Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance
Abstract
The elimination of leukemic stem cells (LSC) is a highly desired therapeutic strategy for treating acute myeloid leukemia (AML). Although the repression of aryl hydrocarbon receptor (AHR) signaling has been demonstrated to support the short-term maintenance of primitive AML cells in culture, there has been no investigation into whether altered AHR signaling has a pathological role in human AML or if it affects the function of endogenous LSCs. Our research reveals that AHR signaling is suppressed in human AML blasts and is particularly downregulated in LSC-enriched populations within leukemias. A specific set of AHR target genes is uniquely repressed in LSCs across various genetic AML subtypes. The administration of the specific AHR agonist FICZ, both in vitro and in vivo, significantly inhibited leukemic growth, encouraged differentiation, and reduced self-renewal. Furthermore, LSCs were found to suppress a group of FICZ-responsive AHR target genes that act as tumor suppressors and differentiation promoters. Notably, FICZ stimulation did not adversely affect the function of normal hematopoietic stem and progenitor cells (HSPC) and did not lead to the upregulation of a significant LSC-specific AHR target in HSPCs, indicating that different mechanisms control FICZ-induced AHR signaling effects in HSCs compared to LSCs. Overall, this study emphasizes the suppression of AHR signaling as a crucial regulatory mechanism for LSCs and provides evidence in a preclinical model that activating the AHR pathway through FICZ can initiate unique transcriptional programs in AML, positioning it as a novel chemotherapeutic strategy to selectively target human LSCs.
Significance: The AHR pathway is suppressed in leukemic stem cells (LSC), indicating that activating AHR signaling could serve as a potential therapeutic approach to target LSCs and treat acute myeloid leukemia.
Introduction
Acute myeloid leukemia (AML) arises from driver gene mutations in hematopoietic stem or progenitor cells (HSPC), leading to the formation of leukemic stem cells (LSC) characterized by extensive self-renewal capacity and limited differentiation ability, which allows for the generation of large quantities of immature myeloid cells. Since LSCs are responsible for initiating the disease and causing relapse, effective and durable treatments against leukemia must focus on inducing LSC death or terminal differentiation. Achieving this goal has proven difficult due to the inadequate understanding of the molecular pathways that underlie the self-renewal program of human AML-LSCs.
Research has shown that LSC-containing populations exhibit distinct gene expression alterations, and the gene signatures of LSCs are predictive of therapy resistance and reduced overall patient survival. Notably, the gene expression profile that characterizes LSC fractions is not restricted to specific genetic subtypes or classifications of AML and bears resemblance to the expression profile of normal hematopoietic stem cells (HSC). These findings suggest that there are fundamental molecular mechanisms inherent to HSCs that could be targeted to disrupt the self-renewal and differentiation processes of LSCs. The challenge lies in selectively targeting these pathways in LSCs while preserving the normal function of HSCs.
This study investigates the AHR pathway as a potential regulatory mechanism that LSCs may exploit to enhance their oncogenic functions. AHR is a ligand-activated transcription factor known for its role in mediating the metabolism of environmental toxins and is increasingly recognized for its significance in various endogenous cellular functions. In mouse models with AHR knockouts, varying genetic backgrounds have resulted in different phenotypes, leading to ambiguity regarding AHR’s precise function. Nevertheless, findings indicate elevated HSPC proliferation in certain mouse models, which aligns with studies in human hematopoietic contexts where AHR antagonists have been shown to promote HSPC expansion ex vivo. Similarly, enhanced maintenance of LSCs in culture through AHR antagonism has been observed in specific patient samples. AHR activation has also been examined in THP-1 acute monocytic leukemia cells and in conjunction with high-dose retinoic acid in acute myeloblastic leukemia contexts, yielding some improvements in leukemic differentiation and proliferation inhibition.
