Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors

The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38− leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38− subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells.


INTRODUCTION
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with a median age of 69 years. 1 The vast majority of patients with AML achieve complete remission after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. [2][3][4] A leukemia stem cell (LSC) paradigm may explain this failure of complete remission to reliably translate into cure. LSCs, like normal hematopoietic stem cells (HSCs), have self-renewal capacity and give rise to partially differentiated progeny that composes the bulk of the leukemia, but possesses only limited proliferative potential. 5 The currently existing treatments in AML, such as cytarabine (ara-C) and anthracycline (for example, daunorubicin), at the cost of a great toxicity, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. 6,7 Therefore, AML remains a clinical challenge and new therapies are urgently needed. [8][9][10] Only a rare population of AML cells enriched for LSCs, characterized by a CD34+CD38 − phenotype is capable of generating leukemia in immunodeficient mice. 11 More recently, evidence has been presented for a clinically relevant population of leukemic cells CD34+CD38 − in AML. This leukemic subpopulation, with a positive aldehyde dehydrogenase activity (ALDH+) in flow cytometry has been shown to be highly enriched in LSCs. [12][13][14] Interest in ALDH is due to its activity as a marker for identification of stem cell in different tissues. 15,16 The different isoforms of ALDHs (ALDH1, 2 and 3) control the levels of three endogenous apoptogenic aldehydes: methional, malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Cancer cells protect themselves from the apoptogenic effect of these aldehydes by the ALDHs that oxidize them to their non-apoptogenic carboxylic acids. 17 Among the family of acetylenic ALDH inhibitors, we identified the dimethyl ampal thiolester (DIMATE), an α,β, acetylenic N-substituted aminothiol ester, as an interesting candidate for cancer treatment. DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDHs 1 and 3. 18,19 It induces apoptosis in the chemoresistant mouse lymphoid cells BAF3bcl2 that are also resistant to disulfiram, a well-characterized inhibitor of ALDH2. 20 Moreover, although DIMATE was apoptogenic on cultures of human prostate cancer cells DU145, it was reversibly cytostatic on normal human prostate epithelial cells. 19 On the basis of these preliminary data on ALDH activity in LSC and differential effects between normal and cancer cells, we hypothesized that DIMATE could be a candidate for targeted therapy on LSC while sparing normal hematopoietic progenitors, thus providing an efficient and safe approach for chemotherapy of acute leukemia aiming at the eradication of minimal residual disease.

Patient samples
Peripheral blood samples from 10 patients with AML (Table 1) were collected before leukemia chemotherapy and after informed consent, and were part of the diagnostic procedures. The study was approved by the institutional review board from the Mediterranean V (Ref. 15.013) and Agence National de la Sécurité du Médicament (Ref. 150054B-11). Control non-leukemic HSCs were collected by apheresis from patients (n = 55) requiring autologous stem cell transplantation for non-myeloid malignancies.

Cell viability
Cells were seeded into 96-well cell culture plates at a concentration of 50 000 cells/well. Cell viability was monitored by AlamarBlue Cell viability protocol (Thermo Fisher Scientific, Illkirch, France) on a TriStar LB 941 Multimode Microplate Reader (Berthold Technologies, Thoiry, France).
The sensitivity toward different drugs DIMATE (Advanced BioDesign, Lyon, France), cytarabine, daunorubicine and azacytidin (Sigma-Aldrich) was determined using different concentrations of the drugs. After 48 h, the growth inhibitory effect of the drug was analyzed using Resasurin (Sigma-Aldrich) according to manufacturer's instruction. The drug response was quantified by the half maximal inhibitory concentration (IC50) for each particular cell line, and determined by non-linear regression analysis of log-dose/response curves.

Quantitative determination of HNE protein and MDA/protein adducts by ELISA
The formation of 4-HNE protein and MDA protein adducts was quantified with the Oxiselect HNE Adduct Elisa kit (Cell Biolabs, San Diego, CA, USA) and the OxiSelect MDA Adduct ELISA Kit (Cell Biolabs), respectively. Briefly, cell lysates were prepared by sonication in reducing SDS Sample Buffer. Homogenates were diluted to 10 μg protein/ml and adsorbed in 96-well protein binding plates by incubation at 37°C for at least 2 h. Wells were washed twice with PBS and incubated for an additional 2 h at room temperature on an orbital shaker. Following three washes in PBS, 100 μl of anti-HNE antibody or anti-MDA were added to the wells and incubated for 1 h at room temperature. Subsequently, goat anti-rabbit secondary antibody-HRP conjugate (diluted 1/1000 with the assay diluent) was added and incubation continued for 1 h. Wells were washed five times in PBS and HRP substrate was added. Reaction was stopped with an acidic solution, and absorbance read on a microplate reader at 450 nm. The amount of HNE protein adducts was determined by comparison with a standard curve prepared from HNE-BSA and MDA-BSA standards supplied by the manufacturer.

