A proof-of-concept study for the pathogenetic role of enhancer hypomethylation of MYBPHL in multiple myeloma.

Enhancer DNA methylation and expression of MYBPHL was studied in multiple myeloma (MM). By bisulfite genomic sequencing, among the three CpGs inside the MYBPHL enhancer, CpG1 was significantly hypomethylated in MM cell lines (6.7-50.0%) than normal plasma cells (37.5-75.0%) (P = 0.007), which was negatively correlated with qPCR-measured MYBPHL expression. In RPMI-8226 and WL-2 cells, bearing the highest CpG1 methylation, 5-azadC caused enhancer demethylation and expression of MYBPHL. In primary samples, higher CpG1 methylation was associated with lower MYBPHL expression. By luciferase assay, luciferase activity was enhanced by MYBPHL enhancer compared with empty vector control, but reduced by site-directed mutagenesis of each CpG. RNA-seq data of newly diagnosed MM patients showed that MYBPHL expression was associated with t(11;14). MOLP-8 cells carrying t(11;14) express the highest levels of MYBPHL, and its knockdown reduced cellular proliferation and increased cell death. Herein, as a proof-of-concept, our data demonstrated that the MYBPHL enhancer, particularly CpG1, was hypomethylated and associated with increased MYBPHL expression in MM, which was implicated in myelomagenesis.

Venetoclax, bortezomib and S63845, an MCL1 inhibitor, in multiple myeloma.

OBJECTIVES: Venetoclax, an orally available BCL2-selective inhibitor, has demonstrated promising single-agent anti-tumour activity in myeloma especially patients with t(11;14). Herein, whether venetoclax sensitivity could be enhanced or restored in combination with bortezomib or S63845, a novel MCL1-selective inhibitor, was examined in human myeloma cell lines (HMCLs), including bortezomib-resistant HMCLs. METHODS: By MTS assay, half-maximal inhibitory concentration (IC50 ) and hence sensitivity/resistance to venetoclax, bortezomib and S63845 were determined. KEY FINDINGS: Venetoclax (IC50 ≥100 nm), bortezomib (IC50 ≥50 nm) and S63845 (IC50 ≥100 nm) resistance was observed in nine (75%), three (25%) and six (50%) HMCLs, respectively. Moreover, venetoclax sensitivity was independent of bortezomib (R2  = 0.1107) or S63845 (R2  = 0.0213) sensitivity. Venetoclax sensitivity correlated with high mRNA ratio of BCL2/MCL1 (P = 0.0091), BCL2/BCL2L1 (P = 0.0182) and low MCL1 expression (P = 0.0091). In HMCLs sensitive to both venetoclax and bortezomib/S63845, venetoclax combined with S63845 showed stronger synergistic effect than combined with bortezomib. Moreover, in venetoclax-resistant HMCLs, S63845, but not bortezomib, significantly restored venetoclax sensitivity. Conversely, bortezomib combined with S63845 did not result in augmented bortezomib sensitivity or abolishment of bortezomib resistance. CONCLUSIONS: Regardless of t(11;14), combination of venetoclax with S63845 is a promising strategy in enhancing venetoclax sensitivity or overcoming venetoclax resistance in myeloma therapy, hence warrant future clinical studies.

Epigenetic silencing of miR-340-5p in multiple myeloma: mechanisms and prognostic impact.

