of last year's progress report was: FH-deficient UOK262 cells do not appear to utilize either glucose or glutamine for carbon, even though UOK262 cells consume glucose and produce lactic acid. The restoration of FH activity in these cells increases OCR, sensitizes them to the hexokinase inhibitor 3-bromopyruvate; protects them from thiaminase in the presence of the LDH-A inhibitor NHI-2; and had no effect on the toxicity of the glutaminase inhibitor CB-839 either in the presence or absence of glutamine. The sum of the experiments indicate that the FH-deficient UOK262 cells simply are unaffected by pertubations of glucose and glutamine metabolism.... The current working hypothesis is that UOK262 FH-deficient cells require a carbon source that may not be not required for the FH replete cells. We are following up these initial results in several ways...One avenue is to identify the critical carbon source for the enzyme deficient cells. If glucose and glutamine are less essential for the UOK262 FH deficient cells than for FH repleted cells, then the FH-deficient cells may be dependent on other sources of carbon. UOK262 and UOK269 cells are grown in medium supplemented with both fetal calf serum and additional non-essential amino acid (NEAA) supplement. In preliminary experiments, we grew both pairs of cell lines in medium without NEAA to determine whether NEAA was essential for growth. Initial experiments indicate that FH-deficient UOK262 cells, but not FH-replete cells, and also not UOK269 paired cell lines, senesced without the NEAA supplement. If confirmed this suggests that the UOK262 cells may require carbon from an amino acid source in the NEAA supplement, that is not required for the enzyme repleted cells. This would open up potential therapeutic possibilities. We followed this path by first determining which of the amino acids in the NEAA supplement was required for cell growth of UOK262 cells. By adding each amino acid individually, we found that the cells appeared to require asparagine for accelerated growth, both in medium containing glutamine and in medium not containing glutamine. In growth experiments performed over a 144 hour time frame we showed that both glutamine and asparagine increased the growth of both UOK262 FH-deplete and UOK262 FH-repleted cells, and that the effects were additive. We also found the same to be true for UOK269 SDH-deplete and replete cell lines and for UOK 268 FH-depleted and repleted cell lines. The growth curves appeared to diverge at 96 hours, and the growth effects of the amino acids was most pronounced at hour 144. By this time point cells lacking both asparagine and glutamine have stopped growing. These studies demonstrate that asparagine is an essential additional carbon source for these UOK cell lines, along with glutamine. Several lines of evidence suggest that asparagine and glutamine are used for complementary metabolic pathways. First, the concentration of asparagine (100 uM) is 20-fold lower than the concentration of glutamine (2 mM). If the amino acids were utilized interchangeably, one would not expect an equal effect of asparagine in comparison to glutamine. Second, the Seahorse metabolic analyzer experiments show different results with the two amino acids. Glutamine increased OCR and ATP production, and decreased glycolysis (ECAR), in both UOK262 FH deplete and FH repleted cells, but the addition of asparagine had no effect on OCR and ATP production or on glycolysis (ECAR). These results suggest that glutamine is used preferentially for energy production and asparagine is used primarily for biomass carbon. The reliance of cells on glutamine and asparagine points to a potential metabolic therapy for these tumors, as therapeutic agents exist for inhibiting glutamine metabolism (CB839) and for deplete systemic asparagine (asparaginase). Our initial results have suggested that individually these agents would not have a beneficial effect, but combined they would inhibit cell growth. Therefore we tested the effects of the glutaminase inhibitor CB839 in the presence and absence of asparagine. We found that the growth inhibitory effects of CB839 were enhanced by the elimination of asparagine from the medium in UOK262 cells, further suggesting that both glutamine and asparagine metabolism must be blocked to see optimal growth inhibition. Further, the effects were most pronounced at Hr 144, consistent with the evidence from the growth experiments described above, that a longer period of incubation is required for the metabolic consequences of asparagine and glutamine supplementation or deprivation to be appreciated. Similar preliminary results were obtained with SDH-replete and deplete UOK269 cells. To determine the cell signaling pathways involved in both asparagine and glutamine exposure, we first conducted a phosphoblot experiment using UOK262 cells that suggested that glutamine and asparagine exposure triggered activation of the MTOR pathway. Western blots subsequently have confirmed that both asparagine and glutamine lead to downstream activation of MTOR pathway, as indicated by p235 S6-RP and p240 S6-RP. A similar effect of glutamine and asparagine on p235 S6-RP is seen in the UOK269 cells. In preliminary experiments, the MTOR inhibitor sapanisertib appeared to reverse the growth effects of asparagine but not of glutamine. Experiments are now ongoing to look at the effects of sapanisertib on CB839 cytotoxicity in UOK262 cells. In summary, we have identified asparagine as a key nutrient for UOK262 and UOK269 cells. Identification of an additional key nutrient was the goal for this year as set out in last year's report. To determine the effects of MTOR signaling activation, have collected RNA for an RNASeq analysis of both UOK262 deplete and repleted cells after 96 hours exposure to glutamine, asparagine or both compared to cells grown without either amino acid supplementation. We chose 96 hours because that was the time point where the growth curves began to diverge. The RNA samples are now at FNLCR and will be available in about 1 month. Later this year we plan 1) to perform radioisotope tracer (SIRM) experiments with labeled glutamine and asparagine to determine how these two amino acids are metabolized and to possibly confirm our initial conclusion that these amino acids are channeled into separate metabolic pathways in these cells; and 2) since our results suggest that simultaneous treatment of UOK262 cells with the glutaminase inhibitor CB839 and either the enzyme asparaginase would have a therapeutic effect, and we plan to test this in a UOK262 xenograft model.
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