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Edward D. Ball - One of the best experts on this subject based on the ideXlab platform.
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5-Azacytidine Potentiates the Anti-Proliferative Activity of Anti-CD33 Monoclonal Antibodies (mAb) in Acute Myeloid Leukemia in Part through Induction of Syk and SHP-1 Expression.
Blood, 2005Co-Authors: L Balaian, Edward D. BallAbstract:We have previously demonstrated that anti-CD33 monoclonal antibodies have anti-proliferative and pro-apoptotic effects on AML cells and that this ability is associated with the expression and functional activity of the Protein tyrosine Kinase Syk and Protein tyrosine phosphatase SHP-1. However, about 30% of primary AML samples are Syk-negative and 15% are SHP-1-negative.Since absence of Protein expression and unresponsiveness of tumor cells could be caused by silencing by hypermethylation, we tested whether the demethylating agent 5-azacytidine (5-aza) could restore Syk and SHP-1 and, therefore, enhance CD33 ligation mediated inhibition of leukemia cell growth. 40 primary AML samples were tested for their response to submaximal anti-proliferative concentrations of 5-aza (100nM for 48 h) and, subsequently, were cultured in the presence of anti-CD33 or control anti-CD13 mAb. In the majority of cases, 5-aza alone induced various degree of inhibition of proliferation (ranging from 0 to 90%). Based on the level of inhibition, samples were divided into 3 groups: low ( 50%). 52.5% of samples were Low Responders, 22.5% -medium R and 25% -high R. The average level of inhibition was determined for each group: (LR-6.4.+1.8, MR-34.5+1.9, HR-69.9+6.9 Incremental doses (0.01–1 mg/ml) of anti-CD33 mAb (alone) inhibited proliferation of AML samples variably (15–35%). No differences between groups were determined. However, the addition of anti-CD33 mAb to the 5-aza pre-treated samples revealed differences between groups. In the LR group response more than doubled (from 20 to 58%), in the MR group the increase was about 30%. However, in the HR group, no difference was observed. The level of expression of Protein Kinase Syk and Protein phosphatase SHP-1 was determined for each sample by Western blot. Of 40 samples 25 (62.5%) were Syk-positive and 15 (37.5%) were Syk-negative. 35 samples (87.5%) were SHP-1-positive and 5 samples (12.5%) were SHP1-negative. The majority of Syk-negative samples (11 of 15) were in LR group, while the remaining 4 samples were distributed between MR and HR groups and demonstrated significant levels of inhibition. This difference was statistically significant (p=0.003) and suggested a correlation between the Syk+/Syk− samples and their response to the inhibitory activity of 5-Aza. Similarly, analysis of SHP-1 expression and the response of samples to 5-aza inhibition revealed an even stronger correlation (p= 0.023) since all 5 SHP-1-negative samples were in LR group. Meanwhile, in 36.3% (4 of 11 Syk-negative cases), and 40% (2 of 5 SHP-1-negative samples) 5-aza treatment resulted in restoration of Protein expression. Moreover, in Syk-positive cases combined treatment induced a 20% increase in inhibition from 36% (5-aza alone) to 55% (5-aza+ a -CD33). In Syk-negative samples, this increase was significant (about 40%). In SHP-1-positive samples combined treatment also induced about a 20% increase in inhibition. However, in SHP-1-negative samples the increase was dramatic (about 70%) In all cases, control a -CD13 mAb did not result in any increases in 5-aza-induced inhibition of proliferation. These data support the notion of a clinical trial of combination 5-aza and anti-CD33 mAb therapy for patients with AML. In addition, biomarkers for response may help select patients in whom the combination therapy will be most efficacious. A phase I/II clinical trial is under development at this time.
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The Anti-Proliferative Effect of Immunotoxin Gemtuzimab Ozogamycin (GO) (Mylotarg) to Acute Myeloid Leukemia (AML) Cells Is Associated with the Level of Protein Kinase Syk Exspression.
Blood, 2004Co-Authors: L Balaian, Edward D. BallAbstract:AML cells express the cell surface antigen CD33 that serves as a down-regulator of cell growth. Anti-CD33 monoclonal antibody (mAb) coupled to a toxin is a licensed drug for the treatment of relapsed AML (Mylotarg). Syk is not only an essential element in several cascades coupling antigen receptors to cell responses, but also Syk is a tumor suppressor gene. Silencing of Syk by hypermethylation results in absence of Syk expression and unresponsiveness of tumor cells to various treatments.. Earlier we demonstrated that about 30% of the AML samples were Syk-negative and the response of leukemia cells to CD33 ligation correlated with Syk expression. Therefore, here we investigated whether or not the response of the AML cells to GO treatment also depends on the level of Syk expression. It is even more intriguing since 50% of Mylotarg is not conjugated to calicheamicin, and hence some of its activity is in fact due to anti-CD33 mAb signaling. Primary leukemia cells were analyzed for their level of the Syk expression (11 Syk positive and 6 Syk-negative) and then treated with GO. In both groups GO mediated dose-dependent inhibition of proliferation, however the level of inhibition in Syk-positive group was significantly higher (p 100 ng/ml) this effect was diminished. Moreover, 8 of 11 (72.7%) samples in Syk-positive group were “good” responders to GO (inhibition>50%), while in Syk-negative group only 1 of 6 (16.6%) samples demonstrated similar response. This difference was statistically significant and suggesting a correlation between the level of Syk expression in AML cells and their response to Mylotarg. After treatment of Syk-negative AML samples with methylation inhibitor 5-aza-cytidine in 2 of 6 samples Syk Protein expression was restored. However, in all 6 samples after 5-aza-CdR treatment GO inhibitory activity was significantly higher (p
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The inhibitory effect of anti-CD33 monoclonal antibodies on AML cell growth correlates with Syk and/or ZAP-70 expression.
Experimental hematology, 2003Co-Authors: Larisa Balaian, Rui-kun Zhong, Edward D. BallAbstract:Abstract Objectives. Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The Protein Kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. Methods. We investigated 25 primary AML samples for their proliferative response ( 3 H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. Results. Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 Protein Kinase. In Syk/ZAP-70 + samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70 − samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 Protein phosphatase constitutively. Conclusions. Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.
L Balaian - One of the best experts on this subject based on the ideXlab platform.
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Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of Protein Kinase Syk
Leukemia, 2006Co-Authors: L Balaian, E D BallAbstract:Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that, upon ligation with a monoclonal antibody (mAb), is a downregulator of cell growth in a Syk-dependent manner. An anti-CD33 mAb coupled to a toxin, gemtuzumab ozogamicin (GO), is used for the treatment of AML (Mylotarg). Therefore, we investigated whether the response of AML cells to GO treatment also depends on Syk expression. Forty primary AML samples (25 Syk-positive and 15 Syk-negative) were tested for their response to the anti-proliferative effects of GO and unmodified anti-CD33 mAb. A correlation between Syk expression and the response of leukemia cells to GO and anti-CD33 mAb was found. ‘Blocking’ of Syk by small interfering RNA resulted in unresponsiveness of AML cells to both GO and anti-CD33 mAb-mediated cytotoxicity. Syk upregulation by the de-methylating agent 5-azacytidine (5-aza) induced re-expression of Syk in some cases, resulting in enhanced GO and anti-CD33-mediated inhibition of leukemia cell growth. Thus, the cytotoxicity of both GO and anti-CD33 in primary AML samples was associated with Syk expression. 5-Aza restored Syk and increased the sensitivity of originally Syk-negative, non-responsive cells to CD33 ligation to levels of Syk-positive cells. These data have clinical significance for predicting response to GO and designing clinical trials.
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5-Azacytidine Potentiates the Anti-Proliferative Activity of Anti-CD33 Monoclonal Antibodies (mAb) in Acute Myeloid Leukemia in Part through Induction of Syk and SHP-1 Expression.
Blood, 2005Co-Authors: L Balaian, Edward D. BallAbstract:We have previously demonstrated that anti-CD33 monoclonal antibodies have anti-proliferative and pro-apoptotic effects on AML cells and that this ability is associated with the expression and functional activity of the Protein tyrosine Kinase Syk and Protein tyrosine phosphatase SHP-1. However, about 30% of primary AML samples are Syk-negative and 15% are SHP-1-negative.Since absence of Protein expression and unresponsiveness of tumor cells could be caused by silencing by hypermethylation, we tested whether the demethylating agent 5-azacytidine (5-aza) could restore Syk and SHP-1 and, therefore, enhance CD33 ligation mediated inhibition of leukemia cell growth. 40 primary AML samples were tested for their response to submaximal anti-proliferative concentrations of 5-aza (100nM for 48 h) and, subsequently, were cultured in the presence of anti-CD33 or control anti-CD13 mAb. In the majority of cases, 5-aza alone induced various degree of inhibition of proliferation (ranging from 0 to 90%). Based on the level of inhibition, samples were divided into 3 groups: low ( 50%). 52.5% of samples were Low Responders, 22.5% -medium R and 25% -high R. The average level of inhibition was determined for each group: (LR-6.4.+1.8, MR-34.5+1.9, HR-69.9+6.9 Incremental doses (0.01–1 mg/ml) of anti-CD33 mAb (alone) inhibited proliferation of AML samples variably (15–35%). No differences between groups were determined. However, the addition of anti-CD33 mAb to the 5-aza pre-treated samples revealed differences between groups. In the LR group response more than doubled (from 20 to 58%), in the MR group the increase was about 30%. However, in the HR group, no difference was observed. The level of expression of Protein Kinase Syk and Protein phosphatase SHP-1 was determined for each sample by Western blot. Of 40 samples 25 (62.5%) were Syk-positive and 15 (37.5%) were Syk-negative. 35 samples (87.5%) were SHP-1-positive and 5 samples (12.5%) were SHP1-negative. The majority of Syk-negative samples (11 of 15) were in LR group, while the remaining 4 samples were distributed between MR and HR groups and demonstrated significant levels of inhibition. This difference was statistically significant (p=0.003) and suggested a correlation between the Syk+/Syk− samples and their response to the inhibitory activity of 5-Aza. Similarly, analysis of SHP-1 expression and the response of samples to 5-aza inhibition revealed an even stronger correlation (p= 0.023) since all 5 SHP-1-negative samples were in LR group. Meanwhile, in 36.3% (4 of 11 Syk-negative cases), and 40% (2 of 5 SHP-1-negative samples) 5-aza treatment resulted in restoration of Protein expression. Moreover, in Syk-positive cases combined treatment induced a 20% increase in inhibition from 36% (5-aza alone) to 55% (5-aza+ a -CD33). In Syk-negative samples, this increase was significant (about 40%). In SHP-1-positive samples combined treatment also induced about a 20% increase in inhibition. However, in SHP-1-negative samples the increase was dramatic (about 70%) In all cases, control a -CD13 mAb did not result in any increases in 5-aza-induced inhibition of proliferation. These data support the notion of a clinical trial of combination 5-aza and anti-CD33 mAb therapy for patients with AML. In addition, biomarkers for response may help select patients in whom the combination therapy will be most efficacious. A phase I/II clinical trial is under development at this time.
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The Anti-Proliferative Effect of Immunotoxin Gemtuzimab Ozogamycin (GO) (Mylotarg) to Acute Myeloid Leukemia (AML) Cells Is Associated with the Level of Protein Kinase Syk Exspression.
Blood, 2004Co-Authors: L Balaian, Edward D. BallAbstract:AML cells express the cell surface antigen CD33 that serves as a down-regulator of cell growth. Anti-CD33 monoclonal antibody (mAb) coupled to a toxin is a licensed drug for the treatment of relapsed AML (Mylotarg). Syk is not only an essential element in several cascades coupling antigen receptors to cell responses, but also Syk is a tumor suppressor gene. Silencing of Syk by hypermethylation results in absence of Syk expression and unresponsiveness of tumor cells to various treatments.. Earlier we demonstrated that about 30% of the AML samples were Syk-negative and the response of leukemia cells to CD33 ligation correlated with Syk expression. Therefore, here we investigated whether or not the response of the AML cells to GO treatment also depends on the level of Syk expression. It is even more intriguing since 50% of Mylotarg is not conjugated to calicheamicin, and hence some of its activity is in fact due to anti-CD33 mAb signaling. Primary leukemia cells were analyzed for their level of the Syk expression (11 Syk positive and 6 Syk-negative) and then treated with GO. In both groups GO mediated dose-dependent inhibition of proliferation, however the level of inhibition in Syk-positive group was significantly higher (p 100 ng/ml) this effect was diminished. Moreover, 8 of 11 (72.7%) samples in Syk-positive group were “good” responders to GO (inhibition>50%), while in Syk-negative group only 1 of 6 (16.6%) samples demonstrated similar response. This difference was statistically significant and suggesting a correlation between the level of Syk expression in AML cells and their response to Mylotarg. After treatment of Syk-negative AML samples with methylation inhibitor 5-aza-cytidine in 2 of 6 samples Syk Protein expression was restored. However, in all 6 samples after 5-aza-CdR treatment GO inhibitory activity was significantly higher (p
E D Ball - One of the best experts on this subject based on the ideXlab platform.
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Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of Protein Kinase Syk
Leukemia, 2006Co-Authors: L Balaian, E D BallAbstract:Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that, upon ligation with a monoclonal antibody (mAb), is a downregulator of cell growth in a Syk-dependent manner. An anti-CD33 mAb coupled to a toxin, gemtuzumab ozogamicin (GO), is used for the treatment of AML (Mylotarg). Therefore, we investigated whether the response of AML cells to GO treatment also depends on Syk expression. Forty primary AML samples (25 Syk-positive and 15 Syk-negative) were tested for their response to the anti-proliferative effects of GO and unmodified anti-CD33 mAb. A correlation between Syk expression and the response of leukemia cells to GO and anti-CD33 mAb was found. ‘Blocking’ of Syk by small interfering RNA resulted in unresponsiveness of AML cells to both GO and anti-CD33 mAb-mediated cytotoxicity. Syk upregulation by the de-methylating agent 5-azacytidine (5-aza) induced re-expression of Syk in some cases, resulting in enhanced GO and anti-CD33-mediated inhibition of leukemia cell growth. Thus, the cytotoxicity of both GO and anti-CD33 in primary AML samples was associated with Syk expression. 5-Aza restored Syk and increased the sensitivity of originally Syk-negative, non-responsive cells to CD33 ligation to levels of Syk-positive cells. These data have clinical significance for predicting response to GO and designing clinical trials.
Larisa Balaian - One of the best experts on this subject based on the ideXlab platform.
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The inhibitory effect of anti-CD33 monoclonal antibodies on AML cell growth correlates with Syk and/or ZAP-70 expression.
Experimental hematology, 2003Co-Authors: Larisa Balaian, Rui-kun Zhong, Edward D. BallAbstract:Abstract Objectives. Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The Protein Kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. Methods. We investigated 25 primary AML samples for their proliferative response ( 3 H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. Results. Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 Protein Kinase. In Syk/ZAP-70 + samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70 − samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 Protein phosphatase constitutively. Conclusions. Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.
Rui-kun Zhong - One of the best experts on this subject based on the ideXlab platform.
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The inhibitory effect of anti-CD33 monoclonal antibodies on AML cell growth correlates with Syk and/or ZAP-70 expression.
Experimental hematology, 2003Co-Authors: Larisa Balaian, Rui-kun Zhong, Edward D. BallAbstract:Abstract Objectives. Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The Protein Kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. Methods. We investigated 25 primary AML samples for their proliferative response ( 3 H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. Results. Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 Protein Kinase. In Syk/ZAP-70 + samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70 − samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 Protein phosphatase constitutively. Conclusions. Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.