BACKGROUND: miR-340-5p, localized to 5q35, is a tumor suppressor miRNA implicated in multiple cancers. As a CpG island is present at the putative promoter region of its host gene, RNF130, we hypothesized that the intronic miR-340-5p is a tumor suppressor miRNA epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma. RESULTS: By pyrosequencing-confirmed methylation-specific PCR, RNF130/miR-340 was methylated in 8/15 (53.3%) myeloma cell lines but not normal plasma cells. Methylation correlated inversely with the expression of both miR-340-5p and RNF130. In completely methylated WL-2 and RPMI-8226R cells, 5-AzadC treatment led to demethylation and re-expression of miR-340-5p. In primary samples, RNF130/miR-340 methylation was detected in 4 (22.2%) monoclonal gammopathy of undetermined significance, 15 (23.8%) diagnostic myeloma, and 7 (23.3%) relapsed myeloma. RNF130/miR-340 methylation at diagnosis was associated with inferior overall survival (median 27 vs. 68 months; P = 3.944E-5). In WL-2 cells, overexpression of miR-340-5p resulted in reduced cellular proliferation [MTS, P = 0.002; verified in KMS-12-PE (P = 0.002) and RPMI-8226R (P = 2.623E-05) cells], increased cell death (trypan blue, P = 0.005), and enhanced apoptosis by annexin V-PI staining. Moreover, by qRT-PCR, overexpression of miR-340-5p led to repression of both known targets (CCND1 and NRAS) and bioinformatically predicted targets in MAPK signaling (MEKK1, MEKK2, and MEKKK3) and apoptosis (MDM4 and XIAP), hence downregulation of phospho-ERK1/2 and XIAP by Western blot. Furthermore, by qRT-PCR, in CD138-sorted primary samples (n = 37), miR-340-5p and XIAP were inversely correlated (P = 0.002). By luciferase assay, XIAP was confirmed as a direct target of miR-340-5p via targeting of the distal but not proximal seed region binding site. CONCLUSIONS: Collectively, tumor-specific methylation-mediated silencing of miR-340-5p is likely an early event in myelomagenesis with adverse survival impact, via targeting multiple oncogenes in MAPK signaling and apoptosis, thereby a tumor suppressive miRNA in myeloma.

Frequent functional activation of RAS signalling not explained by RAS/RAF mutations in relapsed/refractory multiple myeloma.

RAS mutations are frequent in relapsed/refractory multiple myeloma (RRMM) but functional study in primary samples is scanty. Herein, in primary myeloma plasma cells of 17 suspected RRMM, functional activation of RAS signalling was studied by Western blot of phosphorylated ERK1/2 (phospho-ERK1/2). Moreover, activating mutations in KRAS, NRAS, BRAF, and ALK were studied by PCR and bidirectional direct sequencing. Furthermore, methylation of negative RAS signalling regulator genes, RASSF1A and RASD1, were analyzed by methylation-specific PCR. As evidenced by phospho-ERK1/2 over-expression, functional RAS activation was detected in 12 (75.0%) RRMM. Of patients with functional RAS activation, sequencing data showed only seven (58.3%) patients with one each had NRAS Q61H, NRAS Q61K, KRAS G12D, KRAS G12V, KRAS G13D, KRAS Q61P, or BRAF V600E mutation, whereas five (41.7%) patients had no RAS/RAF mutation. Conversely, patients without functional RAS activation had no RAS/RAF mutation. Moreover, none of the patients with functional RAS activation had ALK mutations, or methylation of RASSF1A and RASD1. Collectively, functional activation of RAS signalling was present in majority of RRMM but only about half (58.3%) accountable by RAS/RAF mutations. If verified in larger studies, clinical investigations of MEK inhibitors are warranted regardless of RAS/RAF mutations.

Epigenetic silencing of LPP/miR-28 in multiple myeloma.

AIMS: miR-28-5- is a tumour suppressor microRNA implicated in cancers. As a CpG island is absent in miR-28-5- but present in its host gene, LPP (LIM domain containing preferred translocation partner in lipoma), we hypothesized that miR-28-5p is epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma. METHODS: Methylation-specific PCR, verified by quantitative bisulfite pyrosequencing, was employed to study methylation of LPP/miR-28 in healthy controls (n=10), human myeloma cell lines (HMCLs) (n=15), and primary myeloma marrow samples at diagnosis (n=49) and at relapse (n=18). Quantitative reverse transcription PCR was used to investigate expression of miR-28-5p, LPP and CCND1. RESULTS: LPP/miR-28 was completely unmethylated in all healthy controls and 12 (80%) HMCLs, but partially methylated in three (20%) HMCLs. Methylation of LPP/miR-28 correlated with low expression of miR-285p (p=0.012) and LPP (p=0.037) in HMCLs. In RPMI-8226R cells, in which LPP/miR-28 was partially methylated, 5-AzadC treatment led to demethylation of LPP/miR-28 and re-expression of both miR-28-5p (p=0.0007) and LPP (p=0.0007), whereas continuous culture without 5-AzadC restored LPP/miR-28 methylation and reduced expression of both miR-28-5p (p=0.0013) and LPP (p=0.0025). Moreover, a known miR-28-5p target, CCND1, was expressed at higher levels in HMCLs with LPP/miR-28 methylation than those without, consistent with a tumour suppressor role of miR-28-5p in myeloma. However, in primary samples, LPP/miR-28 was methylated in two (4.1%) at diagnosis, whereas none at relapse. CONCLUSIONS: This is the first report of epigenetic regulation of the intronic miR-28-5p expression by promoter DNA methylation of its host gene, hence warrants further study in different cancers.

