BCP-ALL cells treated with IP display significantly increased cell proliferation and higher stem cell-like and lipid metabolism marker levels
First, we analyzed the expression patterns of proliferation-related genes (cyclin D1, E2F1, and Ki-67), stem cell-like (OCT4, Nanog, and SOX2), and leukemia-associated mutant markers (AML1, ETV6, and Ikaros1) in BCP-ALL cells (REH and Nalm-6) treated with IP to explore the potential functional relationship between IP and BCP-ALL. Cell proliferation marker expression increased in BCP-ALL cells treated with IP in a concentration-dependent manner (Figure 1A and B). OCT4, Nanog, and SOX2 expression was significantly increased in BCP-ALL cells treated with IP (Figure 1C and D), as was AML1, ETV6, and Ikaros1 mRNA expression (Figure 1E and F).
IP promotes BCP-ALL cell proliferation and transformation
We evaluated the proliferation of IP-responsive cells and expression of stem cell-like and leukemia-associated mutant markers in BCP-ALL cells treated with IP to assess potential tumorigenesis. Similar to those observed at the mRNA level, the protein expression of cell proliferation, stem cell-like, and leukemia-associated mutant markers was increased in BCP-ALL cells treated with IP (Figure 1G and H). The cellular and oncogenic activities regulated by IP were further examined regarding their effects on cell proliferation and transformation in vitro. Firstly, BCP-ALL cells treated with IP displayed increased proliferation in the bromodeoxyuridine (BrdU) incorporation assay (Figure 1I and J). Secondly, cellular transformation in BCP-ALL cells was enhanced by IP in an independent soft agar assay, in which treatment with IP and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-established AHR ligand for comparison, significantly increased colony formation (Figure 1K and L).
IP induces AHR expression, cell proliferation, and cell transformation through the JAK and PI3K signaling pathways
We analyzed AHR expression patterns in leukemia cell lines to explore the potential functional relationship among IP, AHR, and BCP-ALL. Analyses of eight leukemia cell lines (Nalm-6, Sup-B15, REH, Molt-4, K562, HL-60, Jurkat, and B-job) revealed higher AHR expression in B-cell leukemia cells (Nalm-6, REH and Sup-B15) than in lymphoblastoid cell lines (Figure 2A and B). Furthermore, REH cells treated with IP were separately co-incubated with different kinase-specific inhibitors to evaluate transcriptional activation. Moreover, we analyzed the expression of AHR and cell proliferation markers via western blotting. The protein expression of AHR and cell proliferation markers induced by IP treatment was concentration-dependently suppressed by the JAK-specific inhibitor AG480 and MAPK-specific inhibitor U0126 (Figure 2C). Meanwhile, IP increased KYN secretion in REH cells, and inhibition of JAK and MAPK in REH cell lines reversed this effect (Figure 2D). Moreover, we examined the functional effects of IP on BCP-ALL cells. The results indicated that IP significantly increased the cell growth (Figure 2E), proliferation (Figure 2F and G), and transformation of BCL-ALL cells (Figure 2H and I), and these effects were reversed by treatment of the cells with AG480 and U0126.
AHR expression is essential for proliferation and transformation in leukemia cells treated with IP
Endogenous AHR was silenced in REH and Nalm-6 cells using four short hairpin RNA interference to determine whether AHR is essential for IP-induced signaling (Figure S1). AHR-silenced cells were then treated with IP to evaluate the regulatory effects of AHR on cell proliferation. IP activated AHR and cell proliferation marker (Cyclin D1, E2F1, and Ki-67) expression at the protein level (Figure 3A). Also, IP activated BCL-ALL oncogenic marker (AML1, c-Myc, ETV6 and Ikarose1) expression at the protein level (Figure 3A) However, IP-induced AHR, cell proliferation and oncogenic marker activation of BCL-ALL was abrogated in AHR-silenced cells compared to the findings in mock-infected cells (Figure 3A). The proliferation and transformation of AHR-silenced leukemia cells were determined using BrdU incorporation and independent soft agar assays to further assess the tumorigenic effect of AHR on leukemia prognosis. IP-induced AHR expression increased BrdU incorporation in REH and Nalm-6 cells, but AHR-silenced cells exhibited decreased proliferation and transformation (Figure 3B, E and F) even higher dose of IP exposure. IP increased KYN secretion in REH and Nalm-6 cells and silenced of AHR in REH and Nalm-6 cell lines reversed this effect (Figure 3C). KYN increased cell proliferation in REH and Nalm-6 cells, but AHR-silenced cells exhibited decreased proliferation (Figure 3D).
