ALBERT

Description: Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Although the therapeutic armamentarium against multiple myeloma has tremendously increased in recent years, it still remains an incurable disease. A highly promising novel anti-tumoral treatment strategy is to target specific non-redundant metabolic achilles heels of individual cancer entities. The semi-essential amino acid arginine can be synthesized from citrulline in most physiological tissues due to expression of the rate-limiting enzyme argininosuccinate synthetase 1 (ASS1). Various tumor entities do not express ASS1, therefore depend on the exogenous availability of arginine and pharmacological approaches to systemically deplete arginine are in phase I-III clinical development for such arginine-auxotrophic cancers. Cell death induction by arginine depletion can be dramatically enhanced by co-application of the arginine analogue canavanine. Canavanine can be used by the respective aminoacyl tRNA synthetase instead of arginine during protein translation and this leads to a highly toxic intracellular accumulation of misfolded proteins. In preliminary work we have seen that myeloma cells are largely arginine-auxotrophic and can be killed by arginine depletion and canavanine supplementation within hours, while ASS1 expressing cells are completely protected by their endogenous arginine rescue capability. Encouraging results of tumor control have already been seen in a murine myeloma model. Methods Human myeloma cell lines (NCI-H929_A2 and FD50, developed in our laboratory) were cultured and treated in RPMI-1640 medium with or without arginine. Protein levels were determinded by western blot analysis. Cell viability was measured by propidium iodide staining and flow cytometry analysis. RNA quantification was done by qRT-PCR. For autophagosome and aggresome quantification we used immunofluorescence staining (IF) and laser scanning microscopy (LSM). Results Arginine depletion and canavanine supplementation led to misfolded protein accumulation which was followed by massive apoptotic cell death. Both processes were further enhanced by co-treatment with the proteasome inhibitor bortezomib, indicated by an increase in intracellular polyubiquitinated proteins as well as higher cleaved caspase 3 levels and propidium-iodide positive cells after only 8-12 h in both tested cell lines. Unexpectedly, the endoplasmic reticulum (ER)-stress response was activated only very moderately. Expression of CHOP, a pro-apoptotic transcription factor that is highly translated under toxic ER stress, was not altered compared to control conditions. Tunicamycin-mediated induction of enhanced ER stress significantly improved the viability of arginine-starved and canavanine treated cells. This suggests that protein accumulation mainly takes place in the cytoplasm rather than the ER and tunicamycin might alleviate cell death by reduction of total protein translation. Despite severe arginine deficiency and induction of misfolded protein stress, the cells were not able to respond by an adequate upregulation of macroautophagy, as determined by an altered LC3 metabolism. The autophagic flux was significantly reduced compared to control conditions after 4-8 h of treatment. There was a strong induction of BAG3 and p62 proteins, which are both associated with chaperone-assisted autophagy as well as aggresome formation and are normally cleared via macroautophagy. Cytoplasmic aggresome formation was not detectable until onset of apoptosis. Also, no relevant modulation of phosphorylation of the autophagy inducer mTORC and the downstream kinase p70S6K1 was noted upon arginine depletion and canavanine co-treatment. Finally, ER stress induction via tunicamycin did not improve autophagic protein turnover, as determined by IF staining, LSM and western blot. Conclusions Arginine starvation in combination with canavanine supplementation induces fast and highly efficient cell death in arginine-auxotrophic myeloma cells. This novel strategy interferes with myeloma cellular metabolism by induction of misfolded protein accumulation. A relevant upregulation of potentially protective cellular strategies like ER stress responses, aggresome formation and autophagy are either not detectable or they remain insufficient. We hypothesize that our novel metabolic anti-tumor strategy is either too potent or too fast for the tumor cells to cope with its consequences. Disclosures No relevant conflicts of interest to declare.