università di sienaDEPARTMENT OF MEDICAL BIOTECHNOLOGIES

MASTER’S DEGREE IN MEDICAL BIOTECHNOLOGIES

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

Supervisor: Prof. Lorenzo LeonciniCorrelator: Dr. Lucia Mundo Degree Thesis of:

Chennuboina Praveen

Academic Year 2014 - 2015

Aim

• Increasing information identifies other essential pathways that are activated in the pathogenesis of Burkitt lymphoma and highlights the fact that MYC translocation alone is insufficient to drive lymphomagenesis. Therefore Burkitt lymphoma cases lacking the typical MYC translocation, but expressing MYC at the protein level, may represent a good model for a more detailed description of MYC regulation.

• In this study we investigated the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level.

Burkitt lymphoma

• Burkitt lymphoma is a high grade B lineage non-Hodgkin lymphoma and 97% of the tumours associated with Epstein - Barr virus (EBV) infection.

• Firstly described by Denis Parsons Burkitt in 1958 in equatorial Africa, and recognized by world health organization (W.H.O) classification of hematopoietic and lymphoid neoplasms as a highly aggressive mature B-cell lymphoma .

• Burkitt lymphoma was commonly found to be occur throughout tropical Africa, and frequently seen in children as well as among immune compromised individuals

Children with Burkitt lymphoma and their involvement in various body sites

Histology

• Histologically, the tumor is composed of monomorphic cells of intermediate size with round and multiple nucleoli relatively abundant in basophilic cytoplasm, which can give cells a cohesiveness appearance.

• This type of tumor shows high proliferative activity associated with high rate of apoptosis and these cells are surrounded by scattered macrophages that engulf apoptotic cells, give to the tissue a characteristic aspect of starry sky. Typical starry sky patterns of Burkitt lymphoma

Clinical variants

• Based on clinical and geographic presentations Burkitt lymphoma can be classified into three clinical variants: Endemic Burkitt lymphoma, Sporadic Burkitt lymphoma and Immunodeficiency associated Burkitt lymphoma.

Endemic burkitt lymphoma:

• Frequently affects children and young people.

• Equatorial Africa, Papua New Guinea and south America has high rate of incidence for this type of tumor.

• Highly associated with EBV infection and co-infection by plasmodium falciparum also plays an significant role in the pathogenesis of endemic Burkitt lymphoma (eBL).

Lymphoma belt

MYC translocation

• At molecular level, Burkitt lymphoma is characterized by translocation between MYC gene and one of immunoglobulin heavy or light chains.

• In 85% of the cases, the translocation involves chromosome 8 and 14 but less frequently the translocation also involves chromosome t(8,2) or chromosome t(8,22).

MYC gene translocation in Burkitt lymphoma

MYC gene

• belongs to the family of MYC proteins which includes c-myc, L-myc, N-myc and S-myc, but only C-myc, L-myc and N-myc has neoplastic potential.

• The product of the MYC gene, c-MYC, is a global regulator of transcription.

• Translocation of the MYC oncogene results in promoting cellular proliferation and down-regulating the expression of human leukocyte antigen (HLA) class I, thus allowing the tumor cells to evade host immune control.

Crystal Structure of the c-Myc (red) in complex with Max (blue) and DNA

N-myc

• N-myc is another member of MYC family proteins, encoded by MYCN gene.

• located on the short arm of chromosome 2.

• It has N-terminal transactivation domain and C-terminal domain.

• with C-myc it plays a key role in embryonic development.

• Over expression and amplification of N-myc causes tumorigenesis.

N-myc gene structure, functional domains and protein regulatory sites

• However, Burkitt lymphoma cases with no detectable MYC translocation but maintaining MYC expression have been identified, there may be an alternative mechanism can be involved

• over-expression of DNA methyl transferases (DNMT) family members was observed in MYC translocation negative burkitt lymphoma cases

DNA methyltransferases

• DNA methyl transferases (DNMTs) are enzymes that catalyze DNA methylation, which is an epigenetic mechanism that alters protein expression.

• Genomic methylation patterns are frequently altered in tumor cells with global hyper methylation.

Structure of DNA methyltransferases (DNMTs) and DNMT-like proteins

microRNAs

• The expression of DNA-methyl transferase family member can be modulated by the expression of specific microRNAs is shown in a recent paper.

• miRNAs are small, non-coding, single-stranded RNA molecules.

• Till now 940 distinct miRNAs were identified in the human genome.

• able to negatively regulate gene expression by mRNA cleavage or transcriptional inhibition at the post transcriptional level. Biogenesis of microRNAs

Epstein-Barr Virus(EBV)

• Almost always endemic Burkitt lymphoma (eBL) is associated with Epstein-Barr Virus (EBV) infection.

• Epstein-Barr Virus(EBV) genome encodes various types of proteins, which can interact with anti-apoptic molecules, cytokines and signal transducers in promoting Epstein-Barr Virus (EBV) infection, immortalization and transformation.

• Epstein-Barr Virus (EBV) virus exhibit 3 types of latencies, selective expression of EBNA1 present in Burkitt lymphoma at Latency type I.

