The Pim Kinase Inhibitor SGI-1776 Chemosensitizes Multidrug Resistant Cells by Both Inhibiting Drug Transport by ABCB1 and ABCG2 and Decreasing ABCB1 and ABCG2 Surface Expression On Cells That Overexpress Pim-1.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2462-2462
Author(s):  
Karthika Natarajan ◽  
Jasjeet Bhullar ◽  
Suneet Shukla ◽  
Mehmet Burcu ◽  
Suresh V. Ambudkar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Drug Transport ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Surface Expression ◽  
Drug Efflux ◽  
Myeloma Cells ◽  
Chemotherapy Drugs ◽  
Surface Translocation

Abstract Abstract 2462 Overexpression of the ATP-binding cassette (ABC) cellular drug efflux proteins ABCB1 and ABCG2 on acute myeloid leukemia (AML) cells is associated with inferior chemotherapy outcomes. Nevertheless, inhibitors of drug transport have not improved treatment outcomes in clinical trials. The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, is expressed in AML and is implicated in regulation of multiple key cellular processes, as well as drug resistance. Our group has shown that Pim-1 phosphorylates ABCB1 and ABCG2 and promotes their translocation to the cell surface, where they mediate drug efflux. The imidazo[1,2-b]pyridazine small molecule SGI-1776 (Tolero Pharmaceuticals, Inc. Salt Lake City, UT) is the first Pim kinase inhibitor to have entered clinical testing. SGI-1776 has been shown to sensitize ABCB1-overexpressing drug-resistant cells to ABCB1 substrate cancer chemotherapy drugs, but chemosensitization was found to be associated with direct inhibition of drug transport mediated by ABCB1. Moreover, while silencing of Pim-1 expression with siRNA was found to sensitize ABCG2-overexpressing cells to ABCG2 substrate chemotherapy drugs, the effects of SGI-1776 on resistance mediated by ABCG2 have not been studied. Therefore we studied the Pim-1-dependent and -independent effects of SGI-1776 on chemosensitivity of cells overexpressing ABCB1 and ABCG2. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of ABCB1 and ABCG2 substrate drugs including daunorubicin and mitoxantrone 2- to 4-fold in leukemia and myeloma cell lines overexpressing ABCB1 and ABCG2, but had no effect on the IC50 of the non-substrate drug cytarabine, and no effect in parental cells. SGI-1776 also increased apoptosis of ABCB1- and ABCG2-overexpressing leukemia and myeloma cells exposed to ABCB1 and ABCG2 substrate chemotherapy drugs, respectively, and decreased their colony formation in the presence of substrate, but not non-substrate, chemotherapy drugs, with no effect on parental cells. We found that SGI-1776 decreased ABCB1 and ABCG2 surface expression, measured by flow cytometry, on K562/ABCB1 (p=0.013) and K562/ABCG2 (p=0.0038) leukemia cells, respectively, both of which express Pim-1 at high levels, without decrease in total cellular ABCB1 and ABCG2 expression, measured by Western blot analysis. In contrast, SGI-1776 had no effect on ABCB1 and ABCG2 surface expression on HL60/VCR leukemia and 8226/MR20 myeloma cells, which express ABCB1 and ABCG2, respectively, but express Pim-1 at lower levels. Thus SGI-1776 decreased ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1, consistent with decreased cell surface translocation of ABCB1 and ABCG2 as a result of inhibition of Pim-1, but also chemosensitized cells expressing ABCB1 and ABCG2 in the absence of effects on ABCB1 and ABCG2 cell surface expression. We found that SGI-1776 indeed inhibited uptake of fluorescent substrates of both ABCB1 and ABCG2, measured by flow cytometry, in a concentration-dependent manner. We further determined that SGI-1776 inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]-IAAP and stimulated ABCB1 and ABCG2 ATPase activity, consistent with binding to drug-binding sites of ABCB1 and ABCG2 and inhibition of substrate transport by both proteins. Thus SGI-1776 both inhibits drug transport by ABCB1 and ABCG2 and decreases ABCB1 and ABCG2 surface expression on cells that overexpress Pim-1. Pim-1 is thought to be a clinically promising therapeutic target in AML and other malignancies, and other Pim kinase inhibitors are in preclinical and clinical development. Subsequent clinically applicable Pim kinase inhibitors should be characterized with regard to interactions with ABCB1 and ABCG2. In particular, while therapeutic strategies based on inhibition of drug transport mediated by ABCB1 with competitive inhibitors including PSC-833, zosuquidar and cyclosporin A have largely been clinically unsuccessful, inhibition of ABCB1 and ABCG2 cell surface translocation by Pim kinase inhibitors may have therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface.

