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FUNCTIONAL PROTEOMICS IDENTIFIES SETD2 AS A CRITICAL EFFECTOR OF MLL FUSION PROTEINS TO SAFEGUARD GENOMIC INTEGRITY.
Author(s): ,
Anna Skucha
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria
,
Jessica Ebner
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Johannes Schmöllerl
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Adrián César Razquin
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria
,
Thomas Eder
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Alexey Stukalov
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria
,
Sarah Vittori
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria
,
Matthias Muhar
Affiliations:
IMP - Research Institute of Molecular Pathology,Vienna,Austria
,
Mareike Roth
Affiliations:
IMP - Research Institute of Molecular Pathology,Vienna,Austria
,
Balazs Györffy
Affiliations:
MTA TTK Lendület Cancer Biomarker Research Group,Budapest,Hungary
,
Peter Valent
Affiliations:
Department of Internal Medicine, Division of Hematology & Hemostaseology, Medical University of Vienna,Vienna,Austria
,
Keiryn Bennett
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria
,
Johannes Zuber
Affiliations:
IMP - Research Institute of Molecular Pathology,Vienna,Austria
,
Giulio Superti-Furga
Affiliations:
CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences,Vienna,Austria;Center for Physiology and Pharmacology,Medical University of Vienna,Vienna,Austria
Florian Grebien
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
(Abstract release date: 05/18/17) EHA Library. Skucha A. 06/23/17; 181420; S133
Mrs. Anna Skucha
Mrs. Anna Skucha
Contributions
Abstract

Abstract: S133

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 11:30 - 11:45

Location: Room N103

Background
Acute Myeloid Leukemia (AML) frequently harbors chromosomal rearrangements involving the Mixed Lineage Leukemia (MLL) gene. More than 65 different MLL fusion genes exist and many of them have been described to act as strong cancer drivers. While critical effectors of several distinct MLL fusion proteins (MLL-FPs) were identified, it is not clear if transforming mechanisms are conserved across the entire family of MLL fusions. 

Aims

We hypothesized that common oncogenic mechanisms are encoded in stable physical and genetic MLL-fusion-specific interaction networks. Thus, we aimed to identify common critical effectors of different MLL fusion proteins that are presumed to employ different mechanisms of oncogenic transformation. 

Methods

Protein complexes of 7 molecularly distinct, affinity-tagged MLL-FPs (MLL-AF4, MLL-AF9, MLL-ENL, MLL-CBP, MLL-EEN, MLL-GAS7 and MLL-AF1p) were purified from stable cell lines allowing for inducible, single-copy transgene expression and characterized by mass spectrometry. Data analysis identified a comprehensive protein-protein interaction network, which was functionally interrogated by a subtractive shRNA screening approach. Validation experiments included detailed RNAi- and CRISPR/Cas9-mediated loss of function experiments in cell lines and primary cells in vitro and in vivo, using read-outs for changes in proliferation, differentiation, apoptosis and DNA damage.

Results
Characterization of the protein complexes nucleated by 7 MLL fusion proteins by affinity purification coupled to mass spectrometry (AP-MS) revealed a densely interconnected protein-protein interaction network of 963 proteins, comprising previously known MLL-interacting protein complexes (such as PRC2 or SWI/SNF), as well as a high number of new interaction partners of MLL. 128 proteins were found to interact with ≥5 of all 7 MLL-fusions. This subset of conserved MLL-interaction partners was highly enriched for proteins with function in chromatin metabolism and transcriptional control. Systematic functional investigation of the conserved MLL-fusion interactome using subtractive shRNA screens identified the methyltransferase SETD2 as a critical effector of MLL fusion proteins. Both RNAi-based suppression and CRISPR/Cas9-mediated mutagenesis of SETD2 induced myeloid differentiation and apoptosis in human and mouse MLL-rearranged cell lines, while having only modest effects on the proliferation of MLL-wild-type leukemia cells. Depletion of Setd2 in MLL-fusion-transformed mouse fetal liver cells resulted in loss of serial re-plating capacity in vitro and prolonged disease onset in vivo. Furthermore, knockdown of SETD2 caused a proliferative disadvantage in primary cells from AML patients with different MLL-rearrangements without affecting MLL-wild-type AML cells. We found that SETD2 was essential for efficient repair of DNA breaks, as SETD2-deficient leukemia cells showed increased levels of DNA damage and activation of p53, leading to the accumulation of mutations.

Conclusion
In summary, our data highlight the functional relevance of combined proteomic-genomic cellular screening to identify critical effectors of MLL-FPs. In addition, our study identifies a novel role for SETD2 in the maintenance of genomic integrity during initiation and progression of MLL-rearranged AML and establishes SETD2 as a therapeutic target in leukemia with low genomic complexity.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): 11q23, RNA interference (RNAi), Protein-protein interaction, MLL

Abstract: S133

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 11:30 - 11:45

Location: Room N103

Background
Acute Myeloid Leukemia (AML) frequently harbors chromosomal rearrangements involving the Mixed Lineage Leukemia (MLL) gene. More than 65 different MLL fusion genes exist and many of them have been described to act as strong cancer drivers. While critical effectors of several distinct MLL fusion proteins (MLL-FPs) were identified, it is not clear if transforming mechanisms are conserved across the entire family of MLL fusions. 

Aims

We hypothesized that common oncogenic mechanisms are encoded in stable physical and genetic MLL-fusion-specific interaction networks. Thus, we aimed to identify common critical effectors of different MLL fusion proteins that are presumed to employ different mechanisms of oncogenic transformation. 

Methods

Protein complexes of 7 molecularly distinct, affinity-tagged MLL-FPs (MLL-AF4, MLL-AF9, MLL-ENL, MLL-CBP, MLL-EEN, MLL-GAS7 and MLL-AF1p) were purified from stable cell lines allowing for inducible, single-copy transgene expression and characterized by mass spectrometry. Data analysis identified a comprehensive protein-protein interaction network, which was functionally interrogated by a subtractive shRNA screening approach. Validation experiments included detailed RNAi- and CRISPR/Cas9-mediated loss of function experiments in cell lines and primary cells in vitro and in vivo, using read-outs for changes in proliferation, differentiation, apoptosis and DNA damage.

Results
Characterization of the protein complexes nucleated by 7 MLL fusion proteins by affinity purification coupled to mass spectrometry (AP-MS) revealed a densely interconnected protein-protein interaction network of 963 proteins, comprising previously known MLL-interacting protein complexes (such as PRC2 or SWI/SNF), as well as a high number of new interaction partners of MLL. 128 proteins were found to interact with ≥5 of all 7 MLL-fusions. This subset of conserved MLL-interaction partners was highly enriched for proteins with function in chromatin metabolism and transcriptional control. Systematic functional investigation of the conserved MLL-fusion interactome using subtractive shRNA screens identified the methyltransferase SETD2 as a critical effector of MLL fusion proteins. Both RNAi-based suppression and CRISPR/Cas9-mediated mutagenesis of SETD2 induced myeloid differentiation and apoptosis in human and mouse MLL-rearranged cell lines, while having only modest effects on the proliferation of MLL-wild-type leukemia cells. Depletion of Setd2 in MLL-fusion-transformed mouse fetal liver cells resulted in loss of serial re-plating capacity in vitro and prolonged disease onset in vivo. Furthermore, knockdown of SETD2 caused a proliferative disadvantage in primary cells from AML patients with different MLL-rearrangements without affecting MLL-wild-type AML cells. We found that SETD2 was essential for efficient repair of DNA breaks, as SETD2-deficient leukemia cells showed increased levels of DNA damage and activation of p53, leading to the accumulation of mutations.

Conclusion
In summary, our data highlight the functional relevance of combined proteomic-genomic cellular screening to identify critical effectors of MLL-FPs. In addition, our study identifies a novel role for SETD2 in the maintenance of genomic integrity during initiation and progression of MLL-rearranged AML and establishes SETD2 as a therapeutic target in leukemia with low genomic complexity.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): 11q23, RNA interference (RNAi), Protein-protein interaction, MLL

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