Night Transplantation and Catecholamine Exposure Accelerates In Vivo Leukemia Development By Enhancing Bone Marrow Homing Capacity in Leukemic Cells

Xenografts in immune suppressed mice are routinely used for in vivostudies on human acute myeloid leukemia (AML). However, a significant proportion of primary AML samples (»50-60% of cases) were engrafting in mice with particularly latency. Here, we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) accelerates the engraftment of human AML cells in NOD/SCID/IL2Rg^null(NSG) mice.

Equal numbers of freshly thawed AML cells from the same donor (n=5 patients) were transplanted into sublethally irradiated gender-matched 6 to 8 weeks old mice at day or at night (n=3-5 mice per condition and patient). Transplanted mice were monitored for leukemic engraftment in peripheral blood (PB), bone marrow (BM) and other hematopoietic organs using flow cytometry and immunohistochemistry. Enhanced leukemic engraftment was observed in night vs. day transplanted mice for each AML case (Fig. A). Interestingly, in 2/5 samples, engraftment was exclusively detected with the night transplant condition. Limiting dilutions showed that night transplantation allows leukemia (Fig. B) induction from lower cell numbers. Results were confirmed in a syngeneic MLL-PTD/FLT3-ITD mouse leukemia model (Fig. C): mice analyzed at the same time-point after night versus day transplantation showed enhanced splenomegaly (401.2 mg vs. 234.5 mg, p<0.05, Fig. D) and abnormal blood counts (white blood cells numbers 55.41x10⁹vs. 18.63x10⁹, p<0.01). To explore underlying mechanisms, homing assays were performed with fluorescently labelled human AML cells injected via tail vein and analyzed 12 hours later. Significantly higher BM colonization was found with cells injected at night versus day times (0.31±0.09 vs. 0.13±0.18, p<0.001, n=7 AML patients, Fig. E).

Intriguingly, mice transplanted at night showed stress-related behavioral abnormalities. We thus hypothesized that night transplantation induced catecholamine release may affect homing and leukemogenesis. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p<0.001) and BM (21.08±1.04 vs. 13.50±3.57 ng/ml, p<0.05) of night vs. day transplanted animals. Adrenergic receptors have been previously described to express in BM cells, and to regulate hematopoietic stem and progenitor cells (HSPCs) mobilization, Here, we show that these pathways are also important during leukemogenesis. Blocking adrenergic signaling with the b1/2-blocker propranolol prior to night transplantation (10mg/kg, intraperitoneal (i.p.) injection, 1h pre-transplant) reduced homing (0.22±0.17 vs. 0.39±0.22% leukemic cells, p=0.09) and long-term engraftment (23±7.42 vs. 80.2±11.1% leukemic blasts in BM, p<0.0001), and enhanced survival (Fig. F) in NSG mice transplanted with human AML cells and in syngeneic murine leukemia models. Consistently, treatment of mice with epinephrine prior to transplantation (2mg/kg, i.p., 1h pre-transplant) enhanced homing (0.38±0.09 vs. 0.16±0.04%, p<0.0001) and accelerated long-term leukemia induction (12.49±5.23 vs. 4.43±3.6%, p<0.01)..

Surprisingly, even though all our AML samples express the homing molecule CXCR4, blocking CXCR4 (anti-CXCR4 antibody, 50mM) prior to transplantation could not counteract epinephrine-driven homing (0.032±0.017 vs. 0.038±0.025% leukemic cells in BM, p=0.53), indicating that the stimulating effect of adrenergic signaling on homing was independent of the SDF1-CXCR4 axis. Notably, transplantations of healthy human cord-blood CD34+ HSPCs or of mouse BM Lin^- cells revealed no difference in the hematopoietic reconstitution in night vs. day transplantations (Fig. G). This indicates that the observed mechanisms are leukemia specific. To explore further potential mechanisms, RNAseq was performed on MS-5 cells, a mouse stromal cell line, treated with either epinephrine or vehicle control. Preliminary analyses indicate upregulated vitamin D receptor (VDR) expression (log2FC=1.18, p=1.32x10¹²) as potentially involved in catecholamine-induced homing of leukemic cells.

We conclude that enhanced catecholamine levels (e.g. induced by night transplantation) accelerate leukemia development from AML cells, by enhancing the homing of leukemic cells to BM niches via SDF1-CXCRR4 independent, leukemic specific mechanisms.

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