AbstractAldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1− subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1− cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1− leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1− leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.
The Aldehyde Dehydrogenase (ALDHs) gene family contains at least 19 isoforms that metabolize retinoids, reactive oxygen species (ROS) and endogenous and exogenous aldehydes.21 High levels of the ALDH1A1 isoform are expressed in hematopoietic stem cells (HSCs) as measured by mRNA analysis and staining with the fluorescent ALDH1A1 substrate Aldefluor.3 Despite this, ALDH1A1 is dispensable in murine HSCs, as it is compensated for by increased expression of the ALDH3A1 isoform and possibly others.54 Murine HSCs deficient in both ALDH1A1 and ALDH3A1 (Aldh1a1/3a1 DKOs) have a blocked B-cell development and reduced numbers of HSCs.4 The early B cells in these mice have increased levels of ROS and reactive aldehydes along with abnormalities in cell cycling, intracellular signaling and gene expression. Notably, HSCs and progenitors in these mice have elevated p38MAPK activity, increased sensitivity to DNA damage, and aberrant cell cycle distribution.4 These findings suggest an important role for ALDHs in metabolizing ROS and reactive aldehydes in primitive and differentiated hematopoietic cells, and show that the ALDHs and their substrates impact a variety of cellular processes in hematopoiesis.
The role that ALDHs play in normal HSCs suggests they may have important roles in leukemia as well.76 Several prior reports support this contention. In Fanconi anemia, loss of activity of the ALDH2 isoform in concert with FANC-D deficiency predisposes to the development of acute myeloid leukemia (AML) and bone marrow failure.98 ALDH activity in human leukemia also mediates resistance to a number of drugs10 and high levels of ALDH activity, as measured by Aldefluor staining, predict for a poor outcome to treatment.1411 Leukemic stem cells (LSCs) that drive leukemia growth and disease relapse may also be identified with Aldefluor staining with either high or intermediate levels of staining.1615
Given these prior observations, in the present study, we sought to further clarify and define the role and significance of ALDH activity in acute leukemia with a particular focus on determining whether ALDH biology could be exploited for new treatment approaches.
Analysis of public databases
Analysis of ALDH family genes for mRNA expression, DNA methylation, and survival outcomes was performed with data available from The Cancer Genome Atlas (TCGA) (cancergenome.nih.gov/cancersselected/acutemyeloidleukemia).17 Analysis of the expression of ALDH genes in leukemic versus normal hematopoietic cells was performed using the GSE9476 data set.18
Cell lines and primary specimens
Acute myeloid leukemia specimens were obtained from apheresis products of patients who gave Institutional Review Board approved informed consent for sample procurement at the University of Rochester and the University of Colorado in Denver. Normal 34 HSCs were enriched from umbilical cord blood (UCB) specimens collected and distributed by the University of Colorado Cord Blood Bank (UCCBB), which is accredited by the American Association of Blood Banks and licensed by the US Food & Drug Administration. Cell lines were commercially supplied by ATCC (Manassas, VA, USA).
Flow cytometry analysis
Human samples were stained with CD34, CD38, CD123 and Aldefluor as previously described.20193 AML cells were routinely gated through the blast window and it was confirmed that pheresis derived samples were not contaminated with significant numbers of normal HSCs based on staining with anti-CD123, a marker that distinguishes AML from normal HSCs together with other AML directed markers including HLA-DR, CD71 and CD7 (Figure 1C and Online Supplementary Figure S4A).2321 Anti-mouse antibodies were as described previously.244 All antibodies and staining reagents were from BD Biosciences (San Jose, CA, USA). Intracellular 4HNE staining and visualization of gamma (γ) H2AX by flow cytometry were performed as previously described.4 All cell staining and analytical procedures were performed either manually or using high content semi-automated flow cytometry, as previously described.2625
Western blot analysis
Lysates were prepared from Kasumi-1 and UCB CD34 enriched populations. Primary mouse monoclonal antibodies to 4HNE adducts (Ab48506, Abcam; Cambridge, MA, USA) were used in a 1:500 dilution. Western blots were performed as previously described.27
For restoring ALDH1A1 gene function in Kasumi-1 cells, the HA-ALDH1A1 gene was excised from Addgene plasmid # 11610 using HindIII/Xba1 and sub-cloned into the pLVX-EF1a-IRES- mCherry Vector from Clontech (Cat# 631987), using EcoR1/Xba1 cloning sites. The sequence was verified using EF1a-Fwd and IRES-Rev sequencing primers. High titer lentiviral production was performed in 293TN cells and AML cells were transduced as previously described.28
To test the drug sensitivity of MOLM-13 and primary AML cells in vivo, NSGS mice29 were treated intraperitoneally (IP) with busulfan (30 mg/kg) then injected intravenously (IV) with 5–7×10 AML cells. After marrow engraftment was confirmed, control mice were treated IP with saline while the rest of the mice were treated IP with 5 μg/g ATO daily for four days together with 150 μg/g Cy at day 1 and day 4. The mice were sacrificed by CO2 asphyxiation and the bone marrow harvested for flow cytometry analysis. Further details are available in the Online Supplementary Methods.
