GO-induced Cytotoxicity Research Articles

CRISPR/Cas9 gene editing clarifies the role of CD33 SNP rs12459419 in gemtuzumab ozogamicin-mediated cytotoxicity.

The single-nucleotide polymorphism (SNP) rs12459419 is located at the intron/exon junction of CD33 exon2. When exon2 is skipped by this CD33 SNP, the full-length CD33 (CD33FL) is converted to a short CD33 isoform (CD33D2). Since gemtuzumab ozogamicin (GO) only recognizes CD33FL, the CD33 SNP may affect the clinical efficacy of GO. To elucidate the significance of CD33 SNP on GO reactivity, we leveraged the CRISPR/Cas9 genome-editing system to create OCI-AML3 cell lines with specifically modified CD33 SNPs. Levels of CD33 D2 mRNA were significantly higher in the T/T clone (p < 0.001), but CD33D2 protein was not detectable in any clones. There was no significant difference in CD33FL mRNA expression across edited clones, and CD33FL protein expression was lowest in T/T clones, followed by T/C and C/C. Cytotoxicity assays revealed that the IC50 of GO was significantly lower in T/C and C/C clones than in the T/T clone (p < 0.001). Our study demonstrated a difference in GO-induced cytotoxicity in CD33 SNP-edited clones, clearly indicating that at least one CD33 SNP allele, rs12459419 C, is important for sensitivity to GO.

Understanding the Role of the Lateral Dimensional Property of Graphene Oxide on Its Interactions with Renal Cells.

Renal excretion is expected to be the major route for the elimination of biomedically applied nanoparticles from the body. Hence, understanding the nanomedicine-kidney interaction is crucially required, but it is still far from being understood. Herein, we explored the lateral dimension- (~70 nm and ~300 nm), dose- (1, 5, and 15 mg/kg in vivo and 0.1~250 μg/mL in vitro), and time-dependent (48 h and 7 d in vivo) deposition and injury of PEGylated graphene oxide sheets (GOs) in the kidney after i.v. injection in mice. We specially investigated the cytotoxic effects on three typical kidney cell types with which GO renal excretion is related: human renal glomerular endothelial cells (HRGECs) and human podocytes, and human proximal tubular epithelial cells (HK-2). By using in vivo fluorescence imaging and in situ Raman imaging and spectroscopic analysis, we revealed that GOs could gradually be eliminated from the kidneys, where the glomeruli and renal tubules are their target deposition sites, but only the high dose of GO injection induced obvious renal histological and ultrastructural changes. We showed that the high-dose GO-induced cytotoxicity included a cell viability decrease and cellular apoptosis increase. GO uptake by renal cells triggered cellular membrane damage (intracellular LDH release) and increased levels of oxidative stress (ROS level elevation and a decrease in the balance of the GSH/GSSG ratio) accompanied by a mitochondrial membrane potential decrease and up-regulation of the expression of pro-inflammatory cytokines TNF-α and IL-18, resulting in cellular apoptosis. GO treatments activated Keap1/Nrf2 signaling; however, the antioxidant function of Nrf2 could be inhibited by apoptotic engagement. GO-induced cytotoxicity was demonstrated to be associated with oxidative stress and an inflammation reaction. Generally, the l-GOs presented more pronounced cytotoxicity and more severe cellular injury than s-GOs did, demonstrating lateral size-dependent toxicity to the renal cells. More importantly, GO-induced cytotoxicity was independent of renal cell type. The results suggest that the dosage of GOs in biomedical applications should be considered and that more attention should be paid to the ability of a high dose of GO to cause renal deposition and potential nephrotoxicity.

Protein corona reduced graphene oxide cytotoxicity by inhibiting endocytosis

Exposure to graphene is increasing with the extensive application and production of graphene and its derivatives. Graphene may be exposed to a variety of biomicroenvironments, in which the concentrations of biomacromolecules vary. Thus, it is essential to study the toxic behaviors of graphene in different biomicroenvironments. In the present study, we investigated the toxic behaviors caused by graphene oxide (GO) in different biomicroenvironments and its potential mechanisms. Our results revealed that GO injured cells through reactive oxygen species (ROS) induction and induced apoptosis in protein-free microenvironment. However, GO-induced cytotoxicity was weakened remarkably in the presence of protein, which attributed to the reduction of GO internalization and ROS generation. GO induced cell cycle arrest in the G0/G1 phase when the protein concentration is low but had no remarkable cytotoxicity when the protein concentration was high. Our findings provide a basis for the prevention graphene toxicity.

