P828: effect of anti-cd38 monoclonal antibodies on the interaction of myeloma cells with bone marrow stromal cells

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) is a malignant disease of plasma cells with a poor prognosis. It has been shown that bone marrow stromal cells (BMSC) of MM patients differ from their healthy counterparts in their secretion profile and gene expression. Interactions between the tumor and BMSC play an important role in the pathogenesis of MM. Through the secretion of maintenance factors, BMSC promote tumor growth and may contribute to the acquisition of drug resistance in MM. Monoclonal antibodies against CD38 are widely used in the treatment of MM. Their mechanisms of action include direct induction of apoptosis, Fc-dependent cytotoxicity and phagocytosis, and immunomodulatory effects. Aims: This in vitro study investigated the effect of an anti-CD38 monoclonal antibody (daratumumab) on BMSC when interacting with MM cells. Methods: For this purpose, primary MM-BMSC and healthy stromal cell line HS-5 were cultured under various conditions: monoculture, co-culture with MM cell lines, co-culture with pretreated MM-cells, treatment with daratumumab. Following the cultivation vital parameters (vitality, absolute and relative growth ratio) were measured. Afterwards the RNA isolation proceeded. Global RNA expression analysis in BMSC from MM patients and in healthy controls prepared under identical conditions were performed using Oxford Nanopore Technology NGS sequencing. Finally, quantitative PCR was performed to confirm the change in expression of genes that showed a significant change in expression using the NGS method: IL-6, CD38, SIRT1, SIRT3, TP53, CCL2, IRF2BP2 and B2M genes in the BMSC. Results: Co-culture with MM cells and treatment with daratumumab reduced the vital parameters of BMSC. But co-cultured MM cells showed significantly higher growth rates compared to monoculture. Both, the treatment, and pretreatment, increased the vital parameters of the MM cells. Global RNA expression analysis revealed that at least 11 BMSC genes were differentially expressed (p-value <0.05). The most up-regulated genes in BMSC co-cultured with treated MM cells are known to be involved in the acute inflammatory response. Ingenuity Pathway Analysis (IPA) of the differentially expressed genes revealed that the most over-represented canonical pathways were the IL-17 signaling, CD40 signaling and NFκB signaling pathways. QPCR results confirmed that, BMSC co-cultured with daratumumab treated MM cells up regulated several proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. Moreover, a significantly higher expression of CD38, SIRT3 and CCL2 was found in co-cultivated BMSC. Summary: Although monoclonal antibodies against CD38 had no significant effect on isolated BMSC in vitro, apparently due to low expression of CD38 on BMSC, stromal cells significantly respond to treatment of MM cells with monoclonal antibodies against CD38 by changing their expression profile and increasing the expression of genes with pro-inflammatory products. Thus, they support myeloma cells to maintain an environment conducive to tumor development. The central role of tumor-stromal cell interactions is confirmed by the significance of differences between mono- and co-cultured cells (both BMSC and MM). This is additional evidence that intercellular interactions play an important role in tumor response to treatment. Thus, this discovery contributes to understanding why these interactions persist throughout the disease course and may also contribute to drug resistance. Keywords: Bone marrow microenvironment, Gene expression, Plasma cells, Multiple myeloma