The bone marrow microenvironment is a significant source of ARQ197 drug resistance for myeloma cells. It provides cytokines this kind of as HGF and IL-6, which stimulate the ARQ197 growth of myeloma cells and add to their resistance to cure . HGF and, to some extent, IL-6 stimulated DNA synthesis in U266 and OPM-two cells (Supplementary Determine 1, A and B). To establish whether or not ARQ 197 overcomes the defense conferred by the bone marrow microenvironment, we treated U266 and OPM-2 cells with 3 μM ARQ 197 in the presence of significant concentrations of exogenous IL-6 or HGF for 48 hrs. ARQ 197 inhibited cell progress in the existence of IL-six or HGF to a very similar extent as treatment with ARQ 197 alone (Determine two, A and C). In U266 cells, when compared with DMSO-handled cells, three μM ARQ 197 substantially inhibited expansion in the presence of 50 ng/ml IL-six (60% inhibition, P < .0001) or 50 ng/ml HGF (64% inhibition, P < .0001). Similarly, in OPM-2 cells, 3 μM ARQ 197 significantly inhibited growth compared to DMSO-treated cells in the presence of 50 ng/ml IL-6 (62.5%, P < .0001) or 50 ng/ml HGF (61% inhibition, P < .0001). Consistent with the growth inhibition results, incubation with ARQ 197 resulted in inhibition of cell survival in both U266 and OPM-2 cells in the presence of these growth factors (Figure 2, B and D).
To further investigate the impact of the microenvironment on drug resistance, we co-cultured U266 or OPM-2 cells with human NKtert stromal cells, which provide conditions similar to the bone marrow microenvironment. Myeloma cells were seeded on NKtert at a concentration 20 × that of NKtert cells. Under these conditions, NKtert cells stimulated DNA synthesis in U266 cells and, to some extent, in OPM-2 cells (Supplementary Figure 1C). Forty-eight hours of treatment with 3 μM ARQ 197 resulted in cell death of 54% in U266 cells (Figure 2E) and 69% in OPM-2 cells (Figure 2F) when co-cultured with NKtert cells these values were similar to that of U266 cells cultured alone (55% annexin V +/PI + cells Figure 2E) and OPM-2 cells cultured alone (82% annexin V +/PI + cells Figure 2F). Importantly, ARQ 197 did not induce a notable increase in NKtert cell death (22% at 3 μM), suggesting that ARQ 197 spares normal stromal cells.
Activity of ARQ 197 in Melphalan- and Dexamethasone-Resistant Cell Lines
Melphalan is an alkylating agent that was one of the earliest treatments introduced for myeloma. We tested ARQ 197 on paired cell lines that were melphalan resistant (8226/LR-5) and melphalan sensitive (8226/S). ARQ 197 inhibited cell growth by 54% at 3 μM in 8226/S cells (Figure 3A). This was associated with 77% cell death (Figure 3B). Despite the melphalan-resistant 8226/LR-5 cells having a high percentage of endogenous cell death (44%), 3 μM ARQ 197 was still able to inhibit cell growth by 54% and induced 98% cell death (Figure 3, A and B).