COMPARATIVE FUNCTIONAL ANALYSIS OF THE COMMON INTERACTORS OF 7 MLL FUSION PROTEINS
(Abstract release date: 05/19/16)
EHA Library. Skucha A. 06/10/16; 133166; P178
Mrs. Anna Skucha
Contributions
Contributions
Abstract
Abstract: P178
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 different fusion partner genes, leading to the production of novel chimeric proteins. Critical effectors of distinct MLL fusion proteins have previously been identified, and some of them were shown to hold great potential for targeted therapies. However, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist for distinct MLL fusion proteins.
Aims
We aimed to identify common critical effectors of 7 selected MLL fusion proteins (MLL-AF1p, MLL-AF4, MLL-AF9, MLL-CBP, MLL-EEN, MLL-ENL, MLL-GAS7), which have been presumed to employ different molecular mechanisms of oncogenic transformation.
Methods
Stable cell lines allowing for inducible expression of 7 different affinity-tagged MLL fusion proteins were prepared and transgene expression was verified by qRT-PCR and Western Blotting. Affinity purification coupled to mass spectrometry (AP-MS) identified novel interactors of the 7 MLL fusions. Functional annotation of the resulting interactome revealed significant enrichment of a large number of protein complexes. To assign functional information to a subset of 128 interactors, which were found to bind to ≥5 MLL fusions, we employed a RNAi screening approach. The MLL-AF9-positive MOLM-13 cell line was transduced with pools of viral vectors allowing for the expression of 6 shRNAs targeting the same candidate gene. We established a screening methodology that is suitable for positive and negative selection readouts. To confirm and further validate selected high-confidence candidates we performed additional RNAi screens in other MLL-rearranged- and MLL-wild-type leukemia cell lines and characterized RNAi-knock down efficiencies in various human and murine cell systems.
Results
Advanced statistical filtering using a novel, improved algorithm developed by us yielded a densely connected protein-protein interaction network of >950 proteins around 7 MLL fusions. Many protein complexes previously shown to be associated with MLL were significantly enriched, such as PAF-, SWI/SNF- and the RNA Pol II containing coactivator complexes. 128 proteins were found to interact with ≥5 of the 7 MLL-fusions. This list of conserved MLL-interactors is highly enriched for proteins with known functions in chromatin metabolism and transcriptional control. Several RNAi screens in different human leukemia cell lines were used to functionally dissect the conserved MLL-fusion interactome. Our screening methodology identified known MLL interacting proteins such as Menin, DPY30 and HCFC1 as critical effectors of MLL fusion proteins, validating the approach. In addition, we selected a small set of novel interactors of MLL fusion proteins for further validation experiments, including a protein involved in cytoskeletal organization.
Conclusion
In conclusion, we developed a robust experimental pipeline allowing for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive and comparative manner, which will contribute to further clarify the molecular mechanisms of MLL-fusion dependent leukemogenesis.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Leukemogenesis, MLL, Protein-protein interaction, RNA interference (RNAi)
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 different fusion partner genes, leading to the production of novel chimeric proteins. Critical effectors of distinct MLL fusion proteins have previously been identified, and some of them were shown to hold great potential for targeted therapies. However, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist for distinct MLL fusion proteins.
Aims
We aimed to identify common critical effectors of 7 selected MLL fusion proteins (MLL-AF1p, MLL-AF4, MLL-AF9, MLL-CBP, MLL-EEN, MLL-ENL, MLL-GAS7), which have been presumed to employ different molecular mechanisms of oncogenic transformation.
Methods
Stable cell lines allowing for inducible expression of 7 different affinity-tagged MLL fusion proteins were prepared and transgene expression was verified by qRT-PCR and Western Blotting. Affinity purification coupled to mass spectrometry (AP-MS) identified novel interactors of the 7 MLL fusions. Functional annotation of the resulting interactome revealed significant enrichment of a large number of protein complexes. To assign functional information to a subset of 128 interactors, which were found to bind to ≥5 MLL fusions, we employed a RNAi screening approach. The MLL-AF9-positive MOLM-13 cell line was transduced with pools of viral vectors allowing for the expression of 6 shRNAs targeting the same candidate gene. We established a screening methodology that is suitable for positive and negative selection readouts. To confirm and further validate selected high-confidence candidates we performed additional RNAi screens in other MLL-rearranged- and MLL-wild-type leukemia cell lines and characterized RNAi-knock down efficiencies in various human and murine cell systems.
Results
Advanced statistical filtering using a novel, improved algorithm developed by us yielded a densely connected protein-protein interaction network of >950 proteins around 7 MLL fusions. Many protein complexes previously shown to be associated with MLL were significantly enriched, such as PAF-, SWI/SNF- and the RNA Pol II containing coactivator complexes. 128 proteins were found to interact with ≥5 of the 7 MLL-fusions. This list of conserved MLL-interactors is highly enriched for proteins with known functions in chromatin metabolism and transcriptional control. Several RNAi screens in different human leukemia cell lines were used to functionally dissect the conserved MLL-fusion interactome. Our screening methodology identified known MLL interacting proteins such as Menin, DPY30 and HCFC1 as critical effectors of MLL fusion proteins, validating the approach. In addition, we selected a small set of novel interactors of MLL fusion proteins for further validation experiments, including a protein involved in cytoskeletal organization.
