Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2'-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2.

Different mechanisms of drug resistance to hypomethylating agents in the treatment of myelodysplastic syndromes and acute myeloid leukemia.

Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.

Highly stereocontrolled total synthesis of racemic codonopsinol B through isoxazolidine-4,5-diol vinylation.

A new highly diastereoselective synthesis of the polyhydroxylated pyrrolidine alkaloid (±)-codonopsinol B and its N-nor-methyl analogue, starting from achiral materials, is presented. The strategy relies on the trans-stereoselective epoxidation of 2,3-dihydroisoxazole with in situ-generated DMDO, the syn-selective α-chelation-controlled addition of vinyl-MgBr/CeCl3 to the isoxazolidine-4,5-diol intermediate, and the substrate-directed epoxidation of the terminal double bond of the corresponding γ-amino-α,ß-diol with aqueous hydrogen peroxide catalyzed by phosphotungstic heteropoly acid. Each of the key reactions proceeded with an excellent diastereoselectivity (dr > 95:5). (±)-Codonopsinol B was prepared in 10 steps with overall 8.4% yield. The antiproliferative effect of (±)-codonopsinol B and its N-nor-methyl analogue was evaluated using several cell line models.

Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents.

We established the following two variants of the MOLM-13 human acute myeloid leukemia (AML) cell line: (i) MOLM-13/DAC cells are resistant to 5-aza-2'-deoxycytidine (DAC), and (ii) MOLM-13/AZA are resistant to 5-azacytidine (AZA). Both cell variants were obtained through a six-month selection/adaptation procedure with a stepwise increase in the concentration of either DAC or AZA. MOLM-13/DAC cells are resistant to DAC, and MOLM-13/AZA cells are resistant to AZA (approximately 50-fold and 20-fold, respectively), but cross-resistance of MOLM-13/DAC to AZA and of MOLM-13/AZA to DAC was not detected. By measuring the cell retention of fluorescein-linked annexin V and propidium iodide, we showed an apoptotic mode of death for MOLM-13 cells after treatment with either DAC or AZA, for MOLM-13/DAC cells after treatment with AZA, and for MOLM-13/AZA cells after treatment with DAC. When cells progressed to apoptosis, via JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) assay, we detected a reduction in the mitochondrial membrane potential. Furthermore, we characterized promoter methylation levels for some genes encoding proteins regulating apoptosis and the relation of this methylation to the expression of the respective genes. In addition, we focused on determining the expression levels and activity of intrinsic and extrinsic apoptosis pathway proteins.