PB2097 METABOLIC STARVATION TRIGGERED BY L‐ASPARAGINASE SENSITIZES MULTIPLE MYELOMA CELLS TO PROTEASOME INHIBITORS BY INDUCING DNA DAMAGE ACCUMULATION

Abstract

Background:Some human tumors exhibit a high requirement for Glutamine (Gln) for metabolic processes, a condition defined “Gln‐addiction” leading to the investigation of Gln‐dependence as therapeutic target. Emerging data suggest efficacy of Gln‐deprivation also in multiple myeloma (MM) cells which are unable to synthesize adequate amounts of Gln. Serum Gln‐deprivation can be obtained by L‐Asparaginase (L‐ASP) that catalyzes the hydrolysis of asparagine and Gln.Aims:We explore the therapeutic relevance of L‐ASP‐induced Gln depletion alone and in combination with proteasome inhibitors (PIs). We analyze the biological mechanisms supporting the effectiveness of the identified therapeutic strategies.MethodsThe IC50 value of Erwinia Chrysanthemi–derived L‐ASP was determined by using tetrazolium salt MTS based assay in a panel of human MM cell lines (HMCLs). Primary human MM cells were obtained from bone marrow samples with high disease load. Mononuclear cells were isolated by Ficoll‐Hypaque gradient and processed by MiniMacs high‐gradient magnetic separation column to obtain CD138+ cells. Cell death analysis was performed by Annexin V/Propidium Iodide (AV/PI) staining followed by flow‐cytometric analysis. L‐ASP effect on mitochondrial membrane potential was evaluated by FACS analysis following TMRE staining. To evaluate the content of intracellular NAD+, cells were lysed in PCA and the neutralized extracts were analyzed by HPLC; NAD+ values were normalized to protein concentrations. Combination of L‐ASP plus PIs was investigated with an isobologram analysis (CalcuSyn software program). Since c‐MYC protein is not expressed in U266 cell line, a stable c‐MYC overexpressing U266 line was generated by lentiviral transduction to investigate the contribution of cMYC downregulation. Western blot (WB) analysis was used to fully elucidate biological features of our findings.Results:L‐ASP treatment showed anti‐tumor activity in HMCLs with an IC50 value ranging from 0,03 U/mL to 0.2 U/mL. Such effect was also observed in primary MM cells (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible “metabolic shutdown” in MM cells treated with L‐ASP. Co‐treatment experiments showed massive increase of L‐ASP anti‐MM activity in presence of low doses of PIs, with Combination Index value less than 1 in almost all tested drugs‐concentrations. Intracellular NAD+ measurement after 24 h treatment showed a massive NAD+ depletion in co‐treated cells compared to single drugs exposure. However, the addition of NAD+ or its precursors to culture medium was not able to revert the efficacy of the combination. In co‐treated cells, WB analysis revealed massive c‐MYC down regulation, however, the effectiveness of the combination resulted independent from the oncogene deregulation since it was confirmed highly effective in cMYC negative U266 HMCL. By using TMRE cell staining, we found that combination induce mitochondrial trans‐membrane potential dissipation in MM cells. Co‐treatment markedly increased the accumulation of the lower‐molecular‐weight protein γH2AX than specific controls at WB analysis. The addition of N‐Acetyl‐L cysteine to co‐treated cells resulted in a complete rescue in term of viability of MM exposed cells, and improved DNA stability, as suggested by reduced γ‐H2A.X levels compared with controls.Summary/Conclusion:Our data suggest that Gln‐depletion by E. chrysantemi L‐ASP results in a massive metabolic program deregulation and makes MM cells more vulnerable to PIs activity through oxidative stress and DNA damage induction.image