Abstract

While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.