Despite increasing evidence of the AHR pathway’s intrinsic roles in regulating HSC self-renewal and differentiation during normal hematopoiesis, no research has addressed the essential function of AHR signaling in the behavior of primary human AML cells or in the in vivo regulation of the most primitive LSCs responsible for this disease. Consequently, the potential of AHR signaling as a prognostic or therapeutic target in human AML remains largely uncharted. In this study, we hypothesize that AHR repression is a critical mechanism that LSCs utilize to maintain their self-renewal. We demonstrate the inherent suppression of AHR signaling in leukemia and particularly in LSCs. The activation of the AHR pathway through administration of the AHR agonist FICZ in xenografted patient AMLs resulted in irreversible anti-leukemic effects. Importantly, we observe that LSCs, unlike their normal HSC counterparts, respond uniquely to enforced AHR signaling via FICZ. Collectively, our findings suggest that the AHR pathway functions as a core signaling module promoting differentiation that LSCs suppress in vivo to sustain their self-renewal, and that activation of this pathway may represent a novel therapeutic strategy for targeting LSCs.
Materials and Methods
Mice
NOD-scid-IL2Rgc—/— (NSG) mice were bred and maintained in the Stem Cell Unit animal barrier facility at McMaster University. All procedures were approved by the Animal Research Ethics Board at McMaster University.
Primary Cord Blood and AML Patient Samples
Cord blood and acute myeloid leukemia (AML) patient samples were collected with written informed consent from participants. The collection process was approved by the local human subject research ethics board at the University Health Network and McMaster University, in accordance with the Canadian Tri-Council Policy Statement on the Ethical Conduct for Research Involving Humans. Following Ficoll-Paque separation, mononuclear cells were preserved in the vapor phase of liquid nitrogen, utilizing a solution containing 10% DMSO, 40% FBS, and alpha-MEM. The primary samples were thawed in phosphate-buffered saline (PBS) that included 10% FBS and 100 mg/mL deoxyribonuclease (DNAse) before being used in both in vitro and in vivo assays.
Cell Culture, Cell Lines, and Flow Cytometry
Primary AML samples were cultured in DMEM supplemented with 15% FBS, beta-mercaptoethanol, stem cell factor, interleukin-3 (IL3), interleukin-6 (IL6), thrombopoietin, and FMS-like tyrosine kinase 3 ligand. AML cell lines such as HL60 and MV-4;11 were maintained in Iscove Modified Dulbecco Medium with 10% FBS, while the NB4 cell line was cultured in RPMI1640 with 10% FBS. Human cord blood samples were grown in StemSpan SFEM supplemented with IL6, SCF, TPO, and FLT3. The MOLM-13, OCI-AML3, and Kasumi-1 cell lines were cultivated in RPMI with 20% FBS. Although cell cultures were not authenticated, they were used at low passages from ATCC stocks, and all cell lines were tested for Mycoplasma contamination. Flow cytometry analysis was performed using a BD LSRII flow cytometer and FlowJo Software, while cell sorting was conducted with MoFlo XDP.
Suspension Cultures with FICZ and RA
AML cell lines were seeded at a density of 1 to 3 x 10^5 cells/mL in medium supplemented with 0.1% DMSO vehicle or 6-formylindolo[3,2-b]carbazole (FICZ) dissolved in DMSO. HL-60 cells were cultured in either DMSO or FICZ at concentrations ranging from 200 to 1,000 nmol/L and were counted on days 3, 5, 7, and 10. On day 10, the DMSO/FICZ treatment was removed, and cells were replated with fresh medium. MV4;11 cells were cultured in medium supplemented with 1, 3, or 5 mmol/L FICZ or 0.1% DMSO control, growing for a total of 9 days. On day 4, cells were counted and replated at 1 x 10^5 cells/mL in freshly supplemented medium. MOLM-13 and OCI-AML3 cells were cultured in medium containing 500 nmol/L or 2.5 mmol/L FICZ or 0.1% DMSO control for a total of 7 days, with cell counts taken on day 3 and replated at 1 x 10^5 cells/mL. Kasumi-1 cells were cultured in 2.5 mmol/L or 5 mmol/L FICZ or 0.1% DMSO control for 7 days, with counts taken on day 3 and replated at 3 x 10^5 cells/mL. NB4 cells treated with FICZ followed a similar culture method as MV4;11 cells, with final cell counts taken after 5 days. For retinoic acid (RA) experiments, NB4 cells were plated at a density of 1 x 10^5 cells/mL in medium with varying doses of all-trans RA (125–1,000 nmol/L) and grown for 5 days to establish a dose-response curve. Alternatively, NB4 cells were plated at a density of 1 x 10^5 cells/mL in medium supplemented with either 0.1% DMSO and 100–125 nmol/L RA or 3 mmol/L FICZ and 100–125 nmol/L RA for 3, 5, and 7 days; or 1,000 nmol/L RA for 48 hours followed by 0.1% DMSO or 3 mmol/L FICZ for 3, 5, and 7 days. Cell counts were conducted on days 3 and 5, and cells were replated at 1 x 10^5 cells/mL.
