Leukemia Cell Line Models Research Articles

A Novel STAT3 Inhibitor Has Potent Activity in Preclinical Models of Acute Myeloid Leukemia That Incorporate the Stromal Environment

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with relapse rates approaching 40% in children. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is implicated in promoting many cancer phenotypes, including AML. Additionally, the important role of STAT3 in microenvironment-mediated chemoresistance is well established. Therefore, STAT3 is an important target for the development of new agents. Like other transcription factors, the structure of the STAT3 protein does not easily lend itself to the development of a small molecule inhibitor that is both potent and specific. Several commercially available and academic STAT3 inhibitors have been reported, but none is yet suitable for broad clinical application. We have developed a novel class of naphthalene sulfonamide small molecule STAT3 inhibitors with efficacy in AML cell lines and primary samples (e.g. C188-9 [Redell, et al, 2011, Blood 117:5701-9]). Rational optimization steps have yielded a new lead compound, MM-206, with improved stability in cellular contexts. Our aim is to evaluate the activity of MM-206 in preclinical AML models that include the bone marrow stromal environment.Inhibition of STAT3 activity: MM-206 potently inhibited the STAT3 SH2 domain-phosphopeptide interaction, with IC50 1.2 μM by surface plasmon resonance assays. Further, MM-206 attenuated phosphorylation of G-CSF-induced STAT3 as measured by intracellular flow cytometry. AML cell lines were treated with increasing doses of MM-206 for 30 minutes, followed by stimulation with G-CSF to induce STAT3 phosphorylation (pY705). The IC50 values for inhibition of phosphorylation were 1-3 µM, indicating potent inhibitory activity in cells. Similarly, MM-206 inhibited G-CSF-induced PIM1 transcription in a dose dependent manner in AML cell lines.Anti-leukemia activity in vitro: MM-206 demonstrated dose-dependent induction of apoptosis in AML cell lines and pediatric AML samples, even in the presence of bone marrow stromal cells. Cell lines and patient samples were cultured alone or co-cultured with mOrange-transduced HS5 or HS27A stromal cells, then treated with increasing doses of MM-206 for 24 hours. Cells were stained with Annexin V-FITC and apoptosis in the mOrange-negative cells was quantified by FACS. EC50 values were consistent with the doses for inhibition of phosphorylation, ranging from 1 - 10 µM. The increase in EC50s in the stroma co-cultured AML cells, compared to AML cells cultured alone, was ~3-fold, indicating only moderate environment-derived protection against this compound. In complementary experiments, luciferase-transduced AML cell lines were cultured alone or with stromal cells, treated with MM-206 for 24 hours, then AML cell viability was assessed by luminescence. Again, IC50s were in the range of 3 µM for all cell lines tested. There was no difference in potency for cells on stroma in the luminescence assay. In contrast to the anti-AML activity, EC50s were >30 µM in cell line models of acute lymphoblastic leukemia, a disease not typically associated with STAT3-dependent survival.Anti-leukemia activity in vivo: We engrafted NSG mice with luciferase-expressing MV4-11 AML cells. After 2 weeks, mice were randomized to receive MM-206 (30 mg/kg) or vehicle, ip daily Monday-Friday, for 2 or 4 weeks. MM-206 treatment delayed disease progression, as evidenced by significantly lower luminescence values (p<0.001, Fig. 1). Mice treated with MM-206 for 4 weeks (n=8) had significantly reduced blast percent in the bone marrow and significantly prolonged survival (p=0.02 at 10 weeks), compared to the control group (n=7).Summary: MM-206 is a novel naphthalene sulfonamide inhibitor of STAT3 activity, with potent anti-AML activity in preclinical models that incorporate the stromal environment, including a mouse xenograft model. Future work will evaluate the activity of this compound in combination with chemotherapy, as a strategy to overcome environment-mediated chemoresistance. [Display omitted] DisclosuresTweardy:StemMed, Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: compounds including MM-206, Research Funding.

The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias. DisclosuresNo relevant conflicts of interest to declare.

Folylpolyglutamate synthetase splicing alterations in acute lymphoblastic leukemia are provoked by methotrexate and other chemotherapeutics and mediate chemoresistance

Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.

Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model.

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug-resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod-like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure-mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux-pump-mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self-assembly (≈5 min) and excellent stability in cell culture conditions.

