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. 2018 Aug 9;13(8):e0200923.
doi: 10.1371/journal.pone.0200923. eCollection 2018.

Aryl Hydrocarbon Receptor (AHR) Is a Novel Druggable Pathway Controlling Malignant Progenitor Proliferation in Chronic Myeloid Leukemia (CML)

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Aryl Hydrocarbon Receptor (AHR) Is a Novel Druggable Pathway Controlling Malignant Progenitor Proliferation in Chronic Myeloid Leukemia (CML)

Melanie Gentil et al. PLoS One. .
Free PMC article

Abstract

Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of AHR in UT-7 cells and UT-7/11 cells.
(A) Evaluation of the expression of BCR-ABL in the UT-7/11 cells as compared to the parental UT-7 cell line using Western blot analysis. As can be seen in this panel, BCR-ABL protein is highly expressed in the UT-7/11 cell line, with presence of some degradation products, which are visible. (B). Evaluation of the expression of the expression of AHR mRNA using Q-RT-PCR analyses. The expression of AHR is strongly downregulated in the BCR-ABL-expressing UT-7/11 cell line as compared to parental UT-7 cells. This difference is highly significant (t-test, p = 0.0247).
Fig 2
Fig 2. Expression of AHR in primary leukemic samples.
(A) AHR transcript was quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) and were found to be very significantly reduced as compared to normal controls (n = 15) (p < 0.0001, ANOVA and Tukey’s post-hoc test). In 8 of these patients, AHR mRNA was quantified during TKI-induced major molecular response (MMR) and were found to increase towards levels found in controls in a statistically significant manner (p < 0.0001). Reported p values were adjusted for multiple comparisons. (B) AHR expression in CD34+ cells (n = 11) as well as in highly purified CD34+ CD38- cell populations (n = 3). Interestingly, AHR expression was reduced in leukemic CD34+ progenitors (n = 11) but not in more primitive leukemic CD34+ CD38- cells (n = 3, p = 0.0076) as compared to normal controls (n = 15).
Fig 3
Fig 3. Analysis of the effects of AHR antagonist SR-1 on leukemic cell growth.
A-In 7-day liquid cultures, the use of SR-1 at 0.75 μM induced a major expansion of leukemic cells by 60-fold as compared to conditions without SR-1 (p < 0.0001). B-SR-1 allowed expansion of leukemic CD34+ cells by 6-fold in 7 days, as compared to controls (p < 0.0001) n = 3 experiments, Mean + SD, Statistical test: ANOVA + Dunnett’s post-hoc test. Reported p values are related to comparison with the 0 uM dose. All p-values are adjusted for multiple comparisons. PBMC: Peripheral blood mononuclear cells.
Fig 4
Fig 4. Analysis of the effects of AHR antagonist SR-1 on progenitor and leukemic stem cell growth.
A-Experimental strategy used to evaluate the role of AHR signaling in expansion of clonogenic cells and LTC-IC-derived progenitors. B-CML CD34+ cells cultured with SR-1 for 7 and 14 days exhibited a massive expansion of leukemic CFC (> 3 fold increase), as shown in this representative experiment of 3. C -CD34+ cells expanded with SR-1 for 4 and 7 days followed by long-term culture initiating cells (LTC-IC) assays, revealed a massive expansion (> 10-Fold) of LTC-IC-derived progenitors. n = 3 experiments, a representative experiment is shown in the Fig.
Fig 5
Fig 5. Evaluation of the role of AHR agonist FICZ in the growth of leukemic CD34+ cells.
CML CD34+ cells were cultured in the presence of indicated concentrations of FICZ and the cell numbers at day7 were compared to day 0. The use of FICZ induced a decrease of the growth of CD34+ cells at day+7 with the most inhibitory effects observed at concentrations of 100–200 nm (p = 0.0001, n = 3 experiments, ANOVA + Dunnett’s post-hoc test). Reported p-values are related to comparisons performed with the 0 nM dose. All p-values are adjusted for multiple comparisons.
Fig 6
Fig 6. Evaluation of the clonogenic potential of day-4 FICZ-cultured CML CD34+ cells.
A significant reduction of leukemic clonogenic potential was observed with a synergistic profile of FICZ with Imatinib (p = 0.0309, Panel A) and Dasatinib (p = 0.0292. (Panel B). Similarly, in three patients, LTC-IC assays started using 3-day FICZ-treated cells showed a significant growth inhibition of LTC-IC-derived clonogenic progenitors without synergistic profile with Imatinib. (Panel C). n = 3 experiments. CFC: Colony forming cells; IM: Imatinib; DASA: Dasatinib; DMSO: Dimethysulfoxide condition in which cells were cultured in the presence of DMSO at 0.01%.
Fig 7
Fig 7. Transcriptome analysis of primitive CML CD34+CD38low hematopoietic cells expressing low or high AHR expression.
A: Boxplot of AHR gene expression in CD34+CD38low cells from CML patients (2 subgroups of patients: AHR-low and AHR-high) in comparison of CD34+CD38low cells from healthy donors (HD), p-values were calculated by comparing sample groups two by two with 2 sided Student t test and Welch correction; B: Heatmap of the predictive gene expression profile between the 2 CML patients subgroups (AHR-dependent: AHR-low and AHR-high) in CD34+CD38low hematopoietic compartment (352 genes) (unsupervised classification was performed with Pearson distances and average linkage); C: Functional enrichment histogram of predictive expression profile correlated to AHR expression in CD34+CD38low cells from CML patients; Biological Process from Gene Ontology database was used to perform the enrichment (red bars represent Z-scores of the enriched functions; blue bars represent number of genes implicated in enriched function); D: functional interaction network of the AHR-related expression profile in CML hematopoietic progenitors (CD34+CD38low): functional relation connecting gene to function were extracted from Biological Process Gene Ontology Database (circle nodes: enriched genes; octagon nodes: enriched functions; color scale from yellow to purple: Z-scores of the functional enrichment).

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