All statistical analyses were performed using GraphPad Prism 7.0. Typically, unpaired two-tail Student t-tests were performed and P<0.05 was considered significant. Aggregate data were presented as means and standard deviations (SD).
ALDH isoform expression is variable between AMLs and correlates with cytogenetic subtype
Variable levels of Aldefluor staining in AML have been reported and may correlate with outcomes to treatment.1311 However, given the large number of ALDH isoforms and the findings that Aldefluor is a substrate for several of them, we sought to more precisely determine if variability in expression of specific individual ALDH isoforms occurs in AML and if so, whether expression of specific isoforms correlated with prognosis.304 First, we analyzed 165 leukemia specimens from The Cancer Genome Atlas (TCGA) dataset for levels of expression of all 19 human ALDH family genes.31 A 4–5 log difference in expression of the ALDH1A1 isoform was observed among this cohort of AML specimens, with 39.4% (65 of 165) expressing ALDH1A1 at less than 1 RPKM (Figure 1A). None of the AMLs expressed significant levels of ALDH3A1; however, a number of other isoforms including ALDH1B1, ALDH2, ALDH3A2, ALDH3B1, ALDH4A1, ALDH5A1, ALDH6A1, ALDH9A1, ALDH16A1 and ALDH18A1 were expressed at abundant levels in a majority of AML samples (Figure 1A). To confirm these findings, qPCR for ALDH isoforms was performed on 7 primary AML samples and 3 of 7 had no detectable ALDH1A1 expression, 5 of 7 had no ALDH3A1 expression and, again, a number of other ALDH isoforms were variably expressed similar to the TCGA data (Online Supplementary Figure S1A). When ALDH1A1 expression in AML was compared to that in normal HSCs and progenitors via analysis of another publicly available dataset,18 ALDH1A1 was consistently found at high levels in normal CD34 cells from all the bone marrow (BM) and peripheral blood (PB) samples while ALDH1A1 levels were again quite variable in AMLs with a minority having low levels of expression (Figure 1B). ALDH1A3, ALDH2, ALDH3B1 and ALDH8A1 expression was also significantly different between normal hematopoietic cells and AMLs (Online Supplementary Figure S1B). Lastly, several ALDH isoforms including ALDH3B1 and ALDH1A3 had significant negative and positive associations, respectively, with ALDH1A1 (Online Supplementary Figure S1B).
Given the utility of using ALDH functional activity as a marker of HSCs and other cancer stem cells, ALDH may be utilized as a marker of LSCs as well.333211 To assess the level of ALDH1A1 expression in LSCs both at diagnosis and at relapse, AML samples from 6 patients were analyzed with RNA-Seq and the level of ALDH1A1 mRNA transcripts measured in both LSCs and non-LSCs as previously described.34 In these AMLs, ALDH1A1 was expressed at significantly lower levels than HSCs controls in both LSCs and non-LSCs and no significant differences in ALDH1A1 expression between LSCs and non-LSCs were noted (Figure 1C). In order to further test whether ALDH1A1 expression increased following treatment with standard chemotherapy as a mechanism of drug resistance, LSCs from relapse samples from the same 6 patients were similarly examined for ALDH1A1 expression. Again, ALDH1A1 expression was significantly lower in relapse LSCs than the HSC controls, and analysis of paired diagnosis and relapse LSC samples did not show consistent or significant increases in ALDH1A1 expression (Figures 1D). In addition, no significant differences were noted in any other ALDH isoforms between diagnosis and relapse in either LSCs or non-LSCs (Online Supplementary Figure S1D).
Together these data demonstrate that at least in a subset of AMLs, ALDH1A1 expression is not elevated in LSCs and does not increase following standard chemotherapy treatment.