RNA Extraction and qRT-PCR
Total cellular RNA was extracted using TRIzol LS reagent according to the manufacturer’s instructions, and complementary DNA (cDNA) was synthesized using the qScript cDNA Synthesis Kit. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicate using PerfeCTa qPCR SuperMix Low ROX with gene-specific probes and primers. The mRNA content of the samples analyzed by qRT-PCR was normalized based on the amplification of GAPDH.
Immunocytochemistry
Immunocytochemistry was conducted on primary AML samples that were transduced with shMSI2 or shControl lentivirus. Live transduced cells were purified by fluorescence-activated cell sorting (FACS) and then fixed and stained for CYP1B1 protein levels.
Western Blotting
Whole-cell lysates were prepared by lysing cells in RIPA buffer, which included sodium chloride, NP-40, deoxycholic acid, sodium dodecyl sulfate, Tris, and EDTA, as well as a protease inhibitor cocktail to maintain protein integrity. The concentrations of proteins were quantified using the Bradford assay, ensuring that samples were consistent in their loading amounts. The samples were normalized to a concentration of 1 mg/mL in NuPAGE LDS sample buffer with beta-mercaptoethanol and subsequently boiled before undergoing electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes, which were then blocked with bovine serum albumin. Following this, the membranes were incubated overnight with primary antibodies. After washing the membranes, secondary antibodies were added for detection, and the samples were then analyzed.
Cell Death and Cell Cycle Assays
To assess cell death, flow cytometry was employed using Annexin V and 7-AAD in an Annexin V binding buffer. For the analysis of the cell cycle, HL60 cells were cultured with and without FICZ for a duration of ten days. After this period, the cells were treated with BrdU for three hours, washed, fixed, and stained according to the manufacturer’s instructions.
Primary AML Clonogenic Progenitor Assay
Thawed primary AML samples were counted and plated in a methylcellulose-based hematopoietic colony formation medium, supplemented with either DMSO or 750 nmol/L FICZ or 750 nmol/L SR1. Colonies were scored between days ten to fourteen. For secondary colony-forming unit (CFU) assays, the cells were recovered from the methylcellulose by diluting and washing with phosphate-buffered saline (PBS) before being plated in fresh complete medium without DMSO or FICZ supplementation. Secondary colonies were scored on days ten to fourteen. Human cord blood samples were plated similarly, at a density of 1 x 10^3 cells per 35-mm plate. AML-CFU assays were also conducted on flow-sorted GFP+ shMSI2 or shControl GFP+ cells. In all cases, cell suspensions were plated in duplicate, and loose colonies consisting of ten or more cells were counted.
Cytospin Preparation and Morphology Staining
Human primary AML cells and AML cell lines grown in suspension were collected and cytospun onto glass slides. After drying, the slides were stained for morphology using Kwik-Diff Stains and subsequently washed with water. The slides were then mounted with Histomount Mounting Solution and prepared for imaging.
Lentivirus Production, AML Transduction, and Xenotransplantation
The production, knockdown, and validation of shMSI2- and shControl-expressing lentiviral particles were carried out as previously described. Before transducing primary AML samples with shMSI2 or shControl lentivirus, the AML samples were flow sorted for CD34 marker expression to enrich for leukemic stem cells (LSCs). The AML cells were transduced at a multiplicity of infection (MOI) of 50 for twenty-four hours in X-VIVO medium supplemented with BIT and L-glutamine, along with growth factors such as IL6, SCF, FLT3, and TPO. Post-transduction, cells were validated for GFP expression, washed, and subsequently transplanted intrafemorally into sublethally irradiated NSG mice. Three months after transplantation, the mice were sacrificed, and bone marrow from various locations was harvested, filtered, and processed for analysis of human AML engraftment.
