B CELL-SPECIFIC CONDITIONAL EXPRESSION OF MYD88P.L252P LEADS TO THE DEVELOPMENT OF DIFFUSE LARGE B CELL LYMPHOMA IN MICE
(Abstract release date: 05/19/16)
EHA Library. Knittel G. 06/11/16; 135245; S489
Mr. Gero Knittel
Contributions
Contributions
Abstract
Abstract: S489
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Auditorium 1
Background
The adaptor protein MYD88 is critical to relay activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B cell malignancies, including diffuse large B cell lymphoma (DLBCL). 29% of activated B cell (ABC)-type DLBCL, which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2.
Aims
Here, we aimed to investigate the potential role of the Myd88p.L265P point mutation in lymphomagenesis.
Methods
We generated a novel mouse model (termed Myd88c-p.L252P), in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88p.L252P (the orthologous position of the human MYD88p.L265P mutation) from the endogenous locus. More precisely, the endogenous exons 2 to 6 of Myd88 (which consists of 6 exons) were flanked by loxP-sites. Downstream of the last exon, a second set of exons 2 to 6 was inserted, harboring the point mutation p.L252P. Cre-mediated recombination leads to the excision of the endogenous exons 2 to 6 and expression of the inserted, mutated set of exons. This system very closely mimicks the situation observed in the clinic, as it allows for the heterozygous expression of Myd88p.L252P from the endogenous locus.
Results
To assess the functionally of our allele, we generated mouse embryonic fibroblasts (MEFs) homozygous for the Myd88c-p.L252P allele, recombined them in vitro by lentiviral transduction. After puromycin selection, cDNA was sequenced and only the mutant transcript was detected. Western blotting verified the expression of this mutant transcript and increased phospho-p65 levels as a marker for NF-kB activation.We activated our allele B cell-specifically by crossing it to the Cd19-Cre mouse. The resulting Myd88c-p.L252P;Cd19Cre/wt animals had a lifespan of around 500 days, significantly shorter than that of the Cd19Cre/wt control. Magnetic resonance imaging and autopsy showed the development of splenomegaly and lymphadenopathy starting at around 60 weeks of age. Further histological and immunohistochemical investigation revealed the existence of lymphoproliferative disease and the sporadic emergence of large B cell lymphoma with an Bcl6-/Mum1+/B220+/Cd138- immunotype. Analysis of V(D)J recombination by southern blotting showed clonal populations in samples from animals diagnosed with lymphoma by histology.BCL-2 is highly expressed in most ABC DLBCL cases. To potentially enhance lymphomagenesis in our mice, we aimed to combine the Myd88p.L252P mutation with Bcl-2 overexpression by making use of a newly genrated LSL.BCL2 allele, where human BCL2 expression is driven by the CAGGs promoter. Indeed, all Myd88c-p.L252P;LSL.BCL2;Cd19Cre/wt mice die of aggressive large B cell lymphoma at a median of 36 weeks. All animals showed splenomegaly and/or lymphadenopathy, accompanied by infiltration of the liver. Bone marrow involvement was only detected once and was locally restricted, indicating a secondary lesion. Clonality analysis by southern blot showed oligoclonality, suggesting a strong oncogenic potential of Myd88p.L252P in combination with BCL2 overexpression. Interestingly, lymphoma cells were of a Bcl6-/Mum1+/B220-/Cd138+ immunotype, in accordance with the plasmoblastic morphology that was histologically observed.
Conclusion
In summary, we generated a mouse model that enables Cre-mediated expression of Myd88p.L252P from the endogenous locus. Myd88c-p.L252P;Cd19Cre/wt mice develop and eventually die of lymphoproliferative disease. The emergence of diffuse large B cell lymphoma (DLBCL) was observed. Combination of B cell-specific Myd88p.L252P with BCL-2 overexpression results in the development of an aggressive lymphoma with plasmoblastic features, most reminiscent of ABC-type DLBCL.
Session topic: Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): DLBCL, Mouse model, NF- B
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Auditorium 1
Background
The adaptor protein MYD88 is critical to relay activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B cell malignancies, including diffuse large B cell lymphoma (DLBCL). 29% of activated B cell (ABC)-type DLBCL, which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2.
Aims
Here, we aimed to investigate the potential role of the Myd88p.L265P point mutation in lymphomagenesis.
Methods
We generated a novel mouse model (termed Myd88c-p.L252P), in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88p.L252P (the orthologous position of the human MYD88p.L265P mutation) from the endogenous locus. More precisely, the endogenous exons 2 to 6 of Myd88 (which consists of 6 exons) were flanked by loxP-sites. Downstream of the last exon, a second set of exons 2 to 6 was inserted, harboring the point mutation p.L252P. Cre-mediated recombination leads to the excision of the endogenous exons 2 to 6 and expression of the inserted, mutated set of exons. This system very closely mimicks the situation observed in the clinic, as it allows for the heterozygous expression of Myd88p.L252P from the endogenous locus.
