Epigenetics in acute promyelocytic leukaemia pathogenesis and treatment response: a TRAnsition to targeted therapies.

Transcriptional deregulation plays a key role in a large array of cancers, and successful targeting of oncogenic transcription factors that sustain diseases has been a holy grail in the field. Acute promyelocytic leukaemia (APL) driven by chimeric transcription factors encoding retinoic acid receptor alpha fusions is the paradigm of targeted cancer therapy, in which the application of all-trans retinoic acid (ATRA) treatments have markedly transformed this highly fatal cancer to a highly manageable disease. The extremely high complete remission rate resulted from targeted therapies using ATRA in combination with arsenic trioxide will likely be able to minimise or even totally eliminate the use of highly toxic chemotherapeutic agents in APL. In this article, we will review the molecular basis and the upcoming challenges of these targeted therapies in APL, and discuss the recent advance in our understanding of epigenetics underlying ATRA response and their potential use to further improve treatment response and overcome resistance.

Methylation of TET2, CBL and CEBPA in Ph-negative myeloproliferative neoplasms.

A loss-of-function mutation of TET2, CBL and CEBPA has been implicated in the pathogenesis or leukaemic transformation of myeloproliferative neoplasm. As tumour suppressor genes may potentially be inactivated by promoter hypermethylation, the authors studied the methylation status of these genes in three cell lines and diagnostic marrow samples from 45 patients with myeloproliferative neoplasm (MPN) (essential thrombocythaemia, N=34; polycythaemia vera, N=7 and primary myelofibrosis, N=4) by methylation-specific PCR. TET2 was heterozygously methylated in MEG-01 and K562 but completely unmethylated in HEL. On the other hand, both CBL and CEBPA were completely unmethylated in all three cell lines. In the primary marrow samples, methylation of TET2 occurred in two (5.9%) patients with essential thrombocythaemia (4.4% of all patients), both without JAK2 V617 mutation, but not in polycythaemia vera or primary myelofibrosis. There was no association between TET2 methylation with the type of MPN (p=0.713). Hypermethylation of CBL or CEBPA was not detected in any patients. In summary, methylation of TET2, CBL and CEBPA is infrequent in MPN at diagnosis. The role of methylation of these genes at the time of leukaemic transformation warrants further study.

Methylation of cyclin-dependent kinase inhibitors, XAF1, JUNB, CDH13 and soluble Wnt inhibitors in essential thrombocythaemia.

BACKGROUND: Methylation of genes regulating cell-cycle check-point (INK4 cyclin-dependent kinase inhibitors), apoptosis (XAF1), adhesion (CDH13), JUNB and Wnt signalling (soluble Wnt inhibitors) has been implicated in pathogenesis of haematological and epithelial cancers. METHOD: The authors studied the methylation status of CDKN2A, CDKN2B, XAF1, CDH13, JUNB and a panel of soluble Wnt inhibitors including WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5 by methylation-specific PCR in 31 bone marrow and 21 peripheral blood samples of patients with essential thrombocythaemia. RESULTS AND DISCUSSION: There was no evidence of hypermethylation of all these genes in both the BM and PB samples. Therefore, in contrast to myeloid leukaemias, methylation of these genes regulating the cell cycle, apoptosis, adhesion and Wnt signalling does not play an important role in the pathogenesis of myeloproliferative diseases. Whether differential methylation may occur in the progenitor or mature blood cell compartments remains to be verified. Our study contributes to the literature on methylation in chronic myeloproliferatve diseases.

Epigenetic dysregulation of Wnt signaling pathway in multiple myeloma.

Wnt signaling has recently been implicated in carcinogenesis. We studied the activity of Wnt signaling and the methylation status of WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 and SFRP5 by methylation-specific PCR in myeloma cell lines and primary myeloma samples. Of the four cell lines, Wnt signaling was constitutively activated in LP1 and WL2, correlating with hypermethylation and hence silencing. Moreover, 5-aza-2'-deoxycytidine treatment of these two cell lines showed progressive demethylation of methylated Wnt inhibitors, re-expression of transcripts and downregulation of Wnt signaling. In both LP1 and WL2 cells, multiple Wnts and Fzs were simultaneously expressed. Treatment of WL2, in which SFRP1 was completely methylated, with recombinant secreted Frizzled-related protein 1 (SFRP1) induced downregulation of Wnt signaling and inhibition of proliferation. In primary myeloma samples, 42% patients had methylation of at least one of these seven genes, of which 61.9% had > or = 2 genes methylated. In conclusion, Wnt signaling is constitutively activated in myeloma, associated with methylation silencing of one or multiple soluble Wnt antagonists. An autocrine loop regulating Wnt signaling was demonstrated in the myeloma plasma cells, in which cellular proliferation was efficiently inhibited by recombinant SFRP1. Methylation study of a panel of genes, regulating a cellular pathway instead of isolated genes, is important.

