Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib’s ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

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.