Epstein-Barr Virus(EBV) genome

Epstein-Barr Virus(EBV) latent proteinsGene

Expressed

EBNA

1

EBNA2 EBNA-

3A

EBNA-

3B

EBNA-

3C

EBNA-

LP

LMP1 LMP

2A

LMP

2B

EBER

Product Protein Protein Protein Protein Protein Protein Protein Protein Protein ncRNA

s

Latency I + - - - - - - - - +

Latency II +   - - - - - - - +

Latency III + + + + + + + + + +

Epstein-Barr Virus(EBV) expression of RNA and Viral Proteins in the three Phases of Latency

Results

MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases express MYC at both mRNA and protein levels

MYC mRNA and protein expression in MYC translocation-positive and -negative Burkitt lymphoma cases

• A. RT-qPCR results show the up- regulation of the MYC gene in both MYC translocation Positive and Negative Burkitt lymphoma cases at mRNA level (A).

• B-C. Immunohistochemistry results shows staining intensity was about 95% neoplastic cells in MYC translocation Positive cases (B) and 60% of neoplastic cells in MYC translocation negative cases at Protein level.

Next generation sequencing

A. Genomic view of the distribution of MYC variants in sequenced sample, and B. Histogram shows the distribution of abundance of the MYC gene in MYC translocation-negative sample (green) and other endemic MYC translocation-positive Burkitt lymphomas RNA-seq samples (red)

• we sought to verify whether cryptic MYC abnormalities might have been missed by FISH analysis. To this aim, we studied by RNA-sequencing of MYC translocation-negative Burkitt lymphoma case. Indeed, analysis of the MYC locus revealed a normal structure of MYC transcripts.

MYC translocation-positive and MYC translocation-negative Burkitt lymphoma

cases present with different microRNA expression patterns

• To ascertain whether there was a distinctive miRNA signature for MYC translocation-positive and negative Burkitt lymphomas, we profiled ten MYC translocation-positive and ten MYC translocation-negative Burkitt lymphoma cases.

Unsupervised analysis of Burkitt lymphomas

• A. Unsupervised hierarchical clustering (HC) of MYC translocation-negative (yellow) and MYC translocation positive (blue) cases roughly discriminated based on the miRNA expression pattern. 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. Principle Component Analysis(PCA) confirmed the distinction between MYC translocation-positive (blue) and MYC translocation-negative (yellow) samples.

Differentially expressed miRNAs beetween MYC translocation-positive and negative Burkitt lymphomas

• Supervised Hierarchical clustering (HC) results shows different expression patterns of 4 specific microRNAs hsa-miR-29a, hsa-miR-29b, hsa-miR-513a-5p, hsa-miR-628-3p in MYC translocation Positive and MYC translocation Negative Burkitt lymphoma cases.

• Validation of miRNA profiling was assessed by RT-qPCR, which confirmed differential expression of these miRNAs in the two groups.

Target ID P value Fold change

(absolute value)

Regulation in MYC-

negative

hsa-miR-513a-5p

hsa-miR-628-3p

hsa-miR-29a

hsa-miR-29b

 

0,031124841

0,004815838

0,0142882

0,001516702

1,02109958

1,01011474

1,086645638

1,5403288

Down

Down

Up

Up

Different microRNA expression patterns of MYC translocation-negative Burkitt lymphoma cases

The microRNA pattern impacts on the gene expression profiling (GEP) of Burkitt lymphoma cases

• After identification of miRNAs differential expression in MYC translocation-positive and MYC translocation-negative samples, we investigated whether they could affect the gene expression pattern of the tumors.

• Since a direct regulation of DNMT family members and MYCN by hsa-miR-29b has been previously demonstrated . DNMT1, DNMT3A, DNMT3B and MYCN mRNA expression analysis was performed in a total of 10 MYC translocation-positive and 10 MYC translocation- negative cases by RT-qPCR.

RT-qPCR validation and immunohistochemical evaluation of DNMT1 in MYC translocation-positive and MYC translocation-negative Burkitt lymphoma primary tumors

• A. 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.

RT-qPCR validation and immunohistochemical evaluation of DNMT3A in MYC translocation-positive and MYC translocation-negative Burkitt lymphoma primary tumors

• A. 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.

RT-qPCR validation and immunohistochemical evaluation of DNMT3B in MYC translocation-positive and MYC translocation-negative Burkitt lymphoma primary tumors

• A. 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.

RT-qPCR validation and immunohistochemical evaluation of N-MYC in MYC translocation-positive and MYC translocation-negative Burkitt lymphoma primary tumors

• A. The expression of NMYC was analysed by RT-qPCR. MYC-translocation negative cases show a dramatic hyper-expression of the gene.

• 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.

DNMT1, DNMT3A, DNMT3B and NMYC protein expression in Burkitt lymphoma tumor samples

• DNMTs and N-MYC protein expression was analyzed in 10 MYC translocation-positive and 10 MYC translocation-negative Burkitt lymphoma tumor samples by immunohistochemistry.

• In MYC translocation-positive Burkitt lymphomas the expression of DNMT1, DNMT3A, DNMT3B proteins was low/intermediate, but in MYC translocation negative Burkitt lymphomas the expression was high.

• N-MYC protein expression was low in all the MYC translocation-positive Burkitt lymphomas and MYC-translocation negative samples demonstrated higher N-MYC positivity.

Conclusion

• The evidences of N- MYC and DNA methyl transferase family member dysregulation point at a more complex scenario involving MYC and other players in Burkitt lymphoma tumorigenesis, and underline the role of a miRNAs- MYC feedback loop. Therefore, MYC translocation- negative Burkitt lymphoma cases can represent a model to understand the intricate molecular pathways responsible for both MYC over-expression and its interaction with complex cellular processes.

Thank youMy special thanks to :

Prof. Lorenzo Leoncini Dr. Lucia Mundo Dr. Sara Gazaneo