KBM7 Cells Research Articles

Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study

IntroductionHigh-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. MethodsKBM7-mu cells were exposed to 200μM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h of treatment. After a 2–3week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. ResultsWe identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. DiscussionThe KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity.

Abstract 3607: Loss-of-function screens using haploid KBM7 and HAP1 cells to identify mechanisms of anti-cancer drug resistance

Abstract The lack of biomarkers to predict anti-cancer therapy response remains a major obstacle in the treatment of various cancers. This handicap not only affects novel targeted therapies. Also for classical chemotherapeutic drugs such as DNA cross-linking agents, topoisomerase inhibitors or microtubule-targeting compounds, which are all frequently used in the clinic, we do not understand well why some patients have a major benefit of the therapy whereas others do not. A decreased intracellular drug accumulation has been proposed as one potential mechanism, but the clinical relevance of known uptake and efflux mechanisms is still controversial for various chemotherapeutic drugs. To identify novel factors that may be relevant for the intracellular concentration of drugs, we carried out loss-of-function screens using haploid KBM7 and HAP1 cells. In particular, we used the PARP inhibitor olaparib, the topoisomerase I inhibitor topotecan, platinum drugs, and the microtubule-targeting compounds docetaxel and vinorelbine to select resistant clones. We will present results from these screens and their validation, including independent cell lines and TCGA data sets. Citation Format: Nora M. Gerhards, Charlotte Guyader, Vincent A. Blomen, Aslı Küçükosmanoğlu, Olaf van Tellingen, Daniel J. Vis, Lodewyk F. Wessels, Thijn R. Brummelkamp, Piet Borst, Sven Rottenberg. Loss-of-function screens using haploid KBM7 and HAP1 cells to identify mechanisms of anti-cancer drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3607. doi:10.1158/1538-7445.AM2015-3607

GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death.

Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death.

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

BackgroundSeveral recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.ResultsWe describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.ConclusionTM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1236-7) contains supplementary material, which is available to authorized users.

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9.

Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.

We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0554-4) contains supplementary material, which is available to authorized users.

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.

Effects of quercetin on hedgehog signaling in chronic myeloid leukemia KBM7 cells.

To investigate the effects of quercetin on Hedgehog (Hh) signaling in chronic myeloid leukemia KBM7 cells. The KBM7 cells were treated with 50, 100 and 200 μmol/L quercetin for 48 h respectively. And then the trypan blue assay was used to examine the proliferative inhibition of quercetin. Apoptotic cells and cell cycle were measured by flow cytometry. The mRNA and protein expression were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot, respectively. Quercetin significantly inhibited KBM7 cell proliferation, induced cell apoptosis, and blocked cell cycle at G1 phase, which were in dose-dependent manners. The mRNA and protein expression of Smoothened and Glioma1 (Gli1), the members of Hh pathway decreased after treatment with quercetin. The Bcl-2 and Cyclin D1, targets of Hh signaling, also decreased after treatment with quercetin, respectively. Quercetin also could increase p53 and Caspase-3 expression. Bcr-abl mRNA copies decreased, but no changes of phosphorylated Bcr-abl and Bcr-abl proteins were observed, after treatment with quercetin. Quercetin could inhibit Hh signaling and its downstream targets in the KBM7 cells. And it might be one of mechanisms of inducing apoptosis and inhibiting cell cycle by quercetin.

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens

We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.

Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-κB

The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors.

P051 Mapping the TNF-induced necroptosis pathway by experimental test-tube genetics in human haploid cells

