Chronic Myelogenous Leukemia Cell Line Research Articles

Olmutinib Reverses Doxorubicin Resistance in ETS1-Overexpressing Leukemia Cells.

BackgroundDrug resistance is a major problem in the treatment of leukemia with doxorubicin (Dox), and the erythroblastosis virus E26 oncogene homolog 1 (ETS1) gene is associated with drug resistance. Olmutinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) reported to play a role in reversing multidrug resistance (MDR) in cancer cells. The objective of this study was to investigate whether olmutinib could reverse Dox resistance in leukemia cells overexpressing ETS1.Material/MethodsHuman chronic myelogenous leukemia cell line K562 and its Dox-resistant cell line K562/ADR were used. Western blot and qPCR detected the expression of ETS1 and ABCB1. Cell proliferation was measured by cell counting kit-8 and methyl thiazolyl tetrazolium. Cell apoptosis was observed by western blot and flow cytometry. A nude mice K562/ADR xenograft model was used to investigate the inhibitory effects of olmutinib on tumor growth in vivo.ResultsThe mRNA and protein expressions of ETS1 and ABCB1 were up-regulated in Dox-resistant leukemia cell line K562/ADR. We overexpressed ETS1 in both cell lines, finding that olmutinib inhibited the cell viability of K562 and K562/ADR in a concentration-dependent manner. The cytotoxicity of Dox to EST1-overexpressing K562/ADR cells was enhanced by olmutinib. Olmutinib also promoted apoptosis of K562 and K562/ADR cells compared with Dox treatment alone. In vivo, olmutinib enhanced the inhibitory effects of Dox on ETS1-overexpressing K562/ADR cell xenograft growth.ConclusionsOur results suggest that the novel EGFR TKI olmutinib enhances the sensitivity of ETS1-overexpressing leukemia cells to Dox.

Quantum dots functionalized with 3-mercaptophenylboronic acids as novel nanoplatforms to evaluate sialic acid content on cell membranes

Sialic acids (SAs) modulate essential physiological and pathological conditions, including cell-cell communication, immune response, neurological disorders, and cancer. Besides, SAs confer negative charges to cell membranes, also contributing to hemorheology. Phenylboronic acids, called as mimetic lectins, have been highlighted to study SA profiles. The association of these interesting molecules with the optical properties of quantum dots (QDs) can provide a deeper/complementary understanding of mechanisms involving SAs. Herein, we explored the thiol affinity to the QD surface to develop a simple, fast and direct attachment procedure to functionalize these nanocrystals with 3-mercaptophenylboronic acids (MPBAs). The functionalization was confirmed by fluorescence correlation spectroscopy and inductively coupled plasma spectrometry. The conjugate specificity/efficiency was proved in experiments using red blood cells (RBCs). A labeling >90% was found for RBCs incubated with conjugates, which reduced to 17% after neuraminidase pretreatment. Moreover, QDs-MPBA conjugates were applied in a comparative study using acute (KG-1) and chronic (K562) myelogenous leukemia cell lines. Results indicated that KG-1 membranes have a greater level of SA, with 100% of cells labeled and a median of fluorescence intensity of ca. 2.5-fold higher when compared to K562 (94%). Therefore, this novel QDs-MPBA conjugate can be considered a promising nanoplatform to evaluate SA contents in a variety of biological systems.

Complex transcriptional regulation of the BCL2L12 gene: Novel, active promoter in K562 cells.

The BCL2L12, one of the latest discovered members of the BCL2 family, has both pro- and anti-apoptotic roles that are cell-type-dependent. Its role in tumorigenesis is highly implicated. Sixty-three splice variants of this gene have been identified so far, with significant differences in expression patterns between various cancer cell lines. Presently, little is known regarding the regulation of expression of the BCL2L12 gene. For the vast majority of BCL2L12 gene splice variants, the 5'- and 3'-untranslated regions as well as their transcriptional regulation have not been determined yet. The aim of this study was to get insight into the regulation of the BCL2L12 gene transcription in human chronic myelogenous leukemia (K562) cell line. Our results point to the activity of novel transcription start site of the BCL2L12 gene and indicate that Sp1 and GATA-1 transcription factors could be involved in the regulation of BCL2L12 gene expression in K562 cells. The previously reported active promoter of BCL2L12 gene differs from the one we described in our study. If this novel BCL2L12 promoter is confirmed to be active in other malignancies, transcripts generated from this region could be considered as new cancer-specific biomarkers. The results of our study contribute to the better understanding of the transcriptional regulation of the BCL2L12 gene.