In vivo experimentation
To determine the antileukemic activity of DIMATE in a clinically relevant setting, we established patient-derived xenografts, or PDX models, in which 3 × 10 6 immunopurified CD34+ leukemic peripheral blood mononuclear cells from AML patient were transplanted intravenously into NOD/ SCID/IL2Rγ null immunodeficient mouse strain (NOG) for expansion (n = 25). For human AML xenograft, a patient with refractory AML after induction (complex karyotype+del(5q), bad prognosis, see UPN2, Table 1) was selected. After injection of primary AML cells, NOG mice were monitored for leukemia development by flow cytometric analysis of peripheral blood for human CD45-positive (hCD45+) cells. Four weeks were required for the development of the models monitored by the h-rate corresponding to the amount of hCD45+cells/mouse CD45-positive cells (mCD45+). Mice were next randomized and treatment with DIMATE (14, 28 mg/kg) or drug vehicle started for 4 weeks. Weekly monitoring of hCD45+ and mCD45+ circulating cells was performed during all treatment period. After treatment, mice were killed and bone marrow and spleen were harvested. hCD45+ and mCD45+ sorting and monitoring, in spleen and bone marrow, and spleen weighing was performed. All in vivo animal studies had been reviewed and approved by the local ethics committee (01_TransCurebio-Services-AB-01).

DISCUSSION
Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure in AML. 10 More precisely, CD34 +CD38 − leukemic cell population, enriched in LSCs, are highly resistant to these and other conventional chemotherapies. 21 Therefore new therapies are urgently needed for this deadly disease. Furthermore, conventional chemotherapy have similar cytotoxic effects on normal or leukemic HSCs. 21 In sharp contrast, we demonstrated that DIMATE, through ALDH inhibition, targeted and eradicated in vitro and in vivo several human myeloid leukemia cell lines and human leukemic cells population highly enriched in LSCs, but spared healthy HSCs. Most interestingly, we have determined a therapeutic zone, between 5 and 9 μmol/l, where DIMATE eradicated all LSCs (100% of lethality) and showed low toxicity (under 3% of lethality) on healthy HSCs. Moreover, with such treatment concentrations, healthy HSCs retained their self-renewing and multi-lineage differentiation capacity. ALDH detoxifying enzymes has been extensively associated with a number of malignancies and drug-resistant phenotypes. In the particular case of AML, the ALDH gene family is found upregulated or amplificated in 42% of cases, impacting also life expectancy, negatively ( Supplementary Figures 2A and B). 22,23 These data suggest that the altered ALDH activity in AML confers advantages for cell proliferation and survival and/or for the progression of the disease. DIMATE is described as an irreversible inhibitor of recombinant ALDH1 and ALDH3. Therefore, we evaluated the capacity of DIMATE to inhibit the ALDH activity in leukemic cells. 18,19 Selective cytotoxicity of DIMATE on cancer cells of human and murine origin has already been demonstrated in vitro on human epithelial cancer cells. Indeed, DIMATE induces an irreversible apoptosis in human prostate epithelial cancer cells DU145, but it is a reversible cytostatic agent on human prostate epithelial normal cells. 18,19 In the specific AML framework, safety of ALDH inhibitor on healthy HSCs has already been demonstrated, and even more remarkable is the inhibition of ALDH and retinoid signaling induces expansion of human HSCs. 24 These hypotheses are supported by our experiments in mice. In NOG mice, xenografted with human AML, enriched in LSCs, DIMATE eradicated in vivo, specifically, human AML cells (hCD45+) in blood, spleen and bone marrow. In contrast, DIMATE spared healthy circulating mouse cells (mCD45+). Moreover, with a humanization rate of 17%, our mouse model could be optimized, to try to obtain more significant results, in spleen and bone marrow, in particular, in the DIMATE 14 mg/kg treatment group.
With very unusual anti-cancer properties, DIMATE seems to be a very promising molecule for the treatment of AML and cancer more generally. Results from our work open new therapeutic perspectives in AML and provide a conceptual support for initiation of a phase I-II clinical trials.
We conclude that DIMATE is a very promising drug that opens new therapeutic perspectives in myeloid malignancies with putative interest in lymphoid malignancies as this drug is also able to inhibit the anti-apoptotic effect of bcl-2. 18 A limitation of our work concerns the LSC definition. Indeed, in our work, to define LSCs we used the CD34+CD38 − ALDH+ phenotype. However, several pathways, specific genes or microRNA, cytometric or transcriptomic signatures have also been proposed to distinguish LSCs from HSCs. [25][26][27][28] There is no consensus on an absolute definition for LSCs and it is more correct to speak of 'leukemic cells population enriched in LSCs'. Furthermore, according to some robust experimental works, LSCs could not be a stem cell disorder but rather a reacquisition of stem cell characteristics by classic leukemic cells. 29 Finally, we should not underestimate the role of the bone marrow environment in the leukemogenesis. Indeed, a pathologic bone marrow 'niche' could lead to permanent generation of LSCs. [30][31][32] Nevertheless, LSCs remain an interesting target for other innovative therapeutic strategies. [33][34][35] Analysis of DIMATE effect on normal and leukemic bone marrow 'niche' is the next step in our study aiming at a better understanding of the mechanisms of action of this innovative drug.