Introduction: Sustained cancer cell growth requires up-regulation of nutrient acquisition mechanisms. Novel approaches can identify "tumor fuel", the way it is obtained and then altered for cellular usage. Autophagy is a conserved catabolic process that is thought to replenish nutrient pool and thereby starvation survival by providing bioenergetic and biosynthetic substrates. Some KRAS-mutated cancers show heightened dependency to glutamine as a source of energy. Since a large proportion of myeloma tumors are KRAS-mutated, we investigated in the present study the contribution of macropinocytosis and autophagy as the cellular mechanisms involved in protein uptake and degradation, respectively, to growth and survival of myeloma cells under glutamine-deprived condition.
Methods: Macropinosomes were visualized using confocal microscopy in KRAS-mutated and WT human myeloma cells utilising TMR-dextran and 5-[N-ethyl-N-isopropyl] amiloride (EIPA) as a marker and specific inhibitor of macropinocytosis, respectively. Internalization and degradation of labelled albumin and its co-localization with TMR-dextran were evaluated in parallel. Bafilomycin was employed to evaluate the dependency of protein degradation on autophagy. Cell sensitivity to glutamine deprivation was examined with an ATP-based viability test. To evaluate the possible role of macropinocytosis and autophagy at glutamine deprivation conditions, the effect of albumin supplementation on cell growth and viability was studied in the presence or absence of EIPA or chloroquine. To test whether the degradation of macropinocytosed albumin results in the production of intracellular glutamine, the intracellular concentrations of glutamine was directly measured.
Results: KRAS-mutated KMS28-PE, KMS18 and MM1S cells showed higher levels of EIPA-sensitive macropinocytosis compared to WT-KRAS KMS34, KMS12-BM and TK1 cells. Macropinocytosis led to the internalization of albumin which subsequently underwent bafilomycin-sensitive proteolytic degradation in KRAS-mutated cells. The viability of WT-KRAS cells was reduced in sub-physiological concentrations of glutamine when compared with KRAS-mutated cells that not only remained viable but could continue to slowly proliferate over a period of 7 days (p˂0.05). The compromised growth rate of KRAS-mutated cells at very low concentrations of glutamine was rescued by albumin and this effect was abrogated by EIPA or chloroquine (p˂0.05). The KRAS-mutated cells demonstrated an EIPA-sensitive increase in intracellular concentration of glutamine following albumin supplementation (p˂0.05) that was abrogated following the treatment with chloroquine. In WT cells, in contrast, the level of intracellular glutamine remained almost unchanged following albumin supplementation.
Conclusions: Autophagy and macropinocytosis maintain cellular growth and survival in the context of nutrient deprivation by provision of glutamine, and potentially other amino acids, in RAS-mutated myeloma cancer. These data suggest that novel anti-cancer strategies interrupting these cellular mechanisms may represent a promising therapeutic approach to myeloma.
Spencer: Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.