Abstract 2701: Characterization of acquired EPZ-5676 resistance in cell line models of MLL rearranged leukemia

Abstract DOT1L inhibitor EPZ-5676 is currently under Phase 1 clinical trial investigation in relapsed/refractory patients with acute leukemia, including those with an MLL-rearrangement (MLL-r). Early clinical results, including complete remissions, support ongoing clinical development and preclinical investigation into mechanisms precipitating EPZ-5676 treatment induced resistance. MLL-r cell lines KOPN-8 (MLL-ENL) and NOMO-1 (MLL-AF9) were exposed to an EPZ-5676 concentration above the pre-determined 14 day proliferation assay IC90. Initial treatment of the cell lines led to the expected inhibition of H3K79 dimethylation (H3K79me2) and MLL-r target genes HOXA9 and MEIS1 as outlined in previous work (Daigle et al, Cancer Cell 2011). Resistance to EPZ-5676 in both cell lines emerged following three weeks of continued treatment with EPZ-5676 and was defined by increased growth rates in the presence of inhibitor. Mechanisms of resistance for both cell lines were investigated using RNASeq and ChIPSeq on parental and resistant cell line pools. Our analysis identified common characteristics between the resistant cell lines, but mechanisms by which they became resistant differed. Global H3K79me2 inhibition was maintained in both refractory cell lines, yet ChIP-seq analysis of resistant pools identified specific loci with H3K79me2 recovery in KOPN-8 cells. In resistant KOPN-8 cells recovery of H3K79me2 was concentrated at the HOXA locus and other MLL-r target genes (e.g. MEIS1 and RUNX2), with the remainder of actively transcribed genes maintaining H3K79me2 inhibition at levels observed in parental cells. In contrast, resistant NOMO-1 cells did not recover H3K79me2 at any actively transcribed genes, including those of the MLL-r signature. Only resistant KOPN-8 cells regained expression of the MLL-r target genes HOXA9 and MEIS1. Of note both resistant NOMO-1 and KOPN-8 cell lines had 8 and 40 fold upregulation of the ABCB1 (MDR1, P-gp) tranporter respectively when compared to a matched control cell line. To explore the role of drug efflux transporter ABCB1 on resistance, we treated cells with Valspodar, a known inhibitor of ABCB1. Following treatment with 1 μM Valspodar, KOPN-8 cells showed decreased cell growth similar to the naïve control cell line. Supporting the presence of an alternative resistance mechanism in NOMO-1 cells in addition to MDR1 upregulation, NOMO-1 cells remained resistant upon Valspodar treatment. Detailed gene expression and pathway analysis will be presented supporting mechanisms of treatment emergent resistance to EPZ-5676. In summary, we have identified two mechanisms of EPZ-5676 resistance in MLL-r cell lines, one mechanism dependent on and the other independent of ABCB1. Further refinement of these mechanisms will aid in providing hypotheses for testing mechanisms of EPZ-5676 treatment emergent resistance in patients and may support designing future rational clinical combinations. Citation Format: Scott R. Daigle, Carly T. Campbell, Nigel J. Waters, Edward J. Olhava, Robert A. Copeland, Stephen J. Blakemore, Roy M. Pollock, Jesse J. Smith. Characterization of acquired EPZ-5676 resistance in cell line models of MLL rearranged leukemia. [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 2701. doi:10.1158/1538-7445.AM2015-2701

The role of topoisomerase II beta on breakage and proximity of RUNX1 to partner alleles RUNX1T1 and EVI1

Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.

PRAME-Induced Inhibition of Retinoic Acid Receptor Signaling-Mediated Differentiation—A Possible Target for ATRA Response in AML without t(15;17)

In acute myeloid leukemia (AML) without retinoic acid receptor (RAR) rearrangement, the effect of all-trans-retinoic acid (ATRA) is still poorly understood despite an association of NPM1 mutation and ATRA response. Recently, preferentially expressed antigen in melanoma (PRAME) has been shown to be a dominant repressor of RAR signaling. Thus, we further investigated ATRA response mechanisms, especially the impact of PRAME expression on ATRA responsiveness. We profiled gene expression in diagnostic samples derived from our AML HD98B trial, in which ATRA was administered in addition to intensive chemotherapy. Our data revealed a PRAME expression-associated gene pattern to be significantly enriched for genes involved in the retinoic acid metabolic process. In leukemia cell line models, we could show that retinoic acid-regulated cell proliferation and differentiation are impacted by PRAME expression. In patients with primary AML, repressor activity of high-PRAME levels might be overcome by the addition of ATRA as indicated by better outcome in 2 independent studies (P = 0.029). PRAME seems to impair differentiation and to increase proliferation likely via blocking RAR signaling, which might be reversed by ATRA. PRAME therefore represents a promising target for both ATRA treatment and possibly future immunotherapeutic approaches in AML.

