LOSS OF PIGH EXPRESSION FREQUENTLY RESULTS IN A GPI-NEGATIVE SUBCLONE LACKING CD52 MEMBRANE EXPRESSION, CONFERRING ALEMTUZUMAB RESISTANCE TO B CELL ACUTE LYMPHOBLASTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Jedema I. 06/10/16; 133150; P162
Assoc. Prof. Inge Jedema
Contributions
Contributions
Abstract
Abstract: P162
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
To improve treatment outcome of patients with B-cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI)-anchored CD52 protein resulted in unsatisfactory clinical efficacy, potentially due to development of escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52-negative escape variants following alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression due to loss of GPI-anchor expression.
Aims
To unravel the mechanism underlying the loss of CD52/GPI expression in B-ALL
Methods
The presence and frequency of CD52/GPI-negative cells was analyzed by flowcytometry in peripheral blood (PB; n=10) and bone marrow (BM; n=8) samples taken with informed consent from B-ALL patients at diagnosis. Diagnosis samples of patients with chronic lymphocytic leukemia (CLL, n= 5), mantle cell leukemia (MCL, n=5) and hairy cell leukemia (HCL, n=6), and PB samples from 5 healthy donors were similarly analyzed in parallel. To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway in GPI positive and GPI negative B-ALL populations purified by flowcytometric cell sorting.
Results
To study the presence and frequency of CD52/GPI-negative cells at diagnosis, we analyzed PB and BM samples from B-ALL patients at diagnosis by flowcytometry. GPI-negative cells were present in 6/10 PB and 5/8 BM samples and comprised between 0.01% and 4.98% of the B-cell population. These obvious GPI-negative B-cell populations were not found in CLL (n=5), MCL (n=5), and HCL (n=6) diagnosis samples, or in healthy donors (n=5). To investigate the mechanism of GPI-anchor loss, gene expression analysis was performed for the 26 GPI biosynthesis pathway genes. In contrast to purified GPI-positive B-ALL populations, loss of PIGH mRNA expression, but not of any of the other genes, was found in all GPI-negative populations (n=9). To validate the relevance of this finding, GPI-negative and GPI-positive B-ALL cell cultures were generated from diagnosis material (n=2) and transduced with a retroviral construct encoding PIGH. Restored GPI-anchor expression and coinciding CD52 membrane expression was observed in the GPI-negative B-ALL cultures upon transduction with PIGH, but not empty vector. To explore the mechanism underlying the loss of PIGH mRNA expression in CD52/GPI-negative B-ALL cells, we performed DNA screening and assessed promoter CpG methylation, comparing GPI-negative with GPI-positive B-ALL cultures from the same individual (n=2). These analyses revealed that in GPI-negative cultures both alleles of the PIGH gene were present, unmutated and intact, but with a heavier methylated promoter region compared to the GPI-positive counterparts. Additionally, a 14 day treatment of GPI-negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI-anchor
Conclusion
In conclusion, the majority of B-ALL patients presented a CD52/GPI-negative, alemtuzumab resistant, B-ALL population already at diagnosis. These cells lost PIGH expression, a key component in GPI-anchor synthesis. This is not due to genomic instability, but to epigenetic downregulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI-negative escape variants in B-ALL
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Alemtuzumab, B cell acute lymphoblastic leukemia, CD52, Resistance
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
To improve treatment outcome of patients with B-cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI)-anchored CD52 protein resulted in unsatisfactory clinical efficacy, potentially due to development of escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52-negative escape variants following alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression due to loss of GPI-anchor expression.
Aims
To unravel the mechanism underlying the loss of CD52/GPI expression in B-ALL
Methods
The presence and frequency of CD52/GPI-negative cells was analyzed by flowcytometry in peripheral blood (PB; n=10) and bone marrow (BM; n=8) samples taken with informed consent from B-ALL patients at diagnosis. Diagnosis samples of patients with chronic lymphocytic leukemia (CLL, n= 5), mantle cell leukemia (MCL, n=5) and hairy cell leukemia (HCL, n=6), and PB samples from 5 healthy donors were similarly analyzed in parallel. To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway in GPI positive and GPI negative B-ALL populations purified by flowcytometric cell sorting.
Results
To study the presence and frequency of CD52/GPI-negative cells at diagnosis, we analyzed PB and BM samples from B-ALL patients at diagnosis by flowcytometry. GPI-negative cells were present in 6/10 PB and 5/8 BM samples and comprised between 0.01% and 4.98% of the B-cell population. These obvious GPI-negative B-cell populations were not found in CLL (n=5), MCL (n=5), and HCL (n=6) diagnosis samples, or in healthy donors (n=5). To investigate the mechanism of GPI-anchor loss, gene expression analysis was performed for the 26 GPI biosynthesis pathway genes. In contrast to purified GPI-positive B-ALL populations, loss of PIGH mRNA expression, but not of any of the other genes, was found in all GPI-negative populations (n=9). To validate the relevance of this finding, GPI-negative and GPI-positive B-ALL cell cultures were generated from diagnosis material (n=2) and transduced with a retroviral construct encoding PIGH. Restored GPI-anchor expression and coinciding CD52 membrane expression was observed in the GPI-negative B-ALL cultures upon transduction with PIGH, but not empty vector. To explore the mechanism underlying the loss of PIGH mRNA expression in CD52/GPI-negative B-ALL cells, we performed DNA screening and assessed promoter CpG methylation, comparing GPI-negative with GPI-positive B-ALL cultures from the same individual (n=2). These analyses revealed that in GPI-negative cultures both alleles of the PIGH gene were present, unmutated and intact, but with a heavier methylated promoter region compared to the GPI-positive counterparts. Additionally, a 14 day treatment of GPI-negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI-anchor
Conclusion
In conclusion, the majority of B-ALL patients presented a CD52/GPI-negative, alemtuzumab resistant, B-ALL population already at diagnosis. These cells lost PIGH expression, a key component in GPI-anchor synthesis. This is not due to genomic instability, but to epigenetic downregulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI-negative escape variants in B-ALL
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Alemtuzumab, B cell acute lymphoblastic leukemia, CD52, Resistance
Abstract: P162
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
To improve treatment outcome of patients with B-cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI)-anchored CD52 protein resulted in unsatisfactory clinical efficacy, potentially due to development of escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52-negative escape variants following alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression due to loss of GPI-anchor expression.
Aims
To unravel the mechanism underlying the loss of CD52/GPI expression in B-ALL
Methods
The presence and frequency of CD52/GPI-negative cells was analyzed by flowcytometry in peripheral blood (PB; n=10) and bone marrow (BM; n=8) samples taken with informed consent from B-ALL patients at diagnosis. Diagnosis samples of patients with chronic lymphocytic leukemia (CLL, n= 5), mantle cell leukemia (MCL, n=5) and hairy cell leukemia (HCL, n=6), and PB samples from 5 healthy donors were similarly analyzed in parallel. To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway in GPI positive and GPI negative B-ALL populations purified by flowcytometric cell sorting.
Results
To study the presence and frequency of CD52/GPI-negative cells at diagnosis, we analyzed PB and BM samples from B-ALL patients at diagnosis by flowcytometry. GPI-negative cells were present in 6/10 PB and 5/8 BM samples and comprised between 0.01% and 4.98% of the B-cell population. These obvious GPI-negative B-cell populations were not found in CLL (n=5), MCL (n=5), and HCL (n=6) diagnosis samples, or in healthy donors (n=5). To investigate the mechanism of GPI-anchor loss, gene expression analysis was performed for the 26 GPI biosynthesis pathway genes. In contrast to purified GPI-positive B-ALL populations, loss of PIGH mRNA expression, but not of any of the other genes, was found in all GPI-negative populations (n=9). To validate the relevance of this finding, GPI-negative and GPI-positive B-ALL cell cultures were generated from diagnosis material (n=2) and transduced with a retroviral construct encoding PIGH. Restored GPI-anchor expression and coinciding CD52 membrane expression was observed in the GPI-negative B-ALL cultures upon transduction with PIGH, but not empty vector. To explore the mechanism underlying the loss of PIGH mRNA expression in CD52/GPI-negative B-ALL cells, we performed DNA screening and assessed promoter CpG methylation, comparing GPI-negative with GPI-positive B-ALL cultures from the same individual (n=2). These analyses revealed that in GPI-negative cultures both alleles of the PIGH gene were present, unmutated and intact, but with a heavier methylated promoter region compared to the GPI-positive counterparts. Additionally, a 14 day treatment of GPI-negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI-anchor
Conclusion
In conclusion, the majority of B-ALL patients presented a CD52/GPI-negative, alemtuzumab resistant, B-ALL population already at diagnosis. These cells lost PIGH expression, a key component in GPI-anchor synthesis. This is not due to genomic instability, but to epigenetic downregulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI-negative escape variants in B-ALL
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Alemtuzumab, B cell acute lymphoblastic leukemia, CD52, Resistance
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
To improve treatment outcome of patients with B-cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI)-anchored CD52 protein resulted in unsatisfactory clinical efficacy, potentially due to development of escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52-negative escape variants following alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression due to loss of GPI-anchor expression.
Aims
To unravel the mechanism underlying the loss of CD52/GPI expression in B-ALL
Methods
The presence and frequency of CD52/GPI-negative cells was analyzed by flowcytometry in peripheral blood (PB; n=10) and bone marrow (BM; n=8) samples taken with informed consent from B-ALL patients at diagnosis. Diagnosis samples of patients with chronic lymphocytic leukemia (CLL, n= 5), mantle cell leukemia (MCL, n=5) and hairy cell leukemia (HCL, n=6), and PB samples from 5 healthy donors were similarly analyzed in parallel. To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway in GPI positive and GPI negative B-ALL populations purified by flowcytometric cell sorting.
Results
To study the presence and frequency of CD52/GPI-negative cells at diagnosis, we analyzed PB and BM samples from B-ALL patients at diagnosis by flowcytometry. GPI-negative cells were present in 6/10 PB and 5/8 BM samples and comprised between 0.01% and 4.98% of the B-cell population. These obvious GPI-negative B-cell populations were not found in CLL (n=5), MCL (n=5), and HCL (n=6) diagnosis samples, or in healthy donors (n=5). To investigate the mechanism of GPI-anchor loss, gene expression analysis was performed for the 26 GPI biosynthesis pathway genes. In contrast to purified GPI-positive B-ALL populations, loss of PIGH mRNA expression, but not of any of the other genes, was found in all GPI-negative populations (n=9). To validate the relevance of this finding, GPI-negative and GPI-positive B-ALL cell cultures were generated from diagnosis material (n=2) and transduced with a retroviral construct encoding PIGH. Restored GPI-anchor expression and coinciding CD52 membrane expression was observed in the GPI-negative B-ALL cultures upon transduction with PIGH, but not empty vector. To explore the mechanism underlying the loss of PIGH mRNA expression in CD52/GPI-negative B-ALL cells, we performed DNA screening and assessed promoter CpG methylation, comparing GPI-negative with GPI-positive B-ALL cultures from the same individual (n=2). These analyses revealed that in GPI-negative cultures both alleles of the PIGH gene were present, unmutated and intact, but with a heavier methylated promoter region compared to the GPI-positive counterparts. Additionally, a 14 day treatment of GPI-negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI-anchor
Conclusion
In conclusion, the majority of B-ALL patients presented a CD52/GPI-negative, alemtuzumab resistant, B-ALL population already at diagnosis. These cells lost PIGH expression, a key component in GPI-anchor synthesis. This is not due to genomic instability, but to epigenetic downregulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI-negative escape variants in B-ALL
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Alemtuzumab, B cell acute lymphoblastic leukemia, CD52, Resistance
{{ help_message }}
{{filter}}