Introduction TNF signaling can result in NF-kB and MAPK activation, apoptosis or necrosis depending on cell type and cell state. Until recently necrosis has been seen as an uncontrolled mode of cell death when compared to the programmed apoptosis. The identification of RIP3 as a crucial mediator of TNF-induced necrosis clearly demonstrates that in many cases necrosis is controlled by a dedicated signaling cascade, leading to the definition of necroptosis [3] , [4] , [6] . Increasing evidences demonstrate that RIP3-mediated necroptosis participates in inflammation and in both innate and adaptive immunity. Despite this, the signaling events downstream of RIP3 kinase activation remain largely obscure. Methods Here we describe the design of a haploid genetic screen in human KBM7 cells that aims to identify new components of TNF-induced RIP3-dependent necroptosis signaling pathways. The KBM7 chronic myeloid leukemia cell line is haploid for all chromosomes except chromosome 8. This allows gene inactivation through random insertional mutagenesis using a retroviral gene-trap vector and subsequent insertion site mapping [1] , [2] . Results Our data demonstrate that the TNF signaling pathway is functional in KBM7 cells resulting in NF-kB and MAPK activation, apoptosis or necroptosis depending on defined experimental settings. Under necroptosis inducing conditions, cell death occurs without detectable caspase activation and is suppressed by the RIP1 kinase inhibitor necrostatin-1. Based on these results, mutagenized KBM7 cells will be submitted to TNF-induced necroptosis and resistant clones mapped to identify genes required for necroptotic cell death. Mixed lineage kinase domain-like protein (MLKL) has been recently shown to participate in TNF-induced necroptosis [5] . An MLKL-deficient KBM7 cell clone is completely resistant to TNF-induced necroptosis while retaining sensitivity to TNF-induced apoptosis. This finding confirms by gene inactivation in human cells that MLKL is crucial for TNF-necroptosis and, importantly, validates our screening approach. Conclusion We expect the screen to genetically define the essential molecular elements required for necroptosis. Identified components will then be characterized using affinity-purification coupled to mass spectrometry. Our study should thus contribute significantly to the molecular understanding of necroptosis, a process whose relevance in inflammation, innate and adaptive immunity has just began to be fully appreciated.

AMN107, a Novel Aminopyrimidine Inhibitor of Bcr-Abl, Has In vitro Activity against Imatinib-Resistant Chronic Myeloid Leukemia

Resistance to or intolerance of imatinib in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) has encouraged the development of more potent Bcr-Abl inhibitors. AMN107 is a novel, orally bioavailable ATP-competitive inhibitor of Bcr-Abl. The effects of AMN107 were compared with those of imatinib on imatinib-sensitive (KBM5 and KBM7) and imatinib-resistant CML cell lines (KBM5-STI571R1.0 and KBM7-STI571R1.0). Compared with the antiproliferative activity of imatinib, AMN107 was 43 times more potent in KBM5 (IC50 of 11.3 versus 480.5 nmol/L) and 60 times more potent in KBM7 (IC50 of 4.3 versus 259.0 nmol/L) cells. IC50 for AMN107 and imatinib were 2,418.3 and 6,361.4 nmol/L, respectively, in KBM5-STI571R1.0, and 97.2 and 2,497.3 nmol/L, respectively, in KBM7-STI571R1.0 cells. AMN107 inhibited autophosphorylation of Bcr-Abl kinase more effectively than imatinib in all cell lines. They had similar effects on cell cycle progression and apoptotic response in these cell lines. Among severe combined immunodeficient mice bearing KBM5 cells, mean survival times of groups treated with 10, 20, and 30 mg/kg/d of AMN107, starting day 20 after leukemic cell grafting and continuing for 20 days, were 144%, 159%, and 182%, respectively, compared with controls. These results strongly support investigation of the clinical efficacy of AMN107 in patients with CML.

AMN107, a Novel Aminopyrimidine Inhibitor of BCR-ABL, Has Pre-Clinical Activity Against Imatinib Mesylate-Resistant Chronic Myeloid Leukemia (CML).

