Key Points• Loss of imprinting occurs at the 14q32 domain in APL.• DNA methylation at the CTCF binding sites correlates with the overexpression of 14q32 miRNAs.Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-a (PML-RARa)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/ remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL. (Blood. 2014;123(13):2066-2074 IntroductionAcute promyelocytic leukemia (APL) is a subclass of acute myeloid leukemia (AML) characterized by the balanced reciprocal translocation t(15;17)(q22;q11-12) resulting in the fusion between the promyelocytic leukemia gene (PML) and the retinoic acid receptor-a (RARA) gene. 1 The chimeric protein PML-RARa leads to a block of myeloid cell differentiation through constitutive repression of retinoic acid responsive genes.2 This is consistent with the typical accumulation of abnormal hematopoietic progenitor cells blocked at the promyelocyte stage. Accordingly, PML-RARa has been shown to induce APL in transgenic mice. 3 However, deregulation of the retinoic acid pathway is insufficient to initiate APL, 4 and several studies have shown additional genetic and epigenetic processes that accompany the expression of the PML-RARa protein, 5,6 also involving master transcription regulators 7 and modulators of chromatin structure. 8,9 MicroRNAs (miRNAs) are single-stranded small noncoding RNAs (sncRNAs) that negatively regulate the expression of target genes.10 They have been extensively associated with cancer as regulators of cell proliferation, differentiation, and apoptosis. 11 We have previously reported a signature of overexpressed miRNAs in APL.12 These miRNAs are clustered in the DLK1-DIO3 imprinted domain on chromosome 14q32, 13,14 and their specific upregulation in primary APL cells was also confirmed by other ...

A hallmark of acute promyelocytic leukemia (APL) is altered nuclear architecture, with disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) mediated by the PML-retinoic acid receptor α (RARα) oncoprotein. To address whether this phenomenon plays a role in disease pathogenesis, we generated a knock-in mouse model with NB disruption mediated by 2 point mutations (C62A/C65A) in the Pml RING domain. Although no leukemias developed in Pml mice, these transgenic mice also expressing RARα linked to a dimerization domain (p50-RARα model) exhibited a doubling in the rate of leukemia, with a reduced latency period. Additionally, we found that response to targeted therapy with all- retinoic acid in vivo was dependent on NB integrity. PML-RARα is recognized to be insufficient for development of APL, requiring acquisition of cooperating mutations. We therefore investigated whether NB disruption might be mutagenic. Compared with wild-type cells, primary Pml cells exhibited increased sister-chromatid exchange and chromosome abnormalities. Moreover, functional assays showed impaired homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways, with defective localization of Brca1 and Rad51 to sites of DNA damage. These data directly demonstrate that Pml NBs are critical for DNA damage responses, and suggest that Pml NB disruption is a central contributor to APL pathogenesis.

The promyelocytic leukemia gene (PML) encodes a protein which localizes to PML-nuclear bodies (NBs), sub-nuclear multi-protein structures, which have been implicated in diverse biological functions such as apoptosis, cell proliferation and senescence. However, the exact biochemical and molecular basis of PML function up until now has not been defined. Strikingly, over a decade ago, PML-NBs were found to be disrupted in acute promyelocytic leukemia (APL) in which PML is fused to the gene encoding retinoic acid receptor alpha (RARA) due to the t(15;17) chromosomal translocation, generating the PML-RARA chimeric protein. The treatment of APL patients with all-transretinoic acid (ATRA) and arsenic trioxide which target the PML-RARA oncoprotein results in clinical remission, associated with blast cell differentiation and reformation of the PML NBs, thus linking NB integrity with disease status. This review focuses on the current theories for molecular and biochemical functions of the PML-NBs, which would imply a role in the pathogenesis of APL, whilst also discussing the intriguing possibility that their disruption may not be in itself a significant oncogenic event.

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