Bone Marrow Neuropathy Prevents Hematopoietic Regeneration

Abstract 139

Chemotherapy regimens commonly produce long-term hematopoietic injury, possibly through a lesion to the hematopoietic stem cell (HSC) and/or its niche but the mechanisms have yet to be elucidated. Nestin-expressing mesenchymal stem cells, under regulation of the sympathetic nervous system (SNS), are a critical component of the HSC niche (Nature. 2010; 466:829). Since peripheral neuropathy is a common complication of chemotherapy, we hypothesized that chemotherapy-induced sympathetic bone marrow (BM) neuropathy could impair BM regeneration. To test this issue, we sympathectomized mice using 6-hydroxydopamine (6OHDA) or diphtheria toxin (DT) injection into TH-Cre-iDTR mice, which express the DT receptor exclusively on catecholaminergic neurons. In both models, SNS ablation led to reduced BM regeneration following 5-fluorouracil (5FU) challenge, as determined by the delayed recovery in BM cellularity (1.7-fold↓*), clonogenic progenitors (CFU-C; 2.5-fold↓*) and Lin⁻Sca-1⁺c-Kit⁺Flt3⁻ cells (LSKF cells 2.5-fold↓*). Further, inhibition of β3 adrenergic signaling with the antagonist SR5920A impaired BM recovery to the same extent as 6OHDA sympathectomy: BM cells (1.6-fold↓*), CFU-C (1.6-fold↓*), LSKF (1.6-fold↓*). We then treated mice with the chemotherapy drug cisplatin, which induces neuropathies in mice (2.8-fold↓* in SNS fibers in BM). Cisplatin-treated mice exhibited reduced survival after BM transplantation (66% vs 100%* for cisplatin-treated and control, respectively) due to delayed regeneration: BM cells (4.6-fold↓*), CFU-C (8.8-fold↓*), LSKF (6.4-fold↓*). To evaluate the potential benefit of neuroprotection, we treated mice with 4-methylcatechol (4-MC), a powerful inducer of Nerve Growth Factor (NGF), together with cisplatin or 6OHDA, which led to the protection of SNS fibers (1.8-fold↑* and 11-fold↑* respectively). This prevented transplantation-associated death and increased BM recovery following transplantation or 5FU administration: BM cells (1.9- and 1.6-fold↑, respectively *), CFU-C (3.6- and 1.4-fold↑*), LSKF (4.5- and 2.4-fold↑*). To ascertain whether 4-MC/NGF acted specifically on the nervous system, rather than on hematopoietic or stromal cells, we used TrkA^neo/neo mice, which are genetically deficient in the NGF receptor in non-neuronal tissues (Development. 2004;131:5185). Administration of 4-MC to sympathectomized TrkA^neo/neo mice enhanced BM regeneration after 5FU: BM cells (2.0-fold↑*), CFU-C (2.0-fold↑*), LSKF (2.2-fold↑*), suggesting that 4-MC acts directly on neurons to rescue hematopoiesis. Since deletion of p53 promotes neuron survival after genotoxic damage (Neurodegeneration. 1996;5:233), we intercrossed mice expressing TH-Cre mice with p53^flox/flox animals to delete p53 specifically in catecholaminergic neurons. SNS fibers of TH-Cre;p53^flox/flox mice were protected after cisplatin treatment compared to TH-Cre;p53^+/flox controls, and this was associated with improved BM recovery after transplantation: BM cells (2.4-fold↑*), CFU-C (2.9-fold↑*), LSKF (6.5-fold↑*) when compared to TH-Cre;p53^+/flox animals. Thus, these various pharmacologic and genetic models strongly suggest that chemotherapy-induced sympathetic neuropathy prevents BM regeneration. To evaluate further the mechanisms, we investigated whether BM neuropathy might lead to dysfunctional HSC niche. SNS injury using 6OHDA or cisplatin treatment led to Nestin⁺ cell expansion and accumulation (1.7- and 3.8-fold↑*, respectively). Treatment with 4-MC reverted this phenotype (1.8-fold↓* for 6OHDA and 1.5-fold↓* for cisplatin), indicating that Nestin⁺ cell expansion is due to reduced SNS signaling. SNS injury also led to increased Nestin⁺ cell sensitivity to genotoxic insult as determined by increased Nestin⁺ cell death and apoptosis (1.4-fold↑*) 24h after 5FU injection, which reduced Nestin⁺ cell numbers during regeneration (2.1-fold↓* 12 days after 5FU). By contrast, the number of osteoblasts and endothelial cells were unaffected. These results indicate that adrenergic signals promote the quiescence of HSC niche cells and that sympathetic neuropathy renders the niche vulnerable to genotoxic insult, impairing hematopoietic regeneration. Neural protection may represent a novel strategy to maintain hematopoietic reserve and enhance transplantation efficiency in chemotherapy-treated patients. *P<0.05