Epigenetic silencing of EVL/miR-342 in multiple myeloma.

miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in multiple cancers. As the promoter region of its host gene, EVL, is embedded in a CpG island, we postulated that miR-342-3p is an intronic miRNA co-regulated with its host gene by promoter DNA methylation in multiple myeloma (MM). By methylation-specific polymerase chain reaction, verified by quantitative bisulfite pyrosequencing, methylation of EVL/miR-342 was absent in all healthy controls (n = 10) and 12 of 15 (80%) human myeloma cell lines (HMCLs), but partially methylated in 3 of 15 (20%) HMCLs, including KMS-12-PE, OCI-MY5, and RPMI-8226R. In HMCLs, by real-time quantitative reverse transcription-polymerase chain reaction, methylation of EVL/miR-342 correlated with lower expression of both EVL (P = 0.013) and miR-342-3p (P = 0.023). Moreover, in KMS-12-PE and RPMI-8226R cells, both partially methylated for EVL/miR-342, 5-AzadC treatment led to demethylation of EVL/miR-342 and re-expression of miR-342-3p. Upon removal of 5-AzadC, continuous culture resulted in restoration of EVL/miR-342 methylation and downregulation of miR-342-3p. In primary samples, methylation of EVL/miR-342 was detected in 1 of 18 (5.6%) monoclonal gammopathy of undetermined significance (MGUS), 8 of 63 (12.7%) diagnostic MM, and 5 of 30 (16.7%) relapsed MM. EVL/miR-342 methylation was preferentially detected in IgD MM but not found to impact survival. Collectively, in MM, miR-342-3p is an intronic miRNA regulated by promoter DNA methylation of its host gene, EVL, in a tumor-specific manner. Methylation of EVL/miR-342 was present in consecutive stages of myelomagenesis including MGUS, diagnostic MM, and relapsed MM.

Epigenetic silencing of tumor suppressor long non-coding RNA BM742401 in chronic lymphocytic leukemia.

BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in chronic lymphocytic leukemia (CLL). The promoter of BM742401 was unmethylated in normal controls including three each of normal bone marrow, peripheral blood buffy coats, and CD19-sorted peripheral B-cells, but methylated in four (57.1%) CLL cell lines. Methylation of BM742401 correlated inversely with expression. In the completely methylated WAC3CD5+ CLL cells, 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic but not caspase-8-dependent extrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (43.9%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (≥ stage 2)(P = 0.002). Furthermore, BM742401 methylation was associated with miR-129-2 methylation (P = 0.05). BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The mechanism of BM742401 as a tumor suppressor warrants further studies.

High applicability of ASO-RQPCR for detection of minimal residual disease in multiple myeloma by entirely patient-specific primers/probes.

Allele-specific oligonucleotide real-time quantitative PCR (ASO-RQPCR) is a standardized technique for detection and monitoring of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) but not multiple myeloma (MM) due to a low applicability inherent with presence of somatic hypermutation. Herein, by a staged PCR approach and sequencing, clonality and tumor-specific complementarity-determining region 3 (CDR3) sequence were identified in 13/13 MM by sequential PCR of IgH VDJ (n = 10), IgH DJ (n = 2), or IgK VJ (n = 1). Using consensus primers/probes conventionally employed in ALL, ASO-RQPCR worked in three (23.1 %) cases only. Conversely, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in eight (61.5 %) cases with a sensitivity of 5 × 10-4-10-5. Moreover, using standard curves constructed by serial dilution of plasmids cloned with patient-specific CDR3, ASO-RQPCR was successful in 12 (92.3 %) cases with a sensitivity of 10-4-10-5, but not in a case lacking an N region, in which design of a tumor-specific ASO primer was precluded. Finally, in a patient in complete response (CR), further reduction of MRD after autologous stem cell transplantation (ASCT) was demonstrated. In summary, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in >90 % MM patients and enabled detection of dynamic changes of MRD before and after ASCT despite conventional CR.