Transcriptomic analysis suggested the involvement of impaired lipid droplet biogenesis in graphene oxide-induced cytotoxicity in human umbilical vein endothelial cells

Previous studies revealed that direct contact with graphene oxide (GO) induced cytotoxic effects, but the importance of involvement of metabolic pathways, in particular lipid metabolism pathways, might be overlooked. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to GO with large size (denoted as GO-L) or small size (denoted as GO-S), and transcriptomics were used to understand the mechanisms of cytotoxicity of GO at systemic levels. It was shown that GO-L more significantly induced cytotoxicity compared with GO-S. Transcriptomic analysis revealed that compared with GO-S, GO-L had larger impact on gene ontology terms related with mitochondrial function as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related with cell death and growth. But GO-S showed greater influence on KEGG pathways related with lipid metabolism. Both types of GO showed minimal impact on oxidative stress but increased de novo lipogenesis protein fatty acid synthase (FASN). However, only GO-S significantly promoted acyl-CoA synthetase 3 (ACSL3), a key enzyme responsible for esterification of free fatty acids and lipid droplet biogenesis. Not surprisingly, GO-L but not GO-S impaired lipid droplet biogenesis, and increasing lipid levels by oleic acid or α-linolenic acid reduced the cytotoxicity of GO-L to HUVECs. Combined, the results from this study suggested that impaired lipid droplet biogenesis was involved in GO-induced cytotoxicity in HUVECs, and inducing lipid droplet biogenesis could prevent the cytotoxicity of GO.

An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells.

Gemtuzumab ozogamicin (GO), an anti-CD33 antibody linked to calicheamicin via an acid-labile linker, is the first antibody-drug conjugate (ADC). The acidic environment inside lysosomes of target cells is an important intracellular determinant of the cytocidal action of GO, as the linker is hydrolyzed under acidic conditions. However, lysosomal activity in acute myeloid leukemia (AML) blasts in GO therapy has been insufficiently evaluated. It has been suggested that lysosome activity is suppressed in AML due to hyperactivation of the phosphoinositide 3-kinase/Akt pathway. We therefore hypothesized that agents which activate lysosomal function would potentiate the cytotoxicity of GO. Here, we found that a clinically useful mTORC1/2 dual inhibitor, AZD2014, reduced pH in the acidic organelles, including lysosomes, as shown by increased LysoTracker fluorescent intensity, and synergistically enhanced the cytotoxic effect of GO in primary leukemia cells. GO-induced cytotoxicity appeared to be enhanced with the increase in lysosomal activity by AZD2014. These results indicate that AZD2014 activated lysosomal function in primary leukemia cells, which in turn enhanced the cytotoxicity of GO. Enhancement of lysosomal activity may represent a new therapeutic strategy in the treatment of GO and other ADCs, particularly in cases with low lysosomal activity.

Live Imaging of Label-Free Graphene Oxide Reveals Critical Factors Causing Oxidative-Stress-Mediated Cellular Responses.

The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and the pulmonary system. The aim of the present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions, s-GO (50-200 nm) and l-GO (5-15 μm), were produced and thoroughly characterized. Exploiting intrinsic fluorescence of graphene oxide (GO) and using confocal live-cell imaging, the behavior of the cells in response to the material was visualized in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated reactive oxygen species (ROS) levels, pro-inflammatory response, and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, the lateral dimension of GO played a more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO-induced cytotoxicity.

Graphene oxide induces cytotoxicity and oxidative stress in bluegill sunfish cells.

Graphene oxide (GO) is considered a promising material for biological application due to its unique properties. However, the potential toxicity of GO to aquatic organism particularly bluegill sun fish cells (BF-2) is unexplored or remains poorly understood. GO-induced cytotoxicity and oxidative stress in BF-2 cells were assessed using a battery of biomarkers. Two different biological assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake were used to evaluate the cytotoxicity of GO on BF-2 cells. It was found that GO induced dose- and time-dependent cytotoxicity on BF-2 cells. BF-2 cells exposed to lower concentration of GO (40μg ml-1 ) for 24 induced morphological changes when compared to their respective controls. As evidence for oxidative stress lipid peroxidation, superoxide dismutase, catalase, reactive oxygen species and 8-hydroxy-2'-deoxyguanosine levels were increased and glutathione levels were found to decline in BF-2 cells after treatment with GO. Our findings demonstrate that GO when exposed to BF-2 fish cells cause oxidative stress.