Novel curcumin derivatives as P-glycoprotein inhibitors: Molecular modeling, synthesis and sensitization of multidrug resistant cells to doxorubicin

The MDR1/P-glycoprotein (Pgp)/ABCB1 multidrug transporter is being investigated as a druggable target for antitumor therapy for decades. The natural product curcumin is known to provide an efficient scaffold for compounds capable of blocking Pgp mediated efflux and sensitization of multidrug resistant (MDR) cells to the Pgp transported drug doxorubicin (Dox). We performed molecular dynamics simulations and docking of curcumin derivatives into the Pgp model. Based on these calculations, a series of pyrazolocurcumin derivatives with predicted metabolic stability and/or improved binding affinity were proposed for synthesis and evaluation of MDR reversal potency against Dox selected K562/4 subline, a derivative of K562 human chronic myelogenous leukemia cell line. Compounds 16 and 19 which are both dimethylcurcumin pyrazole derivatives bearing an N-p-phenylcarboxylic amide substitution, were the most potent Pgp blockers as determined by intracellular Dox accumulation. Furthermore, at non-toxic submicromolar concentrations 16 and 19 dramatically sensitized K562/4 cells to Dox. Together with good water solubility of 16 and 19, these results indicate that the new pyrazolo derivatives of curcumin are a promising scaffold for development of clinically applicable Pgp antagonists.

EIF3b regulates the cell proliferation and apoptosis processes in chronic myelogenous leukemia cell lines via regulating the expression of C3G.

To investigate the functions of eIF3b in chronic myelogenous leukemia (CML). The expression of eIF3b was inhibited by transfecting aspecifically designed shRNA into the CML cell lines of TK-6 and K562. The CCK8 assay was conducted to determine cell viability, and flow cytometry was used to examine the change in the cell cycle and cell apoptosis. RNAsequencing was applied to screen the candidate targets of eIF3b to identify the underlying mechanisms of eIF3b.An in vivo tumour xenograft mouse model was established by injecting shRNA transfected cells into the NCG mice. The tumour size and body weight of mice were monitored every other day. The mice were sacrificed 2weeks after the tumour cell injection. The expression of eIF3b and target genes in the tumour tissues were determined by immunohistochemical staining and Western blotting. The group with inhibited expression of eIF3b led to about 50% lower cell viability compared with that of the control group (P < 0.05). Flow cytometry suggested that the percentage of increase in apoptotic cells was eight times higher than those in control group for TK-6 and K562 cells (P < 0.05). However, the difference between the cell amounts in the S phase for the experiment and control groups was not significant. After RNAsequencing and further validation via qPCR, C3G was screened as the potential target of eIF3b involved in the cell proliferation and apoptosis of CML cell lines. Subsequent in vivo analysis proved that the inhibition of eIF3b suppressed tumour formation and decreased C3G expression, thereby indicating that C3G was the potential target of eIF3b. eIF3b is correlated with the cell proliferation and cell apoptosis of CML. Moreover, eIF3b regulation most probably occurs via regulating the expression of C3G.

Developing a biopolymeric chitosan supported Schiff-base and Cu(II), Ni(II) and Zn(II) complexes and biological evaluation as pro-drug