Conclusion
In conclusion, we developed a robust experimental pipeline allowing for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive and comparative manner, which will contribute to further clarify the molecular mechanisms of MLL-fusion dependent leukemogenesis.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Leukemogenesis, MLL, Protein-protein interaction, RNA interference (RNAi)
Abstract: P178
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 different fusion partner genes, leading to the production of novel chimeric proteins. Critical effectors of distinct MLL fusion proteins have previously been identified, and some of them were shown to hold great potential for targeted therapies. However, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist for distinct MLL fusion proteins.
Aims
We aimed to identify common critical effectors of 7 selected MLL fusion proteins (MLL-AF1p, MLL-AF4, MLL-AF9, MLL-CBP, MLL-EEN, MLL-ENL, MLL-GAS7), which have been presumed to employ different molecular mechanisms of oncogenic transformation.
Methods
Stable cell lines allowing for inducible expression of 7 different affinity-tagged MLL fusion proteins were prepared and transgene expression was verified by qRT-PCR and Western Blotting. Affinity purification coupled to mass spectrometry (AP-MS) identified novel interactors of the 7 MLL fusions. Functional annotation of the resulting interactome revealed significant enrichment of a large number of protein complexes. To assign functional information to a subset of 128 interactors, which were found to bind to ≥5 MLL fusions, we employed a RNAi screening approach. The MLL-AF9-positive MOLM-13 cell line was transduced with pools of viral vectors allowing for the expression of 6 shRNAs targeting the same candidate gene. We established a screening methodology that is suitable for positive and negative selection readouts. To confirm and further validate selected high-confidence candidates we performed additional RNAi screens in other MLL-rearranged- and MLL-wild-type leukemia cell lines and characterized RNAi-knock down efficiencies in various human and murine cell systems.
Results
Advanced statistical filtering using a novel, improved algorithm developed by us yielded a densely connected protein-protein interaction network of >950 proteins around 7 MLL fusions. Many protein complexes previously shown to be associated with MLL were significantly enriched, such as PAF-, SWI/SNF- and the RNA Pol II containing coactivator complexes. 128 proteins were found to interact with ≥5 of the 7 MLL-fusions. This list of conserved MLL-interactors is highly enriched for proteins with known functions in chromatin metabolism and transcriptional control. Several RNAi screens in different human leukemia cell lines were used to functionally dissect the conserved MLL-fusion interactome. Our screening methodology identified known MLL interacting proteins such as Menin, DPY30 and HCFC1 as critical effectors of MLL fusion proteins, validating the approach. In addition, we selected a small set of novel interactors of MLL fusion proteins for further validation experiments, including a protein involved in cytoskeletal organization.
Conclusion
In conclusion, we developed a robust experimental pipeline allowing for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive and comparative manner, which will contribute to further clarify the molecular mechanisms of MLL-fusion dependent leukemogenesis.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Leukemogenesis, MLL, Protein-protein interaction, RNA interference (RNAi)
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 different fusion partner genes, leading to the production of novel chimeric proteins. Critical effectors of distinct MLL fusion proteins have previously been identified, and some of them were shown to hold great potential for targeted therapies. However, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist for distinct MLL fusion proteins.
Aims
We aimed to identify common critical effectors of 7 selected MLL fusion proteins (MLL-AF1p, MLL-AF4, MLL-AF9, MLL-CBP, MLL-EEN, MLL-ENL, MLL-GAS7), which have been presumed to employ different molecular mechanisms of oncogenic transformation.
Methods
Stable cell lines allowing for inducible expression of 7 different affinity-tagged MLL fusion proteins were prepared and transgene expression was verified by qRT-PCR and Western Blotting. Affinity purification coupled to mass spectrometry (AP-MS) identified novel interactors of the 7 MLL fusions. Functional annotation of the resulting interactome revealed significant enrichment of a large number of protein complexes. To assign functional information to a subset of 128 interactors, which were found to bind to ≥5 MLL fusions, we employed a RNAi screening approach. The MLL-AF9-positive MOLM-13 cell line was transduced with pools of viral vectors allowing for the expression of 6 shRNAs targeting the same candidate gene. We established a screening methodology that is suitable for positive and negative selection readouts. To confirm and further validate selected high-confidence candidates we performed additional RNAi screens in other MLL-rearranged- and MLL-wild-type leukemia cell lines and characterized RNAi-knock down efficiencies in various human and murine cell systems.
Results
Advanced statistical filtering using a novel, improved algorithm developed by us yielded a densely connected protein-protein interaction network of >950 proteins around 7 MLL fusions. Many protein complexes previously shown to be associated with MLL were significantly enriched, such as PAF-, SWI/SNF- and the RNA Pol II containing coactivator complexes. 128 proteins were found to interact with ≥5 of the 7 MLL-fusions. This list of conserved MLL-interactors is highly enriched for proteins with known functions in chromatin metabolism and transcriptional control. Several RNAi screens in different human leukemia cell lines were used to functionally dissect the conserved MLL-fusion interactome. Our screening methodology identified known MLL interacting proteins such as Menin, DPY30 and HCFC1 as critical effectors of MLL fusion proteins, validating the approach. In addition, we selected a small set of novel interactors of MLL fusion proteins for further validation experiments, including a protein involved in cytoskeletal organization.
Conclusion
In conclusion, we developed a robust experimental pipeline allowing for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive and comparative manner, which will contribute to further clarify the molecular mechanisms of MLL-fusion dependent leukemogenesis.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Leukemogenesis, MLL, Protein-protein interaction, RNA interference (RNAi)
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