Antileukemic Compounds Research Articles

Preclinical activity of resazurin in acute myeloid leukaemia.

Resazurin, a phenoxazine used in cell viability assays, acts invitro as an anti-leukaemic compound through the production of cellular reactive oxygen species (ROS) resulting in mitochondrial dysfunction and cell death. However, the invivo tolerance and efficacy of resazurin in cancer are unknown. In this study, we investigated the invitro and invivo effects of resazurin in acute myeloid leukaemia (AML). Resazurininduced cell death in a dose-dependent manner in AML cell lines and reduced proliferation and colony formation in exvivo treated patient-derived AML cells. Cells treated with a reduced dose of resazurin for 72 h acquired a more mature immunophenotype suggesting cell differentiation as a mechanism contributing to the anti-leukaemic effect. Invivo optical imaging in healthy mice demonstrated a reduction of resazurin to resorufin within 30 min and non-detectable after 2 h, supporting dosing twice daily as optimal. In subcutaneous and orthotopic models of MV4-11 AML in NOD/SCID IL2rγnull mice, anti-tumour effects and an increased survival were found at a dose level of 75 mg/kg twice daily without observed toxicity. Our results suggest that resazurin represents a novel experimental therapeutic for the treatment of AML.

Point of care CD19 chimeric antigen receptor (CAR) T-cells for relapsed/refractory acute myeloid leukemia (AML) with aberrant CD19 antigen expression

Relapsed/refractory (r/r) acute myeloid leukemia (AML) is associated with poor prognosis. CD19 is a B-cell marker, is aberrantly expressed in AML, mostly with t(8; 21)(q22; q22.1). Here we report the results of a phase 2 study giving point of care produced CD19 CAR T- cells for r/r AML with aberrant expression of CD19 (NCT04257175). Lymphodepletion included fludarabine and cyclophosphamide The response was evaluated by bone marrow (BM) aspiration on day 28. Six patients (5 adults and 1 child) were included. Median number of previous chemotherapy lines was 4 (range, 3–8) and four patients received CAR T-cells 8–18 months post allogeneic hematopoietic stem cell transplantation (allo-HSCT). Cytokine release syndrome (CRS) of any grade occurred in all patients, and 1 patient had grade 3 CRS. Immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 2 patients at low grades. Tocilizumab was administered to 2 patients and corticosteroids to 3 patients. Four patients achieved a complete remission (CR), while 2/6 progressed (PD). Three patients (2 with CR and 1 with PD) underwent allo-HSCT (it was the second transplant in 2) 2–5 months post CAR T-cells infusion. The median duration of response in patients achieving CR was 8.5 (range; 3–14) months. However, all patients eventually died within 5 (1–18) months.In conclusion, CD19 CAR T- cell treatment for AML is feasible and safe. However, the response is short and should be followed by allo-HSCT. Hopefully, future long term results will be improved by combining the CAR T- cell therapy with the emerging novel effective anti-leukemic compounds.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Bioassay-guided isolation of anti-leukemic steroids from Aglaia abbreviata by inducing apoptosis

The strategy of bioactivity-guided isolation is widely used to obtain active compounds as quickly as possible. Thus, the inhibitory effects on human erythroleukemia cells (HEL) were applied to guide the isolation of the anti-leukemic compounds from Aglaia abbreviata. As a result, 19 compounds (16 steroids, two phenol derivatives, and a rare C12 chain nor-sesquiterpenoid), including 13 new compounds, were isolated and identified based on spectroscopic data analysis, single-crystal X-ray diffraction data, and electronic circular dichroism (ECD) calculations. Among them, 9 steroids exhibited good selective anti-leukemic activity against HEL and K562 (human chronic myeloid leukemia cells) cells with IC50 values between 2.29 ± 0.18 μM and 19.58 ± 0.13 μM. Notably, all the active compounds had relatively lower toxicity on the normal human liver cell line (HL-7702). Furthermore, five compounds (1, 4, 8, 10, and 19) displayed good anti-inflammatory effects, with IC50 values between 7.15 ± 0.16 and 27.1 ± 0.37 μM. An α,β-unsaturated ketone or a 5,6Δ double bond was crucial for improving anti-leukemic effect from the structure–activity relationship analysis. The compound with the most potential, 14 was selected for the preliminary mechanistic study. Compound 14 can induce apoptosis and cause cell cycle arrest. The expression of the marker proteins, such as PARP and caspase 3, were notably effected by this compound, thus inducing apoptosis. In conclusion, our investigation implied that compound 14 may serve as a potential anti-leukemia agent.

