Page 247 - 2014 Printable Abstract Book
P. 247
(PS4-21) Radiation modification by a Glut-1 inhibitor. John A. Cook, PhD; William DeGraff, MS; Aparna
H. Kesarwala, MD, PhD; and James B. Mitchell, National Cancer Institute, Bethesda, MD
Considerable interest in targeting metabolism for cancer therapy is based on the dependence of
human tumors on aerobic glycolysis for energy production and the recent identification of a variety of
metabolic mutations in human tumors. We are testing the hypothesis that specific metabolic inhibitors
will function as ionizing radiation (IR) modifiers by interfering with the repair of IR-induced DNA damage,
an energy requiring process. Preliminary screening studies have evaluated cell growth and IR response for
the human tumor cell lines: MCF7 (breast), H460 (lung), HT29 (colon), 786-0 (renal), and UOK262 (renal)
treated with the Glut1 inhibitor STF-31 (5 µM). Cell growth was completely inhibited by STF-31 treatment
in H460, 786-0, UOK262 cells. Survival was assessed by the clonogenic assay. Dose modifying factors
(DMF) were calculated at 10% survival from radiation survival curves. The DMF for exponentially growing
H460 cells pre-treated with 5 µM STF-31 for 24 hr was 1.5. Interestingly, plateau phase H460 cells pre-
treated with STF-31 were not radiosensitized (DMF = 1.0). Whole cell lysates of UOK262 cells treated for
24 hr with 5 µM STF-31 were collected and analyzed by capillary electrophoresis mass spectrometry (CE-
MS) in the cation and anion analysis modes for metabolite analysis (Human Metabolome Technologies,
Inc). 116 compounds were identified and quantified associated with glycolysis, TCA, pentose phosphate,
+
and other cellular metabolic pathways. The major effect observed was a marked decrease in NAD /NADH
+
6
+
6
(control: NAD = 3,862 ± 247 pmol/10 cells versus STF31: NAD = 869 ± 59 pmol/10 cells; control: NADH
6
6
= 179 ± 11 pmol/10 cells versus STF31: NADH = 71 ± 4.2 pmol/10 cells). Thus, STF-31 treatment resulted
in a compromise of the cellular redox status, which may account for the cell growth inhibition. Further
metabolic data will be presented with respect to STF-31 mediated enhancement of radiosensitivity.
(PS4-22) To improve standard of care for glioblastoma patients: mTOR kinase inhibition enhances
radiosensitivity in glioblastoma. Anita T. Tandle; Tamalee Kramp; Cody Schlaff; Philip J. Tofilon; and Kevin
Camphausen, NCI, NIH, Bethesda, MD
Glioblastoma multiforme (GBM) is the most common primary brain tumor. Despite maximal
tumor resection, radiation, and chemotherapy (temozolomide [TMZ]), the high degree of infiltration of
GBM leads to 100% recurrence and overall survival of 12-14 months. Aberrant activation of the PII3K
pathway is observed in nearly 90% of GBMs making the downstream effector, mammalian target of
rapamycin (mTOR), a potential drug target. The allosteric mTOR inhibitor rapamycin has failed in the clinic
as a treatment for GBM patients. Therefore, in the current study, we examined the potential benefit of
MLN0128, a novel potent mTOR ATP competitive inhibitor, as a therapeutic strategy for GBM.We studied
the action of MLN0128 in human GBM tumor cells and patient derived stem cells. We show the inhibitory
effect of MLN0128 in nanomolar concentrations on tumor proliferation in multiple GBM cell lines via the
blockage of phospho-AKT, -ribosomal protein, -4EBP1 inhibiting both mTORC1 and mTORC2 signaling in
GBM. Reduction of invasive potential and disruption of paxillin localization to focal adhesions was
observed in vitro. MLN0128 treatment decreased glucose consumption and lactate production, thus
inhibiting aerobic glycolysis or Warburg effect in GBM cells. On clonogenic survival assay MLN0128
decreased GBM cell survival and increased radiosensitivity. MLN0128 exposure significantly delayed the
dispersal of radiation induced γH2AX foci formation in both tumor and stem cells. In vivo treatment of
mouse xenografts with MLN0128 downregulated mTOR targets and significantly inhibited tumor volume
