PF153 MLL‐AF4 CAUSES ABERRANT UPREGULATION OF PROM1 (CD133) IN ACUTE LYMPHOBLASTIC LEUKEMIA BY CONTROLLING ENHANCER‐PROMOTER INTERACTIONS
Authors: Godfrey L. , O’Byrne S. , Crump N.T. , Lau I.‐J. , Thorne R. , Elliott N. , Buck G. , Rice S. , Connor C. , Thomas R. , Cheng D. , Ancliff P. , Inglott S. , Roberts I. , Roy A. , Milne T.A. 
Published: June 2019

Background:

MLL rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukaemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL‐AF4 fusion protein. MLL‐AF4 is thought to promote leukaemogenesis by activating key target genes, mainly by altering the epigenetic profile of the cell through recruitment of DOT1L and increasing histone H3 lysine 79 methylation (H3K79me2/3). One of these key genes is PROM1 which encodes the cell surface protein CD133 (Prominin‐1; Mak2016). CD133 has previously been shown to be expressed on ALL blasts and is of potential therapeutic interest. However, the mechanism by which MLL‐AF4 dysregulates PROM1 expression and the impact of PROM1 expression on leukaemia cell survival remains unclear.

Aims:

To determine the mechanism by which MLL‐AF4 causes aberrant expression of PROM1 in leukaemia.

Methods:

We characterised normal fetal haematopoietic stem and progenitor cells (HSPC), blasts from patient leukaemia samples, and SEM cells (a MLL‐AF4+ cell line) by immunophenotyping and transcriptome analysis. ChIP‐Seq and Capture C (a high resolution chromosome conformation capture technique) was used to elucidate enhancer‐promoter structure at the PROM1 locus.

Results:

We find that the MLL‐AF4 complex aberrantly upregulates PROM1 transcription by controlling enhancer promoter‐interactions.

Immunophenotyping and transcriptome analysis: Immunophenotypic analysis of normal human fetal bone marrow HSPC confirmed that CD133 expression is confined to the stem cell compartment and is downregulated through B cell commitment. In contrast, MLLr patient blasts and SEM cells showed high levels of surface CD133 expression. Analysis of RNA‐Sequencing data showed that PROM1 gene expression in normal fetal B progenitors, patient blasts and SEM cells reflected the pattern of surface CD133 expression

Functional assays: Upon inhibition of MLL‐AF4 binding by a DOT1L inhibitor, CD133 expression is downregulated in SEM cells and nascent RNA‐Seq shows down‐regulation of PROM1 at the transcriptional level. Direct knock down of PROM1 in SEM cells confers slower growth rates/less colony formation and increased cell death.

ChIP‐Seq and Capture‐C: ChIP‐sequencing identified MLL‐AF4 and associated complex components binding to the PROM1 promoter and within the gene body at putative enhancer elements. Capture C profiles identified intragenic enhancer elements within the PROM1 gene body and within the nearby TAPT1 gene. Inhibition of MLL‐AF4 complex activity upon treatment with DOT1L inhibitors causing loss of H34K79me2/3 disrupted gene expression as well as enhancer‐promoter interactions.

Summary/Conclusion:

Together these results indicate that MLL‐AF4 directly up‐regulates PROM1 expression in ALL cells and promotes their proliferation and survival. Therefore, CD133 expression should not only be a useful marker for diagnosis and residual disease monitoring but might also be exploited as a therapeutic target in MLL re‐arranged ALL.