Methods for modulating and sensitizing to serine and glycine limitation - Patent Application 20070229633

Abstract

Disclosed herein is a formulation for starving cells of nutrients such as amino acids and a method for administering said formulation.The formulation of the present invention can substantially lack one or more amino acids.The formulation of the present invention can be administered in combination with an amino acid biosynthesis inhibitor or radiation therapy.The method disclosed herein can make cells sensitive to the depletion of serine and / or glycine. TIFF2024504260000008.tif60139

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 126,294, filed December 16, 2020; U.S. Provisional Patent Application No. 63 / 168,414, filed March 31, 2021; and U.S. Provisional Patent Application No. 63 / 170,805, filed April 5, 2021, which are incorporated by reference in their entireties herein. [Background technology]

[0002] background Many tumor cells exhibit a dependency on exogenous serine, and starvation of dietary serine and glycine can inhibit the growth and prolong survival of these cancers. However, numerous direct and indirect mechanisms promote resistance to this therapeutic approach, including those that promote increased serine availability (e.g., serine synthesis / serine salvage) or downregulation of serine- or glycine-consuming pathways.

[0003] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0004] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition for a first time period, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; b) radiation therapy for a second time period; and c) after the first time period and the second time period, waiting a third time period, wherein the subject is not administered the pharmaceutical composition or administered radiation therapy during the third time period.

[0005] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; and b) an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor.

[0006] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; and b) a therapeutically effective amount of epacadostat. [Brief description of the drawings]

