Resistance to Nutrient Deprivation Via Metabolic Re-Programming As Mechanism of 'Metastasis' Promotion in Multiple Myeloma

Aim: Multiple Myeloma (MM) is a disease of the intra-medullary (IM) compartment but with progression myeloma cells can survive in nutrient deprived extramedullary (EM) sites. Autophagy mediates the degradation and re-cycling of intra-cellular proteins and may promote tumor resistance to metabolic stressors, including nutrient deprivation. Using the MM cell lines TK1 and TK2, contemporaneously propagated from bone marrow (IM) and peripheral blood (EM), respectively, of a MM patient we investigated the role of autophagy in resistance to glutamine (Gln) deprivation.

Methods & Results:TK2 but not TK1 exhibited up-regulated autophagy under both basal and Gln deprived conditions with higher LC3BII/I turnover on immunoblotting and autophagic vacuole formation on electron microscopy following chloroquine (CQ) exposure. Under conditions of Gln deprivation, TK2 was able to proliferate until day 14, whereas TK1 stopped proliferating at day 3. This proliferative advantage under Gln deprivation was abrogated by autophagy inhibitors (CQ or 3-MA) as determined by both viable cell enumeration (p=0.008 at day 7, inhibitor vs no inhibitor) and Ki67 expression (p=0.0017) only in TK2. Modulation of Gln concentration demonstrated an inverse correlation with the viability of TK1 but not TK2 - TK1 cell death increased from 36% at Gln 8mM to 56% at Gln 0mM, irrespective of autophagy inhibition. In contrast, while Gln deprivation had no significant effect on the viability of TK2, under Gln deprivation CQ exposure induced a 2-fold increase cell death measured by annexin V/PI positivity and 2.5-fold increase of cPARP expression (p=0.0045).

RNAseq and GSEA analysis showed enrichment of the Krige Amino Acid Deprivation Response (AADR) pathway (ES=0.083) for TK1 with up-regulated expression of Gln transporters (SLC7A5, SLC1A5, SLC38A5 and SLC7A11). In contrast, Glutamine Synthetase (GS) expression, which is responsible for Gln synthesis, was up-regulated in TK2 cells. Interestingly, immunoblotting analysis revealed that Gln deprivation was able to induce the expression of GS while further inducing autophagic flux (2.66 fold increase of LC3II expression at Gln=0mM) only in TK2, whereas it inhibited mTOR activity in both cell lines. Additionally, GS chemical inhibition under Gln deprivation induced cell death in TK2 but not TK1 (32.56% vs 2.5% increase, respectively) demonstrating that Gln synthesis enables TK2 to overcome Gln deprivation.

Conclusion. Metabolic reprogramming mitigating against nutrient deprivation may promote a more 'metastatic' phenotype in advanced MM.