ABCA3 Expression Predicts Response to Gemtuzumab Ozogamicin in AML

Background. The ATP binding cassette transporter 3 (ABCA3) has been recently found to induce a significant reduction in cytotoxicity following exposure to anthracyclines, mitoxantrone, etoposide, Ara-C, vincristine, and rituximab. ABCA3 acts through the modulation of multivesicular bodies (MVB) and contributes to drug sequestration in late endosomal organelles, i.e. MVB and lysosomes. Studies having investigated the prognostic impact of ABCA3 expression in AML have yielded conflicting results as ABCA3 expression has both been reported to exert unfavorable or neutral effects on patient outcomes. In addition, the small sample size of these studies precluded the use of multivariate analyses.Methods. Our goal was to investigate the prognostic impact of ABCA3 expression in adult patients with AML treated with IC with or without gemtuzumab ozogamicin (GO). To this end we investigated the relationship between ABCA3 expression and EFS in a representative series of 221 AML homogeneously treated in the ALFA-0701 trial. qRTPCR amplification of conserved ABCA3 mRNA sequences, as identified with FasterDB database, was performed with GUS and ABL as reference genes. Primer sets were complementary to conserved ABCA3 exons 6-7 and exon 19-20 junctions. Patients were given a 3+7 induction course without (control group, n=110) or with fractionated intravenous GO (n=111) (Castaigne S, Lancet 2012; 379:1508-1516).Results. Among the 278 randomized patients, 221 had available bone-marrow diagnostic samples with high-quality RNA. The same benefits associated with GO were observed in the 221 patients from the present study as in the entire trial population. Overall, median age, CR rate, relapse rate, median follow-up, 3-years EFS were 62.1 years, 76.5%, 66%, 47.45 months, 28±3%, respectively. There was no significant difference in the level of ABCA3 expression between responders and non-responders. In the 169 responders, ABCA3 expression at diagnosis was more than 3-fold higher in the 111 remitters who subsequently relapsed than in the 58 patients who remained in persistent CR (p=0.033). The level of ABCA3 expression was significantly lower in ELN favorable group than in intermediate and adverse risk AML (p= 0.004) and negatively correlated with CD33 expression (R=-0.272, p<10-4). Through univariate analysis, higher ABCA3 expression was associated with shorter EFS (3-years: 22±3 vs 45±7 % p=0.002). Multivariate analysis identified age, treatment arm, and ELN risk group as independent prognostic factors for EFS. In the control group, there was no significant association between ABCA3 expression and CR rate, relapse rate, and EFS. In the 111 patients within the GO arm, there was no significant difference in the level of ABCA3 expression between responders and non-responder whereas in the 89 responders, ABCA3 expression at diagnosis was more than 7-fold higher in the 53 remitters who subsequently relapsed than in the 36 patients who remained in persistent CR (p=0.006). Through univariate analysis, higher ABCA3 expression was associated with shorter EFS (3-years: 22±5 vs 64±9 % p=0.0002). Multivariate analysis identified ABCA3 expression, cytogenetics, CD33 expression, and ECOG as independent prognostic factors for EFS (Figure 1).Conclusion. WhileABCB1 has been previously found to attenuate GO-induced cytotoxicity in AML cells (Walter RB, Blood 2003; 102:1466-1473), present results indicate that higher ABCA3 expression independently predicts poor outcome in AML patients treated with fractionated GO and intensive chemotherapy (IC). GO is an anti-CD33 antibody carrying a toxic calicheamicin derivative that, after hydrolytic release within lysosomal vesicles, induces DNA strand breaks, apoptosis, and cell death. Whether the clinical effect of ABCA3 expression relies on the modulation of CD33 internalization, calicheamicin release or combination thereof is under investigation. Finally our results encourage inhibiting ABCA3, such as with indomethacin, in order to overcome drug resistance in AML treated with GO-IC. [Display omitted] DisclosuresThomas:Pfizer: Consultancy.

