Investigation of the relationship of toll like receptors and signal path components with disease pathology in Multiple myeloma

Multiple myeloma (MM) usually occurs over the age of 50. It is a plasma cell malignancy characterized by bone lesions. Treatment is carried out with chemotherapy agents, immunomodulators and proteasome inhibitors (PI). Since MM cells have a mutant form of the proteasome, which is vital in all eukaryotic cells, PIs are very effective and useful in treatment. Carfilzomib and Ixazomib are second generation PIs with different mechanisms of action on the immunoproteasome. On the other hand, recovery from apoptosis stands out as one of the main mechanisms of MM cell survival in therapy. Venetoclax is an agent that selectively binds to anti-apoptotic proteins and inhibits activity on pro-apoptotic proteins. It is approved for use in the treatment of various hematological malignancies. Today, it is aimed to develop more effective and low-toxicity anti-tumor systems with the combinational use of these agents in the treatment of MM. In addition, the production of different options in the treatment of MM is the subject of research. It is important to decipher the function of Toll like receptors (TLR) and to understand their role in the mechanisms that can be used in cancer treatment. Therefore, the use of TLRs in MM theraphy as a drug sensitization strategy in therapy has recently come to the fore. In this context, the aim of the study was to investigate the synergy of combinational usage of TLR1/2 agonist, anti-apoptotic protein inhibitor and next generation proteasome inhibitors in anti-cancer effect. MM model consisted of U266, RPMI 8226 and NCI H929 cell lines. TLR1/2 agonist Pam3CSK4, anti-apoptotic protein inhibitor Venetoclax, new generation proteasome inhibitors Carfilzomib and Ixazomib were applied in experiments. Cell proliferations, gene expression profiles, anti-apoptotic responses and cytotoxic resistance mechanisms were analyzed with agonist and inhibitor combinations. Proliferation, gene expression and protein expressions were investigated respectively with WST, RT-PCR and Western Blot techniques. TLR signaling pathway was activated in 3 cell lines in the presence of TLR1/2 ligand Pam3CSK4. The anti-apoptotic protein inhibitor efficiently induced cell death. Carfilzomib caused more effective inhibition and apoptosis in a dose and time-dependent content compared to Ixasomib. Proliferation was observed to be heterogeneous between inhibitors and cell lines. It was observed that IC50 values obtained under normal culture conditions were lower than combinations with TLR1/2 agonist and anti-apoptotic protein inhibitor. Highly heterogeneous profiles were found in gene expression of adapter proteins and apoptosis-related proteins involved in the TLR1/2 signaling pathway. Similarly, highly heterogeneous profiles were determined in the expression of apoptotic proteins that develops due to TLR1/2 signaling pathway and chemotherapeutic agents. Our findings demonstrated the potential for modulation of proteasome inhibitor-mediated stress in MM cells with both apoptotic protein inhibitors and ligands stimulating the TLR signaling pathway. Our data show that combinations of TLR agonists, new generation proteasome inhibitors and anti-apoptotic protein inhibitors may be effective in the treatment of MM.