1990 ◽  
Vol 172 (3) ◽  
pp. 973-976 ◽  
Author(s):  
T Tsubata ◽  
M Reth
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Biochemical Analysis ◽  
Surface Expression ◽  
Expression Vectors ◽  
Myeloma Cells ◽  
Immunoglobulin Mu ◽  
Chain Form

We constructed expression vectors coding for the two pre-B-specific genes, VpreB and lambda 5, and transfected them together with a mu vector (mu tm) into Ig- myeloma cells. In a transfectant expressing all three introduced genes, the mu tm chain is transported on the cell surface. A biochemical analysis demonstrated that, in these cells, the mu tm chain is associated noncovalently with an 18-kD protein and covalently with a 22-kD protein, which are most likely the products of VpreB and lambda 5, respectively. Our results, thus, strongly suggest that the products of lambda 5 and VpreB bind to mu chains and have the same capacity as conventional Ig L chains to allow surface expression of mu chains.

scholarly journals Physical dissociation of the TCR-CD3 complex accompanies receptor ligation.

1995 ◽  
Vol 182 (6) ◽  
pp. 1997-2006 ◽  
Author(s):  
H Kishimoto ◽  
R T Kubo ◽  
H Yorifuji ◽  
T Nakayama ◽  
Y Asano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Before And After ◽  
Physical Dissociation ◽  
Receptor Ligation

Recent studies indicate that there may be functional uncoupling of the TCR-CD3 complex and suggest that the TCR-CD3 complex is composed of two parallel signal-transducing units, one made of gamma delta epsilon chains and the other of zeta chains. To elucidate the molecular mechanisms that may explain the functional uncoupling of TCR and CD3, we have analyzed their expression by using flow cytometry as well as immunochemical means both before and after stimulation with anti-TCR-beta, anti-CD3 epsilon, anti-CD2, staphylococcal enterotoxin B, and ionomycin. We present evidence that TCR physically dissociates from CD3 after stimulation of the TCR-CD3 complex. Stimulation with anti-CD3 resulted in down-modulation of TCR within 45 min whereas CD3 epsilon was still expressed on the cell surface as detected by flow cytometry. However, the cell surface expression of TCR and CD3 was not affected when cells were stimulated with anti-TCR-beta under the same conditions. In the case of anti-CD3 treatment of T cells, the TCR down-modulation appeared to be due to the internalization of TCR, as determined by immunoelectron microscopy. Immunochemical analysis of cells after stimulation with either anti-TCR or anti-CD3 mAbs revealed that the overall protein levels of TCR and CD3 were similar. More interestingly, the dissociation of the TCR-CD3 complex was observed with both treatments and occurred in a manner that the TCR and the associated TCR-zeta chain dissociated as a unit from CD3. These results provide the first report of physical dissociation of TCR and CD3 after stimulation through the TCR-CD3 complex. The results also suggest that the signal transduction pathway triggered by TCR may differ from that induced by CD3.

Erlotinib Antagonizes Efflux Via ABC Transporters and Decreases P-Gp Cell Surface Expression by Inhibiting SRC Kinase and mTOR Pathways in Acute Myeloid Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2564-2564 ◽  
Author(s):  
Elodie Lainey ◽  
Marie Sebert ◽  
Cyrielle Bouteloup ◽  
Carole Leroy ◽  
Sylvain Thepot ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Atpase Activity ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Hoechst 33342

