JAK-STAT3 Pathway Regulates CD38 Expression on Multiple Myeloma Cells

CD38 is highly expressed on malignant plasma cells from patients with multiple myeloma (MM), and anti-CD38 monoclonal antibodies (i.e. daratumumab, SAR650984) trigger cytotoxicity through antibody-dependent cellular cytotoxicity (ADCC) and activation of complement-dependent cytotoxicity (CDC). Although remarkable response has been observed in relapsed and refractory MM, relapse of disease is common. To improve the clinical efficacy of CD38-targeting immunotherapies, novel combination treatment strategies have been evaluated in preclinical studies and clinical trials. For example, all-trans retinoic acid or histone deacetylase inhibitors enhance daratumumab-induced cytotoxicity via upregulation of CD38 expression. To date, however, the biologic impact of the bone marrow microenvironment on CD38-targeting immunotherapies has not been characterized. In this study, we evaluated the impact of bone marrow stromal cells (BMSCs) from MM patients on CD38 expression and anti-CD38 Antibody-induced ADCC.

We first cultured MM cells (RPMI8226, MM.1S, MOLP8) with culture supernatant from BMSCs and measured CD38 expression by flow cytometry. A significant reduction of CD38 expression on all MM cell lines was noted in a time-dependent fashion. For example, CD38 expression (mean fluorescence intensity) was reduced 44%, 32%, and 42% on RPMI8226, MM.1S and MOLP8 cells, respectively, after 48 h culture with BMSC supernatants. To identify mediators of this effect, we next examined the effect of MM-relevant cytokines (IL-6, IGF-1, IL-1β) on CD38 expression. Importantly, IL-6 significantly downregulated CD38 expression on MM cells whereas IGF-1 or IL-1β had no significant impact. We further performed quantitative real-time PCR to evaluate CD38 mRNA level in RPMI8226 and MOLP8 cells cultured with BMSC supernatant or IL-6 and confirmed that CD38 transcript was downregulated by both treatments in these cells. Since IL-6, but not IGF-1, downregulated CD38, we hypothesized that JAK-STAT3 pathway might mediate this IL-6 triggered downregulation of CD38 expression. As with IL-6 treatment, transfection of MM.1S cells with constitutively active STAT3 significantly reduced CD38 expression. Conversely, knockdown of STAT3 by siRNA partially restored CD38 expression on RPMI8226 cells in the presence of IL-6. Moreover, we cultured RPMI8226 and MM.1S cells with JAK inhibitor ruxolitinib (1, 3, 10 μM) in the presence of BMSC supernatant or IL-6. We observed significant recovery of CD38 expression on MM cells. We next evaluated the impact of these modulations of CD38 expression on ADCC. Natural killer cell-resistant RPMI8226 cells were pre-treated with BMSC supernatant for 48 h and then labeled with calcein-AM for 30 min, followed by co-culture with peripheral blood mononuclear cells from healthy donors (effector/target ratio of 30:1) in the presence of anti-CD38 antibody (1 μg/ml of SAR650984). As expected, ADCC was reduced by BMSC supernatant.

Our studies, therefore, show that BMSC supernatant downregulates CD38 expression through IL-6-STAT3 signaling pathway in MM cells, and thereby reduces sensitivity to CD38-targeted ADCC in MM cells. They provide the rationale for novel treatment strategies of CD38-targeting immunotherapy in combination with JAK-STAT pathway inhibitors to enhance sensitivity or overcome resistant to CD38-targeted ADCC in the context of the MM BM microenvironment.