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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Acute promyelocytic leukemia (APL) is driven by the oncogene PML-RARA which is generated by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, and which strongly interferes with downstream signalling and the architecture of multiprotein structures known as PML nuclear bodies (NBs). NB disruption is a diagnostic hallmark of APL, yet the significance of this phenomenon to disease pathogenesis and treatment response remains poorly understood. The majority of APL patients can now be cured with combination therapy with arsenic trioxide (ATO) and ATRA (All Trans-Retinoic Acid), which synergize promoting re-formation of disrupted Pml NBs. To date, the importance of NB disruption has only been studied in vitro. To address this, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues (C62A/C65A) in the RING domain of Pml. Homozygous PmlC62A/C65A mice are viable, and developmentally normal. At a cellular level, Pml NB disruption was confirmed and treatment with ATO was associated with defective Pml SUMOylation and degradation. A key feature of APL fusion proteins is the capacity to homodimerise (mediated by the fusion partner e.g. PML), which is not a feature of wild-type RARα. This forced homodimerisation of RARα has been shown to be critical for APL pathogenesis. We investigated whether Pml NB disruption could cooperate in vivo with forced RARα homodimerisation (mediated artificially by linking RARα to the dimerisation domain of the NFκB p50 subunit). While no leukemias arose in PmlC62A/C65A mice, p50-RARα mice expressing PmlC62A/C65A presented a doubling in the rate of leukemia development (p<0.0001) compared to PmlWT-p50-RARα, leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency period to onset of leukemia was significantly reduced in the context of NB disruption (p=0.008). ATRA treatment significantly improved the survival of mice transplanted with PmlWT-p50-RARα or Pml-RARα leukemic blasts, but not with PmlC62A/C65A-p50-RARα. These data reveal not only the key role of PML-RARα expression-induced NB disruption in APL development, but also the importance of re-formation of NBs for an effective response to differentiating drug. While formation of the PML-RARA fusion is considered an initiating event in APL pathogenesis, it is insufficient for the full leukemic phenotype. Exome sequencing studies have consistently identified presence of cooperating mutations. Since Pml and Pml NB have established roles in DNA repair and in the maintenance of genomic stability, we speculated that loss of NB integrity could affect these functions. Whole exome sequencing revealed a pattern of higher genomic instability in PmlC62A/C65A-p50-RARα leukemia as compared to PmlWT-p50-RARα, with detection of mutations found in human APL, including Kras, Ptpn11 and Usp9y. Using DNA repair reporter assays, we demonstrated that DNA repair via both non-homologous end joining (NHEJ; p=0.01) and homologous recombination (HR; p=0.006) pathways was less efficient in PmlC62A/C65A primary cells than in PmlWT cells. Importantly, using a PML-RARα-inducible cell line, comparable defects in the NHEJ and HR pathways, which were PML-RARα dependent, were identified. These data were also supported by an increase in sister-chromatid exchange (p<0.0001) and chromosome abnormality (p=0.0002) in the context of PmlC62A/C65A versus PmlWT. Interestingly, the kinetic of repair of ionising radiation (IR)-induced DNA double-strand breaks, assessed by analysis of γH2AX foci formation and clearance, was not affected. None of the DNA repair players analysed (e.g. Blm, Rad51 and 53BP1) failed to form foci in response to IR. However, their basal levels of foci were significantly greater in the presence of PmlC62A/C65A (p<0.04; quantified using Amnis ImageStreamX Mk II imaging flow cytometer). Additionally, we found that Rad51 foci showed a defect in localisation post-IR when PmlC62A/C65A was expressed, with impairment of Rad51 co-localisation and interaction with γH2AX. Altogether, our data therefore highlight the significant contribution of Pml NB to the effectiveness of DNA damage repair processes, and the manner in which their disruption mediated by the PML-RARα oncoprotein can assist APL pathogenesis. Disclosures Hills: TEVA: Honoraria. Grimwade:TEVA: Research Funding.

Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents. Disclosures: No relevant conflicts of interest to declare.