FICZ-Treated AML and Cord Blood Xenografts
Prior to xenotransplantation into NSG mice, primary AML samples were treated in vitro overnight in IMDM supplemented with BIT and various growth factors, along with either DMSO or FICZ. Sublethally irradiated mice were administered a sample-dependent dose of cells via intrafemoral injection. Different treatment regimens were applied to cohorts of mice, with vehicle solutions used for control groups. Intraperitoneal injections of vehicle or FICZ were conducted at specified intervals, and upon completion of the treatment, the mice were sacrificed, and relevant tissues were collected for analysis.
Intracellular Flow Cytometry
For intracellular flow cytometry, primary AML cells were initially stained with anti-CD34 antibody and a viability dye. The cells were then fixed and permeabilized using a specific kit according to the manufacturer’s guidelines. Following this, the fixed cells were immunostained with an anti-MSI2 antibody and detected with a secondary antibody.
Differential Gene Expression Data Analysis
Microarray data was obtained from a public database, and differential gene expression analysis was performed using appropriate software tools. Comparisons were made between individual AML subtypes and healthy bone marrow controls. A statistical approach was employed to filter for significant gene expression changes.
Gene Set Enrichment Analysis
Gene set enrichment analysis was conducted using a software tool to retrieve predicted target genes. The data were analyzed with specified parameters to identify significant gene sets.
AHR ChIP-Seq Comparison to Downregulated and Upregulated Gene Sets
A comprehensive list of genes located near AHR-bound regions was integrated with identified AHR target genes to form a complete dataset for further analysis. The expression changes within these gene sets were compared, and statistical evaluations were conducted to assess their significance.
Survival Analysis
Survival data was sourced from The Cancer Genome Atlas (TCGA) specifically for acute myeloid leukemia (AML) and normalized against normal skin tissue from the same patients. This dataset included a total of 173 RNA sequencing samples from patients with complete data. Patients were categorized based on lower expression levels of AHR and ZFP3L1 in their leukemic blasts compared to their corresponding skin cells (AHRlow/ZFP3L1low). The Kaplan-Meier method was utilized to generate survival curves from this data, and the P value was determined using a right-censored log-rank test.
Bloodspot Analysis
Expression data for ZFP3L1 was obtained from Bloodspot and assessed within the context of normal hematopoiesis alongside AML datasets, using specific probes. The populations analyzed included acute myeloid leukemia (AML), hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP), megakaryocyte-erythroid progenitors (MEP), polymorphonuclear cells (PMN), and monocytes.
Statistical Analysis
All statistical analyses for both in vitro and in vivo studies were conducted using GraphPad Prism Software. Unpaired Student’s t-tests or Mann-Whitney tests were performed, with a significance cutoff set at P < 0.05. All data are presented as mean ± standard error of the mean (SEM).
Results
AHR Signaling is Attenuated in LSCs
To investigate differential gene expression that may influence leukemic stem cell (LSC) function, we analyzed published transcriptional profiling data from validated LSC+ and LSC-devoid (LSC−) fractions of 78 patient samples. Notably, within this dataset, we found that MSI2, a known regulator of asymmetric division and translation in mouse AML, was transcriptionally elevated 1.6-fold in LSC+ compared to LSC− samples. Additionally, we observed that MSI2 knockdown in primary AML LSC+ cells from four primary samples led to a 2.5-fold decrease in leukemia reconstitution, indicating that MSI2 is essential for LSCs. Given that MSI2 can repress the AHR pathway, these findings suggest that dysregulated AHR signaling may play a role in the pathophysiology of AML. Upon MSI2 knockdown in NB4 and THP-1 AML cell lines and in primary samples, levels of CYP1B1, a canonical downstream effector of AHR signaling, were significantly increased. This provides evidence of AHR signaling derepression following MSI2 knockdown across various AML samples and highlights the need for further exploration into the role of AHR signaling dysregulation in AML and LSCs.
Examining the competency of AHR signaling in AML, we found that AHR mRNA levels were detectable and unchanged between patient LSC+ and LSC− populations. Despite this, CYP1B1 was significantly downregulated in LSC+ fractions, indicating a likely difference in AHR pathway signaling between self-renewing and non-self-renewing AML populations. To further analyze AHR pathway attenuation in LSC+ fractions, we performed gene set enrichment analysis (GSEA) with a list of predicted and validated AHR targets. This analysis revealed a significant negative enrichment score, indicating AHR pathway suppression in LSC+ populations compared to LSC−.
We extended our investigation of AHR signaling status beyond intratumor dynamics to include bulk leukemia and healthy bone marrow by examining a global gene expression profiling dataset. GSEA indicated that most AML subtypes exhibited varying degrees of negative AHR target gene enrichment, while complex karyotype AML and myelodysplastic syndromes (MDS) showed positive enrichment scores. The leading edge genes contributing to the significant negative enrichment score in LSC+ populations clustered more closely with specific AML subtypes and represented genes that were downregulated across all AML subtypes compared to healthy bone marrow.
By expanding the list of AHR target genes to include those identified through AHR ChIP-seq, we observed significant overlap with downregulated genes within leukemia and across multiple leukemia subtypes in relation to healthy bone marrow, while overlap with upregulated genes was not significant. This indicates that the downregulation of AHR targets is enriched in the blasts of most AML subtypes and within LSCs.
The list of significantly downregulated genes that are AHR combined targets in LSCs or each AML subtype was compared to assess the degree of overlap. Among the 40 significantly downregulated AHR target genes in LSCs, we identified 21 that were uniquely repressed in LSCs, suggesting that their downregulation does not reach the same level of significance in blasts as it does in LSCs. To validate these 21 LSC-AHR target genes as actively downregulated AHR pathway genes, we examined their expression in cord blood hematopoietic stem and progenitor cells overexpressing MSI2. This analysis revealed that the expression profile of these genes in LSC clustered more closely with that of MSI2 overexpression and less so with various AML subtype blast populations, likely due to the stronger repression exerted by MSI2 through AHR antagonism.
The LSC-AHR gene signature encompasses the downregulation of differentiation-promoting or known tumor suppressors in AML, such as ZFP36L1, CDKN1A, and TLE1. Overall, our analysis indicates that AHR pathway attenuation is evident in the unfractionated blasts of multiple AML subtypes compared to healthy bone marrow, and within AML, LSC+ populations further enrich for a subset of downregulated LSC-AHR target genes that may play crucial roles in maintaining the LSC self-renewal gene expression program.
AHR Pathway Activation Impairs AML Proliferation and Promotes Differentiation In Vitro
To explore the potential antileukemic effects of activating the AHR pathway, we cultured the AML cell line HL-60 in the presence of FICZ, a well-established high-affinity ligand for AHR. The activation of AHR signaling was confirmed by the dose-dependent upregulation of CYP1B1 transcript levels, demonstrating that FICZ acts as an effective antileukemic agent. Treatment with FICZ resulted in significant reductions in cell proliferation, with observed decreases ranging from 1.2 to 1.8-fold. Importantly, FICZ treatment did not significantly alter cell cycle dynamics but did promote substantial increases in apoptosis and enhanced CD11b expression, indicating its role in promoting differentiation. Notably, the detrimental effects of FICZ were found to be irreversible; even after its removal from the culture medium, HL-60 cells exhibited up to a tenfold reduction in their capacity to proliferate.
Interestingly, the acute promyelocytic leukemia (APL) cell line NB4, which carries the t(15;17) translocation, displayed resistance to FICZ treatment, even at concentrations as high as 5 mmol/L. Previous studies have shown that prolonged exposure to high concentrations of retinoic acid (RA) can overcome the differentiation block imposed by PML-RARA. Therefore, we tested the effects of FICZ in combination with a low dose of RA that does not affect proliferation. When NB4 cells were cultured with a low dose of RA alongside FICZ, a reduction in cell proliferation was observed over a week, and the cells exhibited highly differentiated morphologies compared to controls. Furthermore, a 48-hour pulse of high-dose RA followed by FICZ treatment led to significant reductions in cell proliferation, enhanced differentiated cell morphology, and increased Annexin V staining, indicating that even in the presence of impaired RARA signaling, FICZ can sensitize RA to relieve the differentiation block.
To assess the broader effects of FICZ across various AML subtypes, we applied it to several other AML cell lines, including MV4;11, MOLM-13, OCI-AML3, and Kasumi-1. In MV4;11 cells, higher doses of FICZ resulted in a 2.4-fold reduction in proliferation. For MOLM-13, OCI-AML3, and Kasumi-1 cells, we utilized doses of FICZ that were either non-toxic or moderately inhibitory. In each case, we observed dose-dependent impairments in proliferation or enhancements in myeloid differentiation, confirming the effectiveness of FICZ in targeting leukemic cells across a diverse array of genetic and morphological subtypes.
