In MLL rearranged acute leukemia (MLLre) fusions of MLL/KMT2A with over 90 partner genes generate leukemogenic fusion proteins whose activity determines deep transcriptome and proteome alterations. Leukemia-specific fusion circular RNAs (circRNAs) expressed from rearranged genomes of malignant cells can reinforce the oncogenic potential of fusion proteins (Guarnerio et al. Cell 2016). Apart from the generation of fusion circRNAs in THP1 cell line with KMT2A-MLLT3 (MLL-AF9) fusion, circRNAome deregulation in MLLre leukemia is not known. The role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. CircRNAs are abundantly expressed in the haematopoietic compartment with differences across blood cell types and maturation stages.
Starting from observed deregulation in acute leukemia of circRNAs from genes of the MLL recombinome, we aimed to provide a genome-wide picture of circRNA expression specificities of leukemic cells and to prioritize the most promising circRNAs for functional investigation.
CirComPara (Gaffo et al. NcRNA J. 2017) software, combining six different circRNA detection methods, was used to detect and quantify circRNAs from published and proprietary RNA-seq datasets (ribo-depleted RNA; Illumina® HiSeq2000 paired end 100 nt reads). Targeted quantification by qRT-PCR with divergent primers was used for circRNA validation and screening.
CircRNAs expressed in normal haematopoiesis by MLL and translocation partner genes were studied as candidates to be investigated for participation into disease mechanisms and for the formation of fusion transcripts. MLL/KMT2A and 53 translocation partner genes expressed each at least one abundant circRNA in normal haematopoiesis (CD34+ cells, and mature B-, T-cells and monocytes populations; 4 different healthy donors per cell type) for a total of 327 circRNAs, with half of the genes expressing from 2 to 31 isoforms. Examination of exons circularized in normal blood cells in relation to translocation breakpoint regions predicted perturbed circRNA expression in MLLre, confirmed by analysis of the THP1 cell line (Dal Molin et al. Front. In Gen. 2019). Next, expression comparison of 8,948 circRNAs quantified by RNA-seq in mature blood cell populations and in leukemic cells with KMT2A-MLLT1 (MLL-ENL) and KMT2A-AFF1 (MLL-AF4) fusions disclosed significant deregulation of circKMT2A and circAFF1 isoforms in MLLre compared with normal B-cells (Figure 1). Beyond the direct impact of translocations on circRNAs expressed from the fused genes, genome-wide circRNA expression deregulation in each and in both MLLre groups emerged. Targeted screening by qRT-PCR of the most interesting candidates in different leukemia cell lines and in a cohort of 32 BCP-ALL patient-derived xenograft samples including six cytogenetic subgroups (MLLre, BCR-ABL, ETV6-RUNX1, TCF3-PBX1, Hyperdiploid and B-Others) identified circRNAs specific for MLLre, which will undergo functional investigation.
In MLLre patients, circRNA expression can be disrupted by chromosomal translocations. The here uncovered deep circRNAome deregulation complemented previous observations of linear transcriptome and proteome alterations determined by leukemogenic fusion protein activity. Identification of circRNAs specifically altered in MLLre will prompt investigation of their involvement in disease mechanisms.