A potential role of fetal hemoglobin in the development of multidrug resistance

Our previous data from a human leukemic cell line made resistant to the nucleoside analog (NA) 9-β-D-arabinofuranosylguanine (AraG) revealed a massive upregulation of fetal hemoglobin (HbF) genes and the ABCB1 gene coding for the multidrug resistance P-glycoprotein (P-gp). The expression of these genes is regulated through the same mechanisms, with activation of the p38-MAPK pathway and inhibition of methylation making transcription factors more accessible to activate these genes. We could show that AraG, as well as other NAs, and P-gp substrates could induce global DNA demethylation and induction of Hbγ and P-gp both at the mRNA and protein expression level. We speculate that the expression of HbF prior to drug exposure or in drug-resistant cell lines is a strategy of the cancer to gain more oxygen, and thereby survival benefits. We also believe that P-gp may be induced in order to excrete Hb degradation products from the cells that would otherwise be toxic. By using Hbγ siRNA and pharmacological inhibitors of HbF production we here present a possible relationship between HbF induction and multi-drug resistance in a human leukemia cell line model.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70% of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

Downregulation of IRS2 in Myelodysplastic Syndrome (MDS) May Be Related to Impaired Cell Differentiation,

Abstract 3814Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia. Abnormalities in cell differentiation are probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling from upstream activators to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R), the Erythropoietin receptor (EPOR) and Thrombopoietin receptor (MPL). Recently, we reported that IRS2 was downregulated in MDS cells compared to normal cells, and in high risk compared to low-risk MDS. Additionally, IRS2 was significantly upregulated during erythroid differentiation of normal CD34+ cells, but not in MDS CD34+ cells. In view of the role of IRS2 in EPO, IGF1 and TPO signaling, and possibly in erythroid, granulocytic, and megakaryocytic differentiation, we hypothesized that IRS2 might be related to the impaired hematopoiesis of MDS cells. The aim of the present study was to correlate the mRNA expression levels of IRS2 in bone marrow from MDS patients with laboratorial and clinical data. We also elucidated the expression levels of IRS2 during megakaryocytic, granulocytic and erythroid differentiation of established leukemia cell line models. We studied 12 healthy donors and 32 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification). Gene expression was evaluated by Q-PCR in total cells from bone marrow samples and cell lines. IRS2 protein expression and phosphorylation was evaluated by Western blot and immunoprecipitation. K562 cells were induced to megakaryocytic differentiation by treatment with 20nM PMA for 4 days. NB4 cells were induced to granulocytic differentiation by treatment with 10−6 M ATRA for 4 days. KU812 cells were induced to erythroid differentiation by treatment with 50μM HE and 100 μM HU for 4 days. Spearman correlation analysis showed that IRS2 expression demonstrated a significant positive correlation with peripheral blood neutrophils (r =0.43, P =0.01) and platelets counts (r =0.36, P =0.04), and bone marrow granulocytes percentages (r =0.40, P =0.02). In contrast, a significant negative correlation was observed between IRS2 expression with bone marrow erythroblasts (r =-0.360, P =0.04) and myeloid precursor percentages (grouping myeloblast, promyelocytes, myelocytes, metamyelocytes and blasts) (r =-0.60, P <0.01). There was no correlation between IRS2 expression with age, hemoglobin, number of erythrocytes in the peripheral blood, and number of dysplasias in bone marrow. Finally, since IRS2 is involved with TPO, IGF1 and EPO signaling, and IRS2 expression was correlated with platelet, granulocyte and erythroblast numbers in MDS patients, we monitored IRS2 expression during hematopoietic cell differentiation. Interestingly, a significant increase in IRS2 expression and phosphorylation was observed in K562, NB4 and KU812 after megakaryocytic, granulocytic and erythroid differentiation, respectively. In summary, the lower expression of IRS2 in MDS and its upregulation during differentiation may suggest that defective IRS2 expression in MDS contributes to disease progression and impaired hematopoietic cell differentiation, a hallmark of the disease. Disclosures:No relevant conflicts of interest to declare.

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Novel Histone Deacetilase (HDAC) Inhibitors: In Vitro Effects on Leukemic Cells

AbstractAbstract 4946Histone deacetylase inhibitors (HDAC-I) are a class of agents with the capacity to induce acetylation of histone and non-histone proteins. These molecules have been intensively investigated in a variety of malignancies because of their ability to inhibit proliferation, induce differentiation and apoptosis in tumor cells. However, clinical response to clinically available HDAC-I have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds and at defining the molecular mechanisms of response to this class of agents. Acetyl-L-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane, thereby contributing to the regulation of energy production and metabolism. ALCAR activity as a modulator of cellular stress response has prompted its use to protect against chemotherapy-induced neurotoxicity. However, ALCAR effects on neoplastic cells are still not defined, especially in combination with chemotherapy. Here we investigated the effects of MS-275, a HDAC-I, on cell proliferation and apoptosis in cell line models of acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), in comparison with vorinostat also known as SAHA (suberoyl anilid hydroxamic acid), the most widely used HDAC-I in clinical setting. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and selected sub-toxic concentration of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing 50 % cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine- Orange (AO) technique. Results indicated that the tested compound MS-275 significantly inhibited cell growth, as assessed by MTT assay, when used at of 5000nM. Comparative analysis of the efficacy of the two different HDAC-I compounds indicated that MS-275 was the more effective agent and the only one with clear dose-dependent activity, while SAHA displayed a flat dose-response curve, which dropped only at the highest concentration. In particular, the myeloid cell line Molm-13 was strikingly sensitive to MS-275 (IC50: < 15 nM), U937 and HL60 myeloid cell lines, the lymphoid cell line Jurkat and the MM cell line ARH-77 showed intermediate sensitivity (IC50: < 1000 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). SAHA showed no activity in U937 cells when used at concentrations ranging from 100 to 1000 nM, with a dramatic reduction of absorbance at 5000 nM (>80% reduction compared to the control). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR reveled a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction, as determined by the percentage of cells with a sub-G1 DNA content. MS-275 dose-dependently induced apoptosis in HL-60 cells (4.2%, 17.1%, 60.8%, and 87.5% in the presence of 100, 500, 1000, 5000 nM of MS-275, respectively). Conversely, SAHA induced minimal apoptosis (< 10%) at concentration ranging from 100 to 1000 nM, although > 75% of cells became apoptotic after treatment with the compound at 5000 nM. In summary, our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in cell lines derived from myeloid leukemia and MM. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in selected hematological malignancies. Disclosures:Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Pisano: Sigma-Tau: Employment. Tafuri: Sigma-Tau: Research Funding.

The Heatshockprotein-90 Inhibitor IPI-504 Potently Overcomes Tyrosinekinase Inhibitor-Induced Activation of Cell Protection Mechanisms and Degrades HSP-Client Proteins KIT and FLT3 In Acute Leukemia Models

AbstractAbstract 3982Activating mutations of FLT3 and KIT class III receptor tyrosine kinases (TK) are associated with acute leukemias and systemic mastocytosis. Despite the encouraging therapeutic potential of TK inhibitors (TKI), monotherapy often is not sufficient to obtain lasting responses. Here we show that TKI therapy may lead to cellular protection mechanisms including stabilization/repair of DNA, as well as protection of the function of heat shock proteins. We further demonstrate that degradation of heat shock protein 90 (HSP90) client proteins, FLT3 and KIT, by the HSP90 inhibitor IPI-504, leads to antitumor effects in acute leukemia and mastocytosis models; including additive to synergistic antiproliferative and proapoptotic activity after TKI treatment. To explore the immediate downstream signaling effects of mutant FLT3 and KIT inhibition in acute leukemia and mastocytosis models, we performed unbiased phoshoproteomic analyses of cells before and after TKI treatment. Phosphopeptides that displayed a significant difference in phosphorylation before and after FLT3/KIT inhibition with Imatinib or Sunitinib at IC90 were immunoaffinity purified, and identified by tandem mass spectrometry. Degradation of FLT3 and KIT after HSP90 inhibition was studied by immunoblotting. Antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504 alone, or in combination with TKI treatment, were studied in different leukemia and mastocytosis cell models, and in an isogenic BaF/3 model harboring different clinically relevant KIT and FLT3 mutations. Phoshoproteomics revealed a significant upregulation in phosphorylation of DNA and protein function stabilizing proteins, including HSP members in mutant KIT and FLT3 cell lines. Both FLT3 and KIT turned out to be client proteins of HSP90, and IPI-504 was able to degrade FLT3 and KIT protein, resulting in a strong potentiation of the antiproliferative and proapoptotic effects achieved by TK inhibitors alone. Of interest, IPI-504 displayed antitumor activity even as monotherapy in some leukemia and mastocytosis cell line models. Our data suggest that TK inhibition of FLT3 or KIT causes initiation of cell protective mechanisms, including activation of HSPs, which can therapeutically be targeted by HSP90 inhibitors such as IPI-504. These results provide a rationale to combine TKI with HSP inhibitors to optimize TKI therapy in patients with hematopoietic malignancies. Disclosures:Normant:Infinity pharmaceuticals: Employment.

Differentiation-associated miR-22 represses Max expression and inhibits cell cycle progression

Differentiation agents such as 12- O-tetradecanoylphorbol-13-acetate (TPA) engage cell signaling pathways that activate downstream transcriptional programs necessary for cell differentiation. Recent evidence has indicated microRNAs (miRNAs) are an integral part of these transcriptional programs, which target key proteins and impact cell growth thereby facilitating changes required for differentiation. To further investigate the role of miRNAs in cell growth and differentiation, we focused on miR-22, a miRNA induced by TPA in the HL-60 leukemia cell line model of monocytic differentiation. TPA-induced miR-22 transcription was found to be downstream of the protein kinase c (PKC)–extracellular regulated kinase (ERK) signaling module, a pathway central to the growth and differentiation of many different cell types. Enforced miR-22 expression inhibited the growth of several different cancer cell lines, causing an accumulation of cells in the G1 phase of the cell cycle. The mechanism of miR-22’s inhibitory effects involves targeting of the obligate c-Myc binding partner Max. Enforced miR-22 expression presumably lowers Max levels available for Myc binding, which differentially influenced the transcription of downstream targets of the Myc–Max complex. Our study provides additional support for miRNAs targeting key cellular regulatory microcircuits such as those governed by the Myc–Max transcriptional complex as well as their being active participants in cell growth and differentiation.

Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Abstract 2757Poster Board II-733Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML. Disclosures:No relevant conflicts of interest to declare.

Diverse Roles of Hepatocyte Growth Factor (HGF) in Normal Karyotype Acute Myeloid Leukemia (AML).