We also examined the effects of FICZ on genetically diverse primary patient AML samples to validate its ability to activate AHR. Following exposure to FICZ, we measured elevated levels of CYP1B1 in the majority of samples. Additionally, in samples tested for CYP1A1, another key effector of AHR activation, we observed significant increases that were consistently higher than those for CYP1B1. While three of the twelve AML samples showed negligible effects on AHR activation, we could not rule out the possibility of AHR activation occurring as measured by CYP1A1. Due to the rapid cell death of AML samples in culture, interpreting proliferation and apoptosis measurements can be challenging. Nevertheless, in differentiation assays, we noted significant increases in CD14 expression in two of the three samples tested.
To further investigate the effects of FICZ on leukemic progenitors, we assessed its impact on their capacity to generate colonies. We found a significant reduction in primary AML colony output in five out of six samples tested, indicating that FICZ effectively targets AML progenitors. In the one sample that did not respond to FICZ, there was still a notable reduction in colony-forming unit potential upon secondary replating. It is important to emphasize that in all six samples that exhibited negative impacts on progenitors, AHR activation was confirmed through increased CYP expression. Additionally, we tested two AML samples with the AHR antagonist SR1 and observed only slight increases in colony formation compared to controls, suggesting that there was minimal activation of AHR signaling under the conditions used, allowing for further stimulation of the pathway by FICZ.
In summary, FICZ demonstrates potent antileukemic effects across a wide spectrum of leukemia cell lines and patient samples. In the case of acute promyelocytic leukemia, FICZ can synergize with standard therapeutic approaches to enhance antileukemic outcomes.
In vivo administration of the AHR ligand inhibits leukemic growth. FICZ has been tested in mice to treat various disease models, where it has been administered over a wide dose range with virtually no toxicity demonstrated. To define a treatment regimen for FICZ that may effectively impair in vivo leukemic growth, particularly during the early phases of reconstitution when self-renewal is most heavily relied upon, we first incubated four primary AML samples overnight with 750 nmol/L FICZ or vehicle prior to transplantation into NSG mice. After allowing one week for recovery, mice received three doses of 100 mg/kg FICZ or vehicle per week over four weeks. In FICZ-treated mice, we observed decreased leukemic engraftment from one of the samples tested and a trend toward reduced disease burden from another sample.
The effective dose of antileukemic agents in the xenograft setting can depend on the level of leukemic burden in the animal, which we tested next for FICZ. We reduced the input cell dose for one sample to establish a lower level of engraftment and again carried out the in vivo injections. In this case, leukemic cells in FICZ-treated mice were substantially reduced compared to vehicle-treated animals, demonstrating that a high leukemic load could alter sensitivity to FICZ at the 100 mg/kg dose. Importantly, the level of CD34+ cells was also preferentially reduced within the remaining FICZ-treated grafts compared to controls, indicating a detrimental impact on phenotypically primitive AML cells. To assess the targeting of functional leukemic stem cells, we performed secondary transplantations of grafts and found that engraftment derived from FICZ-treated primary bone marrow was reduced by 7.5-fold compared to control secondary grafts. These findings suggest that FICZ is effective at the primitive cell level when dose to AML cell number ratios are optimized.
Next, we tested whether an elevated FICZ dose could exhibit a broader range of effectiveness. By administering 250 mg/kg FICZ in vivo three times per week over four weeks, we found across three AML samples a more consistent impairment in leukemogenic engraftment, with substantial decreases ranging from 2.5- to 10-fold.
Finally, we initiated experiments involving in vivo treatment of established leukemic grafts. We first tested the effects of beginning treatment early post-transplant and followed this with injections of 250 mg/kg FICZ three times per week for one month. Engraftment of one sample treated in this manner was reduced by approximately 3.5-fold, mirroring similar levels of leukemic burden reduction observed in the pretreatment experiments. As FICZ is rapidly metabolized, to provide the most stringent test of its effects, we transplanted two distinct samples and allowed in vivo leukemic growth to progress for one or eight weeks, respectively, which typically yields heightened engraftment levels ranging from 20% to 70%, then treated with FICZ daily for one month. Not surprisingly, as observed with earlier experiments where engraftment levels were also very high, leukemic burden in these experiments was not significantly reduced at the endpoint. Importantly, both of these samples, however, showed striking reductions in the percentage of primitive CD34+ cells in the FICZ versus DMSO-treated grafts. Moreover, in the grafts, we observed increases in the percentage or mean fluorescence intensity of myeloid differentiation antigens, demonstrating an in vivo FICZ-induced promotion of differentiation.