[0007] [Figure 1] Illustrates the serine synthesis pathway. [Diagram 2] Figure 1 illustrates the growth curves of colon cancer cell lines grown in complete medium (CM) or the equivalent medium lacking serine and glycine (-SG) and treated or not with 10 μM PH755. Data are presented as the mean ± SEM of triplicate cultures and are representative of at least two independent experiments (*p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001; two-way ANOVA with Tukey's post-hoc test). [Diagram 3] Growth curves are illustrated for the indicated cell lines grown in complete medium (CM) or the equivalent medium lacking serine and glycine (-SG) and treated or not with 10 μM PH755. Data are presented as the mean ± SEM of triplicate cultures and are representative of at least two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; two-way ANOVA with Tukey's post-hoc test). [Figure 4]Percentage of BrdU positive cells in HCT116 and DLD-1 cells grown for 48 hours in CM or -SG medium + / - 10 μM PH755 followed by incubation with 10 μM BrdU for 5 hours. Data are means ± SEM of three independent experiments (* p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA with Tukey's post-hoc test). [Diagram 5] The gating strategy for determining the percentage of BrdU positive cells (left panel) and the percentage of cells in different phases of the cell cycle (right panel) is shown by way of example for HCT116 and DLD-1 cells grown in CM and incubated with 10 μM BrdU for 30 min. [Figure 6] Figure 1 shows intracellular serine and glycine levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium + / - 10 μM PH755 as measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (*p<0.05, ***p<0.001, ****p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 7] Shown is the percentage of SubG1 cells in HCT116 and DLD-1 cells grown for 48 hours in CM or -SG medium + / - 10 μM PH755. Data are the mean ± SEM of 5 independent experiments (** p<0.01, *** p<0.001, one-way ANOVA with Tukey's post-hoc test). [Figure 8] Cells grown for 2 days (HCT116) or 3 days (DLD-1) in CM or -SG medium supplemented or not with 10 μM PH755 are shown. Western blots show expression of cleaved caspase 3 and caspase 3. Membranes were reprobed with vinculin as a loading control. Data are representative of three independent experiments. [Figure 9]Figure 1 shows intracellular serine levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LC-MS. Metabolite percentages are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 10] Figure 1 shows intracellular glycine levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LC-MS. Metabolite percentages are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (* p<0.05, ** p<0.01; one-way ANOVA with Tukey's post-hoc test). [Figure 11] HT-29 and DLD-1 cells infected with Cas9 / PHGDH single guide RNA (sgRNA) were cultured in CM or -SG medium for 24 h. Western blots show efficient PHGDH ablation in these cells. Vinculin was used as a loading control to redetect the membrane. [Figure 12] Growth curves of HT-29 and DLD-1 cells infected with Cas9 / PHGDH sgRNA (PHGDH) grown in CM or -SG medium. Data are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; two-way ANOVA with Tukey's post-hoc test). [Figure 13]Intestinal tumor organoids derived from Vil1-creER;Apcfl / fl (Apc) and Vil1-creER;Apcfl / fl;KrasG12D / + (Apc Kras) grown in CM or -SG medium supplemented with or without 10 μM PH755. Left panel: Representative pictures of organoids before medium change (day 0) and 2 days after medium change. Right panel: Quantification of organoid diameter 2 days (Apc) or 4 days (Apc Kras) after medium change. Data are presented as mean ± SEM (n = number of organoids measured / condition; Apc:CM: n = 113, CM+PH755: n = 200, -SG: n = 190, -SG+PH755: n = 158; Apc Kras:CM: n = 149, CM+PH755: n = 134, -SG: n = 134, -SG+PH755: n = 78) and representative of at least two independent experiments (***p<0.001, ****p<0.0001; one-way ANOVA with Tukey's post-hoc test). Scale bars are 200 µm. [Figure 14] Intestinal organoids derived from Apc truncated (Apc5) or Villin-CreER;Apcfl / fl;KrasG12D / + mice (Apc Kras 2) grown for 4 days in tumor organoid medium with (CM) or without (-SG) serine and glycine, supplemented with or without 10 μM PH755. Representative pictures of organoids from at least two independent experiments are shown before medium change (day 0), 2 and 4 days after medium change. Scale bar is 200 μm. [Figure 15] Normal organoids derived from the proximal part of healthy small intestine from Villin-CreERT2 mice grown in normal organoid medium (containing Wnt-3a) with (CM) or without (-SG) serine and glycine, supplemented with or without 10 μM PH755. Representative pictures of organoids from three independent experiments 3 days after medium change are shown. Scale bar is 200 μm. [Figure 16]Colorectal organoids from four patients grown in human organoid medium with (CM) or without (-SG) serine and glycine, supplemented with or without 10 µM PH755. Representative pictures of organoids from at least two independent experiments 10-12 days after medium change are shown. Scale bar is 200 µm. [Figure 17] Illustrated is a scheme depicting the fate of uniformly carbon-labeled glucose (m+6) within purine and glutathione synthesis. Glucose is converted to ribose-5-phosphate, a five-carbon sugar (m+5), through the pentose phosphate pathway and added to the purine base to form a purine nucleoside. The purine ring also contains two one-carbon units and an intact glycine, both of which may result from serine metabolism. Serine is synthesized from the glycolytic intermediate 3-PG, which gives rise to the m+3 isotopic isomer from uniformly labeled glucose. Serine (m+3) can give rise to labeled glycine (m+2) and a labeled one-carbon unit (m+1). Thus, the combination of labeled ribose phosphate, glycine, and one-carbon units can give rise to m+5 or higher labeled purines. The m+5 labeled purines represent the contribution of glucose to ribose synthesis alone, while the m+6-9 labeled purines may represent contributions from de novo synthesized serine. Glutathione is made from glycine, glutamic acid (both of which can be m+2 labeled from glucose), and cysteine. The major isotopic form of glutathione (m+2) can be derived from m+2 glycine, while m+4 labeling reflects the incorporation of m+2 glycine and m+2 glutamic acid. [Figure 18]Intracellular ATP levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LCMS. Metabolite percentages are expressed as mean ± SEM of triplicate cultures and are representative of three independent experiments. Statistics were performed comparing the sum of m+6-9% of the metabolite pool for ATP and GTP and the sum of m+2-4% of the metabolite pool for GSH between experimental groups (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 19] Figure 1 shows intracellular glutamate levels in cells grown in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LC-MS. Metabolite percentages are expressed as the mean ± SEM of triplicate wells and are representative of three independent experiments. [Figure 20] Intracellular GSH and GTP levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LCMS. Metabolite percentages are expressed as mean ± SEM of triplicate cultures and are representative of three independent experiments. Statistics were performed comparing the sum of m+6-9% of the metabolite pool for ATP and GTP and the sum of m+2-4% of the metabolite pool for GSH between experimental groups (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 21]Figure 1 shows intracellular ATP, GTP, and GSH levels in HT-29, HCT116, and DLD-1 cells grown for 3 or 6 hours in CM or -SG medium containing U-[13C]-glucose + / - 10 μM PH755 as measured by LC-MS. Metabolite percentages are expressed as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, *** p< 0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 22] Figure 1 shows total levels of ATP, GTP and GSH in cells grown in CM or -SG medium + / - 10 μM PH755 as measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Diagram 23] Illustrated is a proliferation assay of HT-29 and HCT116 cells grown in -SG medium or -SG medium + 10 μM PH755 supplemented with or without 1 mM sodium formate (For), 0.4 mM glycine (Gly), or both (For / Gly). Data are presented as mean ± SEM of triplicate cultures and are representative of three independent experiments (* p<0.05, ** p<0.01; two-way ANOVA with Tukey's post-hoc test). [Figure 24]HCT116 cells grown in -SG medium or -SG medium + 10 μM PH755 supplemented with or without 1 mM sodium formate (For), 0.4 mM glycine (Gly), or both (For / Gly) in the presence of U-[13C]-glucose are shown. ATP and GTP levels were measured by LC-MS. Metabolite percentages are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments. Serine levels were measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Diagram 25] HCT116 cells grown in -SG medium or -SG medium + 10 μM PH755 supplemented with or without 1 mM sodium formate (For), 0.4 mM glycine (Gly), or both (For / Gly) in the presence of U-[13C]-glucose are shown. ATP and GTP levels were measured by LC-MS. Metabolite percentages are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments. Serine levels were measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 26] HT-29, HCT116, and DLD-1 cells grown in -SG medium + 10 μM PH755 supplemented with 1 mM sodium formate and 0.4 mM glycine for 24 h, the last hour in the presence of 13C2 15N1-glycine. 