Chronic Myeloid Leukemia Cell Lines Research Articles

Two new tetralone glycosides from the green walnut husks of Juglans mandshurica Maxim

Two new tetralone glycosides, 4(S)-5-methoxy juglanoside A (1), 4(S)-5-methoxy juglanoside D (2), together with ten known compounds (3–12) have been isolated from the green walnut husks of Juglans mandshurica Maxim. Their structures were elucidated on the basis of their ESI-MS, 1 D and 2 D NMR spectroscopic data. In addition, all compounds were evaluated for their cytotoxic activities against the cancer BGC-823 (human gastric carcinoma), HCT-15 (human colorectal carcinoma) and K562 (human chronic myeloid leukemia) cell lines. The results showed aglycones of naphthoquinones had stronger cytotoxic activities than glycosides of tetralone.

Combined Therapeutic Effects of Arsenic Trioxide and Poly (Adp-Ribose) Polymerase Inhibitor on Chronic Myeloid Leukaemia Cell Line

Combined Therapeutic Effects of Arsenic Trioxide and Poly (Adp-Ribose) Polymerase Inhibitor on Chronic Myeloid Leukaemia Cell Line

(-)-Epigallocatechin-3-gallate induces cell apoptosis in chronic myeloid leukaemia by regulating Bcr/Abl-mediated p38-MAPK/JNK and JAK2/STAT3/AKT signalling pathways.

Epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, possesses remarkable chemopreventive and therapeutic potential against various types of cancer, including leukaemia. However, the molecular mechanism involved in chronic myeloid leukaemia (CML), especially imatinib-resistant CML cells, is not completely understood. In the present study, we investigated the effect of EGCG on the growth of Bcr/Abl+ CML cell lines, including imatinib-resistant cell lines and primary CML cells. The results revealed that EGCG could inhibit cell growth and induce apoptosis in CML cells. The mechanisms involved inhibition of the Bcr/Abl oncoprotein and regulation of its downstream p38-MAPK/JNK and JAK2/STAT3/AKT pathways. In conclusion, we documented the anti-CML effects of EGCG in imatinib-sensitive and imatinib-resistant Bcr/Abl+ cells, especially T315I-mutated cells.

Antibiotic anisomycin selectively targets leukemia cell lines and patient samples through suppressing Wnt/β-catenin signaling

Chronic myeloid leukemia (CML) responds well to BCR-ABL tyrosine kinase inhibitors (TKI), such as imatinib and dasatinib. However, these inhibitors have been less effective as single agents in the blast phase-CML. In this work, we show that anisomycin, a clinically available drug, targets CML cells at all stages of development and enhances BCR-ABL TKIs’ efficacy. Anisomycin at nanomolar concentration inhibits proliferation and induces apoptosis in a panel of CML cell lines in a dose-dependent manner. It induces apoptosis CD34 stem/progenitor cells isolated from patients with blast phase CML. Using colony formation and serial replating assays, we further show that anisomycin inhibits CML CD34 cell differentiation, proliferation and self-renewal. Additionally, anisomycin is less effective in normal bone marrow (NBM) CD34 cells, suggesting the selective anti-leukemia activity of anisomycin. Combination of anisomycin with imatinib or dasatinib achieves significantly better efficacy than TKI alone in leukemia cell lines and patient samples while sparing normal counterparts. Mechanistically, we demonstrate that p38 MAPK/JNK activation is not required for anti-leukemia activities of anisomycin. Instead, anisomycin displays preferential inhibitory effects to Wnt/β-catenin-mediated signaling in CML. Our work provides the preclinical evidence on the potent efficacy of anisomycin in leukemia and its mechanisms of action. Our work suggests that anisomycin is a potential drug to overcome resistance to BCR-ABL TKI treatment in blast phase CML.

Targeting the Extracellular Signal-Regulated Kinase 5 Pathway to Suppress Human Chronic Myeloid Leukemia Stem Cells.

SummaryTyrosine kinase inhibitors (TKi) are effective against chronic myeloid leukemia (CML), but their inefficacy on leukemia stem cells (LSCs) may lead to relapse. To identify new druggable targets alternative to BCR/ABL, we investigated the role of the MEK5/ERK5 pathway in LSC maintenance in low oxygen, a feature of bone marrow stem cell niches. We found that MEK5/ERK5 pathway inhibition reduced the growth of CML patient-derived cells and cell lines in vitro and the number of leukemic cells in vivo. Treatment in vitro of primary CML cells with MEK5/ERK5 inhibitors, but not TKi, strikingly reduced culture repopulation ability (CRA), serial colony formation ability, long-term culture-initiating cells (LTC-ICs), and CD26-expressing cells. Importantly, MEK5/ERK5 inhibition was effective on CML cells regardless of the presence or absence of imatinib, and did not reduce CRA or LTC-ICs of normal CD34+ cells. Thus, targeting MEK/ERK5 may represent an innovative therapeutic approach to suppress CML progenitor/stem cells.

Targeting heme Oxygenase-1 with hybrid compounds to overcome Imatinib resistance in chronic myeloid leukemia cell lines

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme and a survival-enhancing factor in a number of cancers. Chronic myeloid leukemia (CML) is a blood cancer caused by pathological kinase activity of the BCR-ABL protein, currently treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM). However, resistance to TKIs persists in a number of patients and HO-1 overexpression has been linked with the induction of chemoresistance in CML. With this in mind, in this study, we designed and synthesized the first series of hybrid compounds obtained by combining the structures of IM, as BCR-ABL inhibitor, with imidazole-based HO-1 inhibitors. We found that many hybrids were able to inhibit the enzymatic activity of both targets and to reduce the viability of CML-IM resistant cells, showing that a single molecular entity may reduce the resistance phenomenon.

Volatile anesthetics sevoflurane targets leukemia stem/progenitor cells via Wnt/β-catenin inhibition

Most of the studies regarding the direct effect of anesthetics on tumour cells are focused on opioids and voltage-gated sodium channels. Little is known on the effect of volatile anesthetics on tumour progression. In this study, we show that sevoflurane, a volatile anesthetic, negatively affects chronic myeloid leukemia (CML) CD34 stem/progenitor cells’ biological properties. Sevoflurane significantly inhibits the growth of a panel of CML cell lines in a dose-dependent manner without affecting their survival. It also inhibits proliferation, differentiation and self-renewal capacities but not survival of CML CD34 cells. In addition, sevoflurane significantly augments dasatinib’s efficacy in CML cell lines and stem/progenitors. Mechanistically, sevoflurane dose-dependently decreases levels of β-catenin and c-Myc but not phospho-P38 MAPK in K562 and CML CD34 cells. The decreased Wnt/ β-catenin activity and the reduced levels of Wnt/β-catenin-targeted transcriptions are observed in CML cells exposed to sevoflurane. The complete rescue of the inhibitory effects of sevoflurane in K562 and CML CD34 cells by β-catenin stabilization using both genetic and pharmacological approaches further demonstrates that sevoflurane acts on CML cells via a β-catenin-dependent manner. Our results clearly show the direct and negative effects of sevoflurane on the leukemia cell lines as well as leukemia stem/progenitors. Our findings also reveal Wnt/β-catenin as the target of volatile anesthetics.

Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies.

Telomerase has a critical role in cell proliferation, tumor maintaining, and therapy resistance, which act by modifying many signaling pathways. 2-[(E)-3-Naphtalen-2-yl-but-2-enoylamino]-benzoic acid (BIBR1532) is one of the most studied telomerase inhibitors, and it targets telomerase components TERC and TERT. In this novel study, we aimed to investigate the epigenetic effects of BIBR1532 on both hematologic malignancies and solid tumors. K-562 human chronic myeloid leukemia cell line and U87MG glioblastoma cell line were compared with control groups without BIBR1532 treatment. Cytotoxic effects of BIBR1532 were determined by using WST-1 assay. Apoptotic effects of BIBR1532 were detected by using annexin V method. To assess expression changes in the human epigenetic chromatin modification enzyme genes, total RNA was isolated from K-562 and U87MG cells treated with BIBR1532 and untreated control cells. BIBR1532 induced 2.41-fold apoptotic cell death in U87MG cell lines compared with control groups. Apoptosis was slightly induced in K-562 cells with BIBR1532 treatment compared with control cells. We observed that BIBR1532 also regulates similar genes in both cell lines, and it is useful on epigenetic mechanisms. As a result, telomerase inhibitor BIBR1532 has a significant effect on both hematological malignancies and solid tumors.

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL.

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11b+Ly6C+Ly6G+ cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1β, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response

AbstractHeat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38−) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)–resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Abstract 3739: Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML)

Abstract Despite the impressive success of molecular targeted therapy in chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI) such as imatinib (IM), a proportion of patients exhibit suboptimal response and intolerance to the drug. While the mechanism by which resistance or intolerance develops is not completely clear, it is possible that several secondary modifiers could determine disease progression and drug response in CML. Nuclear Hormone Receptors-NHRs are attractive but less explored therapeutic targets in myeloid leukemia. The impressive success of treatment with ATRA, a RAR ligand, in the treatment of APL has not been translated to other myeloid leukemia subtypes. Recently, Prost et al., 2015 showed that Peroxisome proliferator-activated receptor-γ agonist Pioglitazone eliminates the residual CML stem cell pool and these patients remained in complete molecular response even after withdrawal of IM. The role of other NHRs in CML TKI resistance has not been explored extensively. Based on the in-vitro drug sensitivity data, CML cell lines KU812, EM2 were grouped as sensitive (IC50-0.15 & 2.29uM) and Lama84, KCL22 were grouped as resistant (IC50-54.45 & 66uM) to IM. The NHRs and coregulators that are differentially expressed between IM sensitive and resistant cell lines were identified using NHR RT2 PCR profiler array (SAbiosciences). Based on +2 fold cutoff, the differentially expressed NHRs between test and control group were identified. Eight NHRs (AHR, AR, ESR1, ESRRG, PPARγ, RXRA, RXRB & THRA) were upregulated in the sensitive compared to the resistant cell lines, which was further validated by qRT-PCR. To understand the effect of these NHRs in sensitizing resistant CML cells to Imatinib, KCL22 and Lama84 cells were pretreated with NHR ligands, followed by increasing concentrations of Imatinib (5µM-100µM). Ligands of RXR (9-cis retinoic acid), AR (testosterone) and ESRRG1 (GSK4716) significantly improved IM sensitivity by increasing the RNA expression of IM influx transporter hOCT1, which was also reported by Wang et al., 2012; Austin et al., 2014 using ligands for RXR, PXR and PPARγ. Pretreatment with these ligands did not have significant effect on the mRNA expression of BCR-ABL, but there was increased inhibition of the BCR-ABL downstream targets such as pCRKL, pSTAT5 and pAKT at the protein level in combination with IM. The AR, RXR & ESRRG ligands significantly decreased the IC50 to IM (p<0.0001) in primary CML cells (n=22) in combination with IM. We also observed significant sensitization of primary samples (n=7) to IM when used in combination with clinically available RXR and AR ligands isotretinoin (p=0.0011) & stanozolol (p=0.0054). Our finding suggests that NHR ligands, especially RXR, AR & ESRRG when used in combination with IM could overcome resistance. Further studies are warranted to confirm the mechanistic effects of these ligands in modulating drug resistance in CML. Citation Format: Bharathi M. Rajamani, Sreeja Karathedath, Raveen Stephen Illangeswaran, Esther Sathya Bama Benjamin, Vikram Mathews, Aby Abraham, Poonkuzhali Balasubramanian. Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3739.

Abstract 145: Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells

Abstract Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically. Citation Format: Chun Shik Park, Ye Shen, Andrew Lewis, Koramit Suppipat, Monica Puppi, Julie Tomolonis, Taylor Chen, Paul Pang, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Rachel Rau, Daniel Lacorazza. Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 145.

Characterization of imatinib-resistant K562 cell line displaying resistance mechanisms

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.

Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies.

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2.

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs.

Costunolide promotes imatinib-induced apoptosis in chronic myeloid leukemia cells via the Bcr/Abl-Stat5 pathway.

Costunolide, a sesquiterpene lactone, is a small molecular monomer extracted from Inula helenium (Compositae). In the present study, we assessed the antileukemia effects of costunolide on the human chronic myeloid leukemia cell line K562 and its combined activity with imatinib. A Cell Counting Kit-8 assay demonstrated that costunolide significantly inhibited K562 cell proliferation and enhanced imatinib-induced anti-proliferative activity. We found that costunolide significantly induced mitochondrial apoptosis in K562 cells by modulating the protein levels of Bcl-2 family members and by inducing caspase activation. Costunolide promoted imatinib-induced apoptosis via the Bcr/Abl-signal transducer and activator of transcription 5 pathway. Costunolide inhibited proliferation by inducing cell cycle arrest in the G2 /M phase by decreasing cyclin B1 and cyclin-dependent kinase 2 expression and increasing p21 expression. Together, these results demonstrate that costunolide may be a potent therapeutic agent against chronic myeloid leukemia.

In vitro anti-leukemia activity of dual PI3K/mTOR inhibitor Voxtalisib on HL60 and K562 cells, as well as their multidrug resistance counterparts HL60/ADR and K562/A02 cells

Current treatment strategies for leukemia still have some limitations such as severe side effects and drug resistance. Less toxic and more effective drugs for leukemia patients are therefore expected. In the present study, the efficacy of a dual PI3K/mTOR inhibitor, Voxtalisib, on acute myeloid leukemia (AML) cell line HL60 and chronic myeloid leukemia (CML) cell line K562, as well as their Adriamycin (ADR)-selected multi drug resistance (MDR) counterparts HL60/ADR and K562/A02, was investigated. Voxtalisib exhibited potent anti-proliferative activity on these four cell lines dose-dependently, with IC50 values as 2.23 μM for HL60, 4.79 μM for HL60/ADR, 4.20 μM for K562 and 3.90 μM for K562/A02 cells. Voxtalisib arrested cell cycle progression at G1 phase in all cell lines by upregulating p27, downregulating cyclin D1 and p-pRb. When combined with ADR, Voxtalisib reversed the ADR-resistance of HL60/ADR and K562/A02 cells, possibly by reducing MDR1 and MRP1 expression. In conclusion, Voxtalisib showed anti-leukemia activity on AML and CML cell lines as well as their multidrug resistant ones, suggesting Voxtalisib might become a promising drug candidate for therapy of AML and CML in the future.

CD69 partially inhibits apoptosis and erythroid differentiation via CD24, and their knockdown increase imatinib sensitivity in BCR-ABL-positive cells.

Chronic myeloid leukemia (CML) is caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) imatinib and its derivatives represent a breakthrough for CML therapy, but the use of TKI alone is ineffective for many CML patients. CD69, an early activation marker of lymphocytes, participates in immune and inflammatory responses. Previous studies revealed that BCR-ABL upregulates CD69 expression; however, the role of CD69 in CML cells is unknown. Here, we demonstrate that BCR-ABL induced CD69 promoter activity and mRNA and protein expression via the NF-κB pathway. CD69 knockdown partially increased apoptosis and expression of erythroid differentiation markers, α-globin, ζ-globin, and glycophorin A, and increased imatinib sensitivity in K562 and KU812 CML cells. Gene microarray analysis and quantitative real-time PCR verified that CD24, an oncogenic gene, downregulated in K562 cells upon CD69 knockdown. CD69 overexpression increased, whereas CD69 knockdown inhibited CD24 promoter activity and mRNA and protein levels. CD24 knockdown also partially increased apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells, whereas its overexpression inhibited the effects of CD69 knockdown on apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells. Imatinib-induced apoptosis and erythroid differentiation were also inhibited by CD69 or CD24 overexpression in BCR-ABL-expressing CML cell lines and CD34+ cells. Taken together, CD24 is a downstream effector of CD69. CD69 and CD24 partially inhibit apoptosis and erythroid differentiation in CML cells; thus, they may be potential targets to increase imatinib sensitivity.

Modular synthesis of new C-aryl-nucleosides and their anti-CML activity

The C-aryl-ribosyles are of utmost interest for the development of antiviral and anticancer agents. Even if several synthetic pathways have been disclosed for the preparation of these nucleosides, a direct, few steps and modular approaches are still lacking. In line with our previous efforts, we report herein a one step - eco-friendly β-ribosylation of aryles and heteroaryles through a direct Friedel-Craft ribosylation mediated by bismuth triflate, Bi(OTf)3. The resulting carbohydrates have been functionalized by cross-coupling reactions, leading to a series of new C-aryl-nucleosides (32 compounds). Among them, we observed that 5d exerts promising anti-proliferative effects against two human Chronic Myeloid Leukemia (CML) cell lines, both sensitive (K562-S) or resistant (K562-R) to imatinib, the “gold standard of care” used in this pathology. Moreover, we demonstrated that 5d kills CML cells by a non-conventional mechanism of cell death.

Imatinib Affects the Expression of SLC22A1 in a Non-Linear Concentration-Dependent Manner Within 24 Hours.

BackgroundImatinib is actively transported into cells by the organic cation transporter (OCT1), encoded by SLC22A1. As a result, the expression of SLC22A1 is considered a prognostic marker for treatment with imatinib in patients with chronic myeloid leukemia (CML). Although limited, there is conflicting evidence indicating that imatinib may affect the expression of SLC22A1. However, thus far, no studies have investigated the effect of imatinib on SLC22A1 expression in an imatinib-sensitive cell line, which would mimic a typical clinical setting. Changes in the expression of SLC22A1 as a result of imatinib could potentially negate its usefulness as a prognostic marker.Material/MethodsThe K562 CML cell line was exposed to varying concentrations of imatinib for 24, 48, and 72 h and SLC22A1 expression was determined by quantitative real-time PCR.ResultsOur findings suggest that imatinib affects the expression of SLC22A1 within 24 h in a non-linear concentration-dependent manner.ConclusionsThis is the first study to report on the short-term effect of imatinib on the expression of SLC22A1 in an imatinib-sensitive CML cell line. Our results suggest that imatinib affects SLC22A1 mRNA expression in a non-linear dose-dependent manner and that the changes in the expression of SLC22A1 as a result of the concentration of imatinib occur within 24 h of exposure to imatinib and remain stable thereafter for up to 72 h.