It is interesting to note that when a sample responded to FICZ, CYP1B1 was upregulated in the FACS-isolated AML grafts at the end of in vivo treatment, but not upregulated when a sample was nonresponsive. Although more samples will need to be tested to make definitive conclusions, this trend suggests that greater increases in AHR agonism or activation of AHR beyond a certain threshold in vivo may lead to more pronounced leukemic impairment. Along these lines, where both a low and high FICZ dose was tested, we found that the increased FICZ dose further promoted AHR agonism and led to more effective impairments of leukemic engraftment. While MSI2 is likely among several repressors of AHR activity in leukemia, samples that were sensitive to even low-dose in vivo AHR ligand treatment had the highest levels of MSI2 and the lowest of CYP1B1 compared with less responsive samples. These findings highlight the importance of future exploration into the potential for high MSI2-expressing AMLs, which display more significant AHR pathway suppression, to be particularly sensitive to AHR stimulation. Overall, our in vivo FICZ treatment data provides evidence for irreversible antileukemic effects and highlights the potential of AHR pathway stimulation to target leukemic stem cells.
FICZ Administration Does Not Alter Normal HSPC Function
FICZ is recognized for its ability to inhibit proliferation and encourage differentiation in the context of leukemia. Its selective action that minimizes negative impacts on normal hematopoietic stem and progenitor cells (HSPCs) enhances its potential as a clinically relevant therapeutic option for acute myeloid leukemia (AML). To investigate this, CD34+ cord blood (CB) cells were treated with a concentration of 750 nmol/L of FICZ, similar to that used in AML samples. Following treatment, FICZ-stimulated CB cells demonstrated activation of the aryl hydrocarbon receptor (AHR) pathway, indicated by the upregulation of CYP1B1 and a modest increase in proliferation over a three-day culture period. Despite these changes, there were no significant differences observed in the proportions of primitive or mature myeloid cell types generated during this time.
Additionally, a slight increase in cell apoptosis was noted in one of the two CB samples evaluated. Except for a minor increase in burst-forming unit-erythroid cells in one sample, FICZ did not lead to major differences in progenitor outputs in either the first or secondary colony-forming unit assays. In the same high-dose (250 mg/kg) in vivo administration strategy used for treating AML xenografts, mice transplanted with CD34+ CB cells exhibited virtually no changes in the levels of human CD45+ grafts or the proportions of CD33+, CD14+, or CD19+ cells within these grafts after FICZ treatment. One CB sample showed no alterations in the primitive CD34+ compartment, while the other displayed an increased frequency of CD34+ cells. These findings suggest that normal CB cells are minimally affected by FICZ stimulation.
In terms of gene expression, while CYP1B1 levels were similarly elevated by FICZ treatment in both AML and CB cells, this upregulation alone does not account for the selective differentiation and antiproliferative effects observed in AML cells. The distinct epigenomic landscape of primitive AML cells compared to CB cells likely results in different targets being accessible and activated by AHR in these two contexts. To explore the mechanisms responsible for the differential response to FICZ, attention was directed to AHR targets that are preferentially downregulated in leukemic stem cells (LSCs). Among these, ZFP36L1 emerged as a notable target due to its decreased expression in both normal and malignant primitive hematopoietic cells when compared to more mature myeloid cells. Following FICZ stimulation of HL-60, MV4;11, and NOMO-1 cells, a significant upregulation of ZFP36L1 was observed, indicating a response specific to primary AML samples, with no appreciable elevation in primitive CB cells. Notably, ZFP36L1 has been associated with poor prognosis in a subset of AML patients, highlighting its potential role in mediating some of FICZ's differential effects in the context of leukemia.
