Glutamine-Dependence Targeting By Asparaginase Significantly Increases Anti-Myeloma Activity of Proteasome Inhibitors

Human tumors exhibit high dependence on Glutamine (Gln) for their metabolic processes and macromolecule synthesis, condition that has been defined as "Gln-addiction". The role of such vulnerability has been recently investigated also in Multiple Myeloma (MM) cells, with Gln uptake exploited as novel therapeutic target. Indeed, MM cells by exhibiting significant lack of Gln synthetase become highly sensitive to Gln depletion. L-asparaginase (ASNase), a bacteric enzyme that by degradating circulating asparagine and glutamine leads to intracellular aminoacids depletion, has been reported to exert a certain degree of activity on human myeloma cell lines (HMLC).

Based on these data, here we explored the anti-MM activity of Erwinia chrysantemi -derived L. Asparaginase (ASNase; 10-folder higher glutaminase activity than Escherichia Coli -derived formulation) in combination with the proteasome inhibitor (PI) carfilzomib (KAR), since PIs represent the backbone of current induction therapy for most MM patients.

The IC50 value of both E. chrysantemi ASNase and KAR was evaluated in a panel of HMCL carrying a different genetic background. Next, by using Chou-Talalay method we found that low doses of KAR significantly enhances the anti-MM activity of ASNase. The effect of such combination was also tested in presence of IL6 and IGF-1, which mimics Bone Marrow Stromal Cells (BMSCs) milieu. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. The efficacy of co-treatment was confirmed by treating freshly isolated CD138 positive cells obtained from newly diagnosed MM patients (NDMM). Finally, western blot analysis was employed to fully elucidate mechanisms causing the observed synergism.

ASNase treatment showed potent cytotoxic activity in all HMCL lines tested with an IC50 value ranging from 0,03 U/mL to 0.3 U/mL. High sensitivity was also observed on primary CD138⁺ MM cells obtained from NDMM (IC50 =0.3 U/mL). Importantly, anti-MM activity of ASNase was further increased by adding low doses of the irreversible proteasome inhibitor KAR with a Combination Index value less than 1 in almost all tested drug-concentrations. Similar data were observed in tumors cells obtained from NDMM patients. Of note, the combination ASNase plus KAR resulted more potent than single agent treatment also in culture condition of Gln deprivation, suggesting that ASNase toxicity is not only related to its glutaminase activity. IL6 or IGF-1 addition did not reduce anti-MM activity of ASNase plus KAR, indicating that BMSCs microenvironment may not influence its activity. A time and dose-dependent activity of co-treatment was also observed, with higher efficacy measured at 48 h compared with 24h of drugs exposure. The caspase-3 and PARP cleavage observed in co-treated cells, revealed the crucial role of apoptosis in the synergistic effect observed. Also authophagic features, including LC3II cleavage, were observed in tumor cells treated with KAR plus ASNase compared with single agent treatment.

The effect of ASNase treatment on neoplastic cells metabolism has already showed potent antineoplastic activity in a wide range of hematologic malignancies including acute lymphoblastic leukemia and aggressive lymphomas. As result, the Gln dependence represents an attractive therapeutic target to be exploited also in plasma cells disorders. Our preliminary data show that E. chrysantemy derived asparaginase exerts a potent anti-MM activity, which is further increased by addition of the irreversible proteasome inhibitor carfilzomib through a mixed apoptotic/authophagic-mediated mechanism.