Aryl Hydrocarbon Receptor (AhR) Pathway Directly Controls Hematopoietic Proliferation in Chronic Myeloid Leukemia (CML) and Its Inhibition Allows Massive Expansion of Leukemic Progenitors and Stem Cells

AhR is an ubiquitous basic helix-loop-helix protein, mediating cell responses to external ligands and involved in cell division, cell quiescence and inflammatory responses. After ligand binding, AhR, which is an intracytoplasmic receptor associated to HSP60, dissociates from it and translocates to nucleus where it binds to aryl hydrocarbon nuclear translator (ARNT), this dimeric complex activating transcription. It has been shown that AhR is involved in normal hematopoietic progenitor proliferation in human cells. In addition,  AhR K/O mice exhibit a myeloproliferative syndrome-like disease, suggesting a role of AhR in hematopoietic stem cell (HSC) quiescence. The goal of this study was to study the potential role of AhR and its partners in CML progenitors and stem cells. We have first evaluated the expression of AhR in UT7 cell line and UT7-cells expressing BCR-ABL. AhR expression was highly reduced in UT7 cell expressing BCR-ABL as compared to control. AhR transcript was then 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 < 0.05). 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. To determine if AhR expression is differentially expressed in HSC as compared to progenitors, we have analyzed 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) as compared to normal controls (n= 15). These data suggested that AhR, known to be involved in HSC quiescence, could be amenable to pharmacological manipulation in CML cells. We first evaluated the potential effects of StemRegenin (SR1), which is an AhR antagonist on the growth of leukemic progenitors and stem cells. CD34+  CML cells treated with SR-1 had a reduced expression of AHHR, a repressor of AhR. 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 < 0.05). Similarly, SR-1 allowed expansion of leukemic CD34+ cells by 6-fold in 7 days, as compared to controls. CD34+ cells expanded in SR-1, were then used in CFC and LTC-IC assays to determine if SR-1 could allow maintenance and/or expansion of CML progenitors and stem cells. CML CD34+ cells cultured with SR-1 for 7 and 14 days exhibited a massive expansion of leukemic CFC (> 3 fold increase). Similarly, 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.  We then used the 6-Formylindolo (3,2-b) carbazole (FICZ),  a natural agonist of AhR, to determine its effects on leukemic progenitors and stem cells. The agonistic effect of FICZ was validated by the increase of the downstream target of AHR CYP1A1 transcripts after 7 days of culture. As expected, liquid culture of CML CD34+ cells induced a 2-fold decrease of CD34+ cells at day+7 in the presence of 100 nM of FICZ. Day-4 FICZ cultured CD34+ cells showed  a significant reduction of leukemic CFC potential with a synergistic profile with Imatinib and Dasatinib ( p < 0.05). Similarly, in three patients, in LTC-IC assays started after 3-day FICZ-treated cells, 5-week LTC-IC-derived progenitors showed a significant growth inhibition with a synergistic profile with Imatinib. In conclusion, these findings demonstrate for the first time that down-regulation of AhR expression, a major cell cycle regulator, is involved in the MPD phenotype associated with CML. The further inhibition of AhR expression by its antagonist allowed massive expansion of hematopoietic progenitors and stem cells. In contrast, AhR agonists inhibit clonogenic and LTC-IC-derived stem cell growth and could be used in LSC targeting in CML.