Abstract Abstract 2564 Background: Erlotinib (Erlo) was originally developed as an epidermal growth factor receptor inhibitor, yet it also exerts antileukemic “off-target” effects, in vitro and in vivo in MDS and AML (Boehrer et al., Blood, 2008). In a preliminary pre-clinical study, we observed that Erlo increased chemosensitivity to current AML drugs in different AML cell lines and in ex vivo AML patient cells (n=3) (ASH 2010, 2163). Those first results suggested an implication of ABC-transporters in the potentiation of apoptosis. Here, we bring direct evidence for Erlo's ability to hinder efflux pumps and to decrease their expression on AML cells. Methods: Drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ or doxorubicin-Dox), and specific efflux via P-gp (substrates: DioC23 and Rho-123), MRP (s: Calcein and CDCFDA) and BCRP (s: Hoechst 33342) were quantified by FACS following incubation with 10mM Erlo. Intracellular VP-16) content was quantified by Rapid Resolution Liquid Chromatography (RRLC). Biochemical inhibitors of the respective ABC-transporters (CSA (1μM), verapamil (Vera-10μM), MK571 (10μM), KO143 (500nM) served as positive controls. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), Dox (100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assessment of sensitivity to the drug combinations listed above were carried out in KG-1 cells, and its more immature variant KG-1a and in ex vivo CD34+ marrow cells from AML patients (AML post MDS n=5, de novo AML n=5). P-gp's ATPase activity was quantified with the luminescence-based Pgp-Gloä Assay System. Surface expression of P-gp was determined by FACS analysis and total protein expression of MRP, BCRP and P-gp by immunoblot analysis. Functional relevance of signaling pathways was tested using the SRC inhibitor PP2 (10μM) and the mTOR inhibitor Rapamicin (10nM). Results: We found that I) Erlo inhibited efflux via P-gp, MRP and BCRP as demonstrated by increased intracellular retention of DioC23/Rho-123, Calcein/CDCFDA and Hoechst 33342, respectively, andby its ability to retain MTX (300nM) and Dox (200nM) intracellularly II) Inhibition of drug efflux was higher in KG-1 than in KG-1a cellss, in agreement with a lower expression of P-gp and BCRP on KG-1a as compared to KG-1 cells; III) Quantification of VP-16 by RRLC after incubation with or without Erlo showed the ability of Erlo to increase intracellular VP-16 contents by approximately 60%; IV) Erlo increased ATPase activity in a dose-dependant manner, supporting the notion that Erlo is a competitive inhibitor of P-gp; IV) Erlo combined to VP-16 induced synergistic effects on apoptosis in KG-1 cells, and to a lesser extent in KG-1a (48h KG-1: Erlo 20%, VP-16 38%, Erlo+VP16 78%, KG-1a 48h: Erlo 10%, VP-16: 12%, Erlo+VP16: 35%); V) 48h of incubation with Erlo reduced cell surface expression of P-gp in KG-1 cells by 50%, whereas total P-gp protein expression remained unchanged, suggesting that Erlo interferes exclusively with the protein form expressed on the cell surface, VI) Decrease of P-gp cell surface expression was recapitulated upon incubation with PP2 (10μM) or Rapamicin (10nM); VII) the combination of Erlo+VP-16 in 10 AML-patient samples induced synergistic effects on apoptosis in 5 of them and additive effects in 3 of them. Conclusions: We here confirm that Erlo increases sensitivity towards chemotherapeutic agents subjected to drug efflux via ABC-transporters and delineate the molecular pathways conveying these effects. Disclosures: Fenaux: Celgene: Honoraria, Research Funding.

Induction Of Proliferation and Survival Of Multiple Myeloma Cells By CD137 Ligand Reverse Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2537-2537
Author(s):  
Chengcheng Fu ◽  
Hui Liu ◽  
Juan Wang ◽  
Ling Ma ◽  
Songguang Ju ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Counting ◽  
Myeloma Cells ◽  
Reverse Signaling