IDO inhibitors significantly inhibit AHR-mediated KYN expression, cell proliferation, and cell transformation
IDOs control tryptophan catabolism (9), whereas IP influences KYN–AHR oncogenic signaling (12). We measured KYN expression and oncogenic activity in leukemia cells (REH and Nalm-6) to verify the regulatory effects of IP on AHR–IDO–KYN signaling. We induced Nalm-6 cells to express AHR via IP treatment in the presence of IDO-selective inhibitors (INCB024360, 5 and 10 mmol/L; NLG-8189, 15 and 30 mmol/L). Both IDO inhibitors significantly suppressed, albeit at varying degrees, AHR, IDO1, IDO2, TDO2, Ikaros1, ETV6 AML1, and cell proliferation marker (Cyclin D1, E2F1 and Ki-67) expression (Figure 4A). KYN levels were increased in leukemia cells treated with IP, thereby inducing AHR expression. However, the IP-mediated upregulation of its targets and KYN release were inhibited in leukemia cells treated with IP and IDO inhibitors (Figure 4B). Furthermore, IP-induced AHR expression increased BrdU incorporation and transformation activity in B-cell leukemia cells (REH and Nalm-6), but cells treated with IDO inhibitors exhibited decreased proliferation and transformation (Figure 4C–F).
KYN promotes leukemia cell proliferation and transformation
We then examined the functional effects of KYN and its positive feedback loop on leukemia cells. The results indicated that KYN significantly increased cell proliferation marker expression (cyclin D1, E2F1, and Ki-67), as well as IDO1, TDO2, Ikaros1, AML1, and ETV6 expression, in leukemia cells (Figure 5A). Meanwhile, IDO inhibition decreased their expression. Similarly, leukemia cell proliferation and transformation following KYN treatment were examined using BrdU incorporation and independent soft agar assays. The results illustrated that KYN increased leukemia cell proliferation and transformation (Figure 5B–E).
IP significantly induces Nalm-6 cell proliferation in an orthotopic xenograft mouse model
Nalm-6 cells were injected into non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice via the tail vein injection to evaluate the oncogenic activity of IP. After 1 week, the mice were treated with IP (40 ng/g 3 day) for 50 days (Figure 5F). Spleen cells were stained with human and mouse CD45 monoclonal antibodies to reveal the onset of BCP-ALL, and human CD45+ cells were counted as cell oncogenic proliferation markers. IP treatment significantly increased Nalm-6 cell proliferation compared to the effects of vehicle treatment in NOD-SCID mice (Figure 5G). IP-treated mice displayed shorter survival than vehicle-treated mice (Figure 5H). In addition, IP-treated mice had a significantly higher percentage of Nalm-6 cells and higher serum KYN levels than vehicle-treated mice (Figure 5I and J).
AHR–IDO–KYN axis expression is significantly correlated with the clinical outcome of patients with BCP-ALL
To evaluate the clinical potential outcome of AhR, 50 patients with BCP ALL from the Kaohsiung Medical University Hospital were enrolled into the AhR cohort study between August 2006 and November 2020. Forty of the patients with BCP ALL originally enrolled had sufficient follow-up data for analysis. All patients were treated by following the protocols of the Taiwan Pediatric Oncology Group. The baseline characteristics of the patients based on the categorization of AhR expression are shown in Table I. The average follow-up time for all patients was 60 months. Bone marrow cells were harvested from the ALL patients at diagnosis or relapse and the normal controls were obtained after informed consent. Further, to classify the regulation effect on leukemia patients, the expression level of AhR in patients with ALL was defined as the high group, in which AhR mRNA detected was 6-fold higher than that in normal donor. There were significant differences between low or high levels groups of AhR expression and disease relapse (p = 0.01) and disease-free survival (p < 0.01). In contrast, there was no correlation between the AhR expression and age, gender, white blood cell count, hemoglobin levels, platelet count, levels of hepatomegaly or splenomegaly (Table 1). This suggests that AhR expression has a significant regulatory effect on patient relapse and overall survival in patients with BCP ALL.
Further, to characterize the potential correlation between prognosis and AHR–IDO–KYN signaling clinically, the mRNA expression of these genes and level of KYN in 50 BCP-ALL samples were examined. IDO1, KYN, AHR, and TDO2 expression was correlated with clinical outcomes. Patients with primary or relapsed ALL exhibited significantly higher IDO1, AHR, TDO2, and KYN levels than patients without relapse (Figure 6A-D). Moreover, mRNA expression of ISX strongly correlated with those of IDO1 and TDO2 in patients with BCP-ALL (Pearson’s correlation coefficient, r = 0.7037 and 0.8599, respectively, P < 0.0001; Figure 6E and F). In the analyses of disease-free survival, patients with low AHR expression had a significantly longer survival time than those with high expression following treatment with established chemotherapy (Figure 6G; p < 0.001; HR=7.188).