Contributions
Abstract: EP378
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Chromosomal inversion or translocation between 3q21 and 3q26 (inv(3)/t(3;3)) results in the aberrant expression of the proto-oncogenic transcription factor EVI1 located at 3q26, causing the development of Acute Myeloid Leukemia (AML). It has been shown that in those AMLs a single GATA2 enhancer at chromosome 3q21 translocates to 3q26 and turns into a hyperactive super-enhancer, driving overexpression of EVI1. AMLs with inv(3)/t(3;3) are dependent on EVI1 and are characterized by an aggressive course and are refractory to therapy.
Aims
In this study we aimed to investigate how the hijacked GATA2 enhancer leads to EVI1 activation. To this purpose, we identified sequence motifs in this enhancer essential for EVI1 transcription in inv(3)/t(3;3) AML.
Methods
We generated a model to study EVI1 regulation in inv(3)/t(3;3) AML cells by inserting a GFP reporter 3’ of endogenous EVI1 into the inv(3) MUTZ3 cell line. To uncover important transcription factor binding elements essential for driving EVI1 transcription in the GATA2 hijacked enhancer, we applied an unbiased CRISPR/Cas9 enhancer scan.To this end, we constructed a lentiviral library containing all sgRNAs covering the translocated region and transduced it into MUTZ3-EVI1-GFP cells at a low multiplicity of infection to analyze the effect of single mutations in the enhancer on EVI1 expression.
Results
The unbiased CRISPR/Cas9 scan of the translocated GATA2 enhancer in 3q26-rearranged AMLs pinpointed a single regulatory element of approximately 1 kb of open chromatin that is critically required for aberrant EVI1 expression. This element contained a DNA binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele. This motif was required for EVI1 expression but dispensable for GATA2. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the translocated GATA2 enhancer resulted in myeloid differentiation and cell death.
Conclusion
Our data show an essential role for MYB in the regulation of EVI1 expression via the translocated GATA2 enhancer. This is a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are non-functional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity.
Keyword(s): Acute myeloid leukemia, EVI1, Transcriptional regulation
Abstract: EP378
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Chromosomal inversion or translocation between 3q21 and 3q26 (inv(3)/t(3;3)) results in the aberrant expression of the proto-oncogenic transcription factor EVI1 located at 3q26, causing the development of Acute Myeloid Leukemia (AML). It has been shown that in those AMLs a single GATA2 enhancer at chromosome 3q21 translocates to 3q26 and turns into a hyperactive super-enhancer, driving overexpression of EVI1. AMLs with inv(3)/t(3;3) are dependent on EVI1 and are characterized by an aggressive course and are refractory to therapy.
Aims
In this study we aimed to investigate how the hijacked GATA2 enhancer leads to EVI1 activation. To this purpose, we identified sequence motifs in this enhancer essential for EVI1 transcription in inv(3)/t(3;3) AML.
Methods
We generated a model to study EVI1 regulation in inv(3)/t(3;3) AML cells by inserting a GFP reporter 3’ of endogenous EVI1 into the inv(3) MUTZ3 cell line. To uncover important transcription factor binding elements essential for driving EVI1 transcription in the GATA2 hijacked enhancer, we applied an unbiased CRISPR/Cas9 enhancer scan.To this end, we constructed a lentiviral library containing all sgRNAs covering the translocated region and transduced it into MUTZ3-EVI1-GFP cells at a low multiplicity of infection to analyze the effect of single mutations in the enhancer on EVI1 expression.
Results
The unbiased CRISPR/Cas9 scan of the translocated GATA2 enhancer in 3q26-rearranged AMLs pinpointed a single regulatory element of approximately 1 kb of open chromatin that is critically required for aberrant EVI1 expression. This element contained a DNA binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele. This motif was required for EVI1 expression but dispensable for GATA2. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the translocated GATA2 enhancer resulted in myeloid differentiation and cell death.
Conclusion
Our data show an essential role for MYB in the regulation of EVI1 expression via the translocated GATA2 enhancer. This is a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are non-functional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity.
Keyword(s): Acute myeloid leukemia, EVI1, Transcriptional regulation