Evaluation of the toxicity of graphene oxide exposure to the eye

Graphene and its derivatives are the new carbon nanomaterials with the prospect for great applications in electronics, energy storage, biosensors and medicine. However, little is known about the toxicity of graphene or its derivatives in the case of occasional or repeated ocular exposure. We performed in vitro and in vivo studies to evaluate the toxicity of graphene oxide (GO) exposure to the eye. Primary human corneal epithelium cells (hCorECs) and human conjunctiva epithelium cells (hConECs) were exposed to GO (12.5–100 μg/mL). Acute GO exposure (2 h) did not induce cytotoxicity to hCorECs. However, short-term GO exposure (24 h) exerted significant cytotoxicity to hCorECs and hConECs with increased intracellular reactive oxygen species (ROS). Glutathione (GSH) reduced the GO-induced cytotoxicity. We further performed acute eye irritation tests in albino rabbits according to the Organization for Economic Cooperation and Development (OECD) guidelines, and the rabbits did not exhibit corneal opacity, conjunctival redness, abnormality of the iris, or chemosis at any time point after the instillation of 100 μg/mL of GO. However, 5-day repeated GO exposure (50 and 100 μg/mL) caused reversible mild corneal opacity, conjunctival redness and corneal epithelium damage to Sprague-Dawley rats, which was also alleviated by GSH. Therefore, our study suggests that GO-induced time- and dose-dependent cytotoxicity to hCorECs and hConECs via oxidative stress. Occasional GO exposure did not cause acute eye irritation; short-term repeated GO exposure generally resulted in reversible damage to the eye via oxidative stress, which may be alleviated by the antioxidant GSH.

AKT Signaling as a Novel Factor Associated with In Vitro Resistance of Human AML to Gemtuzumab Ozogamicin

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ1 derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ1 -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ1 and, by extrapolation, other DNA damage-based therapeutics.

Mechanisms of Discrepancy Between CD33 Expression and Gemtuzumab Ozogamicin-Induced Cytotoxicity in Leukemia Cells.

Abstract 4812Gemtuzumab ozogamicin (GO) is an immunoconjugate of an anti-CD33 antibody and a toxic calicheamicin-γ1 derivative that facilitates cellular uptake of the latter drug. CD33 is selectively expressed on myeloid cells, especially on acute myeloid leukemia (AML) cells, but usually not on non-hematopoietic cells, and therefore the clinical use is confined to AML. Despite its predominant expression on AML cells, GO induces complete remission only in 30% of recurrent AML. The major side-effects of GO include not only myelosuppression but also hepatotoxicity, although CD33 is not expressed on hepatobiliary cells. It suggests that the efficacy of GO is not strictly regulated by CD33 expression. We investigated relationship between CD33 expression and GO cytotoxicity by using leukemia cell lines, and classified them into four groups; group A defined as CD33(+) and cytotoxicity (+), group B as CD33(+) and cytotoxicity (-), group C as CD33(-) and cytotoxicity (+), and group D as CD33(-) and cytotoxicity (-). We selected four cell lines, HL-60, MEG-01, MOLT-3 and K562 which belong to group A, B, C and D, respectively, and were used for further experiment. CD33 was expressed on HL-60 but not on MOLT-3. However, GO-induced cytotoxicity was seen in both cells. We therefore compared effects of GO in HL-60 and MOLT-3. When incubated with GO for 72 hours, viability of HL-60 significantly decreased at 10 ng/mL of GO, while cytotoxic effect of GO against MOLT-3 became apparent when GO concentration was raised to 100 ng/mL. Viability of HL-60 markedly decreased when treated with GO for 30 minutes and then cultured in medium without containing GO for 72 hours, but no cytotoxic effect was observed in MOLT-3 unless culture with GO was prolonged to more than 6 hours. GO-induced apoptosis was detected in both HL-60 and MOLT-3 by Annexin V assay and TUNEL assay. The pretreatment with unconjugated anti-CD33 antibody (hP67.6) resulted in reduced cytotoxic effects of GO in HL-60, but not in MOLT-3. It indicates that GO induces apoptosis in CD33-non-expressing MOLT-3. Higher doses and longer exposure of GO treatment are necessary to induce cytotoxicity against MOLT-3 when compared with HL-60. GO might cause cytotoxicity against MOLT-3 through drug absorption pathway other than CD33-mediated endocytosis. Although HL-60 and MEG-01 expressed CD33, GO induced cytotoxicity on HL-60, but not on MEG-01. We then investigated internalization of antibody-bound CD33. After incubated with hP67.6 on ice, cells were cultured in antibody-free medium, and remaining cell surface-associated CD33 was measured. The loss of cell surface CD33 was observed in MEG-01 as well as HL-60, showing that internalization of CD33 following antibody binding occurred in MEG-01. It indicates that resistance to GO-induced cytotoxicity in MEG-01 is not associated with internalization property. Drug transport pathway other than receptor-mediated endocytosis was investigated. Cells were cultured in rhodamine 123 (Rh123)-containing medium for 30 minutes and the influx of Rh123 was assessed by the fluorescence activity of Rh123-containing cells. Cells were then cultured in medium without containing Rh123 for further 3 hours, and the efflux of Rh123 was analyzed by measuring the reduction curve of fluorescence activity. Rh123 accumulated in MEG-01 twice higher than HL-60. However, fluorescence activity of Rh123 decreased to 20% after 3 hours, while it remained unchanged in HL-60, indicating that drug efflux was activated in MEG-01. Multidrug resistance (MDR)-1 protein was strongly expressed in MEG-01. When cyclosporin-A, a modulator of MDR-1, was given with GO to MEG-01, GO-induced cytotoxicity was significantly increased, while no such effect was observed in HL-60. It suggests that p-glycoprotein-mediated efflux of GO is one of the resistance mechanisms in MEG-01. Disclosures:No relevant conflicts of interest to declare.

Ubenimex Enhances the Gemtuzumab Ozogamicin-Induced Cytotoxicity Against Myeloid Leukemia Cells.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to a derivative of a tumoricidal antibiotic calicheamicin. GO alone induces complete remission in 26% of AML patients in first relapse. One way to improve the response rate is to combine GO with other agents. A quantitative relationship has been shown between rates and degrees of CD33 expression and GO-induced cytotoxicity in vitro by using gene transfer to manipulate CD33 expression in myeloid cell lines. We first studied CD33 expression on myeloid leukemia cells by stimulating with several agents in vitro, and found that ubenimex, but not G-CSF, M-CSF, or ATRA, increased CD33 expression on both HL-60 and K562 cells. Ubenimex is an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli and commercially available as an oral agent for clinical use for AML treatment. To investigate whether ubenimex enhances the cytotoxicity of GO, HL-60 and K562 cells were cultured with GO and/or ubenimex for 3 days. When GO was administered, cell viabilities of HL-60 and K562 were reduced to 31.5% and 90.3% as compared with control, respectively. Treatment with ubenimex alone did not influence viabilities of either HL-60 or K562. However, when cells were preincubated with ubenimex and then cultured with GO, cell viabilities decreased to 18.5% and 81.4% for HL-60 and K562, respectively, indicating that pretreatment with ubenimex enhanced GO-induced myeloid leukemia cell death in vitro. We next assessed the mechanism of cell death. The treatment with GO alone and ubenimex alone induced apoptosis in 39.2% and 2.9% of HL-60 cells, respectively. When HL-60 cells were preincubated with ubenimex and then cultured with GO, the number of apoptotic cells increased to 62.9%, demonstrating that ubenimex augments GO-induced apoptosis. Our data suggest that the priming of AML cells with ubenimex should improve the clinical efficacy of GO.

Tyrosine Phosphorylation Enhances ITIM-Dependent Internalization of CD33, the Target for the Anti-AML Immunoconjugate, Gemtuzumab Ozogamicin.

Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing.Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33+ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33− Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33+ AML cell lines.Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed.Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.

Relationship between CD33 Expression, P-Glycoprotein-Mediated Drug Efflux, and Clinical Outcome in Patients Treated in Phase II Trials with Gemtuzumab Ozogamicin Monotherapy.

Background: The anti-acute myeloid leukemia (AML) immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains a humanized anti-CD33 antibody (hP67.6) to facilitate uptake of the toxic calicheamicin-γ1 derivative in CD33-positive AML cells. This putative mechanism implies a critical role for the intracellular accumulation of the toxic moiety for GO-induced cytotoxicity. Indeed, drug efflux by P-glycoprotein (Pgp) mediates resistance to GO and correlates with clinical outcome after GO monotherapy. Furthermore, recent in vitro data obtained in human myeloid cell lines have unequivocally demonstrated a quantitative relationship between CD33 expression and GO-induced cytotoxicity. In light of these findings, we have now re-examined the significance of CD33 expression levels on AML blasts and relationship with Pgp activity for clinical outcome of patients treated with GO monotherapy.Methods: Pre-treatment bone marrow samples from patients enrolled in multicenter phase II protocols evaluating the safety and efficacy of GO monotherapy (generally 2 doses of 9 mg/m2 14 days apart) were used for analysis. Relative CD33 expression was quantified by flowcytometry immunophenotyping using the hP67.6 antibody, and linear fluorescence values used for calculations. Pgp function was cytofluorometrically determined by efflux of the fluorescent dye, DiOC2. Results are presented as mean values and 95% confidence intervals. Unpaired t-tests, Pearson correlations, and logistic regression models were used for statistical analysis.Results: Patients achieving a complete remission (CR) or CR with incomplete recovery of platelet counts (CRp) had statistically significantly higher mean CD33 expression levels (71.20 [57.20–85.19], n=69) compared to non-responders (54.44 [48.38–60.51], n=203; p=0.01). There was an inverse relationship between CD33 expression and Pgp efflux (r=−0.23) and this contributed to responders having a statistically significantly lower mean Pgp efflux (1.40 [1.28–1.52], n=57) compared to non-responders (1.83 [1.72–1.95], n=173; p<0.0001). The addition of Pgp statistically significantly improved a logistic regression model containing only CD33 (p<.0001), whereas the addition of CD33 did not lead to a statistically significantly improved logistic regression model containing only Pgp (p=0.14).Conclusion: These data indicate that CD33 expression levels as well as Pgp efflux are associated with clinical outcome of patients treated with GO monotherapy, and that knowledge of Pgp provides important information regarding the probability of achieving a response, even after consideration of CD33 expression level. The inverse relationship between CD33 abundance and Pgp efflux is consistent with the notion of maturation-stage dependent expression of these proteins, and offers the rationale for the use of cell-differentiation-promoting agents, for example cytokines, in combination with GO to enhance GO-induced cytotoxicity and possibly improve clinical outcome of patients undergoing GO-containing AML therapy.

Granulocyte colony-stimulating factor enhances the in vitro cytotoxicity of gemtuzumab ozogamicin against acute myeloid leukemia cell lines and primary blast cells

To evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on the in vitro sensitivity of acute myeloid leukemia (AML) cell lines and primary AML blast cells to gemtuzumab ozogamicin (GO). AML cell lines and primary blasts from 10 patients with AML were first incubated for 72 hours in the presence of G-CSF (5 or 100 ng/mL) and then exposed to increasing concentrations of GO (1-1,000 ng/mL) for an additional 72 hours. Pretreatment with G-CSF translated into significant enhancement of GO-induced cytotoxicity in the GO-sensitive HL-60 and NB-4 cells. Conversely, the response of GO-insensitive KG-1a, TF-1, and K562 cells was unaffected by in vitro priming with G-CSF. In vitro exposure to G-CSF augmented GO-induced apoptosis in 7 of 10 primary AML samples and rendered blast cells from three refractory patients sensitive to killing effect of GO. The G-CSF-induced increase of the cytocidal activity of GO was independent of effects on the cell cycle and on the expression levels of CD33 antigen. Of potential interest, G-CSF induced dose-dependent inhibition of P-glycoprotein (P-gp/ABCB1) function in the GO-sensitive HL-60 and NB-4 cells and in blasts from three patients with AML that we tested. Collectively, our findings point to G-CSF as a potential sensitizing agent that can be exploited therapeutically to improve the clinical efficacy of GO.

Lysosomal Degradation of CD33, the Target for the Anti-Leukemia Immunoconjugate, Gemtuzumab Ozogamicin: Evidence for Cbl-Mediated Monoubiquitination.