Chitosan derivatives are widely used as key classes of medicinal compounds owing to their non- toxic and biodegradable properties. So, in this work, to enhance chitosan biological activities, a new synthesis of a series of Schiff base and its metals complexes (Cu(II), Ni(II) and Zn(II)) of chitosan (CS) was prepared. Moreover, their physicochemical properties were characterized by IR, UV–Vis, SEM, melting point, thermo gravimetric analysis (TGA), X-ray diffraction (XRD), elemental analysis and 1H NMR techniques. Elemental analysis data confirmed the formation of chitosan-Schiff base as well as the coordination reaction with metals ions by increasing the carbon content caused by substitution. By elemental analysis, the degrees of acetylation (DA), deacetylation (DD) and substitution (DS) were acquired 23, 77.63 and 57.90%, respectively. Additionally, the 1H NMR spectroscopy was used for the determination of degree of deacetylation (DD) and Substitution (DS) of chitosan ranging from 87.5 and 85%, respectively. The presence of a new low-field signal at 10.23 ppm in the 1H NMR spectra confirmed the imine proton of Schiff base. The cytotoxicity of Chitosan, Chitosan-Schiff base and its metals complexes was tested against K562 chronic myelogenous leukemia (CML) and MG-63 (osteosarcoma cancer) cell lines by the MTT assay. The results suggested that the anticancer activity of Schiff base and their complexes was much better than that of pure CS against cancer MG63 cell line. Finally, through flow cytometry, we demonstrated that all compounds were efficient in inducing apoptosis effect in K562 and MG63 cell lines except Schiff base- chitosan in K562 cell lines.

Dinuclear copper(II) complex with a benzimidazole derivative: Crystal structure, theoretical calculations, and cytotoxic activity

This work describes a facile synthesis of 5,6‐dimethyl‐2‐(pyridin‐2‐yl)‐1‐[(pyridin‐2‐yl)methyl]‐1H‐benzimidazole (dppb) and the preparation of a dinuclear copper(II) complex bearing this ligand in a ring‐like conformation of formula [Cu(dppb)(acn)ClO4]2(ClO4)2·4acn (1), where acn represents acetonitrile. The crystal structure of the complex was determined by single‐crystal X‐ray diffraction experiment, which showed that dppb acts as a bridge in 1 due to its bidentate–monodentate μ‐κ2NN′:κN″coordination mode, and the coordination sphere of each metal center is completed by one acn molecule and one perchlorate ion. The structure of dinuclear copper(II) complex is symmetric and the two square–pyramidal copper(II) centers are separated by 5.381(1) Å. The geometry optimization, electronic structure, and vibrational spectrum of the [Cu(dppb)(acn)ClO4]22+ cation were calculated using the density functional theory method. The geometrical parameters obtained theoretically were remarkably similar to those observed in the crystal structure of 1 and the calculated vibrational frequencies are in satisfactory coherence with experimental data. In solution, the prepared complex can bind to DNA (Kb = 1.43 × 105 M−1) and inhibit the growth of a human chronic myelogenous leukemia cell line (K562) in a concentration‐dependent manner; importantly, it is almost four times more active than the corresponding free benzimidazole.

Effects of DNA Methylation on Leukemia Cell Proliferation.

In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation. Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1. DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased. These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

Investigating the cytotoxic and apoptotic effects of sunitinib upon K-562 chronic myelogenous leukemia cell line and assessment of gene profiling.

Tyrosine kinase inhibitors (TKIs) which efficiently inhibit BCR-ABL are highly effective for clinical treatment of chronic myeloid leukemia (CML), but development of resistance to TKIs is a big challenge to treatment. Sunitinib is a multitargeted TKI targeting vascular endothelial growth factor receptor and is defined a safe and effective candidate target, but its effect on other signaling pathways is unknown. To investigate the cytotoxic and apoptotic effect of sunitinib in CML cell model K-562 on JAK-STAT signaling pathway components, suppressor genes and oncogenes, hematopoiesis-related genes, cell cycle and VEGF pathway components, and mRNA level expression changes was aimed. Sunitinib's effective dose cytotoxic IC50 was determined by trypan blue and WST-1 cell proliferation assay tests. Expression levels of target genes were determined by quantitative reverse transcriptase polymerase chain reaction simultaneously after sunitinib application. Protein expression analysis was determined by "WesternBreeze Chromogenic Kit-Anti-Rabbit" based on the principles of the application kit by Western blot analysis. Assessing the cytotoxicity of K-562 cells following sunitinib treatment revealed that sunitinib decreased cell proliferation in a time- and dose-dependent manner. According to the sunitinib inhibition curve, IC50 dose was calculated as 3.5 μM at 48th h for K-562 cells and apoptosis assays pointed that sunitinib induces apoptotic cell death of leukemic cells at moderate levels. Our study supports that sunitinib might be used as a novel therapeutic target to trigger apoptosis in CML cells which in turn might accelerate therapeutic response in regard to inhibiting oncogenes and enhancing tumor suppressors in cooperation with cell cycle regulatory genes.