Frequent DAP kinase but not p14 or Apaf-1 hypermethylation in B-cell chronic lymphocytic leukemia.

Dysregulation of apoptosis, and thus the p14/DAP kinase/HDM2/p53/Apaf-1 pathway, is potentially important in carcinogenesis. Chronic lymphocytic leukemia (CLL), uncommon in the Chinese, is a disease characterized by impaired apoptosis, of the neoplastic lymphocytes. Hypermethylation of p14, DAP kinase and Apaf-1 was studied by methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles in 50 diagnostic marrow samples from patients with CLL. Chinese CLL patients had an indolent course similar to Caucasians with median overall survival (OS) of 96 months, which was adversely affected by advanced Rai stage (projected 5-year OS = 72% and 39% for Rai < or = 2 and Rai > 2; P = 0.01). DAP kinase was methylated in 18 (36%) patients while p14 and Apaf-1 were completely unmethylated in all the primary CLL samples. There was no correlation between DAP kinase hypermethylation and age, sex, poor-risk karyotype, lymphocyte count and Rai stage at diagnosis. Projected OS for patients with and without DAP kinase hypermethylation were 59 and 57% (P = 0.91). DAP kinase, but not p14 and Apaf-1, of the DAP kinase/p14/HDM2/p53/Apaf-1 pathway is frequently hypermethylated in CLL, but not of prognostic significance. Moreover Chinese patients with CLL share a similarly indolent clinical course, and this is the first comprehensive study on p14, DAP kinase and Apaf-1 hypermethylation in CLL.

Methylation of INK4 and CIP/KIP families of cyclin-dependent kinase inhibitor in chronic lymphocytic leukaemia in Chinese patients.

BACKGROUND: INK4 (p15, p16, p18 and p19) and CIP/KIP (p21, p27 and p57) are two families of cyclin-dependent kinase inhibitors (CKI) targeting CDK4/6 and CDK2, respectively. AIM: To study the role of methylation in the inactivation of CKI in chronic lymphocytic leukaemia (CLL). MATERIALS AND METHODS: Methylation-specific polymerase chain reaction was carried out on DNA obtained from the bone marrow of 56 newly diagnosed patients with CLL. RESULTS: Similar demographic features and clinical outcome were observed in our patients when compared with Caucasian patients, including an indolent clinical course (10-year overall survival 51%) and advanced Rai stage (p = 0.006), and a high-risk karyotype such as trisomy 12 and complex aberrations (p = 0.03). In the INK4 family, methylation in p15 and p16 occurred in 20 (35.7%) and 8 (14.3%) patients, respectively. In all, 5 (8.9%) CLL samples harboured concurrent methylation of both p15 and p16. Apart from an association of p16 methylation with higher presenting leucocyte count (64.5 x 10(9)/l in methylated p16 and 16.0 x 10(9)/l in unmethylated p16 patients; p = 0.016), there was no association between p15 and p16 methylation and age, sex and Rai stage. No difference was observed in the overall survival for patients with and without p15 and p16 methylation. By contrast, p18 and Rb were unmethylated in all samples. In the CIP/KIP family, apart from infrequent methylation of p57 in 4 (7.1%) patients, methylation of p21 and p27 was uniformly absent. CONCLUSION: p15 and, less frequently, p16 of the INK4 family of CKI, instead of the CIP or KIP family, were targeted by methylation in CLL. p16 methylation was associated with a higher lymphocyte count at presentation. This is the first comprehensive study of the epigenetic dysregulation of the INK4 and CIP/KIP families of CKI in Chinese patients with CLL.