compared to drug and irradiation alone. Combination treatment of GBM cells with MLN0128 and other
245 | P a g e
H. Kesarwala, MD, PhD; and James B. Mitchell, National Cancer Institute, Bethesda, MD
Considerable interest in targeting metabolism for cancer therapy is based on the dependence of
human tumors on aerobic glycolysis for energy production and the recent identification of a variety of
metabolic mutations in human tumors. We are testing the hypothesis that specific metabolic inhibitors
will function as ionizing radiation (IR) modifiers by interfering with the repair of IR-induced DNA damage,
an energy requiring process. Preliminary screening studies have evaluated cell growth and IR response for
the human tumor cell lines: MCF7 (breast), H460 (lung), HT29 (colon), 786-0 (renal), and UOK262 (renal)
treated with the Glut1 inhibitor STF-31 (5 µM). Cell growth was completely inhibited by STF-31 treatment
in H460, 786-0, UOK262 cells. Survival was assessed by the clonogenic assay. Dose modifying factors
(DMF) were calculated at 10% survival from radiation survival curves. The DMF for exponentially growing
H460 cells pre-treated with 5 µM STF-31 for 24 hr was 1.5. Interestingly, plateau phase H460 cells pre-
treated with STF-31 were not radiosensitized (DMF = 1.0). Whole cell lysates of UOK262 cells treated for
24 hr with 5 µM STF-31 were collected and analyzed by capillary electrophoresis mass spectrometry (CE-
MS) in the cation and anion analysis modes for metabolite analysis (Human Metabolome Technologies,
Inc). 116 compounds were identified and quantified associated with glycolysis, TCA, pentose phosphate,
+
and other cellular metabolic pathways. The major effect observed was a marked decrease in NAD /NADH
+
6
+
6
(control: NAD = 3,862 ± 247 pmol/10 cells versus STF31: NAD = 869 ± 59 pmol/10 cells; control: NADH
6
6
= 179 ± 11 pmol/10 cells versus STF31: NADH = 71 ± 4.2 pmol/10 cells). Thus, STF-31 treatment resulted
in a compromise of the cellular redox status, which may account for the cell growth inhibition. Further
metabolic data will be presented with respect to STF-31 mediated enhancement of radiosensitivity.
(PS4-22) To improve standard of care for glioblastoma patients: mTOR kinase inhibition enhances
radiosensitivity in glioblastoma. Anita T. Tandle; Tamalee Kramp; Cody Schlaff; Philip J. Tofilon; and Kevin
Camphausen, NCI, NIH, Bethesda, MD
Glioblastoma multiforme (GBM) is the most common primary brain tumor. Despite maximal
tumor resection, radiation, and chemotherapy (temozolomide [TMZ]), the high degree of infiltration of
GBM leads to 100% recurrence and overall survival of 12-14 months. Aberrant activation of the PII3K
pathway is observed in nearly 90% of GBMs making the downstream effector, mammalian target of
rapamycin (mTOR), a potential drug target. The allosteric mTOR inhibitor rapamycin has failed in the clinic
as a treatment for GBM patients. Therefore, in the current study, we examined the potential benefit of
MLN0128, a novel potent mTOR ATP competitive inhibitor, as a therapeutic strategy for GBM.We studied
the action of MLN0128 in human GBM tumor cells and patient derived stem cells. We show the inhibitory
effect of MLN0128 in nanomolar concentrations on tumor proliferation in multiple GBM cell lines via the
blockage of phospho-AKT, -ribosomal protein, -4EBP1 inhibiting both mTORC1 and mTORC2 signaling in
GBM. Reduction of invasive potential and disruption of paxillin localization to focal adhesions was
observed in vitro. MLN0128 treatment decreased glucose consumption and lactate production, thus
inhibiting aerobic glycolysis or Warburg effect in GBM cells. On clonogenic survival assay MLN0128
decreased GBM cell survival and increased radiosensitivity. MLN0128 exposure significantly delayed the
dispersal of radiation induced γH2AX foci formation in both tumor and stem cells. In vivo treatment of
mouse xenografts with MLN0128 downregulated mTOR targets and significantly inhibited tumor volume
compared to drug and irradiation alone. Combination treatment of GBM cells with MLN0128 and other
245 | P a g e