Given the significant ALDH isoform variability at the mRNA level between different AMLs observed in these studies, we next sought to investigate whether there was similar variability in ALDH enzymatic activity between AML samples. Eighteen primary AMLs were stained with Aldefluor dye and analyzed by flow cytometry. Consistent with the gene expression data, substantial differences in the overall content of Aldefluor staining cells between the different AML samples were observed (Figure 2A and B). As with the gene expression studies, a subset of AMLs had very low or absent numbers of cells with detectable Aldefluor staining [Aldefluor (0–0.1%)] (Figure 2A) demonstrating that they lacked ALDH1A1 and any other ALDH isoforms that may use Aldefluor as a substrate. Together, these findings demonstrate considerable variability in expression of different ALDH isoforms between different AMLs as well as relative to the HSCs and progenitors from which they were presumably derived. The most striking finding was the low to undetectable levels of expression and activity of ALDH1A1 in any cells in a substantial subset of AMLs (25%–40%) despite its universal high-level expression in normal HSCs and progenitors (henceforth termed ALDH1A1 AML).
We next analyzed whether variability in the expression of the different ALDH isoforms, particularly ALDH1A1, had prognostic significance in predicting outcomes to treatment. When TCGA AML samples were grouped into Good, Intermediate and Poor Risk cytogenetic categories and analyzed for levels of ALDH1A1 expression, a significant association between the Good Risk cytogenetic category and lower levels of ALDH1A1 expression was observed (Figure 2C). In addition, low-level expression of the ALDH1A1 isoform was correlated with improved event-free survival (EFS) and overall survival (OS) in the entire cohort of AML patients (Figure 2D). To evaluate whether the prognostic value of ALDH1A1 was independent from cytogenetics, intermediate risk samples were fractionated based on ALDH1A1 expression and analyzed for EFS and OS. A trend to an association between better EFS or OS and lower expression of ALDH1A1 was found within intermediate risk cytogenetic subgroups, although the sample sizes were not big enough to formally confirm this association (Online Supplementary Figure S1E).
Absence of ALDH1A1 in human leukemia cell lines renders them sensitive to toxic ALDH substrates
The finding that there is a subset of AMLs that do not express ALDH1A1 (ALDH1A1 AMLs) while normal HSCs and progenitors express high levels of ALDH1A1 suggested that exploiting low level ALDH1A1 expression may offer a new avenue to selectively target this subgroup of AMLs.35 Previously we had shown that primary murine marrow cells lacking Aldh1a1 and Aldh3a1 were sensitive to the reactive aldehyde 4-hydroxynonenal (4HNE), likely because these ALDHs are the primary metabolizers of this toxic compound.4 In addition, an in silico screen of compounds active in human leukemic stem cells revealed that 4HNE has potent human anti-leukemic activity.36 Together, these findings suggested that human ALDH1A1 AMLs may be particularly sensitive to 4HNE treatment given their predicted limited ability to metabolize this toxic compound. To test this, first, a series of human AML cell lines were screened by Aldefluor staining along with qPCR analysis and both Kasumi-1 and MOLM-13 were identified as representative ALDH1A1 AML cell lines suitable for further analysis (Online Supplementary Figure S2A and B). At baseline, Kasumi-1 had high levels of endogenous reactive aldehyde adducts relative to normal UCB CD34 cells (Figure 3A), which further increased after 4HNE treatment. DNA damage and ROS were also increased in Kasumi-1 cells treated with 4HNE while normal CD34 cells were relatively resistant to these effects (Figure 3B and C). In addition, when Kasumi-1 cells were exposed to increasing concentrations of 4HNE, an LD50 of 4 μM was found, while in contrast, CD34 normal cord blood cells were almost completely resistant to comparable doses of 4HNE treatment (Figure 3D).
