin Converting Enzyme Like 1), Cckbr

(Cholecystokinin B Receptor) and Star (Steroidogenic Acute Regulatory Protein), which have all been associated with altered pain perception, neuropathic pain and neuronal injury in previous studies [11; 30; 34; 46; 49; 51; 83]. The direct and secondary interactions between DEGs following vincristine administration are shown in Supplementary Figure 1.

In contrast, the administration of oxaliplatin caused predominantly downregulation of DEGs (For full list of DEGs following vincristine administration see Supplementary Table S2). Following oxaliplatin treatment, 60 DEGs were up-regulated and 235 DEGs were down-regulated (Figure

2C and D). Among the down-regulated genes (log2fold change < - 1) were Snhg1 (small nucleolar RNA host gene 1), Meg3 (Maternally expressed gene 3), Malat1 (Metastasis- associatedACCEPTED lung adenocarcinoma transcript 1 ),MANUSCRIPT Mirg (miRNA containing gene) and Wasf1 (WAS protein family, member 1). Interestingly, none of these down-regulated genes has been associated with altered pain responses to date. Among the up-regulated genes (log2fold change >

1) were Ptgds (Prostaglandin-H2 D-isomerase), Vtn (Vitronectin) and Cldn11 (Claudin 11) ACCEPTED MANUSCRIPT

(Figure 2C), of which only Prostaglandin-H2 D-isomerase has been linked to painful states [5].

The direct and secondary interactions between DEGs following oxaliplatin administration are shown in Supplementary Figure 2.

Similarly, following cisplatin treatment, 72 DEG were up-regulated and 184 DEG were down- regulated (Figure 2E and F) (For full list of DEGs following vincristine administration see

Supplementary Table S3). Among the down-regulated genes (log2fold change < - 1) were Pclo

(Piccolo (presynaptic cytomatrix protein)), Zfhx2 (Zinc finger homeobox 2), Raph1 (Ras association (RalGDS/AF-6) and pleckstrin homology domains 1) and Syn1 (Synapsin I); some of which have been associated with altered pain perception and neuropathic pain in previous studies

[23; 75; 76; 78]. The highly upregulated DEGs (log2fold change > 1) included Lcn2 (Lipocalin-

2), Hba-a2 (Hemoglobin alpha, adult chain 2) and Alas2 (Aminolevulinic acid synthase 2, erythroid), of which only Lipocalin-2 have been associated with altered pain behavior [29]. The direct and secondary interactions between DEGs following cisplatin administration are shown in

Supplementary Figure 3.

As dorsal root ganglia are a heterogeneous tissue containing the cell bodies of peripheral nerves, epithelial cells, fibroblasts, glial cells and immune cells, DEGs discovered following treatment with vincristine, oxaliplatin and cisplatin cannot be unequivocally assigned to sensory neurons alone. We thus next performed cell type enrichment analysis by CTEN, a web-based analytical platform using a highly expressed, cell specific (HECS) gene database to identify enriched cell types fromACCEPTED transcriptomic data [68], to gain MANUSCRIPT insights into cell-specific effects of vincristine, oxaliplatin or cisplatin.

Cell type enrichment analysis showed that DEGs after vincristine administration significantly overlap with gene profiles of bone marrow and of immune cells (Figure 3A and ACCEPTED MANUSCRIPT

Supplementary Table 4). Specifically, the highest scores were assigned to bone marrow

(157.5), bone (147.0), granulocytes (84.1), macrophages (41.7), spleen (38.8) and microglia

(27.5), suggesting that treatment with vincristine leads to gene expression changes consistent with neuro-inflammatory mechanisms. Surprisingly, DEGs caused by oxaliplatin administration overlapped with gene profiles of neuronal cells, as the highest scores were assigned to cerebral cortex (9.4), hypothalamus (8.2), cerebellum (8.1), amygdala (6.6), spinal cord (5.4) and dorsal root ganglia (4.5). However, oxaliplatin treatment did not result in altered expression of genes that are associated with the immune system, suggesting a predominant neuropathic phenotype

(Figure 3B and Supplementary Table 4). In contrast, DEGs associated with cisplatin treatment showed mixed cell profiles overlapping with those seen in both vincristine- and oxaliplatin- treated animals, with the highest scores assigned to bone marrow (13.6), bone (11.4), granulocytes ((10.5), cerebral cortex (9.4), dorsal root ganglia (9.2) and spinal cord (8.4) (Figure

3C and Supplementary Table 4).