Background: CD33, the target for the anti-leukemia immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), is a transmembrane glycoprotein that contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Although we have previously shown that disruption of the ITIMs prevents effective uptake of antibody-bound CD33 and significantly reduces GO-induced cytotoxicity, the mechanisms underlying this uptake and intracellular trafficking of the antibody-CD33 complex are not known. In this study, we tested whether CD33 is a target for monoubiquitination, a posttranslational modification that marks proteins for lysosomal degradation, and how this modification relates to tyrosine phosphorylation and endocytosis.Methods: CD33− Jurkat and 32D cells transduced with a lentiviral vector expressing either wild-type or mutant CD33 were analyzed for CD33 expression as well as internalization of anti-CD33 antibody by flow cytometry. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33 or TKB domain of Cbl/Cbl-b, using human myeloid cell lysates. For co-immunoprecipitation experiments, constructs encoding wild-type and mutant CD33, ubiquitin, Cbl/Cbl-b, and wild-type Fyn were transfected into HEK293T cells.Results: In engineered Jurkat cells, treatment with either anti-CD33 antibody or the tyrosine phosphatase inhibitor pervandate increased tyrosine phosphorylation of CD33. Pervanadate enhanced uptake of antibody-bound CD33; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2. CD33 interacted with the TKB domains of Cbl and Cbl-b in GST fusion protein pulldown assays. Similarly, Cbl and Cbl-b were co-immunoprecipitated with CD33 in transfected 293T cells. Experiments in 293T cells further showed that CD33 is monoubiquitinated in an ITIM-dependent manner. Cbl or Cbl-b significantly increased the amount of CD33-associated ubiquitination; Cbl/Cbl-b-dependent ubiquitination could further be enhanced by co-transfected Fyn. Finally, 32D cells expressing lysine-to-arginine mutants of CD33 displayed much higher levels of surface CD33 but had reduced internalization of antibody-bound CD33 compared to cells expressing wild-type CD33, consistent with a reduced internalization/degradation of the lysine-to-arginine mutants.Conclusion: These studies indicate that Src-family kinase dependent phosphorylation favors internalization of antibody-bound CD33 and identify Cbl family proteins as potential binding partners of CD33. Importantly, CD33 is a target for ITIM-dependent monoubiquitination, and Cbl family proteins can act as an E3 ligase in this reaction. Our data therefore suggest a model where lysosomal routing and degradation of antibody-bound CD33 is secondary to Src-family kinase-induced phosphorylation of CD33 with subsequent phosphotyrosine-dependent binding of Cbl to and monoubiquitination of CD33.

Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin–induced cytotoxicity

AbstractGemtuzumab ozogamicin (GO; Mylotarg), a novel immunoconjugate used for treatment of acute myeloid leukemia (AML), contains the humanized anti-CD33 antibody (hP67.6) as a carrier to facilitate cellular uptake of the toxic calicheamicin-γ1 derivative. By use of lentivirus-mediated gene transfer to manipulate CD33 expression in myeloid cell lines that normally lack CD33 (murine 32D cells) or have very low levels of CD33 (human OCI-AML3 and KG-1a cells), we here show a quantitative relationship between CD33 expression and GO-induced cytotoxicity. The CD33 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) control internalization of antibody bound to CD33. Disruption of the ITIMs by introduction of point mutations not only prevented effective internalization of antibody-bound CD33 but also significantly reduced GO-induced cytotoxicity. Together, our data imply a pivotal role of both the number of CD33 molecules expressed on the cell surface and the amount of internalization of CD33 following antibody binding for GO-induced cytotoxicity and suggest novel therapeutic approaches for improvement of clinical outcome of patients treated with GO.

CD33-directed therapy with gemtuzumab ozogamicin in acute myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms of drug resistance

CD33 is expressed on the malignant blast cells in most cases of acute myeloid leukemia (AML) but not on normal hematopoietic pluripotent stem cells. Antibody-based therapies for AML have, therefore, focused on CD33 as a suitable tumor-associated target antigen. The most promising results have been obtained with gemtuzumab ozogamicin (GO, Mylotarg), a humanized IgG(4) anti-CD33 monoclonal antibody joined to a calicheamicin-gamma(1) derivative. Engagement of CD33 by GO results in immunoconjugate internalization and hydrolytic release of the toxic calicheamicin moiety, which, in turn, causes DNA damage and cell death. Since 2000, when GO was approved for clinical use, treatment trials and pilot studies have revealed potential expanded applications along with additional limitations. At the same time, correlative biological and in vitro functional studies have further characterized CD33 expression patterns in AML, the significance of CD33-antibody interactions, pathways involved in GO-induced cytotoxicity and potential drug resistance mechanisms. This review summarizes the recent data addressing mechanisms of GO action and discusses their relevance with regard to clinical applications and the limitations of using experimental model systems to mimic in vivo conditions. As the first drug conjugate approved for clinical use, GO serves as an important paradigm for other immunoconjugates against internalizing tumor antigens.

Influence of CD33 Expression Levels and ITIM-Dependent Internalization on Gemtuzumab Ozogamicin-Induced Cytotoxicity.

Background: Gemtuzumab ozogamicin (GO; Mylotarg™), a novel immunoconjugate used for treatment of acute myeloid leukemia (AML), contains the humanized anti-CD33 antibody (hP67.6) as a carrier to facilitate cellular uptake of the toxic calicheamicin derivative. However, little is known about the importance of the quantity of CD33 expression for GO-induced cytotoxicity, and it has been argued that cytotoxic effects of GO can be achieved in the absence of CD33 expression. In addition, neither the endocytic process by which CD33 delivers antibody to the cytosol, nor the necessity of CD33 endocytosis for GO-induced cytotoxicity, has been established. We therefore investigated the quantitative relationship between CD33 expression and GO-mediated cytotoxicity, and determined the requirement for CD33 internalization in GO-induced cytotoxicity by identifying and mutating the cytoplasmic domain motif(s) that control internalization of antibody-bound CD33.Methods: Murine myeloid 32D cells (devoid of CD33) and human myeloid OCI-AML3 and KG-1a cells (weakly CD33+) were transduced with a lentiviral vector expressing either wild-type or mutant CD33 as well as green fluorescent protein (GFP) at a multiplicity of infection (MOI) of 1–100. GFP-positive cells were sorted by flow cytometry, re-cultured, and further analyzed for CD33 expression, internalization of anti-CD33 antibody, and GO susceptibility. CD33 expression was quantified by immunofluorescence staining. Antibody uptake was quantified with a flow-cytometry-based assay, in which cells labeled with unconjugated hP67.6 were allowed to internalize in antibody-free medium for various periods of time, before second and third step reagents were used to measure hP67.6 that remained on the cell surface. To assess GO-induced cytotoxicity, cells were treated continuously with various concentrations of GO for 3 days (+ cyclosporine A in KG-1a sublines), and cytotoxicity then determined by flow cytometry with propidium iodide.Results: In all 3 cell lines, lentivirus-mediated transfer of wild-type CD33 yielded subpopulations of GFP-positive cells that showed a MOI-dependent increase in expression levels of cell surface CD33. Compared to parental cells, transduced cells were significantly more sensitive to GO. Importantly, GO-sensitivity increased in a MOI-dependent manner, i.e. in a direct correlation with the cell surface expression levels of CD33. We could easily detect internalization of antibody-bound CD33 in cells that were transduced with wild-type CD33. Internalization of hP67.6 was almost completely prevented in cells that expressed a CD33 construct in which the proximal CD33 cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) was disrupted by introduction of point mutations (CD33/Y340F or CD33/L343A), whereas internalization was only partially prevented in cells that expressed a mutated distal ITIM (CD33/Y358F). In addition, disruption of the ITIMs not only prevented effective internalization of antibody-bound CD33, but also significantly reduced GO-induced cytotoxicity, when compared to cells expressing wild-type CD33 at matchable levels.Conclusion: These data imply a pivotal role of both the number of CD33 molecules expressed on the cell surface and the rate of internalization of CD33 following antibody binding for GO-induced cytotoxicity, and suggest novel therapeutic approaches for improvement of clinical outcome of patients treated with GO.

Multidrug resistance protein attenuates gemtuzumab ozogamicin–induced cytotoxicity in acute myeloid leukemia cells

AbstractGemtuzumab ozogamicin (GO) is a novel immunoconjugate therapy for acute myeloid leukemia (AML). P-glycoprotein (Pgp) confers resistance to GO and is associated with a worse clinical response. To address whether multidrug resistance protein (MRP) affects GO susceptibility, we characterized Pgp, MRP1, and MRP2 expression in CD33+ cell lines and CD33+ AML samples and analyzed the effect of the Pgp inhibitor cyclosporine (CSA) and the MRP inhibitor MK-571 on GO-induced cytotoxicity. MRP1, but not MRP2, expression correlated with MRP activity. MK-571 enhanced GO-induced cytotoxicity in Pgpnegative/MRP-positive NB4 and HL-60 cells. CSA, but not MK-571 alone, restored GO susceptibility in Pgp-positive/MRP-positive TF1 cells; however, MK-571 enhanced cytotoxicity in the presence of CSA. All patient samples exhibited MRP activity, and 17 of 23 exhibited Pgp activity. CSA increased GO-induced cytotoxicity in 12 Pgp-positive samples, whereas MK-571 alone was effective in only one sample with minimal Pgp activity. In 3 Pgp-positive/MRP-positive samples, MK-571 enhanced GO-induced cytotoxicity in the presence of CSA. Thus, MRP1 may attenuate susceptibility to GO. This effect was comparatively less than that for Pgp and required the inhibition of Pgp for detection in cells that coexpressed both transporters. Because MK-571 and CSA failed to affect cytotoxicity in a portion of Pgp-positive/MRP-positive AML samples, additional resistance mechanisms are likely important.