Upregulation of BCMA expression by selinexor

Abstract

Abstract Introduction Selinexor is a first-in-class, selective inhibitor of nuclear export that targets exportin 1 (XPO1), a key nuclear export protein involved in the transport of tumor suppressor proteins and oncogenic factors from the nucleus to the cytoplasm. Selinexor, Bortezomib, and Dexamethasone was approved based on the phase III BOSTON trial for the treatment of patients with multiple myeloma who have received at least one prior therapy. Beyond direct tumor effects, Selinexor has immunomodulating effects and was reported to upregulate BCMA, potentially augmenting BCMA-directed CAR-T cell function. (Wang et al. J Transl Med. 2023). Nevertheless, these studies relied entirely on flow cytometry, which exhibits only limited sensitivity and does not enable receptor quantification at a single-cell level. Here we provide first insights in BCMA receptor density changes following Selinexor treatment using super-resolution microscopy, complemented by qPCR analysis to assess gene expression levels. Furthermore, we intend to build upon the work of Wang et al. by conducting additional studies not only involving CAR-T cells but also bispecific antibodies and antibody-drug conjugates to investigate whether pre-treatment with Selinexor enhances their cytotoxic efficacy. Methods We applied direct Stochastic Optical Reconstruction Microscopy (dSTORM) to investigate the modulation of BCMA expression by Selinexor in Multiple Myeloma (MM) cells. OPM-2 and AMO1 cells were treated with 10 nM or 50 nM Selinexor and BCMA receptor densities were quantified after 1, 2 and 5 days of treatment. These concentrations were selected based on drug titration curves for their minimal impact on cell viability, ensuring that the observed changes in BCMA expression were a result of Selinexor treatment. In parallel, qPCR was employed to measure gene expression levels, corroborating the dSTORM findings. Statistical comparison of dSTORM receptor densities were conducted using Mann Whitney test. Additionally, we plan to evaluate the correlation between BCMA upregulation and therapeutic efficacy in vitro by performing cytotoxicity assays after 5 days of 50 nM Selinexor treatment, utilizing BCMA-targeting CAR-T cells, Teclistamab, and Belantamab Mafodotin. Results Utilizing dSTORM we monitored BCMA expression in OPM-2 and AMO1 cells during treatment with 10 nM and 50 nM Selinexor. In OPM-2 cells, no significant difference was observed after 1 day of treatment compared to DMSO-treated control cells. However, at day 2, a 1.6-fold increase in BCMA receptor density was detected in 50 nM Selinexor-treated cells relative to controls (4.3±0.2 vs 2.6±0.3 clusters/µm2, p=0.0002). After 5 days, receptor density in control cells remained stable, whereas Selinexor-treated cells exhibited a stark increase to 9.5 ± 1.1 clusters/µm² (p < 0.0001), corresponding to a 4.2-fold elevation in BCMA receptor density. These findings were corroborated by qPCR, which revealed a 1.2- and 1.5-fold increase in BCMA gene expression on days 2 and day 5, respectively. Importantly, cell viability remained above 92% during Selinexor treatment. In AMO1 cells, despite a lower baseline BCMA expression averaging 1.0±0.1 clusters/µm², a comparable trend was observed. Specifically, there was a significant 1.6- and 1.9-fold increase in receptor density, alongside a 1.8- and 1.5-fold increase in gene expression, at days 2 and 5, respectively, following treatment with 50 nM Selinexor. Interestingly, AMO1 cell viability was initially 84% but improved to 93% by day 5, further highlighting the minimum toxicity of the chosen Selinexor concentrations. Conclusion In summary, our findings demonstrate that Selinexor treatment leads to a time- and dose-dependent upregulation of BCMA expression at both the protein and transcript levels in MM cell lines, as revealed by dSTORM imaging and qPCR analysis. The significant increase in BCMA receptor density suggests that Selinexor may enhance target antigen availability on the cell surface, potentially improving the efficacy of BCMA-directed immunotherapies. These initial results support further exploration of Selinexor as a sensitizing agent to enhance the effectiveness of BCMA-directed immunotherapies in MM.