P848: TINOSTAMUSTINE (EDO-S101), AN ALKYLATOR AND HISTONE DEACETYLASE INHIBITOR, ENHANCES THE EFFICACY OF DARATUMUMAB IN PRECLINICAL MODELS OF MULTIPLE MYELOMA

Abstract

Background: Tinostamustine (EDO-S101) is a novel alkylating and deacetylase inhibiting molecule designed to improve drug access to DNA strands, induce DNA damage and counteract its repair in cancer cells. It has shown anti-myeloma (MM) activity in different preclinical models both in monotherapy and in combination with bortezomib. Aims: To evaluate whether tinostamustine enhances the anti-myeloma effect of daratumumab. Methods: Tinostamustine was provided by Mundipharma and daratumumab was obtained from pharmacy surplus of the University Hospital of Salamanca. Mechanisms of action of daratumumab were studied by flow cytometry (FCM) in MM cell lines pretreated with tinostamustine and cultured as specified: 1) with F(ab)2 fragments (direct apoptosis via crosslinking); 2) with 10% human serum (CDC assays); 3) co-cultures of MM and NK cells (ADCC assays). Expression of CD38 and NKG2D ligands (MICA and MICB) after tinostamustine treatment was evaluated by FCM, WB and qPCR. The effects of tinostamustine + daratumumab combination were also studied ex vivo in bone marrow (BM) samples from MM patients, and in vivo in two plasmacytoma models developed in CB17-SCID and NK-cell-humanized NSG mice. Results: Pretreatment of U266, JJN3, MM.1S, NCI-H929, RPMI-8226 and MOLP-8 cell lines with tinostamustine (1-2.5 μM) for 48 h increased CD38 expression. Tinostamustine-pretreated myeloma cells showed a higher level of daratumumab binding, as demonstrated by anti-human-IgG1-AF488 staining. Also, tinostamustine (1-2.5 μM) increased MICA and MICB expression in MM cell lines. In ex vivo cultures, tinostamustine increased expression of CD38 in 4 out of 10 patients and MICA in 3 out of 5 patients. Next, the influence of tinostamustine pretreatment (2.5 μM) on several daratumumab mechanisms was evaluated. In apoptosis via crosslinking, the percentage of apoptotic cells in presence of daratumumab was higher in tinostamustine-pretreated MOLP-8 cells vsDMSO-pretreated cells (90.57%±7.35 vs 60.36%±2.86; p<0.001). Likewise, pretreatment with tinostamustine increased the percentage of Annexin-V+/7AAD+ MOLP-8 cells in CDC assays with daratumumab, although not reaching statistical significance (93.22%±1.05 vs73.39%±11.93). In MM-NK co-cultures in absence of daratumumab (NK cell-mediated direct cytotoxicity), the percentages of apoptotic cells in tinostamustine-pretreated vs DMSO-pretreated cells were: 83.64%±5.28 vs 61.58%±6.81 in MOLP-8 cells (p<0.05); 54.27%±5.99 vs 34.24%±7.37 for RPMI-8226 (p<0.05); and 26.15%±4 vs 12.32%±1.97 in MM.1S (p<0.05). However, in the presence of daratumumab (ADCC) these percentages showed a similar tendency although not significant: 88.57%±3.77 vs 78.89%±5.57 in MOLP-8; 70.75%±5.69 vs 58.28%±7.52 for RPMI-8226; and 56.74%±7.25 vs 45.68%±7.1 in MM.1S. Ex vivo experiments with patients’ BM samples (n = 10) showed that simultaneous combination of daratumumab + tinostamustine significantly increased the percentage of eliminated MM cells compared to individual treatments, with an acceptable toxicity on healthy lymphocytes (Figure 1a). Finally, in the CB17-SCID model, tinostamustine treatment (24 h) followed by daratumumab administration controlled tumor growth significantly better than daratumumab alone (Figure 1b). The combination prolonged the median survival as compared to vehicle, and monotherapy with tinostamustine or daratumumab (Figure 1b). Similar results were obtained in the NSG model. Image:Summary/Conclusion: Our preclinical data demonstrate that tinostamustine increases the anti-myeloma effect of daratumumab presumably due to enhanced expression of CD38 and MICA.