13C2 15N1-serine intracellular levels were measured by LC-MS after (+serine pulse) or without (-serine pulse) addition of unlabeled 1 mM serine in the extracellular medium 1 min prior to metabolite extraction. Data are presented as mean ± SEM of triplicate wells and are representative of three independent experiments. [Figure 27]HT-29 and DLD-1 cells infected with Cas9 / PHGDH sgRNA (PHGDH) grown in CM or -SG medium in the presence of U-[13C]-glucose were shown. Serine, ATP, GTP, and GSH levels were measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; (a) unpaired two-tailed Student's t-test, (b-c) one-way ANOVA with Tukey's post-hoc test). [Figure 28] HT-29 and DLD-1 cells infected with Cas9 / PHGDH sgRNA (PHGDH) grown in CM or -SG medium in the presence of U-[13C]-glucose were shown. Serine, ATP, GTP, and GSH levels were measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; (a) unpaired two-tailed Student's t-test, (b-c) one-way ANOVA with Tukey's post-hoc test). [Figure 29] HT-29 and DLD-1 cells infected with Cas9 / PHGDH sgRNA (PHGDH) grown in CM or -SG medium in the presence of U-[13C]-glucose were shown. Serine, ATP, GTP, and GSH levels were measured by LC-MS. Data are presented as mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; (a) unpaired two-tailed Student's t-test, (b-c) one-way ANOVA with Tukey's post-hoc test). [Diagram 30]Figure 1 shows the growth curves of HT-29, HCT116, and DLD-1 cells transiently depleted of ATF-4 using small interfering RNA (siRNA) and cultured in -SG medium for 4 days. Data are presented as the mean ± SEM of triplicate cultures and are representative of two independent experiments (* p<0.05, ** p<0.01, **** p<0.0001; two-way ANOVA with Sidak's post-hoc test). [Diagram 31] Cells grown for 24 h in CM or -SG medium supplemented or not with 10 μM PH755 are shown. Western blots show expression of the three SSP enzymes PHGDH, PSAT, and PSPH (reprobed on membranes with vinculin as a loading control) or the ATF-4 target ASNS (reprobed on membranes with vinculin as a loading control). Data are representative of at least two independent experiments. [Diagram 32] HT-29 and DLD-1 cells infected with Cas9 / PHGDH single guide RNA (sgRNA) are shown, cultured in CM or -SG medium for 24 hours. Western blots show the expression of PHGDH, PSAT and PSPH (membrane reprobed with vinculin as a loading control) or ATF-4 and ASNS (membrane reprobed with vinculin as a loading control) in these cells. Data are representative of three independent experiments. [Diagram 33] Shown are HT-29 and HCT116 cells grown for 4, 8, 12, 16, and 24 hours in CM or -SG medium supplemented with or without 10 μM PH755. Western blots show the expression of SSP enzymes in these cells. Each membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments. [Diagram 34]Shown are HCT116 and DLD-1 cells grown for 24 h in CM or -SG medium supplemented or not with 10 μM PH755. Western blot shows phospho-GCN2 (Thr899), GCN2, phospho-eIF2α (Ser51), and eIF2α. Membranes were reprobed with vinculin as a loading control. Data representative of two independent experiments. [Diagram 35] Shown are HCT116 and DLD-1 cells grown for 24 hours in CM or -SG medium supplemented with or without 10 μM PH755. Where indicated, cells were treated with 10 μM MG-132, a proteasome inhibitor, 6 hours before harvesting the cells. Western blots show expression of ATF-4 and its targets ASNS and PSAT. Membranes were reprobed with vinculin as a loading control. Data are representative of three independent experiments. [Diagram 36] HT-29, HCT116, and DLD-1 cells grown for 6 or 24 hours in CM or -SG medium supplemented with or without 10 μM PH755 are shown. Relative gene expression of ATF4 and PHGDH was measured by qPCR and normalized to cells grown in CM for 6 hours. Data are presented as mean ± SEM of triplicate cultures and representative of two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 37] HT-29, HCT116, and DLD-1 cells grown for 6 or 24 hours in CM or -SG medium supplemented with or without 10 μM PH755 are shown. Relative gene expression of ASNS, PSAT1, and PSPH was measured by qPCR and normalized to cells grown in CM for 6 hours. Data are presented as mean ± SEM of triplicate cultures and representative of two independent experiments (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 38]Shown are HT-29, HCT116, and DLD-1 cells grown in CM, -SG medium, or -SG medium + 10 μM PH755, supplemented with or without 1 mM sodium formate + 0.4 mM glycine (For / Gly). Western blots show expression of the three SSP enzymes PHGDH, PSAT, and PSPH, or ATF-4 and its canonical target ASNS, after 24 h incubation in these media. Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments. [Figure 39] HCT116 and DLD-1 cells grown for 24 h in CM or -SG medium supplemented or not with 10 μM PH755 are shown. Puromycin (90 μM) was added to the culture medium 10 min before harvesting the cells. Where indicated, cells were treated with 10 μg / mL cycloheximide (CHX), a well-known protein synthesis inhibitor, 5 h before harvesting the cells. Western blots show puromycylated peptides. Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments. [Diagram 40] Shown are HCT116 and DLD-1 cells grown for 24 hours in CM or -SG medium supplemented or not with 10 μM PH755. Where indicated, cells were treated with 10 μM MG-132, a proteasome inhibitor, 6 hours prior to cell harvest. Western blots show expression of c-MYC, HIF1a, and p53. Membranes were reprobed with vinculin as a loading control. Data are representative of three independent experiments. [Diagram 41] Shown are DLD-1 and HT-29 cells grown for 24 hours in CM or -SG medium supplemented with or without 10 μM PH755. Western blot shows phospho-p70S6K (Thr389) and p70S6K. Membranes were reprobed with actin as a loading control. Data are representative of three independent experiments. [Diagram 42]Shown are HT-29, HCT116, and DLD-1 cells grown in CM for 24 h. Where indicated, cells were treated with the ER stress inducer, tunicamycin (5 μg / mL) or 10 μM PH755. Western blots show expression of ATF-4 targets ASNS, PHGDH, PSAT, and PSPH. Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments. [Diagram 43] Shown are HT-29, HCT116, and DLD-1 cells grown in CM, -SG medium, or -SG medium + 10 μM PH755, supplemented with or without 1 mM sodium formate + 0.4 mM glycine (For / Gly). Western blots show expression of the three SSP enzymes PHGDH, PSAT, and PSPH or expression of ATF-4 and its canonical target ASNS after 24 h incubation in these media. Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments. [Diagram 44] Body weights of C57BL / 6J mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755, recorded at regular intervals, are shown. Percentages of body weight were calculated from the initial weight taken on the day of diet change. Arrows indicate the start date of the indicated treatment. Data are presented as mean ± SEM (n=10 mice / group). (**p<0.01, ****p<0.0001; two-way ANOVA with Tukey's post-hoc test). [Diagram 45] Low power magnification of transverse sections taken at the level of the caudal diencephalon and rostral midbrain from C57BL / 6J mice (n=5 mice / group) fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755 are shown. There is no evidence of degeneration or softening on hematoxylin and eosin stained sections. Brain weights for mice in each experimental group are shown as mean ± SEM (n=5 mice / group). [Figure 46]High power magnification of transverse sections taken at the level of the cerebral cortex from C57BL / 6J mice (n=5 mice / group) fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755. Neurons and neuropil were morphologically unremarkable. Scale bar 50 μm. [Figure 47] Plasma collected at sacrifice from C57BL / 6J mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755 for 20 days is shown. AST and ALT activities in plasma were measured with commercial kits. Data are presented as mean ± SEM (n = 10 mice / group). [Figure 48] Plasma collected at sacrifice from C57BL / 6J mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755 for 20 days is shown. LC-MS analysis was performed to assess urea and creatinine content. Data are presented as mean ± SEM (n = 10 mice / group). [Figure 49] Quantification of villi length from C57BL / 6J mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755 for 20 days is shown. Data are presented as mean ± SEM (n = 10 mice / group). (* p < 0.05, **** p < 0.0001; one-way ANOVA with Tukey's post-hoc test). [Figure 50] Representative images of Ki67-stained jejunum from C57BL / 6J mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755 for 20 days are shown (n=5 mice / group). [Figure 51]Figure 1 shows mouse body weights in DLD-1 and HCT116 xenograft studies recorded at regular intervals. Percentage of body weight was calculated from the initial body weight taken on the day of diet change. Arrows indicate the start date of the indicated treatment. Data are presented as mean ± SEM. (ns: not significant, *p<0.05; ***p<0.001; two-way ANOVA + Tukey's post-hoc test) (a) CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9. (b) CTR+Veh: n=8; CTR+PH755 n=7; -SG+Veh: n=8; -SG+PH755 n=7 (n=number of mice). [Figure 52] Figure 1 shows mouse body weights in DLD-1 and HCT116 xenograft studies recorded at regular intervals. Percentage of body weight was calculated from the initial body weight taken on the day of diet change. Arrows indicate the start date of the indicated treatment. Data are presented as mean ± SEM. (ns: not significant, *p<0.05; ***p<0.001; two-way ANOVA + Tukey's post-hoc test) (a) CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9. (b) CTR+Veh: n=8; CTR+PH755 n=7; -SG+Veh: n=8; -SG+PH755 n=7 (n=number of mice). [Diagram 53] Plasma collected at sacrifice from mice subcutaneously injected with DLD-1 cells and fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG), and treated with vehicle (Veh) or PH755. LC-MS analysis was performed to measure absolute levels of serine and glycine in plasma. CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9 (n=number of mice). Data are presented as mean ± SEM. (** p<0.01, **** p<0.0001, unpaired two-tailed Student's t-test). [Figure 54]Plasma collected at sacrifice from mice subcutaneously injected with HCT116 cells and fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG), and treated with vehicle (Veh) or PH755, are shown. LC-MS analysis was performed to assess serine and glycine content. Data are presented as mean ± SEM (n = 8 mice / group). (* p < 0.05; unpaired two-tailed Student's t test). [Figure 55] CD-1 nude mice were subcutaneously injected with DLD-1 cells and fed a diet with (CTR) or without (-SG) serine and glycine 2 days after tumor cell injection. Two days after dietary change, mice were orally administered vehicle (Veh) or PH755 once a day for 20 days. The starting dose of PH755 was 100 mg / kg (7 days), then reduced to 50 mg / kg (6 days) and increased again to 75 mg / kg (7 days). Tumor volumes were measured three times a week by caliper measurement. Data are presented as mean ± SEM. CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9 (n=number of mice). (ns: not significant, **P<0.01; two-way ANOVA+Tukey's post hoc test). [Figure 56] CD-1 nude mice were subcutaneously injected with HCT116 cells and fed a diet with (CTR) or without (-SG) serine and glycine 10 days after tumor cell injection. Four days after dietary change, mice were orally administered vehicle (Veh) or PH755 once a day for 11 days. The starting dose of PH755 was 100 mg / kg (3 days) and then reduced to 50 mg / kg (8 days). Tumor volumes were measured three times a week by caliper measurement. Data are presented as mean ± SEM. CTR+Veh: n=8; CTR+PH755 n=7; -SG+Veh: n=8; -SG+PH755 n=7 (n=number of mice). (ns: not significant, *P<0.05; two-way ANOVA+Tukey's post-hoc test). [Figure 57]Representative immunohistochemical pictures and quantification of active caspase-3 positive cells in DLD-1 tumors collected at endpoint from mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755. CTR+Veh: n=9; CTR+PH755 n=9; -SG+Veh: n=10; -SG+PH755 n=8 (n=number of mice). Data are presented as mean ± SEM. (*P<0.05; unpaired two-tailed Student's t-test with Welch's correction). Scale bar is 50 μm. [Figure 58] Serine, glycine, SAM, and SAH levels measured by LC-MS in tumor lysates collected at termination from animals subcutaneously injected with DLD-1 cells. Peak areas were normalized to total ion count (TIC). (e) CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9. (f) CTR+Veh: n=9; CTR+PH755 n=9; -SG+Veh: n=10; -SG+PH755 n=8 (n=number of mice) (*p<0.05, **p<0.01; unpaired two-tailed Student's t-test, Welch's correction applied when necessary). [Figure 59] Serine and glycine levels measured by LC-MS in tumor lysates collected at termination from animals subcutaneously injected with HCT116 cells. Peak areas were normalized to total ion intensity (TIC). CTR+Veh: n=8; CTR+PH755 n=6; -SG+Veh: n=8; -SG+PH755 n=8 (n=number of mice). Data are presented as mean ± SEM. (*p<0.05, **p<0.01; unpaired two-tailed Student's t-test). [Figure 60]ATP and GTP levels measured by LC-MS in tumor lysates collected at termination from animals subcutaneously injected with DLD-1 cells. Peak areas were normalized to total ion intensity (TIC). CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9 (n=number of mice). Data are presented as mean ± SEM. (ns: not significant; unpaired two-tailed Student's t-test). [Figure 61] Serine, glycine, SAM, and SAH levels measured by LC-MS in tumor lysates collected at termination from animals subcutaneously injected with DLD-1 cells. Peak areas were normalized to total ion intensity (TIC). (e) CTR+Veh: n=10; CTR+PH755 n=10; -SG+Veh: n=10; -SG+PH755 n=9. (f) CTR+Veh: n=9; CTR+PH755 n=9; -SG+Veh: n=10; -SG+PH755 n=8 (n=number of mice) (*p<0.05, **p<0.01; unpaired two-tailed Student's t-test, Welch's correction applied if necessary). [Figure 62] Representative immunohistochemical pictures and quantification of PHGDH and PSAT staining in DLD-1 tumors collected at termination from mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755. CTR+Veh: n=9; CTR+PH755 n=9; -SG+Veh: n=9; -SG+PH755 n=7 (n=number of mice). Data are presented as mean ± SEM. (*p<0.05, ***p<0.001; one-way ANOVA with Tukey's post-hoc test). Scale bar is 50 μm. [Figure 63]Representative immunohistochemical pictures and quantification of PHGDH and PSAT staining in DLD-1 tumors collected at termination from mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755. CTR+Veh: n=9; CTR+PH755 n=9; -SG+Veh: n=9; -SG+PH755 n=7 (n=number of mice). Data are presented as mean ± SEM. (*p<0.05, ***p<0.001; one-way ANOVA with Tukey's post-hoc test). Scale bar is 50 μm. [Figure 64] Panels A-E show how dietary restriction of serine and glycine in combination with PHGDH inhibition synergizes to reduce tumor burden and improve survival in a genetic model of intestinal cancer. [Figure 65] Panels A-D show the metabolomic effects of radiation on pancreatic and colorectal cancer cells in vitro. [Figure 66] Panels A-E show the effect of dietary amino acid restriction in response to targeted radiation therapy in vivo. [Figure 67]Figure 1 shows IDO1 expression in vivo. Panel A is a schematic diagram detailing the method used to analyze IDO1 expression in genetically engineered mouse models (GEMMs) of pancreatic ductal adenocarcinoma (PDAC). Panel B shows the indicated proteins after analysis by immunoblotting. Panel C shows quantification of IDO1 relative to total protein (loading control) (healthy pancreas n=5, Pdx1-cre;KrasG12D / +;Trp53fl / + tumors n=6, Pdx1-cre;KrasG12D / +;Trp53R172H / + tumors n=5, unpaired t-test, p-values ​​shown, error bars are standard deviation). Panel D shows KPC A cells, a