Abstract

Chronic lymphocytic leukemia, the most common form of adult leukemia in the Western world, is characterized by a heterogeneous clinical course, with indolent or progressive forms that entail profoundly different approaches to treatment. The biomarkers useful to predict the clinical course of CLL encompass cytogenetic abnormalities, protein expression (ZAP70, CD38) and IGHV mutation status. Among the many molecular features characterizing biological and clinical aspects of CLL patients, deletion of 13q14 and down-regulation of its related microRNAs (miR-15a and miR-16-1) are the most frequent aberrations in CLL. The involvement of the miR-15a and mir-16-1 in CLL has been extensively described by in vivo and in vitro studies and the genomic region encompassing the miR cluster results frequently deleted, but the mechanisms that regulate the expression of these miRs in CLL are still poorly understood. Here we demonstrate that the G to A single nucleotide polymorphism (SNP) rs115069827 prevents the maturation of miR-15a by reducing the binding of the DROSHA complex to the pri-microRNA 15a/16-1. The study of this functional SNP allowed us to discover a novel mechanism of transcription of the miR-15a/16-1 cluster, independent of DLEU2, its host gene. We demonstrate that the genomic region immediately upstream of the miR-15a/16-1, including the SNP, acts as transcriptional activator. The miR-15a/16-1 cluster is transcribed by allele-specific mechanisms: transcription of one allele is driven by the RNA Polymerase II at the DLEU2 promoter, while the other allele is transcribed by a mechanism that involves occupancy of the newly identified transcription activator region by RNA Polymerase III. Interestingly, the latter mechanism is dominant within CLL cells with mono-allelic 13q14 deletion and high expression of ZAP70 (Fisher's exact test p:0.0128) (Table1). Moreover, in our patient cohorts (training set n=28, validation set n=19) there are CLL samples with cells carrying 13q14 deletions that did not encompass the miR-15a/16-1 locus. Almost all of these show high expression of ZAP70 (Fisher's exact test training set p:0.0055; validation set p:0.0374) and primiR regulation by RPIII, in the presence of both alleles of miR-15a/16-1. By CNV analysis of a more telomeric region (∼30 kb, U59 DLEU2 region), that encompasses the second DLEU2 promoter, we demonstrate that these CLL cells carry a mono-allelic deletion of this region, which entails loss of the primiR regulation by the DLEU2 promoter ( occupied by RPII).

Table 1

primiR 15a/16-1 transcritpion by RPII independent mechanism correlates with ZAP70 expression in 13q deleted CLL.

SampleFISHCopy number variation (CNV)ZAP70IGVHRelative expression after α amanitin treatment (2µg/ml)primiR 15a/16-1
RNA Polymerase
U59 DLEU1miR 15a/16-1primiR
15a/16-1
pretRNAtyrpreACTB
preGAPDH
LLC31 13q  nd ↓ ↓ II 
LLC42 NK  +/- ↓ ↓ III 
LLC18 13q  +/- ↓ ↓ II 
LLC07 13q  +/- ↓ II 
LLC45 NK  +/- ↓ ↓ II 
LLC50 NK  +/- ↓ III 
LLC52A 13q  +/- nd ↓ III 
LLC61 13q  +/- ↓ ↓ II 
LLC81 13q  +/- ↓ ↓ II 
LLC47 13q  +/- ↓ III 
LLC03A NK  +/- ↓ nd II 
CLL4Tw 13q  +/- ↓ ↓ II 
CLL3Tw NK  +/- ↓ III 
LLC04 12tri/13q +/- +/+ ↓ ↓ III 
LLC64 13q  +/- ↓ ↓ III 
LLC70 13q +/+ +/+ ↓ III 
LLC36 13q  +/- ↓ ↓ III 
LLC80 NK +/- +/+ ↓ III 
LLC17A NK +/- +/+ nd nd ↓ III 
SampleFISHCopy number variation (CNV)ZAP70IGVHRelative expression after α amanitin treatment (2µg/ml)primiR 15a/16-1
RNA Polymerase
U59 DLEU1miR 15a/16-1primiR
15a/16-1
pretRNAtyrpreACTB
preGAPDH
LLC31 13q  nd ↓ ↓ II 
LLC42 NK  +/- ↓ ↓ III 
LLC18 13q  +/- ↓ ↓ II 
LLC07 13q  +/- ↓ II 
LLC45 NK  +/- ↓ ↓ II 
LLC50 NK  +/- ↓ III 
LLC52A 13q  +/- nd ↓ III 
LLC61 13q  +/- ↓ ↓ II 
LLC81 13q  +/- ↓ ↓ II 
LLC47 13q  +/- ↓ III 
LLC03A NK  +/- ↓ nd II 
CLL4Tw 13q  +/- ↓ ↓ II 
CLL3Tw NK  +/- ↓ III 
LLC04 12tri/13q +/- +/+ ↓ ↓ III 
LLC64 13q  +/- ↓ ↓ III 
LLC70 13q +/+ +/+ ↓ III 
LLC36 13q  +/- ↓ ↓ III 
LLC80 NK +/- +/+ ↓ III 
LLC17A NK +/- +/+ nd nd ↓ III 

Overall these data may clarify important genetic features of the major subgroup of CLL patients and highlight the concept that regulation of the expression of the miR-15a/16-1 is the critical event in CLL pathogenesis. Being ZAP70 high expression associated with poor prognosis in CLL, we show that the concomitant presence of different mechanisms of transcriptional regulation of miR-15a and miR-16-1 could have different impacts on the pathogenesis/progression of the CLL.

nd: not determined, M: IGVH mutated, U: IGVH unmutated, CNV: Copy number variation, NK, normal karyotype; 13q, deletion of 13q chromosome region; 12tri, 12 trisomy; +/+, presence of both alleles; +/-, monoallelic deletion; -, ZAP70≤20%; +, ZAP70>20%; , downregulation of RNA expression after α amanitin treatment; =, no change in the RNA expression after α amanitin treatment; II, RPII; III, RPIII. Fisher's test: RPIII vs ZAP70, p:0.0128; RPIII vs IGVH, p:0.576

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.