In order to begin to translate this concept into a clinically relevant treatment strategy for ALDH1A1 AML, we sought to identify known drugs that would induce intracellular 4HNE in ALDH1A1 AML cells, as 4HNE itself is not clinically useful due to non-specific toxicity and a very short half-life.37 Arsenic trioxide (ATO) is a well-characterized compound used in the treatment of acute promyelocytic leukemia that has significant pro-oxidant activity, suggesting that it could elevate intracellular reactive aldehyde levels, as these are frequent byproducts of ROS.38 Kasumi-1 cells treated with ATO had elevated intracellular 4HNE adduct levels (Figure 3E)39 and ATO induced a concentration-dependent increase of apoptosis in AML cells (Figure 3F). Next, we sought to determine whether we could further amplify the anti-leukemic effects of ATO in ALDH1A1 AML by combining it with 4-hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide (Cy) and known toxic substrate of ALDH1A1. The combination of ATO and even sublethal levels of 4HC led to higher levels of cell killing than control or either drug alone (Figure 3F).40
To determine whether these effects were dependent on ALDH1A1 expression, Kasumi-1 cells were transduced with a lentiviral vector that over-expressed ALDH1A1 (Online Supplementary Figure S2C) and tested over a wide range of 4HC and ATO concentrations, alone and together. Restoration of ALDH1A1 expression at least partially blocked the effects of 4HNE mediated generation of ROS and reactive aldehyde adducts in Kasumi-1 cells (Figure 4A and B, respectively). Expression of ALDH1A1 also protected Kasumi-1 cells to varying degrees from 4HC and ATO, depending on their concentrations (Figure 4C). Similar effects were noted in MOLM-13 cells transduced to express ALDH1A1 (Online Supplementary Figure S2D and E). Furthermore, Kasumi-3 cells, which express high levels of ALDH1A1, were rendered more sensitive to 4HC and ATO following treatment with an ALDH inhibitor (DEAB) (Figure 4D and Online Supplementary Figure S2F). In addition, differences in DNA damage were also noted between ALDH1A1 and ALDH1A1 Kasumi-1 cells treated in vitro with 4HC and ATO (Figure 4E). Lastly, we tested the in vivo anti-leukemia activity of Cy plus ATO on MOLM-13 AML cells. MOLM-13 were used for these studies as opposed to Kasumi-1 as they consistently engraft NSGS mice, while Kasumi-1 do not engraft well in the conditions tested. Briefly, MOLM-13 cells were transplanted into NSGS mice and then treated daily with ATO for four days followed by Cy at day 1 and day 4. When the marrow content of MOLM-13 was analyzed by flow cytometry, two weeks later, the overall content of MOLM-13 cells was dramatically reduced in Cy plus ATO treated mice relative to placebo-treated controls (Figure 4F). Consequently, these results demonstrate that loss of ALDH1A1 renders AML cell lines more sensitive to 4HC and ATO.
Absence of ALDH1A1 in primary AMLs renders them sensitive to toxic ALDH substrates
To determine if the results obtained from the AML cell line studies were relevant to authentic primary AMLs, first, in vitro sensitivity studies were performed. Peripheral blood samples from apheresis collections of 4 ALDH1A1 AML patients were treated overnight with 4HC and ATO, alone or in combination. The 4HC plus ATO combination generated more DNA damage and cell killing than either agent alone or controls (Figure 5A). In addition, when 3 samples known to have both ALDH1A1 and ALDH1A1 subsets were sorted into Aldefluor and Aldefluor fractions (Online Supplementary Figure S3A–C) and treated in vitro with 4HC plus ATO, the Aldefluor cells were sensitive to treatment while the Aldefluor cells were relatively resistant as measured by cell killing and DNA damage (Figure 5B and C). To further determine whether ALDH1A1 expression modulates the effects of 4HC and ATO in primary AML cells, Aldefluor AML cells were treated with DEAB, an inhibitor of ALDH1A1, and then the 4HC/ATO drug combination was added.41 Again, the Aldefluor controls were relatively resistant to treatment with 4HC/ATO, while in contrast the DEAB-treated cells had a greater sensitivity to 4HC/ATO than control (Figure 5D). Together these data further demonstrate that ALDH1A1, at least in part, mediates resistance to these agents.
Next, to test whether 4HC plus ATO was also effective against human primary AML cells capable of engrafting immunodeficient mice, 3 primary Aldefluor AMLs were treated in vitro overnight with either vehicle control or 4HC plus ATO and transplanted into busulfan-treated NSGS mice. All control-treated AMLs engrafted at levels of 20%–100% while 4HC plus ATO treated AMLs failed to engraft any of 25 mice (Figure 6A). In contrast, when normal UCB derived CD34 cells were treated with 4HC plus ATO in a similar fashion, although engraftment was reduced in NSGS mice, it could be readily detected in 75% of recipients (Figure 6B). Lastly, to replicate the clinical scenario as closely as possible, we tested in vivo the effects of Cy and ATO on an Aldefluor AML that had been previously engrafted in NSGS mice compared to an Aldefluor AML positive control known to express high levels of ALDH1A1. Two weeks following in vivo treatment of Aldefluor AML engrafted mice with Cy plus ATO, the overall marrow content of human AML was significantly reduced in mice relative to placebo-treated controls (Figure 7A). In contrast, the Aldefluor AML engrafted mice showed no differences in engraftment between treated and untreated groups (Figure 7B).