Discussion
Our comprehensive analysis of AHR targets reveals that the AHR pathway is inherently repressed across various subtypes of acute myeloid leukemia (AML) when compared to healthy bone marrow, particularly within the leukemic stem cell (LSC) compartment. By excluding significantly downregulated AHR target genes shared among unfractionated AML cells from different subtypes, we identified 21 genes that are highly downregulated specifically in LSCs. This includes known tumor suppressors and validated AHR targets such as CDKN1A and TLE1, as well as genes that promote differentiation like ZFP36L1. These findings align with our evidence of reduced cell-cycle entry and differentiated cell morphology following FICZ treatment, reinforcing the notion that the AHR pathway serves as a core signaling module for differentiation that LSCs suppress to maintain self-renewal and sustained proliferation. The mechanisms by which LSCs inhibit AHR signaling remain unclear, but several upstream regulators across diverse leukemias likely contribute to this repression. We propose that the upregulation of MSI2 in LSC+ populations may facilitate this suppression, given its established role in attenuating the AHR pathway, along with our demonstration that MSI2 knockdown impairs leukemogenesis and enhances the expression of canonical AHR targets in AML.
Our findings emphasize that the attenuation of the AHR pathway plays a pro-LSC role, which is significant considering that AHR can exhibit both tumor-suppressive and oncogenic functions depending on the specific context and cell type. In various cancers such as glioma, breast cancer, and multiple myeloma, tumor cells can secrete AHR ligands that act autocrinely to support tumor maintenance or modulate immune responses. Additionally, bulk AML cells have been shown to secrete AHR ligands in vitro that impair natural killer (NK) cell function. On the other hand, the emerging concept of activating AHR signaling to inhibit neoplastic growth shows promise for certain cancer types, such as liver and prostate cancers, where a tumor-suppressive role for the AHR pathway has been demonstrated. This supports our exploratory studies on AHR pathway stimulation as a novel approach to directly inhibit AML progression at the level of LSCs.
While the primary AML sample AML-4 exhibited a favorable response to low-dose (100 mg/kg) in vivo FICZ treatment, other patient-derived AML cells showed less susceptibility and only began to respond at higher doses (250 mg/kg). Although this aspect was not the focus of our current study, it would be valuable to investigate whether genetic factors or subtype classifications can predict which patients may respond best to FICZ-induced AHR activation. Notably, the two samples (AML-4 and AML-7) that responded most effectively to the tested FICZ doses had upregulated MSI2 compared to other AML samples, suggesting that leukemias with high MSI2 expression may be more susceptible to AHR ligand therapy. Given that AHR pathway suppression appears to occur across most genetic subtypes, future research aimed at understanding how to target this pathway in AML could have significant clinical implications.
Moreover, our findings regarding the use of FICZ as a retinoic acid (RA) adjuvant in promoting differentiation and apoptosis in NB4 cells highlight its potential as a component of future multimodal therapies. This suggests that FICZ could help reduce the required doses of existing therapies for acute promyelocytic leukemia (APL). The selective ability of FICZ to promote differentiation of primitive AML cells may also indicate its potential in combination treatment strategies. Specifically, using FICZ to effectively target LSCs in conjunction with chemotherapeutics aimed at blast cells could enhance the overall efficacy against the entire leukemic clone.
FICZ possesses several advantageous attributes as an AHR agonist, including rapid metabolism and good tolerance in vivo. In studies where mice were administered comparable doses, the effects on the immune system were primarily limited to T cells, exhibiting only mild immunosuppression without detrimental impacts on long-term hematopoiesis. Additionally, we have shown that FICZ has limited effects on normal CB cells both in vitro and in vivo compared to AML cells. This preferential effect on AML cells may be attributed to elevated expression of AHR regulators in the leukemic context, which could lead to more extensive repression of AHR signaling. As observed in other signaling pathways, this heightened repression of AHR signaling in leukemia may indicate a greater reliance on this pathway for the maintenance of LSCs and the leukemic state.
Another possibility is that specific AHR targets are activated solely in the leukemic and/or LSC context, with their restraint being crucial for continued leukemic propagation. The phenomenon of context-dependent and ligand-specific AHR target transactivation is well documented. We hypothesize that differential ligand-induced conformational changes in the AHR and distinct interactions with downstream coactivators or repressors, along with the unique molecular landscape of AML cells, may allow for the activation of unique downstream targets upon FICZ stimulation compared to their normal counterparts. Our identification of ZFP36L1 as a potential target for a subset of leukemias supports the need for future investigations into the broader transcriptomic effects of FICZ across AML and the role these transcriptional changes may play in mediating the prodifferentiation effects of FICZ on human AML cells.