Resistance to, or intolerance of, imatinib mesylate in patients with CML has encouraged the development of other Bcr-Abl inhibitors. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both imatinib sensitive (KBM5 and KBM7) and corresponding imatinib-resistant (KBM5-STI571R1.0 and KBM7-STI571R1.0) cell lines was studied. KBM5 cells contain multiple copies of the Ph chromosome but lack the normal c-ABL. KBM7 cell line is near haploid. KBM5 and KBM7 cells differ in their response to imatinib exposure: no apoptosis with G0/G1 cell cycle arrest in KBM5 cells in contrast to apoptosis in KBM7 cells. KBM5-STI571R1.0 and KBM7-STI571R1.0 have an imatinib IC50 (concentration that kills 50% of the cells) about twenty times higher then the value in the corresponding parental cell line. KBM5- STI571R1.0 have a marginal increase in the BCR-ABL gene copy number, modest increase in p210 protein expression, but a highly imatinib-resistant Bcr/Abl tyrosine kinase activity associated with the acquisition of a single C-T change at ABL nucleotide 944 (T315I). KBM7-STI571R1.0 cells have no mutations within the ATP-binding domain of the Bcr/Abl, but have amplification of BCR/ABL gene and increased levels of expression of Bcr/Abl p210 protein, with decreased inhibition of the Bcr/Abl tyrosine kinase activity by imatinib, and loss of apoptotic response to imatinib. AMN107 showed higher potency in KBM5 cells than imatinib in suppressing cell growth (MTS assay) with IC50 values of 11.3 nM and 480.5 nM respectively. With KBM5-STI571R1.0, IC50 were 2418.3 nM and 6361.4 nM for AMN107 and imatinib, respectively. The IC50 for AMN107 and imatinib treatment in KBM7 cell line were 4.3 nM and 259.0 nM, respectively. In KBM7-STI571R1.0 IC50 were 97.2 nM and 2497.3 nM for AMN107 and imatinib, respectively. In experiments focused on cell cycle analysis and caspase-3 activity, the cell cycle distribution and apoptotic responses induced by imatinib and AMN107 appeared generally similar in all four ell lines, but at significantly lower concentrations of AMN107 (corresponding to findings in MTS assay). At a concentration of 2.5 mM, imatinib inhibited completely Bcr-Abl phosphorylation in both KBM5 and KBM7 cells, while AMN107 achieved the same effect at 125.0 nM In imatinib-resistant cells imatinib did not completely inhibit Bcr-Abl phosphorylation even when doses as high as 10.0 mM were applied, while AMN107 inhibited Bcr-Abl phosphorylation at 2.5 mM. The survival of SCID mice bearing KBM5 cells treated with AMN107 was significantly extended when compared with controls. Irradiated female five weeks old SCID mice were injected intraperitonealy (ip) with 2.4 x 107 KBM5 cells on Day 0. Treatment with AMN107 started on Day 20 and was given daily ip for 20 days. T/C survival ratios were 144%, 159%, and 182% for groups treated with 10, 20, and 30 mg/kg, respectively. AMN107 has significant activity against imatinib-sensitive and -</DEL><INS cite=mailto:fgiles dateTime=2004-07-28T15:36>i</INS><INS cite=mailto:fgiles dateTime=2004-07-28T15:35>matini</INS><INS cite=mailto:fgiles dateTime=2004-07-28T15:36>b </INS>resistant CML and warrants investigation in patients with CML.

AMN107, Novel Aminopyrimidine Inhibitor of Bcr-Abl, Is Significantly More Potent Than Imatinib Mesylate Against Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Cells.

Imatinib mesylate is effective against Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) but, when used as a single agent, responses are transient and most patients relapse within 4–6 months. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both Ph-positive (Z-119 and Z-181) and Ph-negative (Z-138) ALL cell lines was studied. Z-119 and Z-181 cells were derived from Ph-positive ALL patients and retained typical B-cell characteristics and phenotypes of the original leukemia, including cytogenetic abnormality t(9;22) and p190 Bcr/Abl kinase. Z-138, a Ph-negative cell line, was derived from a patient with chronic lymphocytic leukemia and supervening ALL. Treatment with AMN107 or imatinib for 3 days (MTS assay) inhibited proliferation of Z-119 cells with the IC50 values of 19.3 nM and 620.0 nM, respectively, revealing AMN107 to be 32 fold more potent than imatinib. Treatment of Z-181 cell line lasted for 4 days (MTS assay) because of lower growth rate of these cells: IC50 for AMN107 and imatinib were 1.6 nM and 63.9 nM, respectively, showing AMN107 to be 40 fold more potent than imatinib. Neither drug showed activity against Ph-negative Z-138 cells. We also compared the activity of AMN107 in Ph-positive ALL cell lines expressing p190 Bcr/Abl protein to that in Ph-positive chronic myeloid leukemia cell lines KBM5 and KBM7 expressing p210 Bcr/Abl protein. The activity was similar with IC50 in KBM5 cells of 11.3 nM and in KBM7 cells of 4.3 nM. In experiments focused on cell cycle analysis we found that at equipotent doses (as determined by MTS assay) both drugs induced cell accumulation in G0/G1 phase in Z-119 but not in Z-181. We demonstrated that increasing equipotent concentrations of AMN107 and imatinib induced activation of caspase-3 that resulted in apoptosis, as assessed by propidium iodide staining, in Z-119 cells, while Z-181 cells showed lack of apoptotic response. Following treatment with a broad range of AMN107 and imatinib doses for 3 hrs, Bcr/Abl expression and phosphorylation were determined in Z-119 cells by immunoprecipitation and Western blotting: Bcr/Abl phosphorylation was inhibited completely with AMN107 at 125.0 nM, and with imatinib at 2500 nM, confirming again the higher potency of AMN107. Finally, similar differential effect of AMN107 and imatinib on Bcr/Abl protein expression and phosphorylation was observed in leukemic cells obtained from blood of Ph-positive ALL patients. We conclude that AMN107 has significant activity against Ph-positive ALL cells and warrants investigation in patients with Ph-positive ALL.

Changes associated with the development of resistance to imatinib (STI571) in two leukemia cell lines expressing p210 Bcr/Abl protein.

Although various mechanisms have been recognized as being associated with the development of resistance to imatinib mesylate in vitro and in clinical situations, their relative significance and contributions remain poorly understood, as is the sequence of events leading to the selection of the resistant phenotype. Experimental in vitro systems involving well defined cell lines and conditions can be used to some advantage to answer specific questions and to develop in vitro models of imatinib resistance that would reflect its potential heterogeneity. Two cell lines, KBM5 and KBM7, which expressed p210 Bcr/Abl and which differed in their inherent sensitivity to imatinib, the number of copies of the BCR/ABL fusion gene, and the activation of apoptotic pathways, were grown in vitro in the presence of increasing concentrations of imatinib. The resistant cells were analyzed for cell cycle progression, apoptotic response after exposure to imatinib, expression of Bcr/Abl, tyrosine kinase activity, and the presence of mutations within the adenosine triphosphate (ATP) coding domain of BCR/ABL. At various levels of resistance, the cells were transferred into drug-free media, and the stability of the resistant phenotype was determined in the absence of the drug. In KBM7 cells, the development of resistance was characterized by loss of apoptotic response to the drug, amplification of BCR/ABL, increased levels of expression of p210 Bcr/Abl, and decreased inhibition of Bcr/Abl tyrosine kinase (TK) activity by imatinib. No mutations within the ATP-binding domain of Bcr/Abl were identified, and resistance remained stable in the absence of the drug. In KBM5 cells, which previously were found to be characterized by the acquisition of a single C-T mutation at ABL nucleotide 944 (T315I) at high levels of resistance, this same mutation was detected at an intermediate level, but not at a low level, of resistance. The response of KBM5 cells to imatinib was characterized by a low level of apoptotic response, a marginal increase in BCR/ABL copy number, a modest increase in p210 expression, and a highly imatinib-resistant Bcr/Abl TK. Partial reversal of resistance was observed in highly resistant KBM5-STI571(R1.0) cells, which continued to display the C-T mutation. In KBM5 cells with an intermediate level of resistance, the T315I mutation was no longer detectable upon their reversal to the sensitive phenotype. BCR/ABL amplification with subsequent overexpression of Bcr/Abl protein, loss of apoptotic response, or point mutation of the ATP-binding site of BCR/ABL was associated alternatively with the acquisition of the resistant phenotype, supporting the notion that multiple mechanisms are involved in the induction of resistance to imatinib. The initial number of BCR/ABL copies itself was not related directly to the degree of resistance. The reversibility of the resistance may be complete, partial, or irreversible, depending on the mechanism(s) involved and the degree of resistance. Both cell lines serve as models for further elucidation of various aspects of imatinib-resistance mechanisms.

In vitro effects of STI 571-containing drug combinations on the growth of Philadelphia-positive chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a molecular aberration, a fusion BCR-ABL gene encoding for aberrant tyrosine kinase activity, which is crucial in the pathogenesis of CML. In vitro, inhibition of BCR-ABL protein tyrosine kinase activity by a tyrosine kinase inhibitor, Imatinib mesylate (STI571; formerly CGP57148B), successfully suppressed proliferation/survival of the BCR-ABL positive clones. In clinical studies, hematologic and cytogenetic remissions have been achieved in most patients with chronic phase CML; in accelerated and blastic phases of CML, STI571 appeared less effective. In the current study, the authors tested combinations of STI571 and cytarabine and homoharringtonine (HHT), drugs with documented activity in CML. The single agents and their combinations were studied for in vitro effect on proliferation of BCR-ABL positive cell lines KBM5 and KBM7 by 3(4,5-dimethylthiazol-2yl)-2,5 diphenyl-tetrazolium bromide assay and on primary patient-derived BCR-ABL cells by clonogenic assays. The in vitro additive, synergistic, or antagonistic effects of cytarabine and HHT with STI571 were then investigated by computer-assisted analysis using the CalcuSyn software. STI571 consistently suppressed BCR-ABL positive cell proliferation with a dose-effect correlation. In the model system used, STI571/cytarabine and STI571/HHT combinations were more effective in inhibiting KBM5 and KBM7 cell growth than each drug as single agent. These results were also verified in primary CML-derived clonogenic cells in semisolid cultures. In this experimental system, our studies documented additive or synergistic effects with STI571 plus cytarabine or HHT, supporting the future use of STI571 combinations in clinical trials in patients with Philadelphia chromosome-positive leukemias.

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.