line isolated from a tumor in a mixed background Pdx1-cre;KrasG12D / +;Trp53R172H / + mouse, treated with mouse IFNγ (1 ng / ml) for 24 hours or injected subcutaneously into the flank of a CD1 nude mouse to form a tumor. Panel E shows KPC cells isolated from a pure C567Bl6 / J background Pdx1-cre;KrasG12D / +;Trp53R172H / + mouse and treated in culture with mouse IFNγ (1 ng / mL) for 24 hours or injected subcutaneously into the flank of a C567Bl6 / J mouse to form a tumor. Panel F shows the indicated cell lines treated with human IFNγ (1 ng / ml) for 24 hours and cell lysates blotted for the indicated proteins. [Figure 68] 1 shows data extracted from the MERAV database showing the relative abundance of IDO1 mRNA from microarrays. [Figure 69]Figure 1 shows that IDO expression was upregulated by ultra-low adhesion tissue culture plate (3D) growth of cells and IFNγ via protein and JAK / STAT signaling. Panel A shows a schematic diagram detailing the kynurenine pathway by which tryptophan is metabolized. Panel B shows protein expression cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Panel C shows protein expression treated with rotenone (1 μM) or vehicle only control. Panel D shows protein expression cultured in medium containing either glucose (Glc) (10 mM) or galactose (Gal) (10 mM). Panel E shows protein cultured in 2D or 3D conditions. Panel F shows fluorescence intensity of IDO1 / actin in CFPAC-1 in 2D and 3D conditions quantified (n=4, paired t-test, p-values ​​shown, error bars are SEM). Panel G shows results for CFPAC-1 cells cultured for 24 hours in 2D or 3D conditions and treated with epacadostat (1 μM) or vehicle-only control for 16 hours before media kynurenine was analyzed by LCMS (1ex, triplicate wells, error bars are standard deviation). Panel H shows CFPAC-1 or HPAF-II cells cultured for 24 hours in either 2D or 3D conditions and then treated with JAKi (1 μM) or vehicle-only control (veh.) and / or human IFNγ (1 ng / ml) for 16 hours. Cells were then lysed and the indicated proteins were analyzed by immunoblotting. [Figure 70]CFPAC-1 or HPAF-II cells were grown for 24 hours in normal tissue culture plates (2D) or ultra-low attachment tissue culture plates (3D), or cultured in 2D and treated with 1 ng / ml IFNγ. Lysates (Panel A) were blotted for the indicated proteins, and (Panel B) the fluorescence intensity of IDO1 relative to actin (loading control) was quantified (n=4, paired t-test, p-values ​​shown, error bars are SEM). CFPAC-1 and HPAF-II cells were grown for 24 h in either normal tissue culture plates (2D) or ultra-low attachment tissue culture plates (3D) and cell lysates were immunoblotted for the indicated proteins after 16 h of treatment with MG132 (20 μM) or vehicle only control (panel C); after treatment with bafilomycin A1 (100 nM) or vehicle only control for the indicated time points (panel D); or after 16 h of treatment with JAKi (at the indicated concentrations), vehicle only control, or IFNγ (1 ng / ml) (panel E). [Figure 71] We show that one-carbon units derived from tryptophan are incorporated into serine and nucleotides in pancreatic cancer cells. [Figure 72] CFPAC-1 cells cultured in 2D or 3D for 24 h and then treated with epacadostat (1 μM) or vehicle-only control for 24 h in the presence of either unlabeled (12C) or 13C11 tryptophan were shown and the intracellular content of the indicated nucleotides was analyzed by LCMS (1ex, triplicate wells, error bars are standard deviation). [Figure 73] We show that tryptophan-derived one-carbon units are utilized for serine and nucleotide synthesis in PDAC tumors in vivo. [Figure 74] Data are shown for KPC cells from pure C57BL / J Pdx1-cre;KrasG12D / +;Trp53R172H / + mice expressing IDO1 or empty vector control (EV). KPC cells were injected subcutaneously into the flank of C57BL / J mice; once tumors formed, mice were given 800 μL of 120 mM 13C11 tryptophan by intraperitoneal injection and allowed to sit for 3 hours. [Figure 75] We show that cancer cells liberated formic acid derived from tryptophan, which was consumed by pancreatic stellate cells and incorporated into nucleotides. CFPAC-1 (panel A) or HPAF-II (panel B) cells were cultured in 3D for 4 days and then treated for 24 h with IFNγ (1 ng / ml) or vehicle-only control in the presence of either unlabeled (12C), 13C11 tryptophan, or 13C3 15N1 serine. Media content of formic acid was analyzed by derivatization and GC-MS (1ex, triplicate wells, error bars are standard deviation). Panel C shows a schematic of the experimental approach used in panels D-K. CFPAC-1 cells were treated with vehicle-only control or human IFNγ (1 ng / ml) and epacadostat (epac., 1 μM) or vehicle-only control in the presence of unlabeled (12C) or 13C11 tryptophan. Conditioned medium was collected after 24 hours and ImPSCs were cultured in this medium or in non-conditioned matched medium. After 24 hours, the intracellular content of serine (panel D), ATP (panel E), ADP (panel F), and AMP (panel G) was analyzed by LCMS (fraction of major isotopologues compared to total, 1ex, triplicate wells, error bars are standard deviation). ImPSC-GFP cells were cultured for 24 hours in 2D as monoculture or in coculture with CFPAC-1 cells. Cells were then treated with vehicle only control or human IFNγ (1 ng / ml) and epacadostat (1 μM) or vehicle only control in the presence of 13C11 tryptophan for 24 hours. Cells were then trypsinized and sorted using FACS for GFP-positive cells, and the intracellular content of serine (panel H), ATP (panel I), ADP (panel J), and AMP (panel K) was analyzed by LCMS (fractions of major isotopologues relative to total shown, 1ex, triplicate wells, error bars are standard deviation). Panel L shows a proposed model of tryptophan-derived formate usage in pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells. [Figure 76]Figure 1 shows the cellular uptake of 13C1 formate with ATP, DP, AMP, and GTP in ImPSC #1, ImPSC #2, and ImPSC #3 cells. ImPSC #1, ImPSC #2, and ImPSC #3 cells were cultured in the presence of 13C1 formate for 24 hours, and the intracellular content of all possible destinations of one carbon from formate, ATP (panel A), ADP (panel B), AMP (panel C), and GTP (panel D), was analyzed by LCMS (1ex, triplicate wells, error bars are standard deviation). CFPAC-1 cells were treated with IFNγ (1 ng / ml) and / or epacadostat (1 μM) and / or vehicle-only control in the presence of unlabeled (12C) or 13C11 tryptophan. Conditioned medium was collected after 24 hours, and ImPSC #2 cells were cultured in this medium or in unconditioned treatment-matched medium. After 24 h, the intracellular contents of ATP (panel E), ADP (panel F), and serine (panel G) were analyzed by LCMS (fractions of major isotopologues relative to the total shown, 1ex, triplicate wells, error bars are standard deviation). [Figure 77] The left panel shows cell proliferation over 5 days in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM). The right panel shows the fold change in cell number at day 5 compared to day 0 in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM). [Figure 78] Shown are the cellular carbon-13-derived labeled fractions of AMP, ADP, ATP, GDP, and GMP in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description Direct mechanisms that promote resistance to therapeutic approaches that reduce serine and / or glycine availability include those that promote increased serine availability by serine biosynthesis (at tumor or systemic level) via enhanced expression of de novo serine synthesis pathway (SSP) enzymes, whose expression can also be promoted by certain oncogenic mutations. Another route to increase serine availability is the promotion of serine recycling, for example, by mechanisms such as autophagy. Indirect resistance mechanisms can rely on metabolic adaptations other than those directly involved in serine synthesis, for example, downregulating pathways that consume serine (such as nucleotide synthesis). Combination with other therapeutic agents that target these direct or indirect resistance mechanisms can improve the ability of serine and glycine starvation to inhibit, for example, tumor growth, tumor initiation, or metastasis. In addition, combination with therapeutic agents or interventions that increase the demand of cancer cells or tumors for serine and / or glycine can also sensitize cancer cells or tumors to serine and / or glycine starvation.

