Giulia De Falco

Gene expression analysis uncovers similarity and differences among Burkitt lymphoma subtypes

Burkitt lymphoma (BL) is classified into 3 clinical subsets: endemic, sporadic, and immunodeficiency-associated BL. So far, possible differences in their gene expression profiles (GEPs) have not been investigated. We studied GEPs of BL subtypes, other B-cell lymphomas, and B lymphocytes; first, we found that BL is a unique molecular entity, distinct from other B-cell malignancies. Indeed, by unsupervised analysis all BLs clearly clustered apart of other lymphomas. Second, we found that BL subtypes presented slight differences in GEPs. Particularly, they differed for genes involved in cell cycle control, B-cell receptor signaling, and tumor necrosis factor/nuclear factor κB pathways. Notably, by reverse engineering, we found that endemic and sporadic BLs diverged for genes dependent on RBL2 activity. Furthermore, we found that all BLs were intimately related to germinal center cells, differing from them for molecules involved in cell proliferation, immune response, and signal transduction. Finally, to validate GEP, we applied immunohistochemistry to a large panel of cases and showed that RBL2 can cooperate with MYC in inducing a neoplastic phenotype in vitro and in vivo. In conclusion, our study provided substantial insights on the pathobiology of BLs, by offering novel evidences that may be relevant for its classification and possibly future treatment.

Activation of MyoD-dependent transcription by cdk9/cyclin T2.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101–161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

CDK9: From Basal Transcription to Cancer and AIDS

Cdk9 is a member of the Cdc2-like family of kinases. Its cyclin partners are membersof the family of cyclin T (T1, T2a and T2b) and cyclin K. The Cdk9/cyclin T complexesappear to be involved in regulating several physiological processes. Cdk9/cyclin T1belongs to the P-TEFb complex, and is responsible for the phosphorylation of the carboxylterminaldomain (CTD) of the RNA Polymerase II, thus promoting general elongation.Cdk9 has also been described as the kinase of the TAK complex, which is homologousto the P-TEFb complex and involved in HIV replication. Cdk9 also appears to be involvedin the differentiation program of several cell types, such as muscle cells, monocytes andneurons, suggesting that it may have a function in controlling specific differentiativepathways. In addition, Cdk9 seems to have an anti-apoptotic function in monocytes, thatmay be related to its control over differentiation of monocytes. This data suggeststhe involvement of Cdk9 in several physiological processes in the cell, the deregulationof which may be related to the genesis of transforming events, that may in turn leadto the onset of cancer. In addition, since the complex Cdk9/cyclin T1 is able to bind tothe HIV-1 product Tat, the study of the functions of Cdk9/cyclin T may be of interest inunderstanding the basal mechanisms that regulate HIV replication.

B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression.

Endemic, sporadic and HIV‐associated Burkitt lymphoma (BL) all have a B‐cell phenotype and a MYC translocation, but a variable association with the Epstein‐Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation suggested that EBV‐positive and EBV‐negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV‐negative BLs may originate from early centroblasts, whereas EBV‐positive BLs seem to arise from postgerminal center B cells or memory B cells. The appearance of a germinal center phenotype in EBV‐positive cells might thus derive from a block in B‐cell differentiation. The exit from the germinal center involves a complex series of events, which require the activation of BLIMP‐1, and the consequent downregulation of several target genes. Here, we investigated the expression of specific miRNAs predicted to be involved in B‐cell differentiation and found that hsa‐miR‐127 is differentially expressed between EBV‐positive and EBV‐negative BLs. In particular, it was strongly upregulated only in EBV‐positive BL samples, whereas EBV‐negative cases showed levels of expression similar to normal controls, including microdissected germinal centers (GC) cells. In addition, we found evidence that hsa‐miR‐127 is involved in B‐cell differentiation process through posttranscriptional regulation of BLIMP1 and XBP1. The overexpression of this miRNA may thus represent a key event in the lymphomagenesis of EBV positive BL, by blocking the B‐cell differentiation process.

Alteration of MicroRNAs Regulated by c-Myc in Burkitt Lymphoma

Background Burkitt lymphoma (BL) is an aggressive B-cell lymphoma, with a characteristic clinical presentation, morphology and immunophenotype. Over the past years, the typical translocation t(8;14) and its variants have been considered the molecular hallmark of this tumor. However, BL cases with no detectable MYC rearrangement have been identified. Intriguingly, these cases express MYC at levels comparable with cases carrying the translocation. In normal cells c-Myc expression is tightly regulated through a complex feedback loop mechanism. In cancer, MYC is often dysregulated, commonly due to genomic abnormalities. It has recently emerged that this phenomenon may rely on an alteration of post-transcriptional regulation mediated by microRNAs (miRNAs), whose functional alterations are associated with neoplastic transformation. It is also emerging that c-Myc modulates miRNA expression, revealing an intriguing crosstalk between c-Myc and miRNAs. Principal Findings Here, we investigated the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for the translocation. A common trend of miRNA expression, with the exception of hsa-miR-9*, was observed in all of the cases. Intriguingly, down-regulation of this miRNA seems to specifically identify a particular subset of BL cases, lacking MYC translocation. Here, we provided evidence that hsa-miR-9-1 gene is heavily methylated in those cases. Finally, we showed that hsa-miR-9* is able to modulate E2F1 and c-Myc expression. Conclusions Particularly, this study identifies hsa-miR-9* as potentially relevant for malignant transformation in BL cases with no detectable MYC translocation. Deregulation of hsa-miR-9* may therefore be useful as a diagnostic tool, suggesting it as a promising novel candidate for tumor cell marker.

CDK9 (PITALRE): a multifunctional cdc2-related kinase.

CDK9 is a cdc2‐related kinase protein. Previously named PITALRE, this protein is a serine‐threonine kinase involved in many physiological processes. Unlike most of the cdc2‐like kinases, its activity is not cell cycle–regulated. CDK9 acts preferentially in processes different from cell‐cycle regulation, such as differentiation. Its cyclin partners, cyclins of T family, recently have been isolated. CDK9 immunoprecipitates with several unidentified polypeptides that may regulate its kinase activity. CDK9 has been shown to associate with the HIV‐Tat protein, suggesting a possible involvement in AIDS. CDK9 recently was shown to be responsible for the kinase activity associated with the TAK complex and with the P‐TEFb complex, suggesting activity also in the transcription process. J Cell Physiol 177:501–506, 1998.

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2‐like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B‐ and T‐cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B‐ and T‐cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T‐lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed–Sternberg cells of classical Hodgkins lymphoma also showed strong nuclear staining. Diffuse large B‐cell, Burkitts lymphomas, and peripheral T‐cell lymphomas, among T‐cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B‐cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkins lymphomas, Burkitts lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated. Copyright

Interaction between HIV-1 Tat and pRb2/p130: A possible mechanism in the pathogenesis of AIDS-related neoplasms

Tat protein is an early nonstructural protein necessary for virus replication, which is secreted by infected cells and taken up by uninfected cells. Extensive evidence indicates that Tat may be a cofactor in the development of AIDS-related neoplasms. The molecular mechanism underlying Tats oncogenic activity may include deregulation of cellular genes. Among these genes, it has recently been shown that pRb2/p130 oncosuppressor protein is one of the targets in the interaction between HIV gene product Tat and host proteins. However, whether the HIV-1 gene product Tat may inactivate the oncosuppressive function of pRb2/p130 has not yet been elucidated. Here, we show that mRNA levels of pRb2/p130 increase in the presence of Tat, whereas no change in the phosphorylation status of pRb2/p130 is observed. In addition, Tat can inhibit the growth control activity exerted by pRb2/p130 in the T98G cell line. Finally, Tat does not compete with E2F-4 in binding to pRb2/p130. The interaction between Tat and pRb2/p130 seems to result in the deregulation of the control exerted by pRb2/p130 on the cell cycle. Taken together, these results open a window on the role of pRb2/p130 in AIDS-related oncogenesis.

Role of EBV in microRNA dysregulation in Burkitt lymphoma

Since its discovery as the first human tumor virus, Epstein-Barr virus (EBV) has been implicated in the development of a wide range of B-cell lymphoproliferative disorders, the first being Burkitt lymphoma. However, the exact mechanism by which EBV promotes oncogenesis is still matter of discussion. A role in EBV-mediated transformation has been proposed for a novel described class of small non-coding RNAs, the microRNAs (miRNAs). EBV encodes viral miRNAs, through which it may interfere with the physiological regulation exterted by cellular miRNAs. In addition, EBV-coded proteins may also disturb the well-orchestrated mechanisms of regulation of cellular function. In this review, we will focus on the role of EBV in malignant transformation of Burkitt lymphoma, with a particular insight in the interplay between the virus and cellular miRNAs.

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.