Abstract 3107Poster Board III-44Prior studies have demonstrated that hepatocyte growth factor (HGF) regulates proliferation and differentiation of normal hematopoietic progenitors. HGF activity occurs primarily via interactions with the c-met receptor, a tyrosine kinase receptor found on epithelial and some cancer cells. In solid tumors, HGF/c-met interactions mediate increased neoplastic invasion, metastases, and angiogenesis. However, in vitro, HGF has also been shown to mediate anti-tumor effects in leukemia cell line models. To better elucidate the role of HGF in acute leukemogenesis, we evaluated HGF and c-met gene expression in 91 normal karyotype acute myeloid leukemia (NK-AML) patient samples previously characterized for marrow angiogenesis (CD31+ microvessels), FLT-3/NPM-1 gene mutation, and pro-angiogenic factors and receptors (specifically vascular endothelial growth factors (VEGF-A and C) and their receptors). Median patient age was 66 years (range 21-87) with 49 women and 42 men. AML disease FAB subtypes M2 (37%) and M1 (36%) subtypes were most common. Median presenting white blood cell count (WBC) was 32,000/μL (range 0.43-555,000/μL) with marrow blasts of 70.6% (range 15-95.4%). Fourteen percent presented with extramedullary disease. Median OS was 9.4 months (95% CI 6.7 to 11.5 months), with median EFS of 8 months (95% CI 5.7 to 11.5 months) for all patients. Seventy-nine patients received cytarabine and anthracycline-based induction chemotherapy with 58% (n=46) achieving complete remission (CR). Marrow aspirate samples were evaluated by quantitative real-time polymerase chain reaction with levels expressed relative to normal bone marrow controls (set =1). We found that HGF gene expression was upregulated in most primary NK-AML patient samples, with 88% expressing higher HGF than normal bone marrow. Median HGF expression in AML samples was 7.73 fold higher than normal controls. Multivariate analysis including age, complete remission, marrow blasts, extramedullary disease, and expression of other angiogenic factors and receptors as covariables, showed high HGF expression to be significantly associated with both longer overall and event-free survival. Surprisingly, HGF gene expression was found to be negatively correlated with microvessel density and NPM-1 mutation and positively correlated with the VEGF receptor neuropilin-1 (NRP-1) which has been reported to function as co-mediator of HGF activity. No association between HGF and FLT-3 ITD mutation was noted. The majority of AML samples did not express the HGF receptor, c-met, suggesting that HGF function in AML occurs primarily via paracrine interactions with surrounding vascular and stromal cells and/or HGF/NRP-1 autocrine pathways. Further analysis confirmed no significant correlation between HGF and c-met gene expression in AML samples but did demonstrate a subset of NPM-1+ HGF+ AML samples (n=7) expressing high levels of both HGF and c-met (p=0.0005, r=0.96). To confirm whether HGF/c-met autocrine interactions contributed to leukemogenesis in these cells, we treated immunodeficient mice engrafted with an HGF+ c-met+ human AML cell line (HEL) with vehicle vs. a c-met tyrosine kinase inhibitor, and noted growth inhibitory effects following c-met blockade. ConclusionsHGF gene expression was an independent predictive factor for improved clinical outcome and was associated with NRP-1 expression, lower microvessel density, and NPM-1 negative status in normal karyotype AML patients. A subset of AML samples was identified with high concordant HGF and c-met expression consistent with autocrine pathways. Inhibition of HGF/c-met interactions in a preclinical AML model exerted anti-tumor effects. Additional studies of the diverse roles of HGF in myeloid leukemogenesis are warranted. DisclosuresNo relevant conflicts of interest to declare.

Blocking the Autophagy Gene 5 (ATG5) Impairs ATRA-Induced Myeloid Differentiation, and ATG5 Is Downregulated in AML

Autophagy describes a process of membrane trafficking where specialized compartments (autophagosomes) engulf damaged or dispensable organelles and target them to lysosomic degradation. Thus, autophagy has cytoprotective functions e.g. during starvation, but autophagy can also lead to caspase-independent cell death (PCD type II). ATG5 is a central player in autophagy and inactivating ATG5 severely impairs normal development. In hematopoiesis, ATG5 is essential for maturation of B lymphocytes as well as for T cell survival and proliferation. In recent years, several differentiation and death-inducing agents were shown to activate autophagy in acute myeloid leukemia (AML) cell line models, but the mechanisms involved are still poorly defined. We therefore decided to investigate the role of ATG5 and autophagy in all-trans retinoic acid (ATRA)-induced neutrophil differentiation of AML cells. We found that ATG5 mRNA was upregulated 5.9-, 3.8- and 3.4-fold after 6 days of ATRA-treatment in HL60, NB4 and HT93 AML cells, respectively. In contrast, PMA-induced macrophage differentiation of HL60 and U937 cells only slightly induced ATG5 mRNA by 1.3- and 1.5-fold, respectively, indicating a specific role for ATG5 in granulocyte differentiation. In line with the above observation in leukemic cell lines, we found that ATG5 mRNA levels were increased in 5/5 APL patients upon ATRA therapy. In addition, ATG5-ATG12 conjugates, hallmarks of the autophagic process, were markedly activated in ATRA-treated NB4 and HL60 cells compared to control cells as measured by Western blotting. In agreement, we found increased autophagic activity during myeloid differentiation as evidenced by two additional autophagy markers, i.e. conversion of light chain 3 B (LC3B)-I into LC3B-II and degradation of sequestosome 1 (SQSTM1, p62) protein by Western blotting. Inhibition of autophagy during ATRA treatment of NB4 and HL60 cells with 3-Methyladenine or Bafilomycin A significantly impaired neutrophil differentiation by 80% as measured by CD11b surface expression. Similarly, lentivirus-driven short hairpin (sh)RNA-mediated silencing of ATG5 in NB4 cells resulted in an 85% reduction of ATRA-induced neutrophil differentiation as measured by CD11b surface expression and by quantitative RT-PCR of the myeloid differentiation markers GCSFR, C/EBPε and lactotransferrin. Interestingly, inhibition of autophagy increased overall cell death during the myeloid differentiation process rather than reducing it as measured by reduction of tetrazolium salt (XTT assay). Enhanced cell death and reduced myeloid differentiation upon blocking ATG5 would suggest that autophagy is needed for maintaining myeloid differentiation. Further support for our hypothesis that ATG5 is needed for neutrophil development stems from our survey of ATG5 mRNA expression in primary hematopoietic cells. We found significantly higher ATG5 mRNA levels in granulocytes and macrophages (n=7) as compared to CD34+ hematopoietic progenitor cells from healthy donors (n=4; p=0.0424) as well as compared to primary AML patient samples at diagnosis (n=76; p=0.0003). ATG5 mRNA expression in CD34+ and AML patients was not significantly different (p=0.6669). In summary, we show a correlation of high ATG5 expression with terminal myeloid differentiation and of low ATG5 expression with a myeloid leukemic phenotype. Using chemical inhibitors of autophagy as well as RNAi technology to knockdown ATG5, we further provide evidence that ATG5 and consequently autophagy are essential for neutrophil development.