[0009] Described herein are compositions and methods for the inhibition of phosphoglycerate dehydrogenase (PHGDH), the first step in SSP, in combination with compositions lacking serine and / or glycine. PHGDH cooperates with serine and glycine starvation to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH in combination with serine starvation can result in a global protein synthesis defect, which can block the activation of the ATF-4 response, more broadly affecting the protective stress response to amino acid starvation. In vivo, the combination of diet and inhibitors, together with the reduction of one-carbon availability, can show therapeutic efficacy against tumors that are resistant to diet or drugs alone. Inhibition of PHGDH can enhance the therapeutic efficacy of a serine-depleted diet.

[0010] Also described herein are methods of administering serine and glycine depletion therapy in combination with radiation therapy.

[0011] Cancer cells can adapt their metabolism to support growth and survival, resulting in various dependencies and vulnerabilities that can be targeted therapeutically. These metabolic changes can be driven by numerous factors, including genetic alterations in the tumor and the tumor's environment or tissue of origin, but serine metabolism in supporting cancer cell growth may also be important for these observed metabolic changes. Serine and glycine (produced from serine by the SHMT1 / 2 reaction) contribute to numerous important processes, including the synthesis of proteins, nucleotides, and lipids, the generation of antioxidant defenses through glutathione and NADPH synthesis, and the provision of one-carbon units for the folate cycle and methylation reactions.

[0012] Serine, as a non-essential amino acid, can be taken up from the extracellular environment or synthesized de novo by cells using the serine synthetic pathway (SSP). Cancer cells can be voracious consumers of serine and depend on exogenous sources of serine for optimal growth. Some cancer cells can adapt to serine starvation by activating flux through the SSP. Serine is an activator of PKM2, the final step in glycolysis, and reduced PKM2 activity under serine-depleted conditions can allow the conversion of glycolytic intermediates into the SSP. This response is coordinated with the activation of three enzymes of the SSP that depend on ATF-4 and histone methyltransferase G9A, which may allow most cancer cells to survive and continue to proliferate after serine starvation. The efficacy with which cancer cells can adapt to the loss of exogenous serine depends on several factors. Some cancers acquire amplification or overexpression of PHGDH, the first step in the SSP, and these cells tend to be less affected by serine starvation. Similarly, activation of oncogenes such as KRAS, MYC, MDM2, and NRF210 can lead to increased SSP enzyme expression and allow cells to become resistant to the depletion of exogenous serine. Conversely, p53 tumor suppressor protein can inhibit PHGDH expression, but loss of p53 also renders cells more vulnerable to the increased ROS associated with the switch to de novo serine synthesis, resulting in reduced survival in serine-free medium.

[0013] amino acid The compositions disclosed herein can be devoid of serine. The compositions disclosed herein can be devoid of glycine. The compositions disclosed herein can be devoid of serine and glycine. The compositions disclosed herein can be administered in combination with a PHGDH inhibitor, a PSAT1 inhibitor, or a PSPH inhibitor.

[0014] The compositions of the present disclosure include at least 10 amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 10 amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 14 amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 18 amino acids or salts thereof. The salts of the amino acids disclosed herein can be pharma- ceutically acceptable salts. In some embodiments, the compositions disclosed herein lack serine and glycine. In some embodiments, the compositions disclosed herein lack serine. In some embodiments, the compositions disclosed herein lack glycine.

[0015] In some embodiments, the compositions of the present disclosure include 1, 2, 3, 4, 5, 6, 7, 8, or 9 essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 7, 8, or 9 essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 8 essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 9 essential amino acids or salts thereof. The salts of the amino acids disclosed herein can be pharma- ceutically acceptable salts. In some embodiments, the compositions disclosed herein lack serine and glycine. In some embodiments, the compositions disclosed herein lack serine. In some embodiments, the compositions disclosed herein lack glycine.

[0016] In some embodiments, the compositions of the present disclosure include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 7, 8, 9, 10, or 11 non-essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 7 non-essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 8 non-essential amino acids or salts thereof. In some embodiments, the compositions of the present disclosure include 9 non-essential amino acids or salts thereof. The salts of the amino acids disclosed herein can be pharma- ceutically acceptable salts. In some embodiments, the compositions disclosed herein are devoid of serine and glycine. In some embodiments, the compositions disclosed herein are devoid of serine. In some embodiments, the compositions disclosed herein are devoid of glycine.