Inhibitory effects of ethyl acetate and butanol fractions from Morinda lucida benth on benzene-induced leukemia in mice

Morinda lucida Benth has been traditionally reported as a remedy for the treatment of cancer as well as Leukemia in Africa. Therefore, this study was aimed at screening fractions of M. lucida for potential anti-leukemia constituents and activities in mice. leukemia was induced with intra-peritoneal administration of benzene. Fractions of M. lucida were administered by oral gavaging in mice and screened for anti-leukemia and anti-clastogenic activities. The liver sections were assessed for hepatoprotective activity and possible anti-leukemia compounds in fractions were analysed using GC-MS. Docking analysis was done to understand the mechanism of anti-leukemic activity. Significant ( p < 0.05) anti-leukemic activity was observed in the ethyl acetate and n-butanol fractions while prevention of chromosomal and liver damage was exhibited by all the fractions. GC-MS analysis of the fractions revealed anti-leukemic constituents. The identified constituents showed promising pharmacokinetic properties and inhibitory potential against certain proteins involved in cancer development. Overall, the anti-leukemic activity observed in this study might be attributed to the antioxidant, apoptotic induction, cell cycle control, and anti-inflammatory properties of M. lucida fractions.

Analysis of Plant-Plant Interactions Reveals the Presence of Potent Antileukemic Compounds.

A method to identify anticancer compounds in plants was proposed based on the hypothesis that these compounds are primarily present in plants to provide them with an ecological advantage over neighboring plants and other competitors. According to this view, identifying plants that contain compounds that inhibit or interfere with the development of other plant species may facilitate the discovery of novel anticancer agents. The method was developed and tested using Magnolia grandiflora, Gynoxys verrucosa, Picradeniopsis oppositifolia, and Hedyosmum racemosum, which are plant species known to possess compounds with cytotoxic activities. Plant extracts were screened for growth inhibitory activity, and then a thin-layer chromatography bioautography assay was conducted. This located the major antileukemic compounds 1, 2, 4, and 5 in the extracts. Once the active compounds were located, they were extracted and purified, and their structures were determined. The growth inhibitory activity of the purified compounds showed a significant correlation with their antileukemic activity. The proposed approach is rapid, inexpensive, and can easily be implemented in areas of the world with high biodiversity but with less access to advanced facilities and biological assays.

Sesamin Acts as Anti-leukemic Compound Interacting with Novel Phosphoprotein Targets and Inducing Apoptosis in Leukemic Cells.

Leukemia is one of the high-incidence cancers that is characterized by an abnormal production of immature white blood cells. Subject to many reports on the side effects of conventional chemotherapy, herbs and natural compounds have been studied as an alternative medicine. In this study, sesamin, a lignan in sesame seed with pharmaceutical functions including anti-cancer, was chosen and treated with MOLT-4 and NB4 leukemic cell lines in various concentrations for 24 and 48 hours. The effect of sesamin on cell inhibition and expression levels of apoptotic genes in leukemic cell lines were investigated by MTT assay and real-time PCR, respectively. Moreover, apoptotic proteins were studied by mass spectrometry and bioinformatics tools to investigate the relation between sesamin and targeted proteins. Results showed that sesamin increased cell inhibition in both cell lines in dose- and time-dependent manner. Levels of caspase-3, -7, -8, and -9 gene expressions significantly increased, while BCL-2 decreased drastically in sesamin-treated cells. From bioinformatics study, PARP4, IPPK and caspase family proteins were found to be involved in sesamin that induced apoptosis in leukemic cells. Besides, doxorubicin, a chemotherapeutic drug, also shared the same protein targets as sesamin in apoptosis pathway. Sesamin demonstrates its potential to enhance cell inhibition and promotes cell apoptosis in both MOLT-4 and NB4 leukemic cell lines. This study will benefit the development of sesamin as an effective anti-leukemia drug in the future.

Metformin compounds: A review on the importance and the possible applications

Metformin is a particular compound able to coordinate metal ions and form complexes with Schiff bases that have unique properties and high stability, for a wide range of applications. This review describes and discusses organic and inorganic several reactions of synthesis of metformin complexes that coordinate some metal ions such as Ni, Co, Nd, Cr, Cu. These synthesis methods of metformin complexes are shown by the published results and characterization analyses. In medicine and pharmacy, these complexes are commonly applied and have shown a positive reaction in the fields. These complexes have been recommended to lower blood glucose levels and suggested as anti-cancer, anti-bacterial, and anti-leukemia compounds, and some have been used against leukemia. It has also been documented that the addition of specific ligands to metformin has also improved metformin’s pharmacokinetic activity. Therefore, the preparation of many of the Schiff bases and complexes of metformin, such as [Nd (III) metformin Schiff Base, Co(TMP)(MET)(H2O)Cl2], [Ni(TMP)(MET)(H2O)Cl2, grapheneoxide-metformin-nickel, tetrachloro (metfo-rmin) platinum (IV) dimethyl, Cr(III)-2Mfn. HCl and others are described and discussed here. These complexes have the potential to be used for some biological activities, in addition to their potent as a catalyst of the carbon-carbon coupling reaction.

Ethno Botanical Survey, Anti-leukemia and Anticlastogenic Potential of Medicinal Plants used for Treatment of Leukemia in Oyo State Nigeria

Ethnopharmacological Relevance: Leukemia, a cancer of the blood and bone marrow is responsible for about 222.000 deaths annually. The side effects of chemotherapy in leukemia treatment have necessitated the search for natural products especially medicinal plants as alternative therapy.&#x0D; Aim: This study surveyed common plants used for treating leukemia in Oyo state, Nigeria and assessed the anti-leukemic and anti-clastogenic activities of fractions of Nymphaea lotus.&#x0D; Methods: Semi-structured questionnaire (1000) was used to collect the ethnobotanical data among the traditional healers. Leukemia was induced in albino mice with 400 mg/kg body weight of benzene intraperitoneally. Aqueous extracts (120mg/kg/bw), fractions and 5-florouracil (25mg) was administered to mice of different groups. The anti-leukemic, anti-clastogenic, antioxidant, hepatoprotective activities and hematological parameters were determined. N. lotus was subjected to gas-chromatography mass spectroscopy analysis.&#x0D; Results: The ethnobotanical survey recorded 89 plant species with their local names and parts used in the traditional therapeutic preparations. Seven plants (Pistiastratiotes (2.5%), Nymphaea lotus (1.4%), Piper guineense (1.7%), Securinega virosa (2.5%), Calotropis procera (3.4%), Morinda lucida (2.5%) and Xylopia aethiopica (4.5%) with the highest frequency of citation were selected for anti-leukemic and anti-clastogenic screening. The aqueous extract of N. lotus and M. lucida displayed anti-leukemic potential. M. lucida and X. aethiopica improved the hematological parameters. Ethyl acetate fraction of N. lotus significantly (p&lt;0.05) reduced the number of micronucleated polychromatic erythrocyte in the bone marrow and showed anti-leukemic activity. Fractions of N. lotus restored the hematological parameters and exhibited significant (p&lt;0.05) antioxidant activity. Histological observation revealed improvement in the liver general cyto-architecture of mice treated with ethyl acetate and butanol fractions of N. lotus. Some known compounds were identified in ethyl acetate fraction of N. lotus.&#x0D; Conclusion: Most of the species tested had some anti-leukemic effect in mice, which to some extent supports their traditional inclusion in herbal preparations for treatment of leukemia. The study also identified potential anti-leukemic compounds in N. lotus extract. The study also identified potential anti-leukemic compounds in N. lotus extract.

Machine Learning-Driven Drug Discovery: Prediction of Structure-Cytotoxicity Correlation Leads to Identification of Potential Anti-Leukemia Compounds.

In vitro cytotoxicity screening is a crucial step of anticancer drug discovery. The application of deep learning methodology is gaining increasing attentions in processing drug screening data and studying anticancer mechanisms of chemical compounds. In this work, we explored the utilization of convolutional neural network in modeling the anticancer efficacy of small molecules. In particular, we presented a VGG19 model trained on 2D structural formulae to predict the growth-inhibitory effects of compounds against leukemia cell line CCRF-CEM, without any use of chemical descriptors. The model achieved a normalized RMSE of 15.76% on predicting growth inhibition and a Pearson Correlation Coefficient of 0.72 between predicted and experimental data, demonstrating a strong predictive power in this task. Furthermore, we implemented the Layer-wise Relevance Propagation technique to interpret the network and visualize the chemical groups predicted by the model that contribute to toxicity with human-readable representations.Clinical relevance-This work predicts the cytotoxicity of chemical compounds against human leukemic lymphoblast CCRF-CEM cell lines on a continuous scale, which only requires 2D images of the structural formulae of the compounds as inputs. Knowledge in the structure-toxicity relationship of small molecules will potentially increase the hit rate of primary drug screening assays.

Design, Synthesis, and In Vitro Evaluation of Benzofuro[3,2-c]Quinoline Derivatives as Potential Antileukemia Agents.

Herein, we design and synthesize an array of benzofuro[3,2-c]quinolines starting from 3-(2-methoxyphenyl)quinolin-4(1H)ones via a sequential chlorination/demethylation, intramolecular cyclization pathway. This sequential transformation was efficient, conducted under metal-free and mild reaction conditions, and yielded corresponding benzofuro[3,2-c]quinolines in high yields. In vitro biological evaluation indicated that such type of compounds showed excellent antileukemia activity and selectivity, and therefore may offer a promising hit compound for developing antileukemia compounds.

ASIC1A INHIBITOR MAMBALGIN-2 SUPPRESSES GROWTH OF LEUKEMIA CELLS BY CELL CYCLE ARREST

For the first time the inhibitor of proton-gated channel ASIC1a was proposed as prototype of new anti-leukemic compound

Towards repositioning of quinacrine for treatment of acute myeloid leukemia – Promising synergies and in vivo effects

We previously reported that the anti-malarial drug quinacrine has potential to be repositioned for treatment of acute myeloid leukemia (AML). As a next step towards clinical use, we assessed the efficacy of quinacrine in an AML-PS mouse model and investigated possible synergistic effects when combining quinacrine with nine other antileukemic compounds in two AML cell lines. Furthermore, we explored the in vivo activity of quinacrine in combination with the widely used AML agent cytarabine.The in vivo use of quinacrine (100mg/kg three times per week for two consecutive weeks) significantly suppressed circulating blast cells at days 30/31 and increased the median survival time (MST). The in vitro drug combination analysis yielded promising synergistic interactions when combining quinacrine with cytarabine, azacitidine and geldanamycin. Finally, combining quinacrine with cytarabine in vivo showed a significant decrease in circulating leukemic blast cells and increased MST compared to the effect of either drug used alone, thus supporting the findings from the in vitro combination experiments.Taken together, the repositioning potential of quinacrine for treatment of AML is reinforced by demonstrating significant in vivo activity and promising synergies when quinacrine is combined with different agents, including cytarabine, the hypomethylating agent azacitidine and HSP-90 inhibitor geldanamycin.

Abstract 2023: Artemisinins enhanced the antileukemic efficacy of BCL2 inhibitors

Abstract More effective, less toxic treatments are needed to improve longevity and quality of life for patients with acute leukemias. Artesunate (AS), the current WHO-recommended drug for severe malaria, is a semi-synthetic derivative of the natural compound Artemisinin. AS has broad antineoplastic activity in vitro, and is well tolerated, inexpensive, and can be parenterally or orally administered in humans. Both AS and a more potent synthetic derivative, artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838), substantially inhibited acute leukemia growth in vivo and in vitro, at doses where normal CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected. Moreover, AS and ART-838 synergized with several current antileukemic drugs (Fox et al, Oncotarget, 2015). To identify additional potential antileukemic drug combinations, we screened for the growth inhibitory effects of AS and ART-838 against 3 acute leukemia cell lines co-treated with a panel of 122 antileukemic compounds (mainly emerging antineoplastic drugs). Both AS and ART-838 showed synergistic or additive growth inhibition with several compounds. Two BCL2 protein family inhibitors demonstrated among the highest levels of synergy. One of these, ABT-199 (venetoclax), was recently approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and received breakthrough designation for acute myeloid leukemia (AML). Therefore, we decided to further investigate the combination of ABT-199 with AS or ART-838. Synergy between AS (or ART-838) and ABT-199 was confirmed across several acute leukemia cell lines with varying sensitivities to each drug. Both leukemia cell proliferation and survival were affected synergistically by exposure to the combination of AS (or ART-838) plus ABT-199. Utilizing in vivo imaging, we assessed changes in growth kinetics of tail vein-injected acute leukemia cell lines and primary patient-derived samples constitutively expressing luciferase in mice treated with AS, ABT-199, or the combination. The combination of AS plus ABT-199 had the greatest antileukemic effect. Based on these data, we propose that Artemisinins plus BCL2 inhibitors comprise an antileukemic drug pair with high potential for incorporation into acute leukemia therapeutic regimens. Citation Format: Blake S. Moses, Jennifer M. Fox, Xiaochun Chen, Jeffrey W. Tyner, Gary H. Posner, Patrick Bailey, Curt I. Civin. Artemisinins enhanced the antileukemic efficacy of BCL2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2017-2023

INSILICO MODELLING OF CYTOTOXIC BEHAVIOUR OF ANTI-LEUKEMIA COMPOUNDS ON HL-60 CELL LINE

This research employs multiple linear regression technique in the modelling of some potent anti-leukemic compounds using paDEL molecular descriptor software calculator, to identify the best relationship between the chemical structure and toxicities of the anticancer datasets against some leukemic cell lines (HL-60). Statistical parameters such as Q2 and R2pred (test set) were computed to validate the strength of the model, while Williams plot was used to assess its applicability domain. The mean effects of the molecular descriptors in the models were calculated to illuminate the principal properties of the molecules responsible for their cytotoxicity.

Cyanobacteria as a Source for Novel Anti-Leukemic Compounds.

Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.

Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug resistance-conferring point mutations

Numerous FLT3 inhibitors have been explored as a viable therapy for the treatment of acute myeloid leukemia (AML). However, clinical data have been underwhelming due to incomplete inhibition of FLT3 or the emergence of resistant mutations treated with these older agents. We previously developed a series of 3-phenyl-1H-5-pyrazolylamine derivatives as highly potent and selective FLT3 inhibitors with good in vivo efficacy using an intravenous (IV) route. However, the poor bioavailability of these pyrazole compounds limits the development of these promising antileukemic compounds for clinical use. Herein, we describe a novel class of 5-phenyl-thiazol-2-ylamine compounds that are multi-targeted FLT3 inhibitors. From this class of compounds, compound 7h was very potent against AML cell lines and exhibited excellent oral efficacy in AML xenograft models. In addition, further studies demonstrated that compound 7h exhibited potent in vitro and in vivo activities against clinically relevant AC220 (3)-resistant kinase domain mutants of FLT3-ITD.

Synthesis, anticancer activity and molecular docking studies on a series of heterocyclic trans-cyanocombretastatin analogues as antitubulin agents

A series of heterocyclic combretastatin analogues have been synthesized and evaluated for their anticancer activity against a panel of 60 human cancer cell lines. The most potent compounds were two 3,4,5-trimethoxy phenyl analogues containing either an (Z)-indol-2-yl (8) or (Z)-benzo[b]furan-2-yl (12) moiety; these compounds exhibited GI50 values of <10nM against 74% and 70%, respectively, of the human cancer cell lines in the 60-cell panel. Compounds 8, and 12 and two previously reported compounds in the same structural class, i.e. 29 and 31, also showed potent anti-leukemic activity against leukemia MV4-11 cell lines with LD50 values=44nM, 47nM, 18nM, and 180nM, respectively. From the NCI anti-cancer screening results and the data from the invitro toxicity screening on cultured AML cells, seven compounds: 8, 12, 21, 23, 25, 29 and 31 were screened for their invitro inhibitory activity on tubulin polymerization in MV4-11 AML cells; at 50nM, 8 and 29 inhibited polymerization of tubulin by >50%. The binding modes of the three most active compounds (8, 12 and 29) to tubulin were also investigated utilizing molecular docking studies. All three molecules were observed to bind in the same hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine, and were stabilized by van der Waals' interactions with surrounding tubulin residues. The results from the tubulin polymerization and molecular docking studies indicate that compounds 8 and 29 are the most potent anti-leukemic compounds in this structural class, and are considered lead compounds for further development as anti-leukemic drugs.

High-Throughput Drug Screening Identifies Pyrimethamine As a Potent and Selective Inhibitor of Acute Myeloid Leukemia

Introduction: Hematopoietic stem/progenitor cell differentiation is blocked in acute myeloid leukemia (AML) resulting in cytopenias and high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation fostering compounds. High expression of MN1 confers poor prognosis to AML patients and induces resistance to cytarabine and all-trans-retinoic acid (ATRA) induced differentiation. We thus set out to identify compounds which could potentially overcome the differentiation block in AML.Methods: Based on the above concepts and in an effort to identify novel compounds which are potent inducers of differentiation and apoptosis in AML, high-throughput drug screening was employed in the MN1 leukemic model. A total of 3580 bioactive compounds were tested in duplicate at a concentration of 2.5 µM using alamar blue fluorescence as readout. As MN1 cells are resistant to ATRA (at 1µM and even 10µM ATRA), the drug screening was performed in the presence of a clinically relevant dose of ATRA (1 µM) to identify compounds that concurrently act with the cytotoxic and/or differentiating effects of ATRA. To determine whether a compound was effective as monotherapy or if it synergized with ATRA, we also performed a validation phase study in which the IC50 of each candidate compound was tested alone and in combination with ATRA. Fifty-four inhibitors were chosen from the primary screen for further validation based on presumed mechanism of action and novelty. The shortlisted compound pyrimethamine (PMT) was validated for its differentiation and apoptosis promoting effects in various murine and human AML models.Results: Our high-throughput drug screening identified 117 compounds, which reduced MN1 leukemic cell proliferation by more than 80% above the ATRA-treated control in both replicates (inhibitors), 8 borderline inhibitors (one replicate with more than 80% inhibition and one with 74 to 80% inhibition), and 35 outliers, which inhibited cell proliferation by 80% or more in only one replicate. The biologic processes most frequently targeted by the 117 inhibitors were DNA replication (n=26), microtubule assembly (n=12), NF-kB pathway (n=8), dihydrofolate reductase (DHFR, n=3) and heat shock protein 90 (HSP90). Dihydrofolate reductase inhibitors, pyrimethamine and amethopterin/methotrexate emerged as top hits from the screening and preliminary validation studies. Validation studies identified the antifolate pyrimethamine (PMT) that potently induced apoptosis and differentiation in several murine and human leukemic cell lines when administered as a single agent. The cytotoxic effects of pyrimethamine were reversed by addition of an excess of folic acid whereas induction of myeloid differentiation at higher concentrations of pyrimethamine was not mediated through DHFR inhibition. We further evaluated the effect of pyrimethamine in an in vivo xenograft mouse model by subcutaneously inducing tumors with HL60 and THP1 cell lines. Oral pyrimethamine treatment significantly reduced tumor volumes after 14, 19 and 24 days post-transplantation and at death compared to solvent treated mice (P<0.01). The effect of pyrimethamine was further assessed in primary human AML cells and normal CD34+ cells by CFC assays. Colony numbers from primary AML cells, but not normal CD34+ bone marrow cells, were significantly reduced by pyrimethamine as compared to solvent control. Thus, our study identifies pyrimethamine as a candidate drug that is a potent and specific inducer of apoptosis and differentiation with the property of specifically targeting leukemic cells.Conclusion: Our high-throughput drug screening identified pyrimethamine as a potent and specific antileukemic compound and reinforces targeting of folate metabolism as a treatment strategy in acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Up-regulation of anti-apoptotic genes confers resistance to the novel anti-leukaemic compound PEP005 in primary AML cells.

We showed previously that PEP005 induced apoptosis in leukaemic cell lines and blasts from patients with acute myeloid leukaemia (AML). Here we assess the anti-leukeamic effects of PEP005 in vivo and determine the mechanism of resistance of PEP005 non-responsive cells. We used 2 human xenograft mouse models of AML to assess the anti-leukaemic effects of PEP005 in vivo. Expression microarray analysis of primary AML blasts following treatment with PEP005 was used to determine patterns of gene expression that conferred resistance. PEP005 significantly reduced tumour burden in two human leukaemia mouse xenograft models. We also assessed responsiveness of 33 AML samples to PEP005, with 78% of the samples entering apoptosis at 100nM. Resistance to PEP005 was not restricted to a particular AML subtype. Expression microarray analysis of resistant samples following treatment with PEP005 revealed a significant up regulation of the anti-apoptotic genes Bcl-2A1, Mcl-1, and PHLDA1 which was verified using RT-PCR. We conclude that PEP005 shows broad efficacy against AML subtypes and that up regulation of anti-apoptotic genes underlies resistance to this agent and could be used to screen for patients unlikely to benefit from a therapeutic regime involving PEP005.