Results
To assess the functionally of our allele, we generated mouse embryonic fibroblasts (MEFs) homozygous for the Myd88c-p.L252P allele, recombined them in vitro by lentiviral transduction. After puromycin selection, cDNA was sequenced and only the mutant transcript was detected. Western blotting verified the expression of this mutant transcript and increased phospho-p65 levels as a marker for NF-kB activation.We activated our allele B cell-specifically by crossing it to the Cd19-Cre mouse. The resulting Myd88c-p.L252P;Cd19Cre/wt animals had a lifespan of around 500 days, significantly shorter than that of the Cd19Cre/wt control. Magnetic resonance imaging and autopsy showed the development of splenomegaly and lymphadenopathy starting at around 60 weeks of age. Further histological and immunohistochemical investigation revealed the existence of lymphoproliferative disease and the sporadic emergence of large B cell lymphoma with an Bcl6-/Mum1+/B220+/Cd138- immunotype. Analysis of V(D)J recombination by southern blotting showed clonal populations in samples from animals diagnosed with lymphoma by histology.BCL-2 is highly expressed in most ABC DLBCL cases. To potentially enhance lymphomagenesis in our mice, we aimed to combine the Myd88p.L252P mutation with Bcl-2 overexpression by making use of a newly genrated LSL.BCL2 allele, where human BCL2 expression is driven by the CAGGs promoter. Indeed, all Myd88c-p.L252P;LSL.BCL2;Cd19Cre/wt mice die of aggressive large B cell lymphoma at a median of 36 weeks. All animals showed splenomegaly and/or lymphadenopathy, accompanied by infiltration of the liver. Bone marrow involvement was only detected once and was locally restricted, indicating a secondary lesion. Clonality analysis by southern blot showed oligoclonality, suggesting a strong oncogenic potential of Myd88p.L252P in combination with BCL2 overexpression. Interestingly, lymphoma cells were of a Bcl6-/Mum1+/B220-/Cd138+ immunotype, in accordance with the plasmoblastic morphology that was histologically observed.
Conclusion
In summary, we generated a mouse model that enables Cre-mediated expression of Myd88p.L252P from the endogenous locus. Myd88c-p.L252P;Cd19Cre/wt mice develop and eventually die of lymphoproliferative disease. The emergence of diffuse large B cell lymphoma (DLBCL) was observed. Combination of B cell-specific Myd88p.L252P with BCL-2 overexpression results in the development of an aggressive lymphoma with plasmoblastic features, most reminiscent of ABC-type DLBCL.
Session topic: Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): DLBCL, Mouse model, NF- B
Abstract: S489
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Auditorium 1
Background
The adaptor protein MYD88 is critical to relay activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B cell malignancies, including diffuse large B cell lymphoma (DLBCL). 29% of activated B cell (ABC)-type DLBCL, which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2.
Aims
Here, we aimed to investigate the potential role of the Myd88p.L265P point mutation in lymphomagenesis.
Methods
We generated a novel mouse model (termed Myd88c-p.L252P), in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88p.L252P (the orthologous position of the human MYD88p.L265P mutation) from the endogenous locus. More precisely, the endogenous exons 2 to 6 of Myd88 (which consists of 6 exons) were flanked by loxP-sites. Downstream of the last exon, a second set of exons 2 to 6 was inserted, harboring the point mutation p.L252P. Cre-mediated recombination leads to the excision of the endogenous exons 2 to 6 and expression of the inserted, mutated set of exons. This system very closely mimicks the situation observed in the clinic, as it allows for the heterozygous expression of Myd88p.L252P from the endogenous locus.
Results
To assess the functionally of our allele, we generated mouse embryonic fibroblasts (MEFs) homozygous for the Myd88c-p.L252P allele, recombined them in vitro by lentiviral transduction. After puromycin selection, cDNA was sequenced and only the mutant transcript was detected. Western blotting verified the expression of this mutant transcript and increased phospho-p65 levels as a marker for NF-kB activation.We activated our allele B cell-specifically by crossing it to the Cd19-Cre mouse. The resulting Myd88c-p.L252P;Cd19Cre/wt animals had a lifespan of around 500 days, significantly shorter than that of the Cd19Cre/wt control. Magnetic resonance imaging and autopsy showed the development of splenomegaly and lymphadenopathy starting at around 60 weeks of age. Further histological and immunohistochemical investigation revealed the existence of lymphoproliferative disease and the sporadic emergence of large B cell lymphoma with an Bcl6-/Mum1+/B220+/Cd138- immunotype. Analysis of V(D)J recombination by southern blotting showed clonal populations in samples from animals diagnosed with lymphoma by histology.BCL-2 is highly expressed in most ABC DLBCL cases. To potentially enhance lymphomagenesis in our mice, we aimed to combine the Myd88p.L252P mutation with Bcl-2 overexpression by making use of a newly genrated LSL.BCL2 allele, where human BCL2 expression is driven by the CAGGs promoter. Indeed, all Myd88c-p.L252P;LSL.BCL2;Cd19Cre/wt mice die of aggressive large B cell lymphoma at a median of 36 weeks. All animals showed splenomegaly and/or lymphadenopathy, accompanied by infiltration of the liver. Bone marrow involvement was only detected once and was locally restricted, indicating a secondary lesion. Clonality analysis by southern blot showed oligoclonality, suggesting a strong oncogenic potential of Myd88p.L252P in combination with BCL2 overexpression. Interestingly, lymphoma cells were of a Bcl6-/Mum1+/B220-/Cd138+ immunotype, in accordance with the plasmoblastic morphology that was histologically observed.