[0017] The composition of the present disclosure can include an essential amino acid or a salt thereof, and a non-essential amino acid or a salt thereof. In some embodiments, the composition of the present disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 essential amino acids or salts thereof, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-essential amino acids or salts thereof. In some embodiments, the composition of the present disclosure includes 7, 8, or 9 essential amino acids or salts thereof, and 6, 7, 8, or 9 non-essential amino acids or salts thereof. In some embodiments, the composition of the present disclosure includes 8 or 9 essential amino acids or salts thereof, and 8 or 9 non-essential amino acids or salts thereof. In some embodiments, the composition of the present disclosure includes 9 essential amino acids or salts thereof, and 7 non-essential amino acids or salts thereof. In some embodiments, the composition of the present disclosure includes 9 essential amino acids or salts thereof, and 8 non-essential amino acids or salts thereof. In some embodiments, the composition of the present disclosure includes 9 essential amino acids or salts thereof, and 9 non-essential amino acids or salts thereof. The salt of the amino acid disclosed herein can be a pharmaceutically acceptable salt. In some embodiments, the compositions disclosed herein lack serine and glycine. In some embodiments, the compositions disclosed herein lack serine. In some embodiments, the compositions disclosed herein lack glycine.

[0018] In some embodiments, the composition of the present disclosure comprises histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, arginine, glutamine, alanine, aspartic acid, asparagine, glutamic acid, or proline.In some embodiments, the composition of the present disclosure comprises L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, L-threonine, L-tryptophan, L-valine, L-arginine, L-glutamine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, or L-proline.

[0019] In some embodiments, the composition comprises histidine or a salt thereof, for example, L-histidine or L-histidine hydrochloride. In some embodiments, the composition of the present disclosure comprises isoleucine or a salt thereof, for example, L-isoleucine, L-isoleucine methyl ester hydrochloride, or L-isoleucine ethyl ester hydrochloride. The salt of the amino acid disclosed herein can be a pharmaceutically acceptable salt. In some embodiments, the composition of the present disclosure comprises leucine or a salt thereof, for example, L-leucine, L-leucine methyl ester hydrochloride, or L-leucine ethyl ester hydrochloride. The salt of the amino acid disclosed herein can be a pharmaceutically acceptable salt. In some embodiments, the composition of the present disclosure comprises lysine or a salt thereof, for example, L-lysine, L-lysine hydrochloride, or L-lysine dihydrochloride. The salt of the amino acid disclosed herein can be a pharmaceutically acceptable salt. In some embodiments, the compositions of the present disclosure include methionine or a salt thereof, such as L-methionine, L-methionine methyl ester hydrochloride, or L-methionine hydrochloride. The salts of the amino acids disclosed herein can be pharma- ceutically acceptable salts.

[0020] In some embodiments, the composition of the present disclosure comprises cysteine ​​or a salt thereof, such as L-cysteine, L-cysteine ​​hydrochloride, L-cysteine ​​methyl ester hydrochloride, or L-cysteine ​​ethyl ester hydrochloride. In some embodiments, the composition discloses cystine or a salt thereof, such as L-cystine. The salt of the amino acid disclosed herein can be a pharma- ceutically acceptable salt. In some embodiments, the composition of the present disclosure comprises phenylalanine or a salt thereof, such as L-phenylalanine, DL-phenylalanine, or L-phenylalanine methyl ester hydrochloride. In some embodiments, the composition of the present disclosure comprises tyrosine or a salt thereof, such as L-tyrosine or L-tyrosine hydrochloride. In some embodiments, the composition of the present disclosure comprises threonine or a salt thereof, such as L-threonine or L-threonine methyl ester hydrochloride. In some embodiments, the composition of the present disclosure comprises L-tryptophan. In some embodiments, the compositions of the present disclosure include valine or a salt thereof, such as L-valine, L-valine methyl ester hydrochloride, or L-valine ethyl ester hydrochloride. The salts of the amino acids disclosed herein can be pharma- ceutically acceptable salts.

[0021] In some embodiments, the composition of the present disclosure comprises arginine or a salt thereof, for example, L-arginine or L-arginine hydrochloride. In some embodiments, the composition of the present disclosure comprises glutamine or a salt thereof, for example, L-glutamine or L-glutamine hydrochloride. In some embodiments, the composition of the present disclosure comprises alanine or a salt thereof, for example, L-alanine or β-alanine. In some embodiments, the composition of the present disclosure comprises aspartic acid or a salt thereof, for example, L-aspartic acid, D-aspartic acid, L- or D-aspartic acid potassium salt, L- or D-aspartic acid hydrochloride; L- or D-aspartic acid magnesium salt, or L- or D-aspartic acid calcium salt. In some embodiments, the composition of the present disclosure comprises L-asparagine. In some embodiments, the composition of the present disclosure comprises glutamic acid or a salt thereof, for example, L-glutamic acid or L-glutamic acid hydrochloride. In some embodiments, the composition of the present disclosure comprises proline or a salt thereof, such as L-proline, L-proline hydrochloride, L-proline methyl ester hydrochloride, or L-proline ethyl ester hydrochloride. The salt of the amino acid disclosed herein can be a pharma- ceutically acceptable salt.

[0022] Pharmaceutical excipients The composition of the present disclosure can include at least one pharmaceutical excipient, such as an anti-adhesive agent, a binder, a coating, a coloring agent, a disintegrant, a flavoring agent, a preservative, an adsorbent, a sweetener, or a vehicle.In some embodiments, the composition of the present disclosure includes a coloring agent and a flavoring agent.In some embodiments, the composition of the present disclosure includes a coloring agent, a flavoring agent, and a sweetener.In some embodiments, the composition of the present disclosure includes a flavoring agent, