Biology and Epidemiology of APRIL and BLyS in B-cell and NHL

Principal Investigator: James R. Cerhan, M.D., Ph.D., Mayo Clinic
Co-Principal Investigator: Anne J. Novak, Ph.D., Mayo Clinic

B-cell malignancies are serious and frequently fatal illnesses. Some of the genetic changes that lead to lymphoma (such as the 8:14 translocation which fuses the Bcl-2 proto-oncogene to the immunoglobulin heavy chain locus in some follicular lymphomas) have been discovered, but many of the other contributing mechanistic details underlying transformation events are not yet known. It is clear however, that dysregulation of the balance between cell proliferation and programmed cell death is a central feature. Under normal conditions, lymphocytes must strictly regulate growth and apoptosis to provide adequate immunologic defenses against infections on the one hand and yet not overwhelm the organism with inappropriate cell numbers on the other hand.

A large number of signaling molecules have been implicated in homeostatic control of B cells. Mature B cells normally do not divide until they interact with antigen specific for their particular B cell receptor. Others have shown that activation of the B cell receptor generates second messengers, including calcium influx and activated kinases, leading to activation of transcription factors NF-AT, NF-κB, AP1, and others.

Costimulation through tumor necrosis factor receptor (TNFR) family members such as CD40 are also critical determinants of B cell function. The complete set of molecules that regulate mature B cell homeostasis is still being discovered and we have studied the molecular mechanisms that underlie B cell growth, function, and death based on the notion that these same regulatory signaling pathways may contribute significantly to aberrant proliferation and resistance to apoptosis in leukemia and lymphoma.

There is accumulating evidence that implicate the TNF superfamily members BLyS and APRIL, as well as their receptors, as critical factors for the growth and survival of both normal and malignant B cells. BLyS, which is better characterized than APRIL, has been found to be elevated in a number of immune disease models and there is increasing evidence that it may correlate with pathogenesis of various B cell related disorders, including B-cell malignancies. BLyS and APRIL are expressed in B-cell non-Hodgkin lymphoma (NHL) and the expression of BLyS is associated with an aggressive disease phenotype.

While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in the normal and malignant scenario remains to be fully elucidated. Because serum BLyS levels are elevated in a number of B-cell malignancies known to have a familial incidence, it is possible that dysregulation of BLyS occurs at the genetic level. The environmental, as well as genetic, requirements that mediate BLyS expression remain to be defined, and the promoter for the BLyS gene is poorly characterized. In preliminary work generated from our UI/MC Lymphoma SPORE Developmental Projects, we have found that a polymorphism in the BLyS promoter region correlates with increased serum BLyS levels in patients with B-cell malignancies, particularly those with a family history of B cell-related cancers.

Preliminary studies (funded in part as SPORE Developmental Projects) from the UI/MC Lymphoma SPORE demonstrate that:

• Malignant B cells bind BLyS and express BAFF-R, TACI, and BCMA
• BLyS promotes the growth and survival of malignant B cells
• BLyS is highly expressed in B cells form NHL, CLL, and multiple myeloma patients (DNS)
• Serum BLyS levels are elevated in NHL and CLL patients
• Elevated serum BLyS levels correlate with patient survival and a with the presence of a T at -871 in the BLyS promoter

These results provide the conceptual framework for further investigation of these ligands (including APRIL) and receptors (BAFF-R, TACI, and BCMA) in the context of NHL. Many questions remain, including defining the underlying mechanism of how BLyS expression is controlled. It is likely that factors from the extracellular milieu as well as underlying genetics contribute to its regulation. UI/MC Lymphoma SPORE researchers have identified a polymorphism in the BLyS promoter that results in increased BLyS expression, but the translational significance of this polymorphism with respect to the development of lymphoma as well as how it correlates with clinical outcome and response to therapy remain to be fully defined. With the availability of an established case-control and prognostic cohort study of NHL, the investigators will be able to address the latter issues in an integrated basic and population science project that takes advantage of the UI/MC Lymphoma SPORE Molecular Epidemiology Resource that was established in the last grant cycle.

Upon completion of this project, they hope to be able to determine whether genetic variability in BLyS, APRIL, TACI, BCMA, or BAFF-R, correlates with risk of developing NHL, the response to initial therapy, and the subsequent clinical outcome of patients with NHL. Results from this study may provide a prognostic indicator and a clinical screening tool that will allow physicians to identify patients who are predisposed to NHL and/or a poor clinical outcome. Additionally, the researchers will gain insight into the mechanisms that regulate BLyS expression and expand the medical community's understanding of the significance of APRIL in B-cell malignancies.

Project 3 contains four specific aims. The first two are population science aims that utilize the UI/MC Lymphoma SPORE Molecular Epidemiology Resource, while the second two aims are laboratory aims which will focus on mechanisms by which BLyS and APRIL and their receptors influence B-cell malignancies.

Results from the population science aims will feed back to the lab and vice versa, making this a highly translational research project.

1. To evaluate the association of inherited variability in genes encoding BLyS, APRIL, and their key receptors, with risk of NHL

   The major goal of this aim is to determine whether inherited variability in the BLyS and APRIL genes, along with their receptors BCMA, TACI, and BAFF-R, are associated with risk of NHL and it major subtypes in an ongoing clinic-based case-control study. The team will evaluate a putative functional SNP (-871 C/T polymorphism) in the BLyS promoter (rs9514828). They will also resequence the promoter and exonic regions of BLyS (promoter completed), APRIL, BCMA, TACI, and BAFF-R, and then evaluate the association of these candidate genes with NHL risk using a tagSNP approach which will combine data from resequencing and public databases.

2. To evaluate the association of inherited variability in genes encoding BLyS, APRIL, and their key receptors, with NHL survival

   The major goal of this aim is to determine whether inherited variability in the BLyS and APRIL genes, along with their receptors BCMA, TACI, and BAFF-R, is associated with overall NHL survival for the major subtypes using the UI/MC Lymphoma SPORE Molecular Epidemiology Resource.

   In secondary analyses, the investigators will evaluate event-free survival and response to initial course of therapy. We will evaluate the putative functional SNP (-871 C/T polymorphism) in the BLyS promoter (rs9514828). We will also use the resequencing data generated in Aim 1 for BLyS, APRIL, BCMA, TACI, and BAFF-R, to evaluate the association of these candidate genes with NHL survival as outlined in Aim 1. There are extensive scientific and resource synergies gained by the linked prognosis and case-control study.

3. Functional characterization of the -871 BLyS promoter polymorphism and identification of cellular mechanisms involved in BLyS expression

   In this aim, the research team seeks to identify the cells within the tumor microenvironment responsible for BLyS production and determine the inflammatory mediators that regulate its expression. They also plan to define the functional significance of the -871 polymorphism on BLyS expression and characterize the significance of Pax5 in BLyS expression.

4. To determine the significance of APRIL on the growth and survival of malignant B cells

   UI/MC Lymphoma SPORE researchers hypothesize that APRIL is an important factor in malignant B cell biology and thus the goal of this aim will be to define the role of APRIL on malignant B cell growth and survival and to decipher the downstream signaling target that transmit its effects.