Biology of t(6;11) Fusion Proteins and Their Role in MLL-Rearranged Acute Leukemia Lineage Determination

Abstract

Chromosomal translocations are genetic rearrangements where a chromosomal segment is transferred to a non-homologous chromosome which give rise to novel chimeras. Chromosomal rearrangements play a significant role in the development of acute leukemias (acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)). Chromosomal translocation events occurring at 11q23 involving the KMT2A or Mixed-Lineage Leukemia (MLL) gene (n=102) can be diagnosed in about 5-10% of all acute leukemia patients (Marschalek Ann Lab Med 2016), especially prevalent in infant acute leukemias (up to 70% of cases). Different chromosomal translocation partner genes (such as AF4, AF6, AF9orENL and ELL) account for the majority of leukemia cases and have their genomic breakpoints within a major breakpoint cluster region (BCR intron 9-11; Meyer et. al. Leukemia 2018). Some rearrangements are specifically associated with particular disease phenotype e.g. the majority of ALL patients (~ 90%) are mainly caused by the following gene fusions, MLL-AF4, MLL-AF9, MLL-ENL. We are interested in a rare but yet drastic chromosomal translocation t(6;11)(q27;q23) which fuses KMT2A/MLL to Afadin (AFDN/AF6) gene. This chromosomal rearrangement has a very poor prognosis (survival-rate is ~10%) and is predominantly diagnosed in patients with high-risk AML. In this project, we investigate the molecular consequences of two different MLL-AF6 fusions and their corresponding reciprocal AF6-MLL fusions. MLL-AF6 fusions are mainly occurring within MLL intron 9 to 11 and are associated with an AML disease phenotype, while the same fusion occurring within the minor breakpoints region in MLL intron 21 until exon (ex) 24 are mainly diagnosed with T-ALL (T-cell acute lymphoblastic leukemia) disease phenotype. The molecular mechanism that determines the resulting disease phenotype is yet unknown. Therefore, we cloned all of these t(6;11) fusion proteins in order to investigate the functional consequences of the two different breakpoints (MLLex1-9::AF6ex2-30, AF6ex1::MLLex10-37; MLLex1-21::AF6ex2-30, AF6ex1::MLLex22-37). All 4 fusion genes were introduced into our inducible Sleeping Beauty system (Ivics et. al. Mobile DNA 2010; Kowarz et. al. Biotechnol J. 2015) and stably transfected reporter cell lines. Basically, these 4 fusion proteins differ only in the presence or absence of their Plant homeodomain 1-3/Bromodomain (PHD1-3/BD) domain (see Figure 1). The PHD domain regulates the epigenetic and transcriptional regulatory functions of wildtype MLL. Subsequently, we analyzed gene expression differences by the MACE-Seq (Massive Analyses of cDNA Ends). MACE data revealed fundamental differences in gene expression profiles when analyzing the two different sets of t(6;11) fusion genes. The resulting profiles have similarities to either AML or T-ALL and might give a rational explanation for the different lineages in these t(6;11) patients. Altogether, these results notably indicate that our study will provide a novel insight into this type of high-risk leukemia and subsequently will be useful for developing of novel and appropriate therapeutic strategies against acute leukemia. Disclosures No relevant conflicts of interest to declare.