Abstract CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively, regulate cell activation and proliferation of immune system. CD137L, in addition to its ability to costimulate T cells by triggering CD137 receptor, also signals back into antigen presenting cells inducing proliferation, prolonging survival and enhancing secretion of proinflammatory cytokines. The expression of CD137L and its function on multiple myeloma cells is unknown. We identified the constitutive expression of CD137L by flow cytometry on U266, RPMI 8226, LP1, MY5 and KMS-11 of Multiple myeloma (MM) cell lines as high as 96%, 97.5%, 89%, 93% and 94%.But, CD137 expressed on the cell surface was low as 4%, 5%, 1%, 2%, 5% respectively. Now that, CD137L was expressed very strongly on MM cell lines, next, we investigated CD137L expression of MM cells from 85 BM samples of patients seen in the hematological Dept of the First Affiliated Hosp. of Soochow University between January 2012 and June 2013 and diagnosed of active multiple MM, including the patients of newly-diagnosed (n=35), relapsed (n=5) and after 2- 4 prior therapies (n=45). The BM samples were examined using antibodies against CD45RO PE-Cy7, CD138 APC-H7, CD38 FITC and CD137L PE, according to standard protocols for surface staining. Indeed, CD137L protein was expressed by a select group of CD45-CD38++CD138+cells as higher than 95%, the same, CD38 and CD138 are expressed more than 90% of the cells of CD45-CD137L+.There were 22 samples from 11 cases collected before and after treatment and this was further evidence that CD137L molecule was consistently expressed on the MM cell surface. However, CD137L expression was not or hardly detectable on normal plasma cells confirmed by CD45+CD38++CD138+ CD56- CD19+, indicating that CD137L was ectopically expressed by MM cells and probably a specific marker of MM cells. The ectopic CD137L expression was not a mere epiphenomenon but was selected for, what function of it? We hypothesized that it would also act as a growth stimulus for B cell cancers. Then we selected U266-a MM cell line to explore the biological effect of CD137L reverse signaling and its underlying mechanism. As a result, in vitro study, U266 cells(2X105/ml))were cultured plate pre-coated with mAb 1F1 or irrelevant mouse IgG at l ug/ml in PBS and at 400 ul per well of 24-well plate or 80 ul per well of 96-well plate and washed twice after overnight incubation at 4°C. The proliferation and survival of U266 was enhanced by stimulating- CD137L mAb (1F1) than those induced by control mouse IgG by cell counting (4.2 X105/ml VS 3.3 X105/ml), WST-8(1.15 VS 0.81) and CFSE assay (930 VS 991) at incubation for 48h. In addition, the cell cycle analysis showed that CD137L induces proliferation and increases the number of cells in the S phase from 36.1% to 42.5% after 72h incubation. The percentage of apoptosis cells (Annexin V+ and PI+) was 19.6% VS 21.2% with no statistical significance. In order to explore the mechanism of the function of CD137L on MM cells, we surveyed the cytokine profiles during the incubation of U266 cells cultured for 2 days with different stimuli with mAb 1F1 compared with the control group. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 from 3.8% to 63.9% by Flow cytometry. When neutralizing anti-IL-6 mAb (5 ug/ml) was added to the culture medium, the cells(2X105/ml))were cultured for 48 h in pure medium or plus 10 ng/ml Fc or CD137–Fc protein and the cell proliferation measured by WST-8 was 0.79 VS 0.80 VS 0.72.1F1-induced cell proliferation was effectively inhibited. IL-6 can promote cell proliferation and survival of MM. An increase of these cytokines might explain why CD137L expression could stimulate the proliferation of U266. Finally, the U266 cells were treated with bortezomib and the growth of cells was analyzed by WST-8 assay. It demonstrated that bortezomib could inhibit the function of 1F1 and the inhibition ratio of bortezomib was 22%, 51% and 58% at 24h, 48h and 72h. MM is a B-cell malignancy characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. In our study, CD137L is not only a novel ectopic constitutive marker of MM, but also a promoting proliferation factor. This suggests the possibility that its expression on MM cells may be directly target for immunomodulatory therapy for MM. Disclosures: No relevant conflicts of interest to declare.

Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?

2006 ◽  
Vol 290 (1) ◽  
pp. E123-E128 ◽  
Author(s):  
David C. Wright ◽  
Paige C. Geiger ◽  
Dong-Ho Han ◽  
John O. Holloszy
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Tyrosine Kinase ◽  
Glucose Transport ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Muscle Contractions ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5′-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 μM) and the related compound butein (100 μM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.

The mTOR Inhibitor Rapamycin Inhibits Drug Transport in Multidrug Resistant Cell Lines and in Acute Myeloid Leukemia (AML) Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1512-1512 ◽  
Author(s):  
Attaphol Pawarode ◽  
Kieran L. O’Loughlin ◽  
Nicholas W. Cuviello ◽  
William R. Greco ◽  
Laurie A. Ford ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Multidrug Resistance ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Drug Transport ◽  
Chemotherapy Drugs ◽  
Resistance Protein ◽  
Acute Myeloid

Abstract Mechanisms of drug resistance in acute myeloid leukemia (AML) include cellular drug efflux mediated by the multidrug resistance (MDR)-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp), multidrug resistance protein-1 (MRP-1) and breast cancer resistance protein (BCRP) and impaired cytoplasmic-nuclear drug transport mediated by the major vault protein lung resistance protein (LRP), as well as aberrant signal transduction due to activation of the mammalian target of rapamycin (mTOR) and other signaling molecules. Rapamycin has antiproliferative effects in AML by virtue of inhibition of mTOR and its downstream signaling pathways, and has clinical activity in this disease. Rapamycin is also known to block drug transport mediated by Pgp, and we sought to determine its effects on transport mediated by MRP-1, BCRP and LRP and its effects on cytotoxicity of MDR substrate drugs. Rapamycin effects on cellular uptake and efflux of mitoxantrone, a substrate for Pgp, MRP-1 and BCRP, were studied by flow cytometry in cell lines with MDR mediated by these proteins, and effects on nuclear-cytoplasmic distribution of doxorubicin were studied by confocal microscopy in cell lines with MDR mediated by LRP. Rapamycin cytotoxicity and effects on cytotoxicity of MDR protein substrate drugs were assessed in 96-well microculture survival assays. Additionally, effects on cellular drug transport were studied by flow cytometry in blasts from 30 untreated AML patients. Rapamycin, at ≥0.5 μM, had concentration-dependent inhibitory effects on drug transport mediated by Pgp, MRP-1 and BCRP and, at ≥2.5 μM, by LRP in cell lines with MDR mediated by these proteins. The magnitude of rapamycin effects was similar to those of the Pgp modulator PSC-833, the MRP-1 modulator MK-571 and the broad-spectrum MDR modulator cyclosporine A (CsA) in Pgp+ and MRP-1+ cell lines, and approximately half those of the BCRP modulator fumitremorgin C (FTC) and of CsA in BCRP+ cells. Rapamycin as a single agent was cytotoxic toward all cell lines tested and cytotoxicity was not affected by the presence of MDR proteins. Concurrent treatment with rapamycin and mitoxantrone resulted in a synergistic drug interaction in HL60/VCR and HL60/ADR cells, with 20-fold and 40-fold increases in potency compared with the expected effects of additivity, but synergy was not seen in parental HL60 cells. Finally, rapamycin increased mitoxantrone uptake and reduced efflux in 25 of 30 AML samples, and its effects on uptake were greater than those of CsA (p= 0.05; 2-tailed Wilcoxon signed-ranks test). Thus, in addition to its known effects on signal transduction, rapamycin also modulates drug transport in cell lines expressing Pgp, MRP-1, BCRP and LRP, synergizes with MDR protein substrate chemotherapy drugs in these cell lines, and enhances uptake and decreases efflux of MDR substrate drugs in AML cells. Effects of rapamycin on transport of chemotherapy drugs, as well as on signal transduction, should be considered in the design of combination regimens in AML.

Erlotinib Inhibits ABC Transporters of AML Progenitors with Stem Cell Features and Increases Chemosensitivity to Current AML Drugs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2163-2163
Author(s):  
Marie Sebert ◽  
Elodie Lainey ◽  
Sylvain Thepot ◽  
Maximilien Tailler ◽  
Lionel Ades ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Surface ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Myeloid Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Induced Apoptosis ◽  
Over Time

