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CEBPA-MUTANT ACUTE MYELOID LEUKEMIA IS SENSITIVE TO SMALL-MOLECULE-MEDIATED INHIBITION OF THE MENIN-MLL INTERACTION
Author(s): ,
Luisa Schmidt
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Elizabeth Heyes
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Thomas Eder
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Jolanta Grembecka
Affiliations:
Department of Pathology,University of Michigan,Ann Arbor,United States
Florian Grebien
Affiliations:
Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
(Abstract release date: 05/18/17) EHA Library. Schmidt L. 06/23/17; 181421; S134
Luisa Schmidt
Luisa Schmidt
Contributions
Abstract

Abstract: S134

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 11:45 - 12:00

Location: Room N103

Background

The CEBPA gene - encoding for the transcription factor C/EBPα - is mutated in 9% of patients with acute myeloid leukemia (AML). CEBPA N-terminal mutations lead to selective loss of full length C/EBPα p42 expression without affecting translation of the shorter p30 isoform. As a balanced ratio of C/EBPα isoforms is crucial for hematopoietic homeostasis, depletion of p42 leads to increased cell growth and blocks myeloid differentiation, resulting in the development of AML. We have recently shown that the p30 variant of C/EBPα can act as a gain-of-function allele with distinct molecular properties. However, the mechanistic basis of C/EBPα p30-induced leukemogenesis is incompletely understood.

Aims

We hypothesized that the interaction between the oncogenic C/EBPα p30 isoform and the MLL/SET histone methyltransferase complex is required for p30-dependent epigenetic and transcriptomic changes that contribute to leukemogenesis. Therefore, we aimed to investigate the sensitivity of CEBPA-mutant AML to perturbation of MLL/SET function.

Methods
We used CRISPR/Cas9-mediated mutagenesis to interfere with the MLL/SET complex in myeloid progenitor cells from a Cebpap30/p30 AML mouse model. Cellular competition assays were used to assess changes in proliferative capacity of mutant cells. Further, MLL activity was inhibited by the use of small molecules that block the Menin-MLL interaction. In both cases, proliferation, myeloid differentiation and apoptosis were used as readouts. Global changes in gene expression were measured by RNA-seq.

Results
We initially confirmed, via ChIP, that C/EBPα and MLL co-localize on the promoters of p30 target genes, indicating functional cooperativity in gene regulation.

To investigate the importance of different, annotated functional domains within the MLL protein in the context of C/EBPα p30 expression, we introduced targeted mutations across the Mll gene in Cebpap30/p30 cells using the CRISPR/Cas9 system. This analysis revealed a strong dependence of Cebpap30/p30 cells on the expression of an intact MLL protein. Surprisingly, loss of the enzymatic activity of Mll by mutational targeting of the SET domain did not significantly affect cell survival. In contrast, cells were particularly sensitive to mutagenesis of the Menin-binding motif in MLL. Mll targeting strongly induced myeloid differentiation in Cebpap30/p30 cells as measured by increased levels of myeloid surface markers.
To investigate functional consequences upon pharmacological perturbation of the MLL/SET complex, we used MI-463, a potent small-molecule inhibitor of the Menin-MLL interaction. Inhibitor treatment led to a time- and dose-dependent impairment of proliferation, induction of cell cycle arrest and increased apoptosis in Cebpap30/p30 cells. RNA-seq analysis showed that inhibitor treatment induced the expression of genes associated with myeloid differentiation, which could be confirmed by flow cytometry. Importantly, expression of C/EBPα p30 was associated with hypersensitivity to Menin-MLL inhibition, as Cebpap30/p30 cells were 2-6 fold more sensitive than other leukemia cell lines of mouse and human origin.