To further identify shared genes that may contribute to the pathology of chemotherapy-induced neuropathy irrespective of the causative agent, we compared DEGs identified after vincristine, oxaliplatin or cisplatin administration (Figure 4A-G). Vincristine and cisplatin shared 27 DEGs

(Figure 4E), vincristine and oxaliplatin shared 7 DEGs (Figure 4F); and oxaliplatin and cisplatin shared the highest amount of DEGs, in total 171 (Figure 4D and Supplementary

Table 5). Interestingly, we identified 5 genes that were shared between vincristine, oxaliplatin and cisplatin: aminolevulinic acid synthase 2, erythroid (Alas2), hemoglobin, beta adult t chain

(Hbb-bt),ACCEPTED integrin beta 2-like (Itgb2l), lymphocyte MANUSCRIPT antigen 6 complex, locus C2 (Ly6c2) and nuclear receptor subfamily 4, group A, member 3 (Nr4a3) (Figure 4G and Supplementary ACCEPTED MANUSCRIPT

Table 5). Interestingly, none of the above-mentioned genes have been associated with pain pathology to date, and their functional roles in CIPN are unclear.

To further explore the functional roles of DEGs after vincristine, oxaliplatin or cisplatin administration, we performed Gene Set Enrichment analysis using the GSEA tool [72]. For each drug, the number of genes overlapping with the curated C5 gene set (k) and the false discovery rate q-value as a measure of statistical significance are shown in Table 2 (only top 20 GO terms for each drug are shown). Additionally, we compared the 60 most significant GO terms to identify similarities shared between the three experimental groups (Figure 4B and C). All three experimental groups shared 2 GO terms; regulation of transport and response to external stimulus. Vincristine and oxaliplatin shared 6 GO terms; regulation of cell death, regulation of cell proliferation, regulation of multicellular organismal development, regulation of transport, response to endogenous stimulus and response to external stimulus. Vincristine and cisplatin shared 13 GO terms; including defense response, immune system process, locomotion and response to external stimulus. Oxaliplatin and cisplatin shared 15 GO terms, including cell projection organization, macromolecular complex assembly, neurogenesis and vesicle mediated transport (for full lists of shared GO terms see Table 3).

Remarkably, 46% of DEGs after vincristine administration overlapped with immune system- associated processes, 22.6% with regulation of cellular processes, 4.4% with cellular transport processes and none with neuronal processes or DNA and RNA processing (Figure 4C). In contrast,ACCEPTED 25% of DEGs after oxaliplatin administrationMANUSCRIPT overlapped with DNA and RNA processing, 18% with regulation of cellular processes, 13% with cellular transport processes,

12% with neuronal processes and only 5% of immune system associated processes (Figure 4C).

Moreover, 20% of cisplatin induced DEGs overlap with regulation of cellular processes, 14% ACCEPTED MANUSCRIPT

with immune system associated processes, 10% with cellular transport processes, 9% with neuronal processes and 8% with DNA and RNA processing (Figure 4C).

Discussion

Although chemotherapy-induced peripheral neuropathy (CIPN) is one of the major causes of treatment cessation in patients receiving vincristine, oxaliplatin or cisplatin, no currently available treatments adequately control or prevent the development of this side effect [20; 71].

With the exception of cold-induced hyperalgesia in patients receiving oxaliplatin, the pain- related symptoms of CIPN are similar across all three drugs (Table 1). Importantly, these differences are replicated in animal models of CIPN, with oxaliplatin but not cisplatin and vincristine causing cold allodynia, while all compounds caused mechanical hypersensitivity

(Figure 1). However, the mechanisms leading to development of CIPN are poorly understood, multi-factorial and appear to be drug specific [71]. In the present study, we performed transcriptomic analysis of gene expression changes in dorsal root ganglia from mice treated with vincristine, oxaliplatin or cisplatin, with a view to gain an understanding of the differential and shared mechanisms that contribute to the development of CIPN.

One major finding of our analysis was that vincristine treatment caused predominantly up- regulation of genes, whereas gene expression was mostly down-regulated after treatment with oxaliplatin and cisplatin. This may be due to local recruitment of immune cells to the DRGs following vincristine administration. Vincristine has been shown to cause neuronal inflammation

[9; 81]ACCEPTED and altered excitability of peripheral neuronsMANUSCRIPT by affecting the immune system [8; 9; 31;

33; 55; 79]. Consistent with these neuro-inflammatory effects, cell type enrichment analysis

(Cten) and gene set enrichment analysis (GSEA) showed that vincristine administration is ACCEPTED MANUSCRIPT

accompanied with gene expression changes typical for immune cell recruitment and inflammation.

Among the highly upregulated genes following vincristine administration were Atf3 (Activating

Transcription Factor 3), a general marker for nerve damage and regeneration following injury of

DRG neurons [40], and the neuropeptide neurotensin, which is known to be involved in neuronal damage [25; 48]. One of the highly down-regulated genes was Ms4a1, encoding the Membrane

Spanning 4-Domains A1, also called the CD20 antigen, the target of several monoclonal antibodies (e.g. rituximab) used in the treatment of B cell lymphomas and leukemias. The precise function of CD20 remains unknown, however this putative calcium channel is expressed on the surface of pro-B lymphocytes through to mature B-lymphocytes, where its expression is terminated upon differentiation into plasma cells [74]. As Ms4a1 is only expressed in a very limited number of DRG neurons, the observed gene expression changes likely arise from the population of non-neuronal cells in whole DRGs.

Cisplatin-induced gene expression changes incorporated both neuropathic and inflammatory elements, with CTEN analysis of cisplatin-exposed DRGs identifying overlaps with both neuronal and immune cells. Additionally, GSEA identified overlaps with GO Biological processes involved in neuronal damage and the immune system, supporting a mixed neuro- immune pathology induced by cisplatin. Overlaps of both cisplatin and oxaliplatin, but not vincristine, with DNA processing is consistent with the mechanisms of action of these drugs; with vincristineACCEPTED inhibiting the assembly ofMANUSCRIPT β-tubulin [21], while the platinum derivates oxaliplatin and cisplatin bind to DNA and interfere with replication, transcription and cell cycle

[1; 12; 19; 87]. ACCEPTED MANUSCRIPT

In contrast to vincristine and cisplatin, inflammatory mechanisms did not appear to contribute significantly to oxaliplatin-induced neuropathy. Specifically, GSEA of the oxaliplatin-treated group identified GO biological processes associated with neuronal damage and DNA and RNA processing. Furthermore, CTEN of the oxaliplatin-treated groups identified overlaps with neuronal cells, but not with immune cells. This result was somewhat surprising, as oxaliplatin- induced peripheral neuropathy has previously been associated with changes in immune system activation and regulation. For example; oxaliplatin-induced mechanical hyperalgesia and epidermal nerve fibre loss were prevented by the tetracycline minocycline, an antibiotic known to inhibit macrophages/monocytes and microglia [6; 16; 41; 63]. Oxaliplatin also led to increased levels of pro-inflammatory cytokines (IL-6, IL-8, IL1β, TNF-α), which in turn can lead to sensitization of nociceptors [8; 18; 26; 27; 28; 36; 37; 43; 44; 47; 60; 79; 84; 85]. It is possible that in oxaliplatin-induced neuropathy, neuronal damage and the associated changes in gene expression precede inflammatory processes, which may drive development of more chronic neuropathy symptoms. In addition, further research is required to determine if altered expression of inflammatory genes induced by treatment with vincristine and cisplatin causally contribute to the development of CIPN, or are rather a reflection of glial or immune cell damage in DRGs.

Although the symptoms of peripheral neuropathy following oxaliplatin, cisplatin or vincristine treatment appear to be relatively similar in nature, only few DEGs were shared between all agents. Curiously, these genes included integrin β2 like protein and lymphocyte antigen complex

6, which are predominately expressed on immune cells [22; 64], while – perhaps disappointingly – no painACCEPTED- or neuropathy-related genes were identified.MANUSCRIPT Nonetheless, several DEGs emerging after treatment with individual agents have previously been associated with pain processing. For example, knockout of the Neurotensin receptor 1 or ACCEPTED MANUSCRIPT

Endothelin Converting Enzyme 2 led to altered pain perception in mice [49; 51], while knockout of the Cholecystokinin B receptor was connected to functional TLR4 deficiency and a consequent reduction of neuropathic pain in mice [34]. Additionally, Activating Transcription

Factor 3, Cholecystokinin B Receptor and Steroidogenic Acute Regulatory Protein were all found to be upregulated after neuronal injury, including spinal cord injury, burn injury and after treatment of C57BL6 mice with the chemotherapy agent paclitaxel [10; 31; 46; 83]. Similarly, expression of the gene encoding for Piccolo (presynaptic cytomatrix protein) was significantly increased in rat spinal dorsal horn after vincristine treatment and in a model of orofacial neuropathic pain [75; 76]. Additionally, Zinc finger homeobox 2 knockout mice (Zfhx2 -/- ) show deficits in pain sensitivity and a point mutation in Zfhx2 gene results in deficits in pain sensitivity and is associated with insensitive phenotype in humans [23]. Whether DEGs that have no known association with pain are novel targets for the treatment or prevention of CIPN remains to be determined.

Additionally, oxaliplatin administration caused upregulation of genes including Prostaglandin-

H2 D-isomerase, an enzyme that catalyzes the conversion of Prostaglandin H2 in Prostaglandin

D2. This has previously been found to be upregulated in the serum of neuropathic rats after chronic constriction injury [5]. Interestingly, although treatment with oxaliplatin caused cold allodynia (Figure 2) and previous studies suggested the involvement of Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in oxaliplatin-induced cold allodynia

[2; 52; 57; 82; 86], no changes in expression of thermosensitive TRP channels were observed in the presentACCEPTED study. It is possible that these MANUSCRIPT differences are due to differences in methods, analytical techniques or environmental influences. Alternatively, gene expression changes ACCEPTED MANUSCRIPT

occurring as a consequence of oxaliplatin treatment may enhance neuronal excitability to cold stimuli in the absence of changes in the gene expression of thermosensitive TRP channels.

Conclusion

We present a detailed overview of gene expression changes in dorsal root ganglia induced by treatment with the chemotherapeutic agents vincristine, oxaliplatin and cisplatin. Overall, vincristine predominantly caused dysregulation of genes associated with immune system processes, whereas oxaliplatin caused dysregulation of genes associated with neuronal function, and cisplatin resulted in gene expression changes consistent with a mixed inflammatory and neuropathic pathology. These findings may provide insight into novel drug targets for treatment of CIPN.

Acknowledgements

IV and JD were supported by a NHMRC Research fellowships. HS and AM were supported by

Australian Government research scholarships.

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Figure Legends

Figure 1: Behavioral comparison of CIPN symptoms induced by oxaliplatin, cisplatin and vincristine. Mechanical, heat and cold responses were assessed in C57BL/6 mice treated with oxaliplatin, cisplatin and vincristine. (A) Oxaliplatin, cisplatin and vincristine led to a significant reduction in mechanical paw withdrawal compared with vehicle-treated control. (B) Cisplatin caused heat hyperalgesia while vincristine caused heat hypoalgesia and oxaliplatin had no effect on heat withdrawal thresholds assessed using the Hargreaves test. (C) Only treatment with oxaliplatin caused hypersensitivity to cooling, reflected by an increased number of paw flinches at 10 °C. Data are presented as mean ± S.E.M. from n=6 animals. * P < 0.05 compared with control by One-way ANOVA. Figure 2: Analysis of differentially expressed genes in mouse lumbar dorsal root ganglia L3- L5 following vincristine, oxaliplatin and cisplatin administration. Volcano plot of all DEGs following vincristine (A) , oxaliplatin (B) and cisplatin (C) administration showing the most highly upregulated (log2fold change > 1) or downregulated (log2fold change < - 1) genes. Only genes with Padj < 0.05 and log2-foldchange greater than 1 or smaller than -1 were used for further analysis. Vincristine administration caused the up-regulation of 323 DEG and the down- regulation of 45 DEG (D); oxaliplatin administration caused the up-regulation of 60 DEGs and the down-regulation of 235 DEGs (E) and cisplatin administration caused the up-regulation of 72 DEGs and the down-regulation of 184 DEGs (F). Figure 3: Cell type specific effect of vincristine, oxaliplatin and cisplatin. Cell type enrichment analysis of differentially expressed genes in lumbal dorsal root ganglia L3-5 following vincristine (A) , oxaliplatin (B) and cisplatin (C) administration. Cell type enrichment analysis was performed using the CTen tool. The score is generated using one-sided, Fisher's Exact test for enrichment and it is shown as the -log10 of the Benjamini-Hochberg (BH) adjusted P-values. Scores > 20 optimally minimize the false positive rate (FPR). Figure 4: Venn diagrams of (A) differentially expressed genes (Padj <0.05; log2foldchange ±1) or (B) ACCEPTEDidentified GO biological terms shared betweenMANUSCRIPT vincristine, oxaliplatin or cisplatin. GO terms were identified using the gene set enrichment analysis of DEGs with C5 curated gene set (biological process) and were computed with FDR q value setting < 0.05 and top 60 gene set overlaps were shown. C) The five most significant gene set enrichment signatures of the DEGs ACCEPTED MANUSCRIPT

after vincristine, oxaliplatin or cisplatin administration show predominant inflammatory processes after vincristine treatment, while DEGs following oxaliplatin and cisplatin administration overlap predominantly with DNA and RNA processing. Network analysis, generated with STRING version 10, showing interactions between differentially expressed genes shared between (D) oxaliplatin and cisplatin, (E) vincristine and cisplatin, (F) vincristine and oxaliplatin and (G) vincristine, oxaliplatin and cisplatin. Diagrams are presented in confidence view, and only interactions with at least medium or high confidence of evidence are shown. Proteins were clustered using the Markov Cluster Algorithm with inflation parameter of 1.8. Table 1: Peripheral neuropathy symptoms in humans. Paresthesia: An abnormal sensation that is not unpleasant. Dysesthesia: An abnormal unpleasant sensation. Hyperesthesia: Increased sensitivity to stimulation. Hypoesthesia: Decreased sensitivity to stimulation. Neuralgia: Pain in the distribution of a nerve or nerves. +: Effect observed in humans; - : Effects not or extremely rarely observed in humans. Table 2: Gene set enrichment analysis of differentially expressed genes in mouse lumbar dorsal root ganglia L3-L5 following vincristine, oxaliplatin and cisplatin administration. GSEA overlap analysis of DEGs with C5 curated gene set (biological process). K show the number of DEGs in overlap and FDR q-value indicate the significance of each overlap with the GO term and only 20 most significant overlaps are shown. VC: vincristine, OX: oxaliplatin, CIS: cisplatin, GO: gene ontology, FDR: false discovery rate, red: lowest values, yellow: 50% values, green: highest values. GO terms are alphabetically sorted. Table 3: GO terms shared between vincristine and/or oxaliplatin and/or cisplatin. GO terms were identified using the gene set enrichment analysis of DEGs with C5 curated gene set (biological process) and were computed with FDR q value setting < 0.05 and top 60 gene set overlaps were shown.