Acquisition of Mutations in the PSMB5 Gene during Chronic Exposure of Human Acute Lymphoblastic Leukemia CCRF-CEM Cells to the Proteasome Inhibitor Bortezomib

Despite the good prognosis of children suffering from acute lymphoblastic leukemia (ALL), approximately 25% of the patients develop a relapse. The proteasome inhibitor bortezomib (BTZ) is a representative of a novel generation of therapeutic drugs that revealed promising activity in patients with multiple myeloma (MM) and certain types of lymphomas, and is being studied in the treatment of relapsed ALL. Whereas most research on the mechanism of action of BTZ has focused on MM or lymphoma, the present study was designed to examine the activity of BTZ in an ALL model and in particular to assess the propensity to develop resistance to BTZ after prolonged drug exposure. Previously, we have shown (Franke et al., ASH 2007) that BTZ-resistant subclones of the human T-ALL cell line CCRF-CEM could emerge relatively rapid (within 2–3 months) after chronic exposure to stepwise increasing concentrations (0.1–200 nM) of BTZ. These BTZ-resistant cells (CEM/BTZ) showed cross-resistance against other proteasome inhibitors while remaining completely sensitive to a broad spectrum of other anticancer drugs. Subsequent analysis of these BTZ-resistant subclones showed an upregulation of the catalytic proteasomal β-subunits at both mRNA and protein level. In the present study we further elucidated the molecular basis for BTZ-resistance in a panel of CEM/BTZ cells selected at 7 nM BTZ (CEM/BTZ7) up to 200 nM BTZ (CEM/BTZ200). Since the proteasome β5 subunit is the primary target of BTZ, we screened whether mutations in the PSMB5 gene could be involved in conferring resistance to BTZ. Indeed, sequencing of exon 2 of the PSMB5 gene for CEM/BTZ7 cells revealed a single G to T nucleotide shift at position 332 of this gene. In the mature and functional β5 proteasome subunit, this mutation introduces a Cys to Phe substitution at amino acid 52, residing in a highly conserved substrate/inhibitor binding domain of the β5 subunit. In this context, it was of interest to note that CEM/BTZ7 cells already displayed a marked level (28-fold) of cross-resistance to 4A6, a 6-mer peptide that specifically targets the proteasome β5 catalytic subunit, thus suggesting that this mutation is responsible for diminished inhibitor binding. Upon screening for mutations in CEM cells with higher BTZ resistance levels, an additional mutation in the PSMB5 gene was revealed in CEM/BTZ30 cells. Herein, an additional C to T nucleotide shift at position 323 of the PSMB5 gene was identified, introducing an Ala to Val amino acid substitution at amino acid 49 of the mature and functional β5 subunit protein. This amino acid also resides in a highly conserved substrate/inhibitor binding domain of the β5 subunit. Both Cys52Phe and Ala49Val mutations were retained, and no others revealed, in BTZ-resistant CEM cells up to CEM/BTZ200 cells. Of note, recently we have identified an Ala49Thr mutation in the BTZ binding domain of the proteasome β5 subunit in a BTZ-resistant subclone of the human acute myeloid leukemic cell line THP-1 (Oerlemans & Franke, Blood 2008). Moreover, an Ala49Thr mutation was also recently described in a BTZ-resistant Jurkat cell line (Lü et al., J Pharmacol Exp Ther 2008). Together, this study demonstrates that mutations in the PSMB5 gene are provoked relatively fast in ALL cells, already at low levels of BTZ resistance. Furthermore, given the notion that independent studies in 3 different leukemia cell line models demonstrated that BTZ-resistance is associated with a mutation introducing amino acid substitutions at position Ala49 in the highly conserved substrate/inhibitor binding domain of the β5 subunit, this position may be considered as a hot spot for mutations in the PSMB5 gene through which acquired resistance to BTZ may be facilitated. Analysis of mutational status of the PSMB5 gene in future clinical BTZ studies in ALL patients should further confirm the clinical relevance of our finding, and might be exploited in the development of next generation proteasome inhibitors, designed to overcome resistance.