Conclusion
In summary, we generated a mouse model that enables Cre-mediated expression of Myd88p.L252P from the endogenous locus. Myd88c-p.L252P;Cd19Cre/wt mice develop and eventually die of lymphoproliferative disease. The emergence of diffuse large B cell lymphoma (DLBCL) was observed. Combination of B cell-specific Myd88p.L252P with BCL-2 overexpression results in the development of an aggressive lymphoma with plasmoblastic features, most reminiscent of ABC-type DLBCL.
Session topic: Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): DLBCL, Mouse model, NF- B
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Auditorium 1
Background
The adaptor protein MYD88 is critical to relay activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B cell malignancies, including diffuse large B cell lymphoma (DLBCL). 29% of activated B cell (ABC)-type DLBCL, which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2.
Aims
Here, we aimed to investigate the potential role of the Myd88p.L265P point mutation in lymphomagenesis.
Methods
We generated a novel mouse model (termed Myd88c-p.L252P), in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88p.L252P (the orthologous position of the human MYD88p.L265P mutation) from the endogenous locus. More precisely, the endogenous exons 2 to 6 of Myd88 (which consists of 6 exons) were flanked by loxP-sites. Downstream of the last exon, a second set of exons 2 to 6 was inserted, harboring the point mutation p.L252P. Cre-mediated recombination leads to the excision of the endogenous exons 2 to 6 and expression of the inserted, mutated set of exons. This system very closely mimicks the situation observed in the clinic, as it allows for the heterozygous expression of Myd88p.L252P from the endogenous locus.
Results
To assess the functionally of our allele, we generated mouse embryonic fibroblasts (MEFs) homozygous for the Myd88c-p.L252P allele, recombined them in vitro by lentiviral transduction. After puromycin selection, cDNA was sequenced and only the mutant transcript was detected. Western blotting verified the expression of this mutant transcript and increased phospho-p65 levels as a marker for NF-kB activation.We activated our allele B cell-specifically by crossing it to the Cd19-Cre mouse. The resulting Myd88c-p.L252P;Cd19Cre/wt animals had a lifespan of around 500 days, significantly shorter than that of the Cd19Cre/wt control. Magnetic resonance imaging and autopsy showed the development of splenomegaly and lymphadenopathy starting at around 60 weeks of age. Further histological and immunohistochemical investigation revealed the existence of lymphoproliferative disease and the sporadic emergence of large B cell lymphoma with an Bcl6-/Mum1+/B220+/Cd138- immunotype. Analysis of V(D)J recombination by southern blotting showed clonal populations in samples from animals diagnosed with lymphoma by histology.BCL-2 is highly expressed in most ABC DLBCL cases. To potentially enhance lymphomagenesis in our mice, we aimed to combine the Myd88p.L252P mutation with Bcl-2 overexpression by making use of a newly genrated LSL.BCL2 allele, where human BCL2 expression is driven by the CAGGs promoter. Indeed, all Myd88c-p.L252P;LSL.BCL2;Cd19Cre/wt mice die of aggressive large B cell lymphoma at a median of 36 weeks. All animals showed splenomegaly and/or lymphadenopathy, accompanied by infiltration of the liver. Bone marrow involvement was only detected once and was locally restricted, indicating a secondary lesion. Clonality analysis by southern blot showed oligoclonality, suggesting a strong oncogenic potential of Myd88p.L252P in combination with BCL2 overexpression. Interestingly, lymphoma cells were of a Bcl6-/Mum1+/B220-/Cd138+ immunotype, in accordance with the plasmoblastic morphology that was histologically observed.
Conclusion
In summary, we generated a mouse model that enables Cre-mediated expression of Myd88p.L252P from the endogenous locus. Myd88c-p.L252P;Cd19Cre/wt mice develop and eventually die of lymphoproliferative disease. The emergence of diffuse large B cell lymphoma (DLBCL) was observed. Combination of B cell-specific Myd88p.L252P with BCL-2 overexpression results in the development of an aggressive lymphoma with plasmoblastic features, most reminiscent of ABC-type DLBCL.
Session topic: Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): DLBCL, Mouse model, NF- B
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