Conclusion
We show that CEBPA-mutated AML is highly sensitive to perturbation of the MLL/SET complex, either via genetic ablation of MLL or through pharmacological inhibition of the Menin-MLL interaction. Our data indicate that leukemic mutations of C/EBPα selectively cooperate with the SET/MLL complex to regulate gene expression. These findings expand our understanding of and may inform new therapeutic strategies for N-terminal CEBPA mutated AML.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Leukemogenesis, Inhibitor, CCAAT/enhancer binding protein alpha (C/EBPa)

Abstract: S134

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 11:45 - 12:00

Location: Room N103

Background

The CEBPA gene - encoding for the transcription factor C/EBPα - is mutated in 9% of patients with acute myeloid leukemia (AML). CEBPA N-terminal mutations lead to selective loss of full length C/EBPα p42 expression without affecting translation of the shorter p30 isoform. As a balanced ratio of C/EBPα isoforms is crucial for hematopoietic homeostasis, depletion of p42 leads to increased cell growth and blocks myeloid differentiation, resulting in the development of AML. We have recently shown that the p30 variant of C/EBPα can act as a gain-of-function allele with distinct molecular properties. However, the mechanistic basis of C/EBPα p30-induced leukemogenesis is incompletely understood.

Aims

We hypothesized that the interaction between the oncogenic C/EBPα p30 isoform and the MLL/SET histone methyltransferase complex is required for p30-dependent epigenetic and transcriptomic changes that contribute to leukemogenesis. Therefore, we aimed to investigate the sensitivity of CEBPA-mutant AML to perturbation of MLL/SET function.

Methods
We used CRISPR/Cas9-mediated mutagenesis to interfere with the MLL/SET complex in myeloid progenitor cells from a Cebpap30/p30 AML mouse model. Cellular competition assays were used to assess changes in proliferative capacity of mutant cells. Further, MLL activity was inhibited by the use of small molecules that block the Menin-MLL interaction. In both cases, proliferation, myeloid differentiation and apoptosis were used as readouts. Global changes in gene expression were measured by RNA-seq.

Results
We initially confirmed, via ChIP, that C/EBPα and MLL co-localize on the promoters of p30 target genes, indicating functional cooperativity in gene regulation.

To investigate the importance of different, annotated functional domains within the MLL protein in the context of C/EBPα p30 expression, we introduced targeted mutations across the Mll gene in Cebpap30/p30 cells using the CRISPR/Cas9 system. This analysis revealed a strong dependence of Cebpap30/p30 cells on the expression of an intact MLL protein. Surprisingly, loss of the enzymatic activity of Mll by mutational targeting of the SET domain did not significantly affect cell survival. In contrast, cells were particularly sensitive to mutagenesis of the Menin-binding motif in MLL. Mll targeting strongly induced myeloid differentiation in Cebpap30/p30 cells as measured by increased levels of myeloid surface markers.
To investigate functional consequences upon pharmacological perturbation of the MLL/SET complex, we used MI-463, a potent small-molecule inhibitor of the Menin-MLL interaction. Inhibitor treatment led to a time- and dose-dependent impairment of proliferation, induction of cell cycle arrest and increased apoptosis in Cebpap30/p30 cells. RNA-seq analysis showed that inhibitor treatment induced the expression of genes associated with myeloid differentiation, which could be confirmed by flow cytometry. Importantly, expression of C/EBPα p30 was associated with hypersensitivity to Menin-MLL inhibition, as Cebpap30/p30 cells were 2-6 fold more sensitive than other leukemia cell lines of mouse and human origin.

Conclusion
We show that CEBPA-mutated AML is highly sensitive to perturbation of the MLL/SET complex, either via genetic ablation of MLL or through pharmacological inhibition of the Menin-MLL interaction. Our data indicate that leukemic mutations of C/EBPα selectively cooperate with the SET/MLL complex to regulate gene expression. These findings expand our understanding of and may inform new therapeutic strategies for N-terminal CEBPA mutated AML.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Leukemogenesis, Inhibitor, CCAAT/enhancer binding protein alpha (C/EBPa)

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