C-Met Oncogene Enhances Fas-Mediated Apoptosis in B-ALL t(12;21) Cells.

c-Met, the tyrosine-kinase receptor for HGF, has been related to cell motility, proliferation and protection from apoptosis in epithelial cancers (Cytokine Growth Factor Rev 2002; 13:41). On the other hand it has been proposed that c-Met could also stimulate apoptosis: association with the pro-apoptotic protein FAS has been shown in some cell types (Biochem Biophys Res Commun 1997; 230:89). So far no data are available on a conceivable role of c-Met in pediatric B-ALL.We observed that c-Met is expressed at higher levels in pediatric t(12;21) B-ALL, that better respond to therapy, and in normal B cells compared to B-ALL t(12;21)-, while the latter leukemias do not differ for Fas expression (Haematologica 2004; 89:113). Here, we studied c-MET/FAS association in t(12;21) B-ALL, and the role of this complex in drug-induced apoptosis.We performed IP/WB experiments in REH (B-ALL t(12;21)) cells and in healthy CD19+ cells: for the first time we observed in B cells the co-precipitation of c-MET and FAS.To investigate if the HGF/c-MET pathway could be activated, we stimulated REH cells with HGF for 48 hours. The phosphorylation of c-MET increased 5 minutes after stimulation, and lasted after 48 hours. In HGF stimulated cells we observed a higher proliferation rate compared to non-stimulated cells (p=0.01). We also observed that the c-MET/FAS complex was preseved during HGF stimulation. These results indicate that HGF stimulation does not disrupt the protein complex, while HGF activates the c-MET signaling pathway despite of FAS binding.To investigate the role of complex formation on FAS activity, REH cells were stimulated with HGF and then treated with Doxorubicin that acts specifically through the FAS apoptosis pathway. After 24 hours of Doxorubicin treatment, we observed in HGF pretreated cells an apoptotic rate higher than in not-pretreated cells (p=0.03). These results indicate that the complex c-MET/FAS does not inhibit c-MET or FAS functions, and importantly show that c-MET activation enhances FAS-mediated apoptosis.To demonstrate that the higher apoptotic sensibility after HGF stimulation is not related to the increased proliferation rate, we treated cells with L-Asparaginase that does not act through the FAS pathway: we did not find an increase of apoptosis after pre-stimulating c-MET with HGF. This is an important result because it confirms that the higher apoptotic rate observed after Doxorubicin treatment in pre-stimulated cells is directly related to increased activation of the FAS pathway. We also verified that the c-MET/FAS complex remains stable after 24 hours from Doxorubicin treatment in apoptotic cells, and results show that it is even more pronounced in HGF treated cells.To confirm FAS involvement in Doxorubicin treated cells, we checked the cleavage of Caspase-8: cleavage is more apparent in HGF pre-treated cells, demonstrating that the FAS pathway is more effective in c-MET activated cells. After L-Asparaginase treatment Caspase-8 is not cleaved.The observation that the activation of the c-MET pathway results in increased FAS mediated apoptosis strongly suggests that the c-MET/FAS complex has functional implications for understanding drug mechanisms. The finding that the c-MET/FAS complex is present in normal B cells and in the leukemia cell line model for patients that better respond to therapy implies that this protein complex may have an important role in cellular homeostasis and in enhanced pharmacological response of tumoral cells during therapy.

MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models

Inappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 microM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC(50)) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 microM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of the BCR-ABL(+) AR230 cell line with the MDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.