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Table 1: Peripheral neuropathy symptoms in humans. Paresthesia: An abnormal sensation that is not unpleasant. Dysesthesia: An abnormal unpleasant sensation. Hyperesthesia: Increased sensitivity to stimulation. Hypoesthesia: Decreased sensitivity to stimulation. Neuralgia: Pain in the distribution of a nerve or nerves. +: Effect observed in humans; - : Effects not or extremely rarely observed in humans.

Vincristine Oxaliplatin Cisplatin

Symptom

Ref. Ref. Ref.

Effect Effect Effect

Hypoesthesia / numbness + [32; 54] + [56] + [42]

+ (esp to Hyperesthesia / hypersensitivity + [39] [56] - cold) + (often cold- Paresthesia / dysesthesia + [32; 54] [56; 65] + [42] related)

Burning sensation + [4] + [50] + [61]

Neuralgia / pain + [54] + [56] + [42]

Jaw pain + [24] + [80] -

Arthralgia (joint pain) or bone + [24] + [35] - pain

Myalgia (muscle pain) or muscle + [24] + [56] + [69] cramps

Decreased vibration sense + [77] + [58] + [42]

Hyporeflexia / areflexia + [54] + [58] + [42]

Muscle weakness + [32; 54] + [35] - Gait abnormalities (difficulties + [24; 77] + [58] + [42] ACCEPTEDwalking) MANUSCRIPT

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Table 2: Gene set enrichment analysis of differentially expressed genes in mouse lumbar dorsal root ganglia L3-L5 following vincristine, oxaliplatin and cisplatin administration. GSEA overlap analysis of DEGs with C5 curated gene set (biological process). K show the number of DEGs in overlap and FDR q-value indicate the significance of each overlap with the GO term and only 20 most significant overlaps are shown. VC: vincristine, OX: oxaliplatin, CIS: cisplatin, GO: gene ontology, FDR: false discovery rate, red: lowest values, yellow: 50% values, green: highest values. GO terms are alphabetically sorted.

VC # OX CIS # CIS VC OX # Genes in FDR Genes in FDR GO term FDR q- Genes in Overlap q- Overlap q- value Overlap (k) (k) value (k) value 2.35E- GO_CELL_MOTILITY 34 0 0 0 0 15 GO_CELLULAR_MACROMOLECULAR 4.12E _ 0 0 0 0 18 COMPLEX_ASSEMBLY -07 GO_CELLULAR_RESPONSE_TO_ORGA 4.53E- NIC_ 56 0 0 0 0 SUBSTANCE 20 GO_CENTRAL_NERVOUS_SYSTEM_ 9.11 0 0 18 0 0 DEVELOPMENT E-06 GO_CHROMATIN_ASSEMBLY_OR_ 6.45E 0 0 0 0 12 DISASSEMBLY -09 4.46E GO_CYTOSKELETON_ORGANIZATION 0 0 0 0 22 -09 3.47E- 2.14E GO_DEFENSE_RESPONSE 65 0 0 28 37 -10 GO_DEFENSE_RESPONSE_TO_OTHER_ 3.19E 0 0 0 0 18 ORGANISM -09 4.27E GO_DNA_CONFORMATION_CHANGE 0 0 0 0 12 -07 1.78E GO_DNA_PACKAGING 0 0 0 0 11 -07 7.07 GO_FAT_CELL_DIFFERENTIATION 0 0 7 0 0 ACCEPTED MANUSCRIPTE-05 2.87E- GO_HOMEOSTATIC_PROCESS 42 0 0 0 0 15 6.68E- GO_IMMUNE_RESPONSE 64 0 0 0 0 39 GO_IMMUNE_SYSTEM_DEVELOPMEN 31 4.52E- 0 0 0 0 T ACCEPTED MANUSCRIPT