Abstract Abstract 2163 Background: Treatment failure in AML is attributed to the persistence of AML progenitors able, among others, to efflux chemotherapeutic drugs via ABC-transporters. Increased efflux capacity is considered a stem cell feature, and therapeutic inhibition may increase chemosensitivity and help eradicate this progenitor population. Nevertheless, clinical studies assessing a potential benefit of ABC-inhibitors in AML treatment showed no significant survival advantage, possibly because AML cells express different ABC-transporters and classical inhibitors target only a restricted type of efflux channels. We assessed the efficacy of the TKI erlotinib (Erlo) to antagonize drug efflux via most important AML-associated efflux channels, ie P-gp, MRP and BCRP. Methods: Overall drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ), and specific efflux via P-gp (substrates: DioC23 and rhodamine-123), MRP (substrates: calcein and CDCFDA) and BCRP (substrate: Hoechst 33342) were quantified by FACS at 1h and 6h following incubation with 10mM Erlo. Biochemical inhibitors of the respective ABC-transporters (CSA, verapamil, MK-571, KO143) served as controls. Surface expression of P-gp, MRP and BCRP was quantified by FACS. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), doxorubicine (Dox, 100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assays were carried out in myeloid cell lines (KG-1, MOLM-13, HL-60) and ex vivo AML cells (n=3). Immaturity of AML cells was determined in 2 samples by comparing CD34+ versus CD34- cells, and in one pt by co-staining for CD34, CD38, CD123 and CD133. Results: We found that I) Erlo inhibited efflux via P-gp and MRP as demonstrated by increased intracellular retention of DioC23/Rho-123, and calcein/CDCFDA, respectively; II) this degree of inhibition was higher in KG-1 cells than in MOLM-13 or HL-60 cells; III) inhibition of drug efflux was observed already at 1h of incubation, increased over time (6h); IV) Erlo increased intracellular retention of MTZ faster (at 1h with a further increase at 6h) and at least to the same extent than a combination of all three biochemical efflux inhibitors, showing that Erlo's capacity to hinder drug efflux is not restricted to a single ABC-transporter: V) surface expression of P-gp, MRP and BCRP was strongest on KG-1 cells and not altered upon 1h and 6h of Erlo incubation VI) Erlo increased Dox- and VP16-induced apoptosis (48h KG-1: Erlo alone 20%, Dox alone 10%, VP-16 alone 20%, Erlo+Dox: 40%, VP-16+Erlo: 70%), while having no impact on AraC-induced apoptosis; VI) this pattern of chemosensitization was observed in all myeloid cell lines, but once more most pronounced in KG-1 cells. To test the hypothesis that Erlo has comparable effects in pt-derived AML cells ex vivo, we showed by concomitant cell surface staining that I) immature AML subpopulations had a higher efflux capacity (notably via P-gp) than their more mature counterparts (i.e. in one pt with chemoresistant AML: DioC23/Rho-123 fluorescence twice as high in the CD34-/CD38+, CD123+, CD133- than in the CD34+/CD38dim, CD123-, CD133+ subpopulation); II) cell surface expression of P-gp is twice as high in this more immature population (CD34+/CD38dim, CD123-, CD133+) than in CD34-/CD38+, CD123+, CD133+ cells; III) Erlo antagonizes drug efflux via P-gp and MRP at 1h (increasing further at 6h) of incubation; IV) this effect is most pronounced in the immature progenitor cells (1h: decrease of DioC23/Rho-123 efflux in CD34-/CD38+, CD123+, CD133- cells by about 50% and in the more immature CD34-/CD38+, CD123-, CD133+ cells by about 70%); V) Erlo diminishes cell surface expression of P-gp (48h), most effectively in the progenitor populations (by 30% in the CD34-/CD38+, CD123+, CD133- cells versus 50% in CD34-/CD38+, CD123+, CD133- cells); VI) Erlo is able to retain MTZ in both CD34- and CD34+ AML-subpopulations; VII) these effects are accompanied by an increased sensitivity towards Dox and VP-16; VIII) Erlo-induced chemosensitization is higher in the CD34+ than in CD34- AML cells. Conclusions: We here provide novel evidence that erlotinib is able to overcome the stem cell features of increased expression and functionality of ABC-transporters thereby antagonizing the intrinsic chemoresistance of (immature) AML cells. Those results suggest a potential clinical interest of combining erlotinib to chemotherapy in AML Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

Multiple Myeloma Cells Modulate ICAM-3 To Evade Natural Killer Cell-Mediated Lysis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3105-3105
Author(s):  
Tarun K. Garg ◽  
Ricky D Edmondson ◽  
Shweta S. Chavan ◽  
Junaid Khan ◽  
Susann Szmania ◽  
...  
Keyword(s):  
Amino Acids ◽  
Flow Cytometry ◽  
High Risk ◽  
Cell Surface ◽  
Natural Killer ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Differentially Expressed ◽  
Myeloma Cells ◽  
Chromium Release

