A Single Oncogenic Enhancer Rearrangement Causes Concomitant EVI1 and GATA2 Deregulation in Leukemia

Abstract

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

The New Clinicopathologic and Molecular Findings in Myeloid Neoplasms With inv(3)(q21q26)/t(3;3)(q21;q26.2)

“…It is of great interest to note that positioning of an enhancer element was noted by Suzukawa et al 51 back in 1994, but regrettably, the hypothesized enhancer element was wrongly proposed to be associated with RPN1 gene rather than the GATA2 gene because of the lack of further functional studies. 51 As clearly demonstrated by Gröschel et al, 59 there is no new fusion transcript generated, as had been previously thought. 51 By using a completely different approach, namely, bacterial artificial chromosome transgenic mouse model recapitulating the inv(3), Yamazaki et al 60 have shown that the transgenic mice only developed leukemias when EVI1 was ectopically activated by G2DHE, but this did not happen in mice missing GATA2 enhancer, further corroborating the findings by Gröschel and associates.…”

“…As such, this enhancer is named G2DHE. Gröschel et al 59 have showed that the rearrangements of 3q21q26.2 in a form of inversion [inv(3)] or translocation [t(3;3)] result in reallocation of G2DHE to physically interact with the promoter of EVI1, leading to 2 simultaneous events: an ectopically activating EVI1 oncogene, and the concomitant loss of enhancer activity for endogenous GATA2 function with resultant GATA2 haploinsufficiency 59 ( Figure 3, C). It is of great interest to note that positioning of an enhancer element was noted by Suzukawa et al 51 back in 1994, but regrettably, the hypothesized enhancer element was wrongly proposed to be associated with RPN1 gene rather than the GATA2 gene because of the lack of further functional studies.…”

“…51 As has been well commented on by Koche and Armstrong, 58 the exciting breakthrough regarding the molecular event in inv(3)/t(3;3) came from 2 independent groups of investigators who both convincingly demonstrated that the reallocation of GATA2 distal hematopoietic enhancer (G2DHE) to the proximity of the promoter of EVI, and the subsequent activation of the EVI expression are the fundamental molecular events underlying the inv(3)/t(3;3). 59,60 By employing next-generation sequencing and high-resolution chromosome conformation capture sequencing, Gröschel et al 59 have mapped a 9-kb EVI1-promoter-contact fragment within the 18-kb commonly translocated segment in AML and MDS patients with inv(3)/t(3;3). By using functional genomics, including genomic editing and genome engineering, they elegantly identified this 9-kb EVI1-promoter-contact fragment at the enhancer originally located 110 kb upstream of GATA2 rather than RPN1, although RPN1 lies within immediate proximity of this enhancer.…”

“…51 By using a completely different approach, namely, bacterial artificial chromosome transgenic mouse model recapitulating the inv(3), Yamazaki et al 60 have shown that the transgenic mice only developed leukemias when EVI1 was ectopically activated by G2DHE, but this did not happen in mice missing GATA2 enhancer, further corroborating the findings by Gröschel and associates. 59 POTENTIAL THERAPEUTIC APPLICATIONS Because MDS and AML patients with inv(3)/t(3;3) exhibit poor prognosis and currently available treatment regimens do not achieve desirable outcomes, a search for better treatment options is needed. At the experimental level, Zhang et al 61 have showed that blocking of EVI1 DNA binding by pyrrole-imidazole polyamide 1 completely blocked EVI1-responsive reporter activity, and the growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited.…”

2016 Revision to the WHO classification of acute myeloid leukemia

“…Stefen et al analyzed 41 samples using targeted next generation sequencing (NGS), functional genomics and genome-engineering, with confirmed EVI1 overexpression and harboring an inv(3)(q21.3q26.2) or a t(3;3)(q21.3q26.2), including 38 primary bone marrow samples from patients, that is AML (n=33), chronic myeloid leukemia -blast crisis (CML-BC, n=2), and myelodysplastic syndromes (MDS, n=3), as well as three cell lines (MUTZ-3, molm-1 and UCSD-AML1). [1] The data showed that inv(3)/t(3;3) chromosomal rearrangements cause dysregulation of two specific AML predisposition genes, EVI1 and GATA2, by aberrant activity of a single enhancer element in its ectopic chromatin environment, without the formation of an oncogenic fusion product. The identified enhancer appears to originally control the transcription of 110 kb distant GATA2 gene at 3q21, and not the nearby gene RPN1.…”