Functional Study of Novel Gene Fusions and Patterns of Epigenetic Regulator Mutations in Adult and Childhood B-Cell Acute Lymphoblastic Leukemia

Background.

In previous study of adult and childhood B-cell acute lymphoblastic leukemia, through whole exome and transcriptome sequencing we identified three new categories of fusion genes with prognostic significance and much more mutations of epigenetic regulators in adult versus pediatric B-ALL cases. To address how these gene alterations contribute to leukemogenesis and affect prognosis, we performed detailed data analysis and functional study of the three new categories of B-ALL. In addition, the changes of epigenetic regulator were scrutinized in a systematic way.

Methods.

Differentially expressed genes analysis and gene set enrichment analysis (GSEA) were performed. The regulation of target genes by distinct fusions was further verified through lentivirus mediated overexpression, shRNA induced knockdown, quantitative real-time PCR as well as transcriptional reporter assays. The modification of histone acetylation was evaluated by Western bloting. To observe the effect of fusion genes in vivo, murine retroviral bone marrow transplantation models were established.

Results.

Through in-depth data analysis, differentially expressed genes and signaling pathways related to fusion genes were identified. Alternative splicing of transcription factor ERG was seen in most of the cases with DUX4 fusions. In some of the cases, both ERG deletion leading to frameshift of the open reading frame and an abnormal short transcript of ERG were also observed. This group can also be characterized by some outlier genes (AGAP1, STAP1, PTPRM1, PCDH17 and PLEKHA6). Overexpression of DUX4-IGH in human B-ALL cell line induced the appearance of the short form of ERG and upregulation of these outlier genes. In mouse models, both DUX4-IGH and the short ERG inhibited the development of B-cell lineage. NT5E, also known as CD73, which has been reported to promote anthracycline resistance, was significantly downregulated in this group, which could explain the favorable prognosis of this type of B-ALL. Pre-BCR signaling pathway maintaining the survival of leukemic cells at the pre-B stage was activated in patients with MEF2D fusions. Genes related to protein amimo acid phosphorylation and cell morphogenesis involved in differentiation were significantly upregulated in this group. Notably, this group exhibited rather poor survival rate, and the expression of CD73 in this group was as high as that in BCR-ABL1 and BCR-ABL1 like group. When juxtaposed to the upstream of ZNF384, EP300 and CREBBP lost the histone acetylation domain, which could reduce the histone acetylation level and be involved in the leukemogenesis. Indeed, EP300-ZNF384 induced the rapid emergence of acute leukemia in mouse with a median survival of 100 days.

In addition, we checked the sequence variations in our whole exome/genome dataset containing 203 cases. When mutations of different gene categories were scrutinized, abnormalities of epigenetic regulators (ER) drew our attention. Using the ER database that St. Jude Children's Research Hospital established, we found a total of 140 somatic mutations in 80 ER classified into eight classes. Forty two percent of the 203 patients (56.5% of adult and 30.6% of children) harbored at least one gene mutation among the eight classes of ER. The fact that abnormalities of MLL2 and SETD2, the two ER genes with highest mutation rates, were mutually exclusive in most affected cases suggests that they should bear somehow functional resemblance.

Conclusions.

Detailed study of MEF2D, ZNF384 and DUX4-IGH gene fusions expands the extant knowledge about molecular subtypes of B-ALL, and provides more potential targets as well as promotes further personalized treatment in both childhood and adult B-ALL. The overview of ER alterations in adult and childhood B-ALL proves the importance of these genes in the pathogenesis of B-ALL, especially in adult cases.