Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation

Abstract

Background: The oncogenic transcription factor MYC is pathologically activated in many human malignancies. A paradigm for MYC dysregulation is offered by Burkitt lymphoma, where chromosomal translocations leading to Immunoglobulin gene-MYC fusion are the crucial initiating oncogenic events. However, Burkitt lymphoma cases with no detectable MYC rearrangement but maintaining MYC expression have been identified and alternative mechanisms can be involved in MYC dysregulation in these cases.

Methods: We studied the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level. Data was validated at the mRNA and protein level by quantitative Real-Time polymerase chain reaction and immunohistochemistry, respectively.

Results: We identified four microRNAs differentially expressed between the two groups. The impact of these microRNAs on the expression of selected genes was then investigated. Interestingly, in MYC translocation-negative cases we found over-expression of DNA-methyl transferase family members, consistent to hypo-expression of the hsa-miR-29 family. This finding suggests an alternative way for the activation of lymphomagenesis in these cases, based on global changes in methylation landscape, aberrant DNA hypermethylation, lack of epigenetic control on transcription of targeted genes, and increase of genomic instability. In addition, we observed an over-expression of another MYC family gene member, MYCN that may therefore represent a cooperating mechanism of MYC in driving the malignant transformation in those cases lacking an identifiable MYC translocation but expressing the gene at the mRNA and protein levels.

Conclusions: Collectively, our results showed that MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases are slightly different in terms of microRNA and gene expression. MYC translocation-negative Burkitt lymphoma, similarly to other aggressive B-cell non Hodgkin's lymphomas, may represent a model to understand the intricate molecular pathway responsible for MYC dysregulation in cancer.

Fig. 1

MYC mRNA and protein expression in MYC translocation-positive and -negative BL cases. a Quantitative-RT-PCR. The expression of MYC was analysed at the mRNA level in cases either carrying or lacking the translocation. RT-qPCR results show the up-regulation of the gene also in the absence of MYC translocation; (b-c) Immunohistochemistry. In the exemplifying MYC translocation-positive case (b), a strong staining in about 95 % of neoplastic cells is shown in contrast to the MYC translocation-negative one (c), in which the staining intensity was present in about 60 % of cells. b-c: MYC stain. Original Magnification (O.M): 20x

Fig. 2

a Genomic view of the distribution of MYC variants in sequenced sample. Sequence alignments of paired end reads are displayed as greybars spanning exonic sequence of different MYC isoforms (blue segments below the reads alignment). Above the reads alignment section, the coverage histogram shows the read depth distribution of the MYC gene base per base. b Histogram shows the distribution of abundance of the MYC gene calculated in transcripts parts per million (TPM), in MYC translocation-negative sample (green) and other endemic MYC translocation-positive Burkitt lymphomas RNA-seq samples (red)

Fig. 3

Unsupervised analysis of Burkitt lymphomas. a The heat map diagram shows the result of the two-way unsupervised HC of miRNAs and samples based on the expression of 1,375 miRNAs. HC, roughly discriminated MYC translocation-negative (yellow) and MYC translocation-positive (blue) cases based on the miRNA expression pattern. In the matrix, each row represents a miRNA and each column represents a sample. The color scale illustrates the relative expression level of a miRNA across all samples: red represents an expression level above the mean and green represents expression lower than the mean. b PCA confirmed the distinction between MYC translocation-positive (blue) and MYC translocation-negative (yellow) samples

Fig. 4

Differentially expressed miRNAs beetween MYC translocation-positive and negative Burkitt lymphomas a Volcano plot on T-test for different miRNA expression between MYC translocation-positive and MYC translocation-negative. Volcano plot representing filtering threshold for one-tailed T-test for differential expression analysis between MYC translocation-positive and MYC translocation-negative. The plot shows the difference between the means of MYC translocation-positive and MYC translocation-negative for each miRNA plotted against the negative log10 p-value associated with the T-test. Black horizontal shows the threshold for p-value = 0,05 and red vertical lines are used for filtering miRNAs on fold change value of 1 and −1. All of the 4 points of the plot highlighted in red represent differentially expressed miRNAs that pass the filtering thresholds on p-value and fold change. b Hierarchical clustering on 4 differentially expressed miRNAs between MYC translocation-positive and MYC translocation-negative. Hierarchical cluster in samples and miRNAs for 4 differentially expressed miRNAs that passed filtering thresholds. Each row represents a miRNA and each column represents a sample. Similar samples and miRNAs of the experiment are connected by a series of branches. The length of each branch represents the distance in terms of Pearson correlation of log2(Hy3/Hy5) between connected samples or miRNAs. The miRNA clustering tree is shown on the left. The color scale shown at the top illustrates the relative expression level of a miRNA across all samples: red represents an expression level above the mean, green represents expression lower than the mean. The samples are colour coded according to the groups; yellow are the MYC translocation-positive (BL1-10), blue are the MYC translocation-negative. c Validation of miRNA profiling was assessed by RT-qPCR, which confirmed differential expression of these miRNAs in the two groups, being hsa-miR-29a and hsa-miR-29b down-regulated and hsa-miR-513a-5p, and hsa-miR-628-3p up-regulated in MYC translocation-negative BL cases

Fig. 5

RT-qPCR validation and immunohistochemical evaluation of DNMT1 in MYC translocation-positive and MYC translocation-negative BL primary tumors. a Quantitative-RT-PCR. The expression of DNMT1 was analysed at the mRNA level by RT-qPCR. The results show up-regulation of DNMT1 in cases lacking the translocation; (b-c) Immunohistochemistry. In the exemplifying MYC translocation-positive case (b), the staining is present in about 30 % of neoplastic cells, in contrast to the MYC translocation-negative one (c), in which the positivity is depicted in about 80 % of cells. b-c: DNMT1 stain. O.M: 20x

Fig. 6

RT-qPCR validation and immunohistochemical evaluation of DNMT3A in MYC translocation-positive and MYC translocation-negative BL primary tumors. a Quantitative-RT-PCR The expression of DNMT3A was analysed at the mRNA level by RT-qPCR. As for DNMT1, DNMT3A resulted up-regulated in cases lacking the translocation; (b-c) Immunohistochemistry. In the exemplifying MYC translocation-positive case (b), the staining is shown in 40 % of neoplastic cells in contrast to the MYC translocation-negative one (c), in which about 60 % of cells are positive. b-c: DNMT3A stain. O.M: 20x

Fig. 7

RT-qPCR validation and immunohistochemical evaluation of DNMT3B in MYC translocation-positive and MYC translocation-negative BL primary tumors. a Quantitative-RT-PCR The expression of DNMT3B was analysed at the mRNA level by RT-qPCR. As for DNMT3A, DNMT3B resulted up-regulated in cases lacking the translocation; (b-c) Immunohistochemistry. In the exemplifying MYC translocation-positive case (b), the staining is shown in 5 % of neoplastic cells in contrast to the MYC translocation-negative one (c), in which about 70 % of cells are positive. b-c: DNMT3B stain. O.M: 20x

Fig. 8

RT-qPCR validation and immunohistochemical evaluation of N-MYC in MYC translocation-positive and MYC translocation-negative BL primary tumors. a Quantitative-RT-PCR The expression of NMYC was analysed RT-qPCR. MYC-translocation negative cases show a dramatic hyper-expression of the gene; altogether RT-qPCR results confirmed the bioinformatics predictions, which suggest a regulation of these by the miR29 family. Over-expression of the selected genes is in accordance with down-regulation of the miR-29 family observed in MYC-translocation negative cases; (b-c) Immunohistochemistry. In the exemplifying MYC translocation-positive case (b), the staining is present only in 5 % of neoplastic cells in contrast to the MYC translocation-negative one (c), in which the positivity is detectable in about 90 % of cells. b: H&E, c: NMYC stain. b-c, O.M: 40x