Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion

Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy.

Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death.

Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis.

Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM.