Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

Highlights

• •Exogenous and dietary selenium supplementation-induced endogenous CyPGs target LICs
• •CyPG-dependent activation of GPR44 leads to apoptosis of LICs, sparing normal HSCs
• •GPR44 deletion in LICs enhances RTK-activated KRAS-MAPK and -PI3K/AKT/mTORC axes
• •Loss of GPR44 increases aggressiveness of AML

Summary

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ¹²-PGJ₂, and 15d-PGJ₂, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44^−/− LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.

Introduction

Acute myeloid leukemia (AML) represents a malignancy characterized by infiltration of abnormally proliferating hematopoietic progenitor and stem cells (HPSCs) in the bone marrow, blood, and other tissues.

 Cytogenetic and molecular heterogeneity of AML has led to refractoriness and relapse.

 A key impediment in the treatment is the minimal residual disease (MRD), characterized by the presence of leukemia-initiating stem cells (LICs) that are, in part, crucial for the relapse of disease. These cells resemble normal HPSCs in terms of self-renewal, proliferation, and differentiation. Therefore, alternative therapies that target LICs open new avenues to treat AML and other malignancies as the cancer stem cell component continues to be better defined. Early studies showed that selenium (Se) cystine effectively treated AML and chronic myeloid leukemia (CML) patients, resulting in improved leukocytosis and decreased spleen size. Since then, in vitro and in vivo studies have confirmed the anti-leukemic effect of trace element Se. Previously, we reported that Se at supraphysiological levels affected the viability of LICs in CML via skewing the arachidonic acid metabolism toward the anti-inflammatory cyclopentenone prostaglandins (CyPGs), while decreasing the pro-inflammatory PGE₂ and thromboxane B₂. Previous studies reported that CyPGs enhanced the ATM-p53 axis via the peroxisome proliferator activated receptor gamma (PPARγ) activation in CML-LICs. Recently we showed that CyPGs induced by exogenous interleukin-4 benefit AML via activating PPARγ. Interestingly, apart from PPARγ, CyPGs also bind with much greater affinity to GPR44 (also called CRTH2 or DP2), a G protein coupled receptor (GPCR). GPR44 is expressed on multiple cell types but mainly on Th2 effector cells and most of the literature on GPR44 pertains to its role in autoimmune diseases and type 2 immunity with a significant lack of research into its role in hematologic malignancies.

Here, we show that activation of GPR44 mediates the anti-leukemic effect of endogenous CyPGs generated upon Se supplementation in LICs in a murine AML model and patient-derived AML cells. Furthermore, activation of GPR44 by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, enhancing apoptosis of AML LICs. These studies highlight an important therapeutic role for GPR44 in leukemia and provide mechanistic underpinnings for the chemopreventive properties of Se and CyPGs in AML.

Results

Se supplementation improves the outcome of AML

To examine the effect of Se supplementation at supraphysiological levels on the outcome of AML, CD45.2 mice maintained on an AIN-76 diet containing Se (sodium selenite, Na₂SeO₃) at either 0.08 ppm (Se-A, Se-adequate) or 0.4 ppm (Se-S, Se-supplemented) were transplanted with CD45.1⁺ primary (1°) WT AML donor cells (Figure 1A) to generate secondary AML mice as described in Figures S1A–S1D. Mice maintained on the Se-S diet showed decreased leukocytosis (Figure S1E) as well as lower average spleen weight than their Se-A counterparts at the endpoint (Figure S1F and S1G). Se-S mice also showed significant reduction in tumor burden in the bone marrow (Figure S1H) and spleen (Figure S1I), indicated by CD45.1⁺ cells in the Lin⁻ population compared with their Se-A counterparts. WT LICs, identified as CD45.1⁺Lin⁻Sca-1⁻c-Kit⁺ cells (Figure S1J), were reduced in the Se-S group (Figures 1B and 1C). Analysis of recipient bone marrow cells in the form of Lin⁻ population (CD45.1⁻) (Figure S1K) and hematopoietic stem cells (HSCs) (CD45.1⁻Lin⁻Sca-1⁺c-Kit⁺) (Figures S1J and S1L) indicated that normal HSCs were unaffected by Se treatment. Importantly, Se supplementation contributed to a significantly prolonged survival of AML mice (Figure 1D).

We next compared the impact of different forms of dietary Se on the disease, particularly in targeting LICs in the AML mice. CD45.2-recipient mice maintained on Se-deficient (Se-D) diet (with no detectable levels of Se < 0.01 ppm) or other diets supplemented with 0.4 or 1.0 ppm of Na₂SeO₃ (inorganic), or 0.8 or 3.0 ppm of selenomethionine (SeMet; organic) prepared using the Se-D diet. These mice were transplanted with CD45.1⁺ WT AML donor cells and euthanized at 3 weeks post transplantation (Figure 1E). Compared with the Se-D diet, all Se-enriched groups except 0.8 ppm SeMet had significantly improved leukocytosis with an overall reduction of leukocytes (Figure 1F). Mice exhibited decreased splenomegaly (Figures 1G and S1M) and hepatomegaly (Figures 1H and S1N) upon supplementation with Na₂SeO₃ or SeMet. AML cells in the Lin⁻ population and LICs were greatly reduced in the bone marrow and spleen upon Se supplementation with Na₂SeO₃ or SeMet (Figures 1I–1L). In vitro Na₂SeO₃ also exerted an inhibitory effect on the viability of MOLM13 cells, an immortalized human cell line expressing MLL-AF9 fusion gene as in our murine AML model (Figure S1O).

²¹

 Between Na₂SeO₃ and SeMet diet groups, we found that 0.4 ppm Na₂SeO₃ was sufficient, while 3 ppm of SeMet was required to achieve the optimal anti-leukemic effect, indicating a preventive advantage of Na₂SeO₃ over SeMet in AML.

Se supplementation induces endogenous production of CyPGs in AML

Analysis of serum 15d-PGJ₂ of AML mice indicated a Se dose-dependent increase in 15d-PGJ₂ in Na₂SeO₃-fed mice compared with those on the Se-D diet (Figure 2A), which corroborated well with the enhanced expression of upstream enzymes, cyclooxygenase-1 (COX-1) and hematopoietic PGD synthase (H-PGDS) (Figure 2B). A significant increase of serum 15d-PGJ₂ was only observed in 0.8 ppm SeMet-fed mice, but not in the 3.0 ppm SeMet group compared with the Se-D group (Figure 2C). While COX-1 expression showed an increased trend with SeMet, H-PGDS was only significantly increased in the 3.0 ppm SeMet group (Figure 2D). Since Na₂SeO₃ diets gave us more consistent results, we focused on this form of dietary Se for the remainder of this work. Analysis of the bone marrow and spleen from AML mice fed on commercially available Se-A or Se-S diets (Se added as Na₂SeO₃) showed a Se-dependent increase in the expression of H-PGDS and CyPGs in the Se-S group (Figures 2E–2G)…