Epigenetic silencing of tumor suppressor miR-3151 contributes to Chinese chronic lymphocytic leukemia by constitutive activation of MADD/ERK and PIK3R2/AKT signaling pathways.

We hypothesize that miR-3151, localized to a GWAS-identified chronic lymphocytic leukemia (CLL) risk locus (8q22.3), is a tumor suppressor miRNA silenced by promoter DNA methylation in CLL. The promoter of miR-3151 was methylated in 5/7 (71%) CLL cell lines, 30/98 (31%) diagnostic primary samples, but not normal controls. Methylation of miR-3151 correlated inversely with expression. Treatment with 5-Aza-2'-deoxycytidine led to promoter demethylation and miR-3151 re-expression. Luciferase assay confirmed MAP-kinase activating death domain (MADD) and phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2) as direct targets of miR-3151. Moreover, restoration of miR-3151 resulted in inhibition of cellular proliferation and enhanced apoptosis, repression of MADD and PIK3R2, downregulation of MEK/ERK and PI3K/AKT signaling, and repression of MCL1. Lastly, miR-3151 methylation was significantly associated with methylation of miR-203 and miR-34b/c in primary CLL samples. Therefore, this study showed that miR-3151 is a tumor suppressive miRNA frequently hypermethylated and hence silenced in CLL. miR-3151 silencing by DNA methylation protected CLL cells from apoptosis through over-expression of its direct targets MADD and PIK3R2, hence constitutive activation of MEK/ERK and PI3K/AKT signaling respectively, and consequently over-expression of MCL1.

DNA methylation of tumor suppressor protein-coding and non-coding genes in multiple myeloma.

Multiple myeloma is an incurable hematological malignancy arising from immortalized plasma cells in the bone marrow. DNA methylation refers to the catalytic addition of a methyl group to the cytosine ring of a CpG dinucleotide. Methylation of a promoter-associated CpG island, a cluster of CpG dinucleotides, may lead to silencing of the associated gene. In carcinogenesis, methylation of protein-coding or non-coding tumor suppressor genes/miRNAs is associated with transcriptional silencing, loss of tumor suppressor function and prognostic significance. This review first introduces pathogenesis of myeloma and DNA methylation in cancer. Then, it summarizes methylation of protein-coding tumor suppressor genes, especially, the latest genome-wide methylation studies in myeloma, followed by the latest findings of methylation of non-coding tumor suppressor miRNAs in myeloma.

Epigenetic silencing of a long non-coding RNA KIAA0495 in multiple myeloma.

In multiple myeloma, a long non-coding RNA, KIAA0495 (alias PDAM/TP73-AS1), had been found progressively downregulated from normal plasma cell to benign monoclonal gammopathy of undetermined significance to symptomatic myeloma. Herein, by methylation-specific PCR, the putative KIAA0495 promoter was found unmethylated in all healthy controls (N = 14) but methylated in 50 % of myeloma cell lines (N = 10). KIAA0495 methylation was shown inversely correlated with KIAA0495 expression. However, KIAA0495 methylation was detected in none of both primary myeloma samples at diagnosis (N = 61) and at relapse/progression (N = 16). Collectively, despite frequently methylated in cell lines, KIAA0495 methylation appeared unimportant in the pathogenesis or progression of myeloma.

Infrequent DNA methylation of miR-9-1 and miR-9-3 in multiple myeloma.

