EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies.

Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019).

Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance.

We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression.

Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38^low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38^neg and CD38^low cell lines compared to CD38^highcells.

In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38^low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3).

Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro.

Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.