Abstract 454: The role of autophagy in multiple myeloma disease progression

Abstract

Abstract Multiple Myeloma (MM) is an incurable hematologic neoplasm characterized by accumulation of malignant plasma cells in the bone marrow (BM). MM cells establish close ties with the BM, however at late stages they utilize as yet unknown mechanisms to acquire independence from the microenvironment and survive in extramedullary/nutrient deprived sites. Autophagy, a process responsible for degradation of proteins and organelles under stress conditions, has been suggested as a mechanism whereby tumor cells can withstand metabolic stress. Using two newly acquired human myeloma cell lines (HMCL), TK1 and TK2, from the medullary and extramedullary sites of a MM patient respectively, we investigated the ability of autophagy to confer stress/glutamine deprivation tolerance. Results HMCLs require exogenous L-Glutamine (Gln) for sustained proliferation in vitro. However, when TK1 and TK2 were grown under Gln deprivation conditions (8mM, 4mM, 2mM, 0mM) we observed striking differences in their growth response. The TK2 (peripheral blood) cell line continued proliferating until day 14 (p=0.0046), whereas proliferation of the TK1 cell line significantly decreased in the absence of Gln (p=0.0028 between 8mM and 0mM). Furthermore, the proliferative advantage of TK2 was abrogated at 2mM and 0mM of Gln by the use of an autophagy inhibitor (chloroquine [CQ] (p=0.000235 and p=0.000069 respectively), implicating this process in Gln deprivation tolerance of TK2. Additionally, Gln concentration was inversely correlated with TK1 survival, but not TK2, as measured by annexin-V/PI staining. Cell death in TK1 increased at 24h, from 36.022% at Gln 8mM to 56.2% at Gln 0mM, irrespective of autophagy inhibition. In TK2 cells, Gln deprivation did not cause a significant increase in cell death, whereas inhibition of autophagy increased cell death by two-fold. Accordingly, apoptosis (as measured by PARP cleavage) was induced by the addition of CQ only in TK2 under Gln deprivation. Resistance of TK2 cells to metabolic stress also correlated with up-regulation of the autophagic pathway upon CQ exposure, as demonstrated by a higher LC3BII/I turnover and the presence of autophagic vacuoles in TK2 (but not TK1) cells. In addition to upregulation of autophagy, Gln deprivation also induced the expression of glutamine synthetase in TK2 but not TK1 cells. Chemical inhibition of this enzyme by methionine-sulfoximine (5mM) under Gln deprivation conditions was able to induce significant cell death in TK2 and not TK1 (32.57% and 2.53% increase respectively). These data demonstrate that Gln synthesis is an additional mechanism whereby TK2 cells tolerate Gln starvation. In conclusion we have shown that MM survival outside the BM can be attributed to the ability of cells to overcome “glutamine addiction” due to up-regulation of autophagy and ability to synthesise Gln. Thus, autophagy inhibition is an attractive alternative approach to target late-stage, Gln independent, metastatic cells. Citation Format: Ioanna Savvidou, Sahan Chandrasekara, Tiffany Khong, Andrew Spencer. The role of autophagy in multiple myeloma disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 454.