Erwinia Chrysantemi-Derived L-Asparaginase Strongly Enhances Proteasome Inhibitors Activity By Deregulating Cell Metabolic Programs

Background: Human tumours rely on Glutamine (Gln) metabolism to sustain wide range of metabolic processes and macromolecules synthesis. Such dependence defined as "Gln-addiction" has been recently observed also for Multiple Myeloma (MM) cells, where Gln synthetase deficiency make them more sensitive to Gln depletion. L-Asparaginase (L-ASP), the enzyme that catalyzes asparagine (Asn) and Gln hydrolysis, represents a key drug for B-cells derived malignancies treatment such as acute lymphoblastic leukemia.

Aims: The aim of the present study was to screen MM cells sensitivity to pharmacological Gln depletion achieved by Erwinia chrysantemi-derived L-ASP treatment as single agent as well as in combination with proteasome inhibitors (PIs). Based on its high glutaminase activity, E. chrysantemi L-ASP was selected for this screening.

Materials and methods: The IC50 value of L-ASP was determined by MTS assay in a panel of human MM cell lines (HMCLs) harboring different genetic background as well as on primary CD138+ cells derived from newly diagnosed (NDMM) and and relapsed/refractory (RRMM) MM patients. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. L-ASP treatment effect on cell viability and mitochondrial membrane potential was evaluated by FACS analysis following PI and TMRE staining, respectively. Intracellular ATP levels was also measured by HPLC-based method. Drugs combination screening with L-ASP plus PIs including bortezomib (BZ) or carfilzomib (KAR) was investigated by using isobologram analysis (CalcuSyn software program). Finally, western blot analysis was employed to fully elucidate biological mechanisms of our findings.

Results: L-ASP treatment showed potent anti-MM activity in all HMCLs tested with an IC50 values ranging from 0,03 U/mL to 0.2 U/mL. Importantly, such drug preserved its effect also on primary tumor cells obtained from NDMM and RRMM patients (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible "metabolic shutdown" in MM cells treated with L-ASP which led to late mitochondrial stress (48-72h). To further confirm Gln specific role in the observed finding, MM cells viability was measured in presence of different culture media conditions. Asn depletion alone did not modify significantly MM cells viability, instead it strongly contributed to L-ASP induced toxicity of Gln-starved cells. Co-treatment experiments showed massive increase of its anti-MM activity when L-ASP was used in presence of low doses of BZ and KAR, with a combination index below than 1 in all tested drugs-concentrations. Accordingly, caspase-3 and PARP cleavage showed apoptotic cell death features of this co-treatment. Importantly, IL6 or IGF-1 addition did not reduce anti-MM activity of this combination suggesting that MM microenvironment does not reduce efficacy of this strategy. Finally, mechanistic studies revealed higher ATP shortage in co-treated cells together with c-MYC down-regulation, genomic instability and misfolded proteins accumulation, suggesting a massive proteasome activity impairment triggered by these drugs-combination. Ongoing experiment will help to further elucidate this hypothesis.

Conclusions: Our preliminary data show that E. chrysantemi L-ASP exerts a potent anti-MM activity mainly due to its glutaminase activity, which results in a massive metabolic program deregulation. The combination with low-doses of PIs further increases anti-MM activity of this small molecule likely due to huge proteasome activity injury.