An in vitro testing of chemoresistance in leukaemic patients

The fact that leukaemic cells are primarily or secondarily resistant to cytostatics is a serious phenomenon, which leads to the failure of chemotherapy of malignant diseases in clinical practise. Some detoxification and transporting systems are responsible for the generation of chemoresistance on the cellular level and the decrease of effectiveness in treatment. In vitro testing of chemoresistance of leukaemic cells is presently an inseparable component of “tailoring” therapy in the developing field of predictive oncology. The aim of this work was to estimate profiles of drug resistance, based on the predictive in vitro test, and to help in choosing the most effective cytostatic. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoline (MTT) assay was used, based on the direct effect of cytostatics on the viability of leukaemic cells in vitro. The number of living leukaemic cells was evaluated by a computer program, where LC₅₀ (concentration of cytostatics lethal to 50% of leukaemic cells) was established from the achieved dose-relation curves. Seventy-one samples of leukaemic cells isolated from the patients’ peripheral blood or bone marrow were examined. All samples were tested to 3 cytostatics minimally. It was found by the in vitro assay, that resistance to dexamethasone, prednisolone, etoposide and vincristine is increased in patients with acute myeloid leukaemia disease, compared to the acute lymphoblastic leukaemia patients. In patients with a relapsed disease population, leukaemic cells are highly heterogeneous in the MTT assay. It was concluded that the MTT assay can be used to study drug interactions in vitro in leukaemia samples. The type of interaction was highly different between patients, and depended on drug concentrations.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

Immunotherapy of myeloid leukaemia

The treatment of myeloid leukaemia has progressed in recent years with the advent of donor leukocyte infusions (DLI), haemopoietic stem cell transplants (HSCTs) and targeted therapies. However, relapse has a high associated morbidity rate and a method for removing diseased cells in first remission, when a minimal residual disease state is achieved and tumour load is low, has the potential to extend remission times and prevent relapse especially when used in combination with conventional treatments. Acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) are heterogeneous diseases which lack one common molecular target while chronic myeloid leukaemia (CML) patients have experienced prolonged remissions through the use of targeted therapies which remove BCR-ABL⁺ cells effectively in early chronic phase. However, escape mutants have arisen and this therapy has little effectivity in the late chronic phase. Here we review the immune therapies which are close to or in clinical trials for the myeloid leukaemias and describe their potential advantages and disadvantages.     

Epigenetic dysregulation of ID4 predicts disease progression and treatment outcome in myeloid malignancies

Promoter hypermethylation‐mediated inactivation of ID4 plays a crucial role in the development of solid tumours. This study aimed to investigate ID4 methylation and its clinical relevance in myeloid malignancies. ID4 hypermethylation was associated with higher IPSS scores, but was not an independent prognostic biomarker affecting overall survival (OS) in myelodysplastic syndrome (MDS). However, ID4 hypermethylation correlated with shorter OS and leukaemia‐free survival (LFS) time and acted as an independent risk factor affecting OS in acute myeloid leukaemia (AML). Moreover, ID4 methylation was significantly decreased in the follow‐up paired AML patients who achieved complete remission (CR) after induction therapy. Importantly, ID4 methylation was increased during MDS progression to AML and chronic phase (CP) progression to blast crisis (BC) in chronic myeloid leukaemia (CML). Epigenetic studies showed that ID4 methylation might be one of the mechanisms silencing ID4 expression in myeloid leukaemia. Functional studies in vitro showed that restoration of ID4 expression could inhibit cell proliferation and promote apoptosis in both K562 and HL60 cells. These findings indicate that ID4 acts as a tumour suppressor in myeloid malignancies, and ID4 methylation is a potential biomarker in predicting disease progression and treatment outcome.     

Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells

Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC₅₀ for DOX-TRF was lower than the IC₅₀ value for the free drug in both leukaemia cell lines. The IC₅₀ values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC₅₀ values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.     

Drug resistance and DNA repair in leukaemia

Most cytotoxic agents exert their action via damage of DNA. Therefore, the repair of such lesions is of major importance for the sensitivity of malignant cells to chemotherapeutic agents. The underlying mechanisms of various DNA repair pathways have extensively been studied in yeast, bacteria and mammalian cells. Sensitive and drug resistant cancer cell lines have provided models for analysis of the contribution of DNA repair to chemosensitivity. However, the validity of results obtained by laboratory experiments with regard to the clinical situation is limited. In both acute and chronic leukaemias, the emergence of drug resistant cells is a major cause for treatment failure. Recently, assays have become available to measure cellular DNA repair capacity in clinical specimens at the single-cell level. Application of these assays to isolated lymphocytes from patients with chronic lymphatic leukaemia (CLL) revealed large interindividual differences in DNA repair rates. Accelerated O⁶-ethylguanine elimination from DNA and faster processing of repair-induced single-strand breaks were found in CLL lymphocytes from patients nonresponsive to chemotherapy with alkylating agents compared to untreated or treated sensitive patients. Moreover, modulators of DNA repair with different target mechanisms were identified which also influence the sensitivity of cancer cells to alkylating agents. In this article, we review the current knowledge about the contribution of DNA repair to drug resistance in human leukaemia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cartilage polysaccharide induces apoptosis in human leukemia K562 cells

In this study, we extracted a polysaccharide (short-chain polysaccharide [PS]) from porcine cartilage and examined its function in chronic myeloid leukaemia by using human K562 cells and mouse L1210 cells. Results of cell proliferation assay indicated that PS inhibited cancer cell growth at different concentrations, while it had little effect on normal cells. The presence of morphological aspects of apoptosis, such as nuclear shrinkage, was shown in H&E stained sections. The occurrence of PS-induced apoptosis was confirmed by TUNEL assay and cell cycle analysis. The results of immunofluorescent staining indicated the molecular mechanism underlying. Through interfering with the cell cycle of tumor cells, PS may induce apoptosis by downregulating the expression level of cyclin D1 and upregulating the level of p21 protein. Correlation analysis of apoptosis and MAPK suggested that inactivation of ERK was crucial for PS induced apoptosis, while JNK phosphorylation had a small effect and p38 was not involved. In vivo assay showed that PS inhibited L1210 cell growth in vivo and prolonged the life span of L1210-bearing mice. We conclude that PS is a polysaccharide with anticancer effects and induced apoptosis in human K562 cells.     

Acriflavine targets oncogenic STAT5 signaling in myeloid leukemia cells

Acriflavine (ACF) is an antiseptic with anticancer properties, blocking the growth of solid and haematopoietic tumour cells. Moreover, this compound has been also shown to overcome the resistance of cancer cells to chemotherapeutic agents. ACF has been shown to target hypoxia‐inducible factors (HIFs) activity, which are key effectors of hypoxia‐mediated chemoresistance. In this study, we showed that ACF inhibits the growth and survival of chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cell lines in normoxic conditions. We further demonstrated that ACF down‐regulates STAT5 expression in CML and AML cells but activates STAT3 in CML cells in a HIF‐independent manner. In addition, we demonstrated that ACF suppresses the resistance of CML cells to tyrosine kinase inhibitors, such as imatinib. Our data suggest that the dual effect of ACF might be exploited to eradicate de novo or acquired resistance of myeloid leukaemia cells to chemotherapy.     

Mouse sarcoma L1 cell line holoclones have a stemness signature

Accumulating data suggest that cancers contain a fraction of cells called cancer stem cells (CSCs), that may be responsible for upkeep and relapses of disease. In experimental settings, CSCs are regarded as most effective at tumour initiation in in vivo assays. Since the first isolation of cancer stem cells from acute myeloid leukaemia in 1994, cancer stem cells have been identified in human solid tumours and they have also been found in the established cell lines, based on ability of CSCs to form in vitro colonies of a specific morphology, called holoclones. Our study examined the ability of a mouse sarcoma cell line, derived from a lung metastasis of a BALB/c mouse and established as a stably growing line (L1), to produce holoclones in vitro. We aimed to verify a stemness signature of the holoclone cells. The L1 cell line was found to form holoclone colonies in vitro, which were shown to contain a percentage of CSC‐like cells. A fraction of the L1 cells was able to repopulate the original cell line, and presented an increased clonogenic and metastatic potential (18th passage). In addition, MTT assay and flow cytometry of the side population fraction revealed that these cells were more resistant to chemotherapeutic drugs than the original cell line, and over‐expressed the anti‐apoptotic genes, GRP78 and GADD153. We conclude that mouse L1 sarcoma cell line contains CSC‐like cells.     

Airborne cytotoxicity in the DiSC assay caused by solutions of treosulfan but not busulphan

Treosulfan and busulphan are similar molecules, the former used in the treatment of ovarian cancer and the latter in chronic myelogenous leukaemia. We have used both in the differential staining cytotoxicity (DiSC) assay forin vitro drug sensitivity testing to aid in the choice of chemotherapy for individual patients.It was observed that occasionally the viability of control cells in one assay box was reduced compared with control cells in other boxes from the same assay. Treosulfan was suspected as the cause because cells throughout the microtitre box containing treosulfan had reduced viability in 28/62 (45%) experiments and in 9 of these, total kill of all cells in the box was observed.We tested the hypothesis that a metabolite of treosulfan might be the cause of this airborne cytotoxicity, and found that whilst 10 mg ml^–1 of either methane sulphonic acid or tetrahydrofuran had no airborne cytotoxic effect, 1 mg ml^–1 diepoxybutane killed over 95% of cells in all tubes in the same box.Treosulfan is another chemical (cf. azide, mafosfamide and possibly other cytotoxic agents) that can cause airborne cytotoxicity.Abbreviations ALL acute lymphoblastic leukaemia - AML acute non-lymphocytic leukaemia - CLL chronic lymphocytic leukaemia - NHL non-Hodgkin's lymphoma - DiSC assay differential staining cytotoxicity assay - MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay - PBS phosphate buffered saline     

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.     

Strongylocentrotus nudos Egg Polysaccharide induces autophagy and apoptosis in leukaemia cells by regulating mitochondrial function

In this study, we investigated the ability of the Polysaccharide from the Eggs of Strongylocentrotus nudus (SEP) to regulate cellular autophagy and apoptosis in leukaemia cells. Human acute myeloid leukaemia (AML) cells (HL60) and murine AML cells (L1210) treated with SEP were used to assess viability using Cell Counting Kit-8, cytotoxicity by measuring lactate dehydrogenase release, the generation of reactive oxygen species (ROS) by DCFH-DA staining. In addition, we utilized a mouse model of leukaemia in which L1210 cells were injected into DBA/2 mice by sub-axillary injection. Treatment with SEP decreased cell viability, increased in cytotoxicity and increased the release of ROS in a dose-dependent manner. SEP treatment was also associated with the activation of pro-apoptotic proteins cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Activation of the apoptotic pathway led to the release of cytochrome C (CytoC) into the cytosol of the cell resulting in decreased membrane potential. The effect of SEP treatment was depended on the activation of the nuclear factor kappa-B (NF-κB) signalling pathway as SEP treatment led to an increase in NF-κB phosphorylation, and inhibition of NF-κB signalling using PDTC blocked SEP-mediated activation of apoptosis. Treatment with SEP also prolonged survival time in our leukaemia mouse model and was associated with diminished tumour volume, increased leucocyte and lymphocyte proliferation, promoted pro-inflammatory factor release in serum and enhanced immune function. Taken together, these data suggest that SEP inhibits the progression of leukaemia by initiating mitochondrial dysfunction, autophagy, and apoptosis via the NF-κB signalling pathway.     

Multidrug resistance (MDR) genes in haematological malignancies

The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies. Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs. Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR). Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistantin vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype. P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value. Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas. Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation. Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors. However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.Abbreviations ALL acute lymphocytic leukaemia - AML acute myelocytic leukaemia - BM bone marrow - CAT chloramphenicol acetyltransferase - CLL chronic lymphocytic leukaemia - CML chronic myelocytic leukaemia - CR complete remission - HCL hairy cell leukaemia - MDR multidrug resistance - MDS myelodysplastic syndrome - MM multiple myeloma - MoAb monoclonal antibody - NHL non-Hodgkin's lymphoma - PB peripheral blood - PCR polymerase chain reaction - PLL prolymphocytic leukaemia - RMA resistance modifying agent - VAD vincristine, doxorubicin, dexamethasone     

Bis(4-hydroxy-2H-chromen-2-one): Synthesis and effects on leukemic cell lines proliferation and NF-κB regulation

Synthesis of the bis-4-hydroxycoumarin-type compound, 3,3′-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-one), was performed by two alternative pathways, either involving a basic organocatalyzed 1,4-conjugate addition tandem reaction of 4-hydroxycoumarin on chromone-3-carboxylic acid, or a double condensation of 4-hydroxycoumarin on ω-formyl-2′-hydroxyacetophenone. The anti-proliferative effects of the bis-4-hydroxycoumarin-type compound on human K-562 (chronic myeloid leukaemia) and JURKAT (acute T-cell leukaemia) cell lines using trypan blue staining, as well as its involvement in nuclear factor-kappa B (NF-κB) regulation analyzed by luciferase reporter gene assay, gene expression analysis and western blots were analysed. This compound inhibited TNFα-induced NF-κB activation in K-562 (IC₅₀ 17.5 μM) and JURKAT (IC₅₀ 19.0 μM) cell lines, after 8 h of incubation. Interestingly, it exerted mainly cytostatic effects at low doses on both cell lines tested, whereas it decreased JURKAT cell viability starting at 50 μM from 24 h of treatment. Importantly, it did not affect the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors, even at concentrations above 100 μM.     

Cytopathological spectrum of unusual malignant pleural effusions at a tertiary care centre in north India

BACKGROUND: Cytological examination of pleural fluid is one of the most informative laboratory procedures in the diagnosis of pleural effusions. Although tuberculosis is the commonest cause of pleural effusions in developing countries, tumours, including grade ones, can present with effusions. OBJECTIVE: The aim of the present study was to evaluate the uncommon causes of malignant pleural effusion. METHODS: A 2-year retrospective analysis of pleural fluid cytological specimens submitted to the Department of Cytopathology, PGIMER, Chandigarh between January 2003 and December 2004 was performed to retrieve unusual metastases. Out of a total of 898 samples reviewed, 710 were negative for malignancy and 24 cases were suspicious for malignancy. The remaining 164 cases were positive for malignancy, out of which 38 cases revealed malignancies other than adenocarcinoma. RESULTS: The 38 unusual malignancies metastasizing to the pleural cavity included 29 haematological malignancies (non-Hodgkin's lymphoma, acute lymphoid leukaemia, multiple myeloma and chronic myeloid leukaemia) and nine non-haematological malignancies (Ewing's sarcoma, neuroblastoma, Wilms' tumour, squamous cell carcinoma, small-cell carcinoma and malignant fibrous histiocytoma). CONCLUSION: Although metastatic adenocarcinoma was the commonest aetiology of malignant pleural effusions, a significant number of unusual causes of malignant pleural effusion were also encountered.     

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

UDP-GlcNAc:GlcNAc 1-2Man1-6R (GlcNAc to Man) 1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc1-2Man1-6Glc/Man-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man1-3 residue and the 4-hydroxyl of the Man- residue of the Man1-6(Man1-3)Man-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAc1-2(4,6-di-O-methyl-)Man1-6Glc-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.Abbreviations all allyl - AML acute myeloid leukaemia - BSA bovine serum albumin - CML chronic myelogenous leukaemia - Gal G,d-galactose - Glc d-glucose - GlcNAc Gn,N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man M,d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈COOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - oct octyl - pnp p-nitrophenyl - T transferase     

Synthesis and biological evaluation of dithiocarbamate esters of parthenolide as potential anti-acute myelogenous leukaemia agents

A series of dithiocarbamate esters of parthenolide (PTL) was designed, synthesised, and evaluated for their anti- acute myelogenous leukaemia (AML) activities. The most promising compound 7l showed greatly improved potency against AML progenitor cell line KG1a with IC₅₀ value of 0.7?μM, and the efficacy was 8.7-folds comparing to that of PTL (IC₅₀?=?6.1?μM). Compound 7l induced apoptosis of total primary human AML cells and leukaemia stem cell (LSCs) of primary AML cells while sparing normal cells. Furthermore, 7l suppressed the colony formation of primary human leukaemia cells. Moreover, compound 12, the salt form of 7l, prolonged the lifespan of mice in two patient-derived xenograft models and had no observable toxicity. The preliminary molecular mechanism study revealed that 7l-mediated apoptosis is associated with mitogen-activated protein kinase signal pathway. On the basis of these investigations, we propose that 12 might be a promising drug candidate for ultimate discovery of anti-LSCs drug.