Alpha 2-Adrenergic Agonist As a Novel Microenvironment-Directed Agent for Acute Myeloid Leukemia Via Notch Signaling Inhibition

Background: Despite intensive research, therapeutic progress in clinical practice for acute myeloid leukemia (AML) has been limited, and prognosis remains poor. The “niche” in the bone marrow (BM), a specialized microenvironment composed of various hematopoietic and non-hematopoietic cell populations, plays a pivotal role in regulating the maintenance and differentiation of hematopoietic stem cells (HSCs). In contrast, AML remodels the BM microenvironment where leukemia stem cells (LSCs) reside to support its own survival. Activation of β2- or β3-adrenergic receptors (ARs) has been shown to decrease LSCs by improving the BM microenvironment (Hanoun et al., 2014; Arranz et al., 2014). On the other hand, α2-AR (especially α2α-AR) activation has recently been demonstrated to suppress tumor cell growth in several immunocompetent solid tumor murine models by modifying immune cell function (Zhu et al., 2023). Therefore, in this study, we investigated the anti-leukemic effect of α2a-AR signaling in murine AML model. Methods: To evaluate whether the α2-AR agonist (clonidine, CLD) could be a novel agent for AML, we utilized MLL-AF9+ AML model mice. Our experiments included flow cytometry, fluorescence activated cell sorting, quantitative real-time PCR (qPCR), and high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Results: We sorted various cells representative of the BM in AML model mice and measured Adra2a expression (relative to GAPDH), which was highest in MSCs (0.016 ± 0.0047, n = 3), slightly present in BM macrophages and leukemia cells (0.0022 ± 0.0017; 0.000029 ± 0.000023, n = 3) but not detected in other cells such as T cells and myeloid-derived suppressor cells. There was no direct anti-tumor effect of CLD on leukemia cells in vitro; however, AML model mice treated with CLD from day 3 to day 20 post-leukemic cell transplantation exhibited significantly lower infiltration by leukemia cells in the BM (control [CT] / CLD, 88.3 ± 2.5 / 68.8 ± 4.1%, p = 0.0002, n = 18) and significantly less egress of leukemia cells into the peripheral blood and spleen (CT / CLD, 37.2 ± 5.0 / 9.6 ± 1.0%, p < 0.0001; 73.6 ± 1.9 / 67.2 ± 2.3%, p = 0.04, n = 18) compared to CT mice treated with phosphate-buffered saline (PBS) on day 21. This therapeutic effect was associated with a significant improvement in the survival of AML model mice (CT / CLD, median of 24 / 26 days, n = 15, p = 0.03). Administration of CLD is known to inhibit norepinephrine release from the sympathetic nervous system (SNS). However, measurements of norepinephrine levels in the BM 24 hours after administration of either PBS or CLD (from day 3 to day 20 post-transplant) in AML model mice revealed no significant differences. This suggests that the effects of CLD are not mediated by changes in norepinephrine levels. Furthermore, the anti-tumor effect was not attenuated by depletion of BM macrophages with clodronate liposomes, and no synergistic effect of CLD with anti-PD-1 antibody was observed, as previously reported in solid tumors. These results suggest that the anti-tumor effects of CLD administration were mediated by MSCs. To elucidate the molecular mechanism, we focused on Notch signaling, which has been shown to be more highly expressed in AML MSCs compared to healthy MSCs, as revealed by RNA microarrays, and is associated with cell proliferation and chemotherapy resistance in leukemia cells (Takam Kamga et al., 2016; Ahmed et al., 2022). We sorted MSCs from AML model mice treated with CLD or PBS. qPCR analyses demonstrated that the expression of Notch signaling-related genes (Notch1, Notch2, Jag1, and Hes1) was decreased following CLD administration (CT / CLD, 0.010 ± 0.0022 / 0.0049 ± 0.0012, p = 0.07; 0.015 ± 0.0029 / 0.0039 ± 0.0012, p = 0.0037; 0.061 ± 0.012 / 0.036 ± 0.0034, p = 0.09; 1.10 ± 0.13 / 0.71 ± 0.10, p = 0.04, n = 4-10). These results suggest that anti-tumor effect of CLD was mediated by the suppression of Notch signaling in AML MSCs. Conclusion: Our research demonstrates that α2-AR activation in AML MSCs suppresses Notch signaling, which plays a significant role in AML cell-MSC interactions. This suppression contributes to a reduction in AML progression and improvement in survival. These results suggest that α2-AR agonists hold potential as novel therapeutic agents for AML and warrants further investigation for clinical application.