Gene Expression of Gamma Secretase (GS) Complex-Related Proteins, the Enzyme That Sheds B-Cell Maturation Antigen (BCMA), Among Patients with Multiple Myeloma (MM) and Effects of the GS Inhibitor LSN424354 on Solubilized Bcma in MM and Chronic Lymphocytic Leukemia

Introduction: B-cell maturation antigen (BCMA) has been shown to be highly expressed on the surface of tumor cells from patients (pts) with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) and is the target for therapeutic approaches in these diseases. Our group has shown that serum and plasma levels of BCMA correlate with disease status and survival in MM and CLL patients. We have recently shown that solubilized BCMA prevents its ligand B cell activating factor (BAFF) from binding and stimulating B-cells to produce polyclonal antibody. Recently, γ-secretase has been identified as the enzyme that leads to shedding of BCMA from off of B-cells. Gamma secretase is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 is as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 in MM pts with progressive (PD) and in complete remission (CR). We also determined the effects of the gamma secretase inhibitor (GSI) LSN424354 (Eli Lilly), a selective small-molecule inhibitor, on solubilized BCMA levels in MM and CLL tumor cells.

Methods: Bone marrow (BM) mononuclear cells (MCs) were isolated from MM pts following Western Institutional Review Board (WIRB) approval and informed consent in accordance with the Declaration of Helsinki. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY 40219) following the manufacturer's instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. qPCR was performed using TaqMan technology on an OneStepPlus instrument (Life Technology, Grand Island, NY 14072) and followed the standard procedure. Peripheral blood MCs were obtained from patients with CLL. The human MM LAGκ-1A xenograft was grown in the left superficial gluteal muscle of SCID mice for six weeks and removed. Single-cell suspensions were prepared. LAGκ-1A or CLL tumor cells were treated with the GSI LSN424354 in a concentration dependent fashion, and BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay. MM tumor cells were treated with the LSN424354 and performed non-radioactive MTS cell proliferation assay following a standard MTS protocol.

Results: qPCR showed PEN-2 gene expression was slightly increased among MM patients in PD compared to those in CR or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared to those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we determined the effect of the GSI LSN424354 on cultured MM or CLL tumor cells. The GSI inhibitor LSN424354 markedly reduced BCMA levels (> 90%) in supernatants from human MM LAGκ-1A cells after 5 days of tissue culture in a dose dependent fashion at concentrations ranging from 100 pM to 10 nM. Similarly, freshly obtained CLL tumor cells exposed to LSN424354 at concentrations similarly from 100 pM to 10 nM also showed a marked reduction (also > 90%) in supernatant BCMA levels. Notably, MTS assay results showed LSN424354 did not inhibit cell proliferation in MM or CLL tumor cells at concentrations up to 100 nM.

Conclusion: Gamma secretase sheds BCMA off of B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression in MM pts with PD compared to those in CR or MGUS individuals. The GSI LSN424354 reduces solubilized BCMA from tumor cells from CLL patients and human MM xenografts. Since CD147 has been shown to be present in serum, we are currently evaluating CD147 in serum samples from pts with MM and other plasma cell dyscrasias. In addition, we are examining the expression of BCMA on the surface of tumor cells from MM and CLL pts following exposure to BCMA, whether this GSI by reducing solubilized BCMA levels can reverse the immune dysfunction in mice bearing MM, and improve the efficacy of anti-BCMA antibody therapies.