AIMS: The miR-9 family microRNAs (miRNAs) are tumour suppressor miRNAs implicated in carcinogenesis. We postulated that miR-9-1, miR-9-2 and miR-9-3 may be inactivated by aberrant promoter methylation in multiple myeloma (MM). METHODS: Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied by methylation-specific PCR (MSP) in six normal controls, including three each of healthy peripheral blood (PB) or bone marrow buffy coat, 10 MM cell lines, 62 primary MM marrow samples at diagnosis and 22 at relapse/progression. RESULTS: MSP, verified by quantitative pyrosequencing, showed that the promoters of miR-9-3 and miR-9-1 were unmethylated in normal controls but methylated in 4 (40%) and 5 (50%) of 10 MM cell lines, respectively. However, the promoter of miR-9-2 was methylated in three normal PB buffy coat controls and in CD138-sorted healthy marrow plasma cells, indicating possibly tissue specific rather than tumour-specific methylation of miR-9-2, which was thus not studied further. In WL-2 cells, which were completely methylated for miR-9-3, 5-aza-2'-deoxycytidine treatment caused miR-9-3 promoter demethylation and pri-miR-9-3 re-expression. In primary samples, methylation of miR-9-3 was detected in 1 of 62 patients at diagnosis and 1 of 22 patients at relapse/progression. However, miR-9-1 methylation was absent in both primary samples at diagnosis and at relapse/progression. CONCLUSIONS: Hypermethylation of miR-9-3 and miR-9-1 is tumour-specific in MM, leading to reversible miRNA silencing. Frequent methylation of miR-9-3 and miR-9-1 in cell lines, but not in primary samples, may be acquired during in vitro culture, and indicates an unimportant role of miR-9 methylation in myelomagenesis.

Methylation of miR-155-3p in mantle cell lymphoma and other non-Hodgkin's lymphomas.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma (NHL). In cancers, tumor suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling of representative EBV-negative MCL cell lines before and after demethylation treatment, miR-155-3p was found significantly restored. Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-aza-2'-deoxycytidine treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumor suppressive properties. By luciferase assay, lymphotoxin-beta (LT-ß) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.024), and LT-ß upregulation (p=0.043). Collectively, miR-155-3p is a potential tumor suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-ß, which is an upstream activator of the non-canonical NF-kB signaling, miR-155-3p methylation is potentially important in lymphomagenesis.

Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia--implications on constitutive activation of NFκB pathway.

BACKGROUND: The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL). METHODS: Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction. RESULTS: The promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2'-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04). CONCLUSIONS: Of the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.

Establishment of a bortezomib-resistant Chinese human multiple myeloma cell line: MMLAL.

BACKGROUND: A new human myeloma cell line, MMLAL, was established from the myelomatous pleural effusion of a 73-year-old Chinese patient suffering from symptomatic International stage III IgG/lambda myeloma. After a brief period of complete remission, he developed aggressive systemic relapse complicated by malignant pleural effusion with exclusive plasma cell infiltration. His disease remained chemo-refractory, and died six months after relapse. METHODS: Purified mononuclear cells from the pleural effusion of the patient were cultured in the presence of IL-6. Continually growing cells were characterized by morphological, immunophenotypic, cytogenetic, fluorescence in situ hybridization (FISH) and TP53 mutation analyses. Cell proliferation was measured and compared with other myeloma cell lines by cell counting at day 3, 6, 9, and 12. Drug resistance against bortezomib, a proteasome inhibitor approved as a frontline chemotherapy for eligible myeloma patients, was evaluated and compared with other myeloma cell lines by MTT assay. RESULTS: Immunophenotypic analysis of the myeloma cells confirmed strong expression of plasma cell markers CD38 and CD138 but not T-cell or natural killer-cell marker CD56. Cytogenetic analysis of the myeloma cells showed a hypodiploid composite karyotype including loss of chromosome 13 and 17 or deletion of the short arm of chromosome 17, i.e. del(17p), in the form of isochromosome 17q10. FISH confirmed a hypodiploid karyotype with TP53 deletion but absence of t(4;14). Sequencing analysis of the TP53 gene indicated absence of mutation. Cell counting revealed that the maximum viable cell density was about 2.5 X 106 cells/ml. Upon bortezomib treatment, MTT assay reported an IC50 of 72.17nM, suggesting a strong bortezomib resistance. CONCLUSION: A hypodiploid with loss of chromosome 13 and loss or del(17p) human myeloma cell line, MMLAL, was established from the pleural effusion of a Chinese myeloma patient.

