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Letters to the Editor
Derepression of retroelements in acute myeloid
leukemia with 3q aberrations
Acute myeloid leukemia (AML) with chromosomal rearrangements inv(3)/t(3;3) (3q-AML) is a rare but high- ly fatal subtype of leukemia. It is characterized by an aberrant transcription of the proto-oncogene EVI1 (ecotropic viral integration site 1, MECOM) as a result of the chromosomal 3q21q26 rearrangements that lead to the relocation of a master GATA2 distal hematopoietic enhancer to the EVI1 locus and deregulation of both genes.1,2 To date, little is known about what triggers chro- mosomal rearrangements in 3q-AML that ultimately lead to the deregulation of EVI1 via the repositioning of G2DHE. However, recent studies have shown evidence for the involvement of endogenous transposable elements of RNA family (retroelements [RE]) in the formation of com- plex chromosomal aberrations, including translocations, large-scale duplications and amplifications through retro- transposition across different cancer entities.3 Furthermore, hypomethylation of RE has been linked to their pathogenic mobility in epithelial tumors.4–6
Here we present the results of functional genomics analysis of a cohort of 3q-AML patients. Based on our data, we hypothesized that breakpoint-associated RE (breakpoint-RE) could play an important regulatory and activating role in this AML subtype. Therefore, we per- formed an array of in vitro studies using CRISPR-Cas9 approach to dissect their role in 3q-AML.
Targeted chromosome 3q-capture sequencing of 3q- AML patient samples and cell lines with EVI1 overex- pression previously revealed a characteristic 3q21q26 pattern of patient-specific breakpoints, demarcating a leukemogenic EVI1-activating super-enhancer that is found uniquely in inv(3)/t(3;3) AML and contains G2DHE.1 In order to identify a commonality between breakpoints relevant for super-enhancer formation, we reanalyzed 3q-capture sequencing data and found that in 38 of 41 samples, chromosomal breakpoints at 3q21.3 and 3q26.2 mapped to sequences of RE, including long interspersed elements (LINE), short interspersed ele- ments (SINE) and long terminal repeats (LTR) (Figure 1A). Of note, RNA sequencing (RNA-Seq) of 3q-AML patients revealed a characteristic RNA readthrough spanning the large super-enhancer region at 3q21.3 (Figure 1B, top panel). A similar enhancer RNA (eRNA) signature was observed in non-3q-rearranged AML cases (Figure 1B, top panel; Online Supplementary Figure S3), indicating active G2DHE regulating GATA2 in its native environment. In 3q-AML, however, the RNA readthrough frequently orig- inated at 3q21.3 breakpoint sites, extending beyond the super-enhancer region. Additionally, allele-specific bisul- fite amplicon sequencing performed on selected AML cases revealed focal demethylation of CpG sites around the chromosomal breakpoints exclusively on the rearranged allele, whereas the intact allele in 3q-AML and both alleles in non-rearranged leukemic cell lines did not show any hypomethylation pattern (Figure 1B, bot- tom panel). Focal hypomethylation around breakpoints on the rearranged allele could be the consequence of chromosomal rearrangements and super-enhancer-relat- ed epigenetic reprogramming, including the deposition of active chromatin marks and physical interaction between the EVI1 promoter and G2DHE.1
Based on our RNA-Seq and bisulfite sequencing data, we investigated whether derepression of breakpoint-RE could possibly represent a priming event for an enhancer rearrangement by relaxation of the local chromatin com-
paction and may play a role in the ectopic activation of EVI1 by the super-enhancer. In order to test this hypoth- esis, we performed a CRISPR-Cas9 gene editing experi- ment using a homology-directed repair (HDR) template to insert selected 3q21.3 breakpoint-RE sequences or G2DHE in the vicinity of the EVI1 locus in the EVI1-pos- itive myeloid leukemia reporter cell line K562 that does not harbor inv(3)/t(3;3) rearrangements (Figure 2A, top panel) (Ottema et al., 2021, under review). The presence of a T2A-eGFP fusion sequence inserted downstream of EVI1 allows for correlation of EVI1 expression with the synchronously expressed green fluorescent protein (GFP). The parental reporter cell line is tolerant of increased EVI1 levels given that its baseline expression is already increased in K562. The insertion sequences were derived from 3q21.3 breakpoints of two leukemia cases: AML 3071, a patient with inv(3) AML and MOLM-1, a near- triploid myeloid leukemia cell line harboring two chro- mosome 3 alleles with inv(3) (Figure 2A, bottom panel).7 The HDR templates were inserted in the corresponding 3q26.2 breakpoint loci as found in AML 3071 and MOLM-1, that is downstream of EVI1 and within the last EVI1 intron, respectively.
Single-cell clones validated by polymerase chain reac- tion (PCR) and Sanger sequencing (Online Supplementary Figure S1A) harboring the ectopic G2DHE showed a shift in GFP fluorescence indicating successful EVI1 activation, whereas clones with 3q21.3 breakpoint-RE sequences showed no change in the GFP signal compared with untreated cells (Figure 2B). Furthermore, single-cell clones were analyzed by quantitative PCR (qPCR) and western blot, which showed results consistent with the flow cytometry analysis (Figure 2C and D, respectively), suggesting that the ectopic activation of EVI1 occurs via G2DHE, whereas breakpoint-RE themselves are insuffi- cient to induce EVI1 transcriptional activation in the K562 reporter cell line.
In order to further dissect a potential regulatory role of breakpoint-RE in 3q-AML, a reciprocal experimental CRISPR-Cas9 approach was applied to delete the original breakpoint-RE in MOLM-1 and UCSD-AML1, the latter being a t(3;3) AML cell line. We expressed pairs of single guide RNA (sgRNA) in stably Cas9-expressing cells to induce a segmental deletion of a fragment containing either the inverted (MOLM-1) or translocated (UCSD- AML1) breakpoint-RE at 3q26.2 on the rearranged alleles located within the last EVI1 intron in MOLM-1, and upstream of the EVI1 promoter in UCSD-AML1 (Figure 3A). Targeting on the non-rearranged allele was expected to result only in generation of indels at the 3q21.3 and 3q26.2 site but not segmental RE deletions. In total, two MOLM-1 and six UCSD-AML1 clones harboring the desired deletion validated by PCR and Sanger sequencing (Online Supplementary Figure S1B) were derived success- fully from single cells. Together with the nontargeting control (NTC) clones (targeted with sgRNA against mCherry and eGFP) and the wild-type (WT) cell line, we performed phenotypic analysis of obtained deletion clones. We observed no differences in proliferation between deletion and control samples (Figure 3B). Slightly reduced EVI1 expression on mRNA and protein level was observed exclusively in the MOLM-1 deletion clones (Figure 3C, left panel).
In order to identify potential genome-wide effects of CRISPR-Cas9-induced RE deletion, we performed genomic and epigenomic analyses of the MOLM-1 and UCSD-AML1 deletion and control clones using circular- ized chromatin conformation capture sequencing (4C- Seq) and chromatin immunoprecipitation followed by
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