In this report, we demonstrate that there is expression of a number of ALDH isoforms in AML and several have substantial variability in expression. The most prominent of these is ALDH1A1, which has low levels of expression and activity in a subset of AMLs, despite its nearly universal expression in normal HSCs and progenitors. In fact, in 25%–40% of AMLs we could find few or no cells expressing any ALDH1A1 as determined by Aldefluor staining and flow cytometry as well as qPCR analysis (termed ALDH1A1AML). This includes finding low to absent levels of ALDH1A1 in LSCs as well. The factors governing the variability of ALDH1A1 expression in AMLs are unknown; however, the majority of ALDH1A1 AMLs in public databases do not have obvious ALDH1A1 gene deletions (data not shown) suggesting that loss of ALDH1A1 expression may occur as a consequence of gene regulation either through epigenetic or gene expression/repression mechanisms.42 Further studying the underlying mechanisms of ALDH1A1 gene regulation in HSCs, as well as AMLs, may provide useful insights into the biology of each. We also found that loss of ALDH1A1 may have prognostic implications as ALDH1A1 AMLs correlate with good prognosis cytogenetics and there was a trend to improved outcomes in intermediate risk cytogenetic AML patients based on level of ALDH1A1 expression, although we could not formally prove an association. It would be interesting to seek to extend and confirm these observations in larger prospective studies. If this trend is confirmed, then ALDH1A1 status may become a useful tool to stratify patients to different treatment approaches. These observations also raise additional questions regarding why ALDH1A1 expression would be associated with cytogenetic status. In addition, if outcomes are indeed impacted by ALDH1A1 status, it would be important to determine whether this was because loss of ALDH1A1 renders ALDH1A1 AMLs more sensitive to chemotherapies that induce ROS and/or reactive aldehydes or whether this acts through a different mechanism.
Since a substantial subgroup of AMLs is deficient in ALDH1A1 expression at different stages of disease (diagnosis and relapse), we speculated that these ALDH1A1 AMLs may specifically be more sensitive to compounds that directly or indirectly generate toxic ALDH1A1 substrates. First, 4HNE, a prototypical toxic reactive aldehyde substrate of ALDH1A1 was quite effective at killing ALDH1A1 cell lines while CD34 UCB, which expresses high levels of ALDH1A1, was quite resistant to this compound. Of more clinical relevance, we found that ATO, a widely used drug in the treatment of APL, could induce the intracellular production of 4HNE, leading to selective killing of ALDH1A1 AMLs. This effect was at least partially blocked by restoration of ALDH1A1 activity through gene transfer, further supporting a role for ALDH1A1 in this effect. The finding that ALDH1A1 gene transfer did not fully block the effect of ATO is likely because ATO potentially works through a variety of mechanisms, of which generating 4HNE is only one.43 To amplify the effect of ATO in ALDH1A1 AML, it was combined with 4HC for in vitro studies and Cy for in vivo studies as these generate a toxic intermediate substrate termed aldophosphamide that is primarily metabolized by ALDH1A1. ALDH1A1 AML cell lines as well as primary ALDH1A1 AMLs were particularly sensitive to the ATO and 4HC/Cy combinations both in vitro and in vivo, while, again, normal HSCs were relatively resistant. The in vivo studies ultimately may be extended to determine mechanisms of resistance to ALDH-directed therapy.
The finding that there is an apparent therapeutic window between normal HSCs and ALDH1A1 AMLs treated with toxic ALDH1A1 substrates, suggests a novel targeted therapy strategy for this newly defined AML subset (modeled in Figure 8). In this strategy, we propose that AMLs lacking ALDH1A1 are more sensitive to compounds that directly or indirectly generate toxic ALDH substrates including ROS, reactive aldehydes and others, while normal HSCs with high levels of ALDH1A1 are relatively resistant. Consequently, rational drug combinations focusing on these agents may be particularly effective in ALDH1A1 AML patients. As a first step towards exploring this treatment strategy, the findings in this report provide a rationale for performing a phase I/II clinical trial of Cy plus ATO in ALDH1A1 AML patients to determine efficacy and toxicity. In addition, these observations suggest the utility of further pre-clinical studies to identify even more effective and less toxic combinations of compounds that exploit the differences in ALDH1A1 expression between normal HSCs and AMLs. Ultimately, it will be interesting to determine whether therapeutic targeting of the differences in ALDH isoform expression between malignant cells and normal tissue stem cells can be extended to other hematologic cancers such as myeloma and lymphoma, and solid tumors as well.
The authors gratefully acknowledge technical assistance by the Flow Cytometry Cores (CFAR and UCCC) and the Animal Housing & Care Vivarium of the University of Colorado Denver (Anschutz Medical Campus).
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/102/6/1054
- Received November 2, 2016.
- Accepted March 8, 2017.
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