Epigenetic inactivation of the MIR129-2 in hematological malignancies.

BACKGROUND: MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers. PATIENTS AND METHODS: Epigenetic inactivation of MIR129-2 was studied by methylation-specific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression of MIR129 and its target, SOX4, in cell lines was measured before and after hypomethylating treatment and MIR129 overexpression. MIR129 expression was correlated with MIR129-2 methylation status in primary lymphoma samples. Tumor suppressor function of MIR129 was demonstrated by MTT and trypan blue exclusion assay after MIR129 overexpression. RESULTS: The sensitivity of the methylated-MSP was one in 10(3). Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression (p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated for MIR129-2, led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation. CONCLUSIONS: MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis.

DNA Methylation of Tumor Suppressive miRNAs in Non-Hodgkin's Lymphomas.

DNA methylation is an epigenetic alteration leading to heritable phenotypic changes of cells with functional consequences. It is important in early embryonic development, stem cell differentiation, and tissue-specific gene expression. In normal cells, promoter-associated CpG islands (CGI) are generally unmethylated except in X-chromosome inactivation or genomic imprinting. In cancer, tumor cells are characterized by global hypomethylation but locus-specific hypermethylation of promoter-associated CGI, resulting in gene silencing. MicroRNAs (miRNAs) are short, non-coding RNA sequences of 18-25 nucleotides, which can repress the translational of multiple protein-coding mRNAs by sequence-specific binding to the 3'untranslated region. Depending on the genes targeted, miRNA can be tumor suppressive if an oncogene is repressed, or it can be oncogenic when a tumor suppressive gene is repressed. Recently, aberrant methylation of tumor suppressive miRNAs has been reported in different types of cancers including lymphomas. Herein, we review the recent literature of methylation of tumor suppressive miRNAs in different histopathologic subtypes of lymphomas, and discuss its potential diagnostic, prognostic, and therapeutic significance.

DNA methylation of microRNA genes in multiple myeloma.

DNA methylation is one of the heritable epigenetic modifications, leading to repressed gene expressions and consequent phenotypic alterations without changing the DNA sequence. MicroRNA (miRNA) is a novel class of short non-coding RNA molecules regulating a wide range of cellular functions through translational repression of their target genes. Recently, epigenetic dysregulation of tumor-suppressor miRNA genes by promoter DNA methylation has been implicated in human cancers, including multiple myeloma (MM). This article presents a brief overview of the pathogenesis of MM, the role of DNA methylation in cancer biology, methods of DNA methylation analysis, miRNA biology and dysregulation of miRNAs in MM and summaries the current data on the role of DNA methylation of tumor-suppressive miRNAs in MM.

DNA methylation of tumor suppressor miRNA genes: a lesson from the miR-34 family.

miRNA is a small ncRNA of 22-25 nucleotides, which leads to mRNA degradation or translational inhibition of its target genes. miRNAs are involved in multiple cellular processes, including cellular differentiation, proliferation and apoptosis, and hence miRNA deregulation has been implicated in disease states, including cancer. On the other hand, DNA methylation leads to gene silencing, and serves as an alternative mechanism of gene inactivation. The aberrant DNA methylation of gene promoters has been shown to result in the inactivation of tumor suppressor genes, and therefore is also implicated in carcinogenesis. This article focuses on the role of miRNA methylation, in particular miR-34a, in cancer. The article begins with an overview of DNA methylation in normal and cancer cells and deregulation of miRNA expression by DNA methylation. These discussions are followed by a description of the gene structure of the miR-34 family of miRNA genes, the tumor suppressor role of miR-34a and the deregulation of miR-34a by DNA methylation in both epithelial and hematological cancers. Moreover, the methylation of miR-34b/c in cancer is also described. Finally, the potential role of miRNA methylation as a biomarker for diagnosis, prognosis (and hence the potential of developing a risk-stratified approach) and a therapeutic target is discussed.

Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms.

BACKGROUND: MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis. METHODS: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs. RESULTS: Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation. CONCLUSION: This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.