The Purine Metabolic Enzyme AMPD1 Is a Novel Therapeutic Target for Multiple Myeloma

Introduction. Novel agents have improved the prognosis of multiple myeloma. However, side effects of novel agents have been a huge issue for especially elderly and frail patients. Additionally, despite the high remission rate by novel agents, multiple myeloma is still an incurable disease. In order to improve those issues, it is necessary to develop a new therapeutic strategy which is highly specific to myeloma cells and which targets a different pathway from the present anti-myeloma agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and myeloma cells, and to examine whether it can be a novel target for multiple myeloma therapy.

Materials and methods. A public available gene expression website GenomicScape (http://www.genomicscape.com/) and Genevestigator (https://genevestigator.com/gv/index.jsp) were utilized in order to study genes specifically expressed in human plasma cells and myeloma cells. To examine gene expression in myeloma cell lines, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression was analyzed by westernblot and immunohistochemistry. Genes co-expressed with AMPD1 in human myeloma cells were identified using public available gene expression datasets (GSE 4581, GSE 9782). Molecular pathways associated with genes co-expressed with AMPD1 were analyzed using Molecular Signatures Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp). Cell viability of myeloma cell lines and peripheral blood mononuclear cells (PBMCs) treated by AMPD inhibitors (compound #3, #4) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed using 7AAD dye and flow-cytometry.

Results. Public gene expression screening analysis identified several genes specifically expressed in human plasma cells and myeloma cells. Among the identified genes, we focused on AMPD1, which has not been previously studied in multiple myeloma. CCLE analysis and RT-PCR analysis showed that AMPD1 is specifically expressed in bone marrow plasma cells, myeloma cell lines and patient derived myeloma cells. AMPD1 protein expression was limited to myeloma cell lines, human bone marrow myeloma cells and extramedullary plasmacytomas. Genes co-expressed with AMPD1 in human myeloma cells were associated with hypoxic pathways. Myeloma cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to cell lines cultured under normoxic condition. AMPD inhibitors induced cell death in myeloma cell lines from around 50 uM, while the effect against PBMCs were minimal. AMPD inhibitors were more effective against myeloma cell lines under hypoxic condition compared to normoxic condition, reflecting the higher AMPD1 expression under hypoxia.

Conclusions. AMPD1 is a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), freeing ammonia during the process. AMPD1 has been previously reported that its expression is limited to skeletal muscles. This is the first report so far that among leukocytes and hematological malignancies, AMPD1 is specifically expressed in bone marrow plasma cells and myeloma cells. We also showed that AMPD1 expression in myeloma cells are increased under hypoxia. This indicates that AMPD1 plays a significant role in myeloma cells surviving under hypoxic conditions such as the bone marrow microenvironment. AMPD inhibitors showed cytotoxicity on myeloma cell lines in vitro, while PBMCs were not affected. Additionally, AMPD inhibitors were more effective under hypoxic condition, suggesting that AMPD1 inhibition works more specifically in the bone marrow microenvironment. Our report raise the possibility that AMPD1 inhibition can be a novel therapeutic strategy for multiple myeloma. Detailed analysis of myeloma cell death by AMPD1 inhibition is now undergoing.