Abstract Introduction Ex vivo activated/expanded natural killer (ENK) cells can induce myeloma cell lysis both in vitro and in murine models and are currently being studied clinically in the setting of high-risk relapsing disease and asymptomatic disease at high risk of progression. This prompted us to study, in myeloma cell lines, whether intrinsic resistance to ENK cell lysis exists, whether repeated challenge with ENK leads to increased resistance, and what the underlying mechanisms of resistance are. Of 11 myeloma cell lines tested in standard 4h chromium release assays, 8 were avidly killed (78-89% lysis, E:T Ratio 10:1) whereas 3 lines were less sensitive (41-65% lysis). Repeated exposure to ENK challenge decreased sensitivity in 4 of 11 lines, that was at least in part due to down-regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors on the myeloma cell surface (Garg et al, Blood 2012, 120:4020). In this study we investigated the resistance issue further via metabolomics, gene expression profiling (GEP) and flow cytometry analysis of OPM2, which was intrinsically resistant and developed further resistance after challenge with ENK cells. Methods Metabolomics was studied using a quantitative proteomic strategy entailing stable isotope labeling with amino acids in cell culture – mass spectrometry (SILAC-MS). Resistant and parental OPM2 cells were grown either in medium with heavy amino acids (13C6 L-Lysine and 13C6 L-Arginine) or with light amino acids (12C6 L-Lysine and 12C6L-Arginine). Reverse labeling with heavy or light amino acids was also done to confirm the results. Cell lysates from heavy and light amino acid labeled cells were pooled, simultaneously resolved on SDS-PAGE, protein bands were excised and analyzed on a mass spectrometer after trypsin digestion. GEP was performed using the Affymetrix U133 Plus 2.0 microarray platform (Santa Clara, CA). The fold change of signal intensity for genes and proteins in resistant vs. parental OPM2 was calculated. The most differentially expressed genes (top 150-fold up or down) and proteins (up or down by 1.3-fold) were compared for commonality. Cell surface protein expression was determined via flow cytometry. The ability of ENK to lyse myeloma cell targets in the presence of isotype control or ICAM-3 blocking antibody was tested in 4h chromium release assays. Results Metabolomics identified >3800 proteins and revealed that the abundance of 352 proteins was significantly altered in resistant myeloma cells. These altered proteins were mainly associated with cell cycle, morphology, organization, cellular compromise, immune response, and survival. Further, a comparison of these differentially expressed proteins with GEP data revealed 3 commonly up-regulated molecules: TBC1D8B, HSPA1A and IFI16; and 2 down-regulated molecules: intercellular adhesion molecule (ICAM-3) and BAI3. Of these, ICAM-3, a ligand for leukocyte function-associated antigen-1 (LFA-1) and a potent signaling molecule, was selected for further studies. Flow cytometry confirmed that ICAM-3 cell surface expression was > 8-fold lower on resistant versus parental OPM2 cells. Further, blocking of ICAM-3 in cytotoxicity assays resulted in decreased lysis (43% blocked, E:T ratio 5:1), suggesting that this molecule is functionally important and takes part in ENK cell-mediated killing. Conclusion In conclusion, quantitative proteomic analysis demonstrated dynamic changes in the ENK-resistant OPM2 myeloma cells that correlated with GEP and differences in ICAM-3 expression may have functional implications. Studies evaluating the expression of ICAM-3 in myeloma patients at diagnosis and relapse are in progress. Myeloma cells may down-regulate ICAM-3 as a mechanism of escape from immune surveillance and therefore, ICAM-3 may be a useful biomarker to predict sensitivity to ENK cell-mediated killing and aid in the selection of patients most likely to benefit from ENK cell therapy. Disclosures: Barlogie: Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

scholarly journals The Second-Generation PIM Kinase Inhibitor TP-3654 Resensitizes ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs

2021 ◽  
Vol 22 (17) ◽  
pp. 9440
Author(s):  
Chung-Pu Wu ◽  
Yan-Qing Li ◽  
Ya-Chen Chi ◽  
Yang-Hui Huang ◽  
Tai-Ho Hung ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Anticancer Drugs ◽  
Drug Transport ◽  
Second Generation ◽  
Kinase Inhibitor ◽  
Multidrug Resistant ◽  
Drug Efflux ◽  
Transport Function ◽  
Therapy Option ◽  
Induction Of Resistance

Human ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) mediates the transport of a wide variety of conventional cytotoxic anticancer drugs and molecular targeted agents. Consequently, the overexpression of ABCG2 in cancer cells is linked to the development of the multidrug resistance (MDR) phenotype. TP-3654 is an experimental second-generation inhibitor of PIM kinase that is currently under investigation in clinical trials to treat advanced solid tumors and myelofibrosis. In this study, we discovered that by attenuating the drug transport function of ABCG2, TP-3654 resensitizes ABCG2-overexpressing multidrug-resistant cancer cells to cytotoxic ABCG2 substrate drugs topotecan, SN-38 and mitoxantrone. Moreover, our results indicate that ABCG2 does not mediate resistance to TP-3654 and may not play a major role in the induction of resistance to TP-3654 in cancer patients. Taken together, our findings reveal that TP-3654 is a selective, potent modulator of ABCG2 drug efflux function that may offer an additional combination therapy option for the treatment of multidrug-resistant cancers.

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