Since there was a difference in the OCR and glycolytic reserve between the deficient and restored cell lines, and evidence that restoration of enzyme activity altered metabolic signaling in both pairs of cells, we believed that these pairs of cell lines offered models to examine strategies that would exploit the Krebs cycle enzyme deficiencies of the SDH-deplete and FH-deplete cells. We first examined the fate of pyruvate in these cells, looking for evidence that disrupting the metabolism of pyruvate could provide a therapeutic window between the deplete and the replete cells. Pyruvate can take one of four metabolic routes: pyruvate can enter the Krebs cycle either through thiamine-dependent PDH, or through the anapleurotic pathway via pyruvate carboxylase, which is a biotin-dependent enzyme; pyruvate can take the last step in glycolysis through conversion to lactate by LDH; and it can be metabolized to alanine by ALT. We found: - No difference in UOK262 or UOK269 deficient or restored in sensitivity to thiaminase, suggesting that PDH did not play an important role distinguishing deplete from replete cells. - No effect of thiaminase on UOK262 and UOK269 restored cells on OCR, also indicating that transit into and through the Krebs cycle is independent of PDH. - No difference in UOK262 or UOK269 deplete or restored in sensitivity to dichloroacetate, an inhibitor of PDH kinase, which negatively regulates PDH, also indicating that PDH may not be a critical node in the metabolism of these cells. - We devised a procedure to deplete serum of biotin, and then compared the UOK 262 and UOK269 cells grown in biotin depleted serum to biotin repleted serum. The cells can grow up to 5 passages in depleted serum before they stop growing and appear to undergo apoptosis. Initial results indicate that biotin depletion did not sensitize UOK262 cell lines to thiaminase, but did sensitize UOK 269 SDH deficient cells but not SDH repleted cells to thiaminase, indicating that pyruvate carboxylase may play an important role in UOK269 SDH deficient cells than in the UOK262 FH-deficient cells. - There was no difference in the sensitivity of the pairs of cell lines to NHI-2, an inhibitor of LDH-A, which regulates one of the other fates of pyruvate, conversion into lactate. However, NHI-2 completely protected against the cytotoxicity of thiaminase in UOK262 FH repleted cells, but had not effect on the FH deficient cells. This suggests that cellular pyruvate is important in the repleted cells but not important in the FH-deficient cells when PDH is inhibited by thiaminase. - No difference in UOK262 or UOK266 deficient or restored cells in sensitivity to CB339, the glutaminase inhibitor; no difference in the dose response to CB839 whether glutamine is present in the medium or not; no difference in thiaminase growth inhibition in the presence of CB839; all suggesting that glutamine is not a critical nutrient. - No difference in UOK262 or UOK266 deficient or restored cells in sensitivity to lonidamine, a putative inhibitor of both hexokinase and the mitochondrial pyruvate carrier. Hexokinase catalyzes the first step in glycolysis. We also found no difference in thiaminase growth inhibition in the presence of lonidamine. - However, we found that restoration of FH activity of UOK262 cells, but not SDH activity of UOK269 cells, sensitizes the cells to 3-bromopyruvate, another inhibitor of hexokinase II. This suggests that restoration of FH activity actually increases flux through the glycolytic pathway. - No difference in UOK262 or UOK269 deficient or restored cells in sensitivity to AZD3965, an inhibitor of the monocarboxylate transporters that mediate transport of lactate and pyruvate - in fact AZD3965 was essentially inert in these cell lines, with no growth inhibition in concentrations up to 100 uM, suggesting that the fate of lactate and possibly pyruvate may not be relevant in these cells. - No difference in UOK262 or UOK266 deficient or restored cells in sensitivity to inhibitors of IGFR1, OSI-906 and BMS-754807, and no difference in thiaminase growth inhibition in the presence of OSI-906 and BMS-754807, all indicating that the uptake of glucose by IGFR was not a critical factor. - Since the OCR of the enzyme restored cells was higher than the OCR of the deficient cells, we tested thiaminase in hypoxic vs normoxic conditions, and did not find that hypoxia increased the growth inhibitory activity of thiaminase in any of the cell lines. In summary, although FH and SDH deficient cell lines have enzymatic deficiencies adjacent to each other in the Krebs cycle, the restoration of enzyme activity in the two cell lines reveals different metabolic consequences of the metabolic lesions. 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. SDH-deficient UOK269 cells appear to utilize the anapleurotic pathway. The current working hypothesis is that 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. UOK262 and UOK269 cells are grown in medium supplemented with both fetal calf serum and additional non-essential amino acid (NEAA) supplement. We undertook withdrawal of NEAA from both pairs of cell lines 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 be obtaining carbon from an amino acid source, that is not required for the enzyme repleted cells. This would open up potential therapeutic possibilities. - We are also investigating another strategy with these cells using the metabolic agent b-lapachone, which depletes cellular NADPH by providing a substrate for the enzyme NQO1 that is constantly replenished because the products of the forward reaction are highly unstable and revert to the substrate. We hypothesized that NADPH levels in these cells would be low given the low OCR and low apparent flux through the Krebs cycle that generates NADPH, so they would be unusually sensitive to further depletion of NADPH. We also collaborated with Jim Mitchell in the Radiation Branch to determine whether thiaminase would sensitize MCF-7 cells to radiation. Although the first experiments were promising and appeared to show a radiation sensitizing effect, subsequent experiments could not confirm this. Cross-programmatic activity I have used my joint appointment at CTEP and in the UOB to try to provide a bridge to the two programs. When I heard that the UOB was conducting a clinical trial of erlotinib plus bevacizumab, I discussed testing the third generation EGFR inhibitor rociletinib, an agent in my CTEP portfolio, with Dr Carole Sourbier in the UOB, since this agent had IGF1R and IR inhibitory activity in addition to EGFR activity. An MTA was arranged, and Dr Soubier has found promising results that have led to additional experiments and an expansion of the MTA. Also, my role at CTEP has allowed me to become familiar with novel anti-metabolite agents to test in the context of SDH and FH deficient RCC, diseases that are not on the radar of the companies developing these agents. For example, I earned about b-lapachone from my activities in CTEP. Brief contextual summary I received permission to open a lab in the UOB and hire one staff scientist in the fall of 2014. I hired a 'displaced' staff scientist Dr. Yongmin Liu full time in March, 2015 and he resigned in August. In November, 2015 I hired Dr Rony Panarsky in a post-doc position.

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Salgado, Roberto; Moore, Helen; Martens, John W M et al. (2018) Steps forward for cancer precision medicine. Nat Rev Drug Discov 17:1-2
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