Effects of the Gamma Secretase Inhibitors (GSI) Dapt (GSI-IX) and LSN424354 on Gene Expression and Shedding of Bcma from Multiple Myeloma and Chronic Lymphocytic Leukemia Tumor Cells

Introduction: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have high levels of solubilized (s) BCMA in the blood, and this protein is responsible for the immune deficiency of MM. sBCMA may also interfere with the efficacy of anti-BCMA-CAR-transduced T cells or other anti-BCMA antibody-based immunotherapy for multiple myeloma patients. Recently, γ-secretase has been identified as the enzyme that leads to the shedding of BCMA from B-cells. Gamma secretase (GS) 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 acts as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 among MM pts with progressive disease (PD) and complete remission (CR). We also determined the effects of the GS inhibitors (i) DAPT (GSI-IX) and LSN424354 (Eli Lilly, Indianapolis, IN) on solubilized BCMA levels from cultured human MM and CLL tumor cells.

Study design: Bone marrow (BM) mononuclear cells (MCs) and serum were collected from monoclonal gammopathy of undetermined significance (MGUS) individuals, MM patients and healthy subjects after obtaining IRB approval. BMMCs from MM patients were treated with or without DAPT (GSI-IX) in a concentration dependent fashion for 24 hours. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY) 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. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the left superficial gluteal muscle of SCID mice. Cells were cultured and treated with the LSN424354 in a concentration dependent fashion. Supernatant BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay.

Results and Discussion: qPCR showed PEN-2 gene expression was only slightly increased among MM patients with progressive disease (PD) compared to those in complete response 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 with those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we examined the effect of the DAPT (GSI-IX) on primary human MM tumor cells. Supernatants from primary human MM cells cultured for 24 hours in DAPT showed markedly reduced BCMA in a concentration dependent fashion (range,0.2 nM-2µM). Using qPCR, DAPT markedly reduced PEN-2 gene expression in a concentration dependent fashion. On the other hand, this GSI did not effect BCMA gene expression. DAPT reduces shedding of BCMA from B-cells but not its gene expression. We also determined BCMA levels in supernatants from cultured tumor cells derived from the human MM LAGκ-1A xenograft following 5 days of tissue culture with LSN424354 at concentrations ranging from 100 pM to 10 nM. Supernatant sBCMA levels were reduced > 90% at the highest concentrations (10nM). Similarly, freshly obtained CLL tumor cells cultured with LSN424354 at concentrations ranging from 100 pM to 10 nM also showed a marked reduction in supernatant BCMA levels at the higher concentrations.

Conclusion: Gamma secretase sheds BCMA off B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression among MM pts with PD compared with those in CR or MGUS individuals. The GSIs DAPT and LSN424354 reduce shedding of BCMA from malignant cells from MM and CLL patients. Since GSIs reduce solubilized BCMA levels, it is possible that treatment with these agents can reverse the immune dysfunction caused by this solubilized protein, and also improve the efficacy of anti-BCMA immunotherapies for treating patients with multiple myeloma and CLL.