Infrequent Wnt inhibitory factor-1 (Wif-1) methylation in chronic lymphocytic leukemia.

The Wnt pathway has been shown recently, to be activated in patients with chronic lymphocytic leukemia (CLL). This is the first study to examine the role of Wnt inhibitory factor-1 (Wif-1) methylation in the pathogenesis of haematolymphoid malignancies. Wif-1, a putative tumor suppressor, is a soluble negative regulator of the Wnt pathway activated in CLL. We studied the role of methylation of Wif-1 in 43 Chinese patients with CLL. At diagnosis, Wif-1 methylation was detected in 5/43 (11.6%) CLL marrow samples. Wif-1 methylation occurred more frequently in patients with advanced age (p = 0.059) but there was no correlation between Wif-1 methylation and sex, lymphocyte count and Rai stage at diagnosis. In conclusion, Wif-1 is infrequently methylated in CLL. Other factors leading to activation of the Wnt pathway warrant further study.

Methylation profiling in multiple myeloma.

BACKGROUND: We analysed the methylation status of a panel of 10 genes including p15, p16, DAPK, p73, VHL, E-CAD, MGMT, RARbeta, RIZ1, and ER. METHODS: The gene promoter methylation status was studied by methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) DNA in the bone marrow of 13 patients with myeloma, and one patient with plasmacytoma. RESULT: None of the 10 genes tested were methylated in eight normal bone marrow samples. For the positive control, the sensitivity of M-MSP ranged from 1 x 10(-2) for E-CAD and MGMT, to 1 x 10(-4) for p73. Of the eight diagnostic myeloma marrow samples, hypermethylation of p15, p16, E-CAD, DAPK and ER occurred in six (75%), four (50%), seven (87.5%), eight (100%), and six (75%) patients. Similarly, of the five samples from patients who progressed from plateau phase, hypermethylation of p15, p16, E-CAD, DAPK, and ER occurred in five (80%), two (40%), five (100%), five (100%), and three (60%). None of the cases had hypermethylation of RIZ1, p73, VHL, RARbeta, and MGMT. At diagnosis, all patients had concurrent hypermethylation of at least three genes, and five (62%) had concurrent methylation of four or more genes. One patient with plasmacytoma had methylation of E-CAD, ER, and DAPK. CONCLUSION: p15, p16, ER, DAPK, and E-CAD (but not RARbeta, p73, VHL, RIZ1, and MGMT) were frequently methylated in MM at both diagnosis and disease progression. Future studies of larger scale are needed to identify the genes responsible for disease progression.

Cyclin A in cell cycle control and cancer.

Cyclin A is particularly interesting among the cyclin family because it can activate two different cyclin-dependent kinases (CDKs) and functions in both S phase and mitosis. An embryonic form of cyclin A that is only essential for spermatogenesis is also present in some organisms. In S phase, phosphorylation of components of the DNA replication machinery such as CDC6 by cyclin A-CDK is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. In mitosis, the precise role of cyclin A is still obscure, but it may contribute to the control of cyclin B stability. Cyclin A starts to accumulate during S phase and is abruptly destroyed before metaphase. The synthesis of cyclin A is mainly controlled at the transcription level, involving E2F and other transcription factors. Removal of cyclin A is carried out by ubiquitin-mediated proteolysis, but whether the same anaphase-promoting complex/cyclosome targeting subunits are used as for cyclin B is debatable. Consistent with its role as a key cell cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.

Isolation and characterization of two distinct growth hormone cDNAs from the goldfish, Carassius auratus.

As a first step toward the development of a ribonuclease protection assay for studying the regulation of growth hormone (GH) gene expression in pituitary cells of the goldfish, Carassius auratus, we report the isolation of two cDNA clones encoding goldfish GH from a cDNA library prepared from pituitary poly(A)+ RNA. The complete nucleotide sequences of these two GH cDNA clones have been determined and both of them were predicted to encode a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 aa. One of the GH cDNAs encodes a polypeptide (gfGHI) with five cysteine residues (similar to other carp Ms), whereas another encodes a polypeptide (gfGHII) with four cysteine residues (similar to most teleostean GHs). Because these two GH cDNAs have distinct nucleotide sequences at their coding and 3' untranslated regions, they are likely to be encoded by two different genes.