Diterpenoids from Roots of Salvia lachnocalyx; In-silico and In-vitro Toxicity against Human Cancer Cell Lines.

Further investigations on phytochemical constituents of dichloromethane extract from roots of Salvia lachnocalyx (S. lachnocalyx) led to the isolation and identification of eight known diterpenoids from this plant for the first time. The chemical structures of the purified compounds were elucidated using spectroscopic analyses including EI-MS, 1H and 13C NMR and by comparison of the resulting spectra with those reported in the literature. Then, the cytotoxic activity of identified compounds was examined against two human cancer cell lines MCF-7 (human breast adenocarcinoma) and K562 (human chronic myelogenous leukemia). Molecular docking of promising cytotoxic compounds were performed by AutoDock Tools 1.5.4 program in the active site of Topoisomerase I. Eight known diterpenoids; 12-hydroxysapriparaquinone (1), 15-deoxyfuerstione (2), horminon (3), 7α-acetoxyroyleanone (4), 11β-hydroxymanoyl oxide (5), microstegiol (6), 1-keto-aethiopinone (7) and 14-deoxycoleon U (8) were isolated of dichloromethane extract from roots of salvia lachnocalyx. Compounds 2, 3, 6, and 8 showed cytotoxic activity against MCF-7 (human breast adenocarcinoma) and K562 (human chronic myelogenous leukemia) cell lines with IC50 values in the range of 2.63-11.83 µg/mL. The inhibition of” topoisomerase I” was suggested by molecular docking calculations as the mechanism of cytotoxicity of the tested compounds. According to cytotoxic assay and docking results, it is suggested that compounds 2, 3, 6, and 8 have good potential as anticancer agents.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Cycloplatinated(II) Derivatives of Mercaptopurine Capable of Binding Interactions with HSA/DNA.

In this study, two new bis-cyclometalated Pt(II) complexes, [Pt(C^N)(S^N)] [S^N = deprotonated 6-mercaptopurine (6-MP) and C^N = deprotonated 2-phenylpyridine (ppy), 2a; C^N = deprotonated benzo[h]quinoline (bhq), 2b], are synthesized by the reaction of [PtR(SMe2)(C^N)] (R = Me or p-MeC6H4) with 1 equiv of 6-mercaptopurine (6-HMP) at room temperature. The complexes are fully characterized using 1H and 13C NMR spectroscopies, electrospray ionization mass spectrometry, and elemental analysis. Biomolecular interaction of complex 2a with human serum albumin (HSA) is studied by fluorescence, UV-vis, and circular dichroism (CD) spectroscopies. The binding constants (Kb) and number of binding sites (n) are evaluated using the Stern-Volmer equation. The intrinsic fluorescence of protein is quenched by a static quenching mechanism, with a binding constant of Kb ∼ 105 reflecting a high affinity of complex 2a for HSA. The thermodynamic parameters (ΔH°, ΔG°, and ΔS°) indicate that the interaction is a spontaneous process and hydrophobic forces play a main role in the reaction. The displacement experiments demonstrate that the reactive binding sites of HSA to complex 2a are mainly located within its hydrophobic cavity in subdomain IIA (site I). Synchronous fluorescence spectra reveal that complex 2a affected the microenvironment of tryptophan-214 residues in subdomain IIA of HSA. In the case of interaction of complex 2b and HSA, because of overlapping of the emission spectra of complex 2b with HSA, chemometric approaches are applied. The results indicate significant interaction between the tryptophan residue of HSA and complex 2b. Moreover, the binding of Pt(II) complexes 2a and 2b causes a reduction of the α-helix content of HSA, as obtained by far-UV CD spectroscopy. The average binding distance (r) between Pt(II) complexes and HSA is obtained by Förster's resonance energy-transfer theory. Also, a molecular docking simulation reveals that π-π-stacking and hydrophobic interactions between these complexes and HSA are significant. Furthermore, the interactions of platinum complexes, 2, with calf-thymus DNA (CT-DNA) are investigated. The UV-vis results and ethidium bromide competitive studies support an intercalative interaction of both Pt(II) complexes with DNA. The new complexes 2 are also screened for anticancer activities. The results show that complexes 2 exhibit significant anticancer activity against the K562 (chronic myelogenous leukemia) cell line.

Synthesis, characterization and in vitro evaluation of anticancer activity of a new water-soluble thiosemicarbazone ligand and its complexes

A new water-soluble thiosemicarbazone ligand, 4-hydroxyl-3-({[(methylamino)carbonothioyl] hydrazono}methyl)benzoic acid (H3LCOOH) and its complexes with Cu(II), Ni(II), Zn(II), Fe(III) and Mn(III) were synthesized and characterized. These compounds were characterized by conductivity, magnetic susceptibility, elemental analysis, FT-IR, 1H NMR, 13C NMR, and UV–Vis spectroscopy. All the compounds have sufficient solubility and stability in water as a safe, green, and biologic solvent. The compounds were evaluated for their anticancer activities against the human chronic myelogenous K562 leukemia and human breast carcinoma MCF-7 cell lines. All the complexes significantly showed more anticancer activity than the ligand alone and the Cu(II) complex showed considerable cytotoxicity against the tested cell lines. The mechanism of action of the most potent complex, Cu(HLCOO) against K562 cell line was further explored by fluorescence microscopy, flow cytometry, cell cycle and reactive oxygen species (ROS) assays. Hoechst staining and flow cytometry indicated the apoptosis induction ability of Cu(HLCOO) on K562 cells. Mechanistic studies indicated that Cu(HLCOO) complex caused cell cycle arrest at G0/G1 phase and this complex was able to inhibit the cell cycle in a time-dependent manner. Cu(HLCOO) can also increase the reactive oxygen species levels in the K562 cells. The obtained results suggest that the studied compounds can be helpful candidates for further evaluations as chemotherapeutic agents in the treatment of cancer.

Bio-Guided Fractionation of Ethanol Extract of Leaves of Esenbeckia alata Kunt (Rutaceae) Led to the Isolation of Two Cytotoxic Quinoline Alkaloids: Evidence of Selectivity Against Leukemia Cells.

Bio-guided fractionation performed on the leaves-derived ethanol extract of Esenbeckia alata (Rutaceae), a plant used in traditional medicine, led to the isolation of two alkaloids, kokusaginine 1 and flindersiamine 2, as main cytotoxic agents. Primary ethanolic extract and raw fractions exhibited cell inhibition against five cancer cell lines at different levels (25–97% inhibition at 50 µg/mL) as well as isolated alkaloids 1–2 (30–90% inhibition at 20 µM). Although alkaloid 2 generally was the most active compound, both alkaloids showed a selective effect on K562, a human chronic myelogenous leukemia cell line. The E1-like ubiquitin-activating enzymes (e.g., UBA5) have been recently described as important targets for future treatment of cancer progression, such as leukemia, among others. Therefore, as a rationale to the observed cytotoxic selectivity, an in-silico evaluation by molecular docking and molecular dynamics was also explored. Compounds 1–2 exhibited good performance on the interaction within the active site of UBA5.

Design, Synthesis and Biological Evaluation of Pentacyclic Triterpene Derivatives: Optimization of Anti-ABL Kinase Activity.

Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new inhibitors of ABL kinase are needed. Our previous study showed that the benzyl ester of gypsogenin (1c), a pentacyclic triterpene, has anti-ABL kinase and a subsequent anti-CML activity. To optimize its activities, benzyl esters of carefully selected triterpenes (PT1–PT6), from different classes comprising oleanane, ursane and lupane, and new substituted benzyl esters of gypsogenin (GP1–GP5) were synthesized. All of the synthesized compounds were purified and charachterized by different spectroscopic methods. Cytotoxicity of the parent triterpenes and the synthesized compounds against CML cell line K562 was examined; revealing three promising compounds PT5, GP2 and GP5 (IC50 5.46, 4.78 and 3.19 μM, respectively). These compounds were shown to inhibit extracellular signal-regulated kinase (ERK) downstream signaling, and induce apoptosis in K562 cells. Among them, PT5 was identified to have in vitro activity (IC50 = 1.44 μM) against ABL1 kinase, about sixfold of 1c, which was justified by molecular docking. The in vitro activities of GP2 and GP5 are less than PT5, hence they were supposed to possess other more mechanisms of cytotoxicity. In general, our design and derivatizations resulted in enhancing the activity against ABL1 kinase and CML cells.

Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia

Abstract Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib. Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML. Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity. Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML. Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.

Pt(II) complexes of the type trans-[PtCl2(DMSO) (hydrazide)] and cis-[PtCl2(hydrazide)2]: Solvolysis and cytotoxic activity

The present work describes the synthesis, solvolysis and cytotoxic activity of Pt (II) complexes of the type trans-[PtCl2 (DMSO) (L)] and cis-[PtCl2(L)2], where L = benzhydrazide (BH), 4-nitrobenzhydrazide (4NH) and 4-(trifluoromethyl)benzhydrazide (4 TF). The structures of these complexes were proposed using spectroscopic methods. All complexes undergo fast solvolysis generating four identical species in DMSO, being that the species cis- and trans-[PtCl2 (DMSO) (L)] are the most important. As to the stability in DMF, Pt (II) complexes of the type cis-[PtCl2(L)2] are very stable, while Pt (II) complexes of the type trans-[PtCl2 (DMSO) (L)] react at a slower rate than those verified in DMSO. The cytotoxic activity of the compounds was evaluated in a chronic myelogenous leukemia cell line. A relationship between solvolysis and cytotoxic activity was verified. Moreover, our results showed that some Pt (II) complexes were more cytotoxic than the free ligands, carboplatin and cisplatin.

Investigation of the role of RCAN1 Gene in apoptosis

Our lab is interested in understanding the molecular basis of chemotherapeutic evoked apoptosis and resistance in leukemic lymphoblasts. The long‐term goal of our research is to improve the therapeutic effectiveness of chemotherapeutic agents. Antileukemic agents and signaling pathway inhibitors induce apoptosis by regulating gene expression; a drawback of this treatment is the development of resistance to these agents. The RCAN1 (Regulator of Calcineurin 1) gene is located on chromosome 21 and is one of many genes implicated in apoptosis. Upregulation of RCAN1 correlates with sensitivity to antileukemic agents and signaling pathway inhibitors. The protein product of this gene, RCAN1, binds to the protein phosphatase calcineurin, inhibiting its activity, and altering cellular responses to survival and death triggers. We are testing the hypothesis that if the protein function of the RCAN1 is knocked out, cells will be protected from apoptosis. We are screening a series of antileukemic agents and signaling pathway inhibitors, utilizing paired wild‐type and RCAN1 knockout HAP1 cell lines, to determine which agents might mediate an apoptotic response via RCAN1 involvement. HAP1 is a human near‐haploid cell line derived from the male chronic myelogenous leukemia (CML) cell line KBM‐7, allowing for efficient CRISPR‐mediated genome editing. Viability of cells exposed to various agents was evaluated by MTT assays and transcript levels were monitored via reverse‐transcription‐PCR analysis of RNA extracted from treated cells. MTT assays show that RCAN1 knockout offered the greatest protection from apoptosis caused by the AKT inhibitor MK‐2206, when compared to wildtype cells. Upregulation of BIM, a proapoptotic protein, by MK‐2206 was reduced in RCAN1 knockout HAP1 cells compared to the wild‐type HAP1 cells. Our results suggest that RCAN1 may influence the AKT pathway to mediate apoptosis.Support or Funding InformationThis research is supported by GM081099 awarded to Rheem Medh and NIH GM008395 for student support of Alejandra Fausto.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.