The role of the bone marrow microenvironment in multiple myeloma

Multiple Myeloma (MM) is an incurable B cell malignancy localised to the bone marrow (BM) that is prone to drug resistance (DR). Here it is supported by the bone marrow microenvironment (BMME) through soluble and contact based mechanisms. Many soluble factors have been implicated in MM DR, including IL-6 and IGF-1. Interactions between MM cells and extracellular proteins such as fibronectin (FN) or CD138 have also been implicated in DR. Overall these supportive signals induce multiple redundant signalling pathways that allow the MM cells to grow and survive, even when treated with chemotherapy. The relationship between the MM cells and the BMME can be studied using human myeloma cell lines (HMCL) and bone marrow stromal cells (BMSC) cocultured (CC) together in vitro to simulate the in vivo environment. These models of the BMME are commonly used to evaluate novel therapies, but few have successfully studied the supportive signals provided by the BMME to MM cells. In this study, a serum-free and serum-containing CC model was established that allowed the study of the effects of soluble and contact based interactions both together and individually on HMCL. The CC system was validated by demonstrating DR to the common anti-MM agent melphalan, when studied further it was determined that both soluble and contact based support was involved. The soluble BMME was investigated using the serum-free CC model that allowed the exchange of soluble factors, contact based support, or both. A panel of 28 soluble factors were measured from cultures of two diverse HMCL – NCI-H929 and OCI- MY1 with or without HS5 stromal cells, in various culture conditions. This demonstrated that contact can modulate the soluble environment and that the soluble factors produced by different HMCL can be quite different. It was then possible to determine the effects of CC and individual soluble factors on JAK/STAT, PI3K/AKT and Ras/MAPK signalling in HMCL. In 3/3 HMCL JAK/STAT was strongly upregulated in response to CC or IL-6 stimulation, PI3K/AKT and Ras/MAPK could also be activated in some conditions. Given the importance of signalling for the survival of MM cells, inhibitors of AKT, HSP90, JAK, MEK and PI3K were investigated for their effects on HMCL signalling and induction of apoptosis. Treatment with the AKT inhibitor GSK2110183B (183) induced phosphorylation of AKT, treatment with the MEK inhibitor GSK1120212B (212) prevented cytokine induced phosphorylation of ERK1/2. The JAK inhibitor CYT387 was able to prevent cytokine induced phosphorylation of AKT, ERK and STAT3. Inhibition of AKT, JAK and MEK with these inhibitors as well as HSP90 with HSP990, and PI3K with a range of inhibitors was able to induce variable amount of apoptosis in HMCL as single agents. When combined AKT and MEK (16 combinations, mean combination index (CI) = 0.104), AKT, JAK and MEK (32 combinations, mean CI 0.239), and HSP90 and JAK (16 combinations, mean CI = 0.703) inhibition demonstrated synergism in 4 HMCL, at all dose and time combinations as determined by CI values less than 1. After determining synergistic combinations the inhibition of AKT, HSP90, JAK and MEK was investigated in CC with single agents or in combinations. DR was demonstrated against all inhibitors except HSP990 when NCI-H929 cells were cultured with HS5 stromal cells. Combinations with HSP90 inhibition show clinical promise, including the combination of CYT387 and HSP990. Because CYT387 synergised well with novel agents, and modulated multiple signalling pathways it was investigated further. CYT387 prevented HMCL proliferation and cell cycling, and induced apoptosis in primary MM cells alone and in combination with current MM therapies melphalan and bortezomib. In summary, a CC system was designed and optimised to investigate the effects of the BMME on the MM cells. Many soluble factors were detected in the BMME, and individual soluble factors, or CC itself was able to induce multiple signalling pathways in HMCL. Novel inhibitors were used to modulate these pathways and induce apoptosis in highly synergistic combinations in HMCL. Individual inhibitors and combinations were tried in CC and DR was demonstrated in most conditions with some, but not all drugs. The combined inhibition of JAK and HSP90 was demonstrated to be synergistic and importantly, was still effective in the presence of the BMME