Acute promyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15;17) which results in the expression of the chimeric protein PML- RARα. Compared to the wild type retinoic acid receptor α (RARα), the fusion protein acquires dominant oncogenic properties and the chromosomal rearrangement is identified as the trigger of APL. However the pathogenesis of APL cannot be explained by the sole failure of RARα regulation and additional genetic and epigenetic alterations are required. We and others have shown that the microRNAs (miRNAs) clustered in the chromosome 14q32 imprinted domain and epigenetically regulated by the upstream differentially methylated regions (DMRs) are overexpressed only in APL (Dixon-McIver et al., 2008; Li et al., 2008; Valleron et al., 2012). Here, using high-throughput amplicon bisulfite sequencing (Roche 454), we characterized the DNA methylation profile of the DMRs in bone marrow/peripheral blood samples from patients with APL, other subclasses of acute myeloid leukemia (AML) and from healthy donors. Sequence reads were quality filtered and a total of 923,981 used to determine the methylation status of 202 CpGs. We identified an APL-specific hypermethylation signature (Fig. 1) at the DMR that spans the promoter of the MEG3 gene (MEG3-DMR) and partially overlaps the miRNA cluster. Hypermethylation encompassed the binding site motifs for the enhancer blocking protein CTCF. Consistent with the CTCF insulating activity, CpG methylation at the CTCF binding sites positively correlated with the expression of miRNAs (Fig. 2). Notably, no significant DNA methylation changes were detected at the intergenic imprinting control region (IG-DMR). Indeed, consistent with a scenario whereby only the genes regulated by the MEG3-DMR would be affected by the aberrant methylation, the gene expression profile performed on a cohort of 97 AML patients showed that among the imprinted genes of the domain only MEG3 was distinctively up-regulated in APL. Taking advantage of the long sequence reads obtained, we performed the haplotype analysis of the DNA methylation changes in diagnostic/remission sample pairs and demonstrated that hypermethylation arises in a mono-allelic manner in APL (Fig. 3). As the expression of the 14q32 miRNAs in the adult is normally restricted to the brain, we propose a model in which loss of imprinting (LOI) at 14q32 leads to aberrant expression of the miRNAs in APL cells. This study provides novel insights into the epigenetic characterization of APL and the mechanism underlying the deregulation of a specific cluster of miRNAs in this subtype of leukemia. The 14q32 miRNAs include species with oncogene and tumor-suppressor activity and their up-regulation may play a role in the APL pathogenesis. Further investigations are required to determine whether LOI is involved in the cancer initiation or it occurs at a later stage, possibly in association with the expression of the chimeric protein PML-RARα.Figure 1Unsupervised hierarchical cluster analysis of the CpG methylation levels. Each row represents a CpG site and each column a sample. The percentage of CpG methylation is depicted using color scales of red (CpG methylation > 50%) and green (CpG methylation < 50%). Sample group labels are indicated (APL; Control; Remission; AMLs).Figure 1. Unsupervised hierarchical cluster analysis of the CpG methylation levels. Each row represents a CpG site and each column a sample. The percentage of CpG methylation is depicted using color scales of red (CpG methylation > 50%) and green (CpG methylation < 50%). Sample group labels are indicated (APL; Control; Remission; AMLs).Figure 2Correlation between DNA methylation levels and miRNAs expression. The expression of 14q32 miRNAs was correlated with the DNA methylation level at the DMRs. The position of CpG islands, CTCF binding sites (A-B-C-D-E-F-G) and amplicons is labeled with green, red and blue horizontal bars, respectively. Amplicons 1-9 reside in the MEG3-DMR. The correlation is represented with red and green vertical bars indicating positive and negative values, respectively.Figure 2. Correlation between DNA methylation levels and miRNAs expression. The expression of 14q32 miRNAs was correlated with the DNA methylation level at the DMRs. The position of CpG islands, CTCF binding sites (A-B-C-D-E-F-G) and amplicons is labeled with green, red and blue horizontal bars, respectively. Amplicons 1-9 reside in the MEG3-DMR. The correlation is represented with red and green vertical bars indicating positive and negative values, respectively.Figure 3Haplotype analysis. The sequenced amplicons were interrogated using the available SNPs (UCSC database). When a heterozygous SNP was observed, sequence reads were separated accordingly to the SNP genotype and unsupervised cluster analysis performed on the allelic CpG methylation pattern. Allelic DNA methylation profiles at the MEG3-DMR: the diagnostic (A) and complete remission (B) stages of a patient with APL; (C) healthy donor. D) Allelic DNA-methylation profile at the IG-DMR, healthy donor. Each column represents a CpG site and each row the methylation pattern of a single sequence read. Blue, methylated; yellow not methylated.Figure 3. Haplotype analysis. The sequenced amplicons were interrogated using the available SNPs (UCSC database). When a heterozygous SNP was observed, sequence reads were separated accordingly to the SNP genotype and unsupervised cluster analysis performed on the allelic CpG methylation pattern. Allelic DNA methylation profiles at the MEG3-DMR: the diagnostic (A) and complete remission (B) stages of a patient with APL; (C) healthy donor. D) Allelic DNA-methylation profile at the IG-DMR, healthy donor. Each column represents a CpG site and each row the methylation pattern of a single sequence read. Blue, methylated; yellow not methylated. Disclosures: No relevant conflicts of interest to declare.