17 1.61E- 8.98E GO_IMMUNE_SYSTEM_PROCESS 103 0 0 38 61 -12 3.6E- GO_INFLAMMATORY_RESPONSE 33 0 0 0 0 22 3.75E- GO_INNATE_IMMUNE_RESPONSE 35 0 0 0 0 20 1.25E- GO_LEUKOCYTE_MIGRATION 25 0 0 0 0 19 2.69E- GO_LOCOMOTION 46 0 0 0 0 21 GO_MACROMOLECULAR_COMPLEX_ 6.49E 0 0 0 0 26 ASSEMBLY -08 GO_MOVEMENT_OF_CELL_OR_ 3.82E- 1.54E 51 0 0 29 SUBCELLULAR_COMPONENT 23 -10 2.98 GO_MRNA_METABOLIC_PROCESS 0 0 16 0 0 E-06 2.90 GO_MRNA_PROCESSING 0 0 15 0 0 E-07 GO_NEGATIVE_REGULATION_OF_CE 4.15 LL_ 0 0 17 0 0 DEATH E-05 GO_NEGATIVE_REGULATION_OF_GE 6.81 NE_ 0 0 22 0 0 EXPRESSION E-05 2.77 6.49E GO_NEUROGENESIS 0 0 22 26 E-05 -08 GO_POSITIVE_REGULATION_OF_ 2.90 0 0 29 0 0 BIOSYNTHETIC_PROCESS E-07 GO_POSITIVE_REGULATION_OF_GEN 1.88 6.49E E_ 0 0 29 29 EXPRESSION E-07 -08 GO_POSITIVE_REGULATION_OF_ 1.15E- 44 0 0 0 0 IMMUNE_SYSTEM_PROCESS 23 GO_POSITIVE_REGULATION_OF_MUL 5.67E- TICELLULAR_ORGANISMAL_PROCES 49 0 0 0 0 S 20 GO_POSITIVE_REGULATION_OF_ 1.22E- 58 0 0 0 0 RESPONSE_TO_STIMULUS 20 GO_POSITIVE_REGULATION_OF_ 2.21 TRANSCRIPTION_FROM_RNA_ 0 0 22 0 0 POLYMERASE_II_PROMOTER E-07 1.21E GO_PROTEIN_COMPLEX_BIOGENESIS 0 0 0 0 23 -07 GO_PROTEIN_COMPLEX_SUBUNIT_ 2.14E 0 0 0 0 31 ORGANIZATIONACCEPTED MANUSCRIPT -10 GO_PROTEIN_DNA_COMPLEX_SUBUN 8.26E IT_ 0 0 0 0 12 ORGANIZATION -08 GO_REGULATION_OF_ANATOMICAL_ 1.75 0 0 19 0 0 STRUCTURE_MORPHOGENESIS E-05 ACCEPTED MANUSCRIPT

1.44 GO_REGULATION_OF_CELL_DEATH 0 0 25 0 0 E-06 GO_REGULATION_OF_CELLULAR_ 2.03E 0 0 0 0 24 LOCALIZATION -07 GO_REGULATION_OF_IMMUNE_ 1.77E- 38 0 0 0 0 RESPONSE 18 GO_REGULATION_OF_IMMUNE_ 1.66E- 56 0 0 0 0 SYSTEM_PROCESS 25 GO_REGULATION_OF_TRANSCRIPTIO N_ 7.54 0 0 35 0 0 FROM_RNA_POLYMERASE_II_ E-11 PROMOTER 1.81 8.98E GO_REGULATION_OF_TRANSPORT 0 0 30 36 E-07 -12 GO_REGULATION_OF_VESICLE_ 1.81 2.82E 0 0 16 15 MEDIATED_TRANSPORT E-07 -07 GO_RESPONSE_TO_BIOTIC_STIMULU 6.49E 0 0 0 0 21 S -08 1.56E- GO_RESPONSE_TO_CYTOKINE 32 0 0 0 0 15 GO_RESPONSE_TO_ENDOGENOUS_ 3.68 0 0 24 0 0 STIMULUS E-06 GO_RESPONSE_TO_EXTERNAL_ 1.97E- 1.91E 64 0 0 34 STIMULUS 26 -10 5.09 GO_RNA_PROCESSING 0 0 18 0 0 E-06 2.67 GO_RNA_SPLICING 0 0 13 0 0 E-06 GO_RNA_SPLICING_VIA_ 4.84 0 0 10 0 0 TRANSESTERIFICATION_REACTIONS E-05 1.01E- GO_TAXIS 28 0 0 0 0 16 GO_TRANSCRIPTION_FROM_RNA_ 4.10 0 0 17 0 0 POLYMERASE_II_PROMOTER E-06

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Table 3: GO terms shared between vincristine and/or oxaliplatin and/or cisplatin. GO terms were identified using the gene set enrichment analysis of DEGs with C5 curated gene set (biological process) and were computed with FDR q value setting < 0.05 and top 60 gene set overlaps were shown.

Vincristine & Oxaliplatin & Cisplatin GO term GO_REGULATION_OF_TRANSPORT

GO_RESPONSE_TO_EXTERNAL_STIMULUS

Vincristine & Oxaliplatin GO term GO_REGULATION_OF_CELL_DEATH GO_REGULATION_OF_CELL_PROLIFERATION GO_REGULATION_OF_MULTICELLULAR_ORGANISMAL_DEVELOPMENT GO_REGULATION_OF_TRANSPORT GO_RESPONSE_TO_ENDOGENOUS_STIMULUS GO_RESPONSE_TO_EXTERNAL_STIMULUS

Vincristine & Cisplatin GO term GO_DEFENSE_RESPONSE GO_DEFENSE_RESPONSE_TO_OTHER_ORGANISM GO_IMMUNE_SYSTEM_PROCESS GO_LOCOMOTION GO_MOVEMENT_OF_CELL_OR_SUBCELLULAR_COMPONENT GO_POSITIVE_REGULATION_OF_CELL_COMMUNICATIONACCEPTED MANUSCRIPT GO_POSITIVE_REGULATION_OF_RESPONSE_TO_STIMULUS GO_REGULATION_OF_IMMUNE_SYSTEM_PROCESS GO_REGULATION_OF_SECRETION ACCEPTED MANUSCRIPT

GO_REGULATION_OF_TRANSPORT GO_RESPONSE_TO_BACTERIUM GO_RESPONSE_TO_BIOTIC_STIMULUS GO_RESPONSE_TO_EXTERNAL_STIMULUS

Oxaliplatin & Cisplatin GO term GO_CELL_PROJECTION_ORGANIZATION GO_MACROMOLECULAR_COMPLEX_ASSEMBLY GO_NEUROGENESIS GO_POSITIVE_REGULATION_OF_BIOSYNTHETIC_PROCESS GO_POSITIVE_REGULATION_OF_GENE_EXPRESSION GO_POSITIVE_REGULATION_OF_TRANSPORT GO_PROTEIN_COMPLEX_BIOGENESIS GO_PROTEIN_COMPLEX_SUBUNIT_ORGANIZATION GO_REGULATION_OF_CELLULAR_LOCALIZATION GO_REGULATION_OF_CLATHRIN_MEDIATED_ENDOCYTOSIS GO_REGULATION_OF_ORGANELLE_ORGANIZATION GO_REGULATION_OF_TRANSPORT GO_REGULATION_OF_VESICLE_MEDIATED_TRANSPORT GO_RESPONSE_TO_EXTERNAL_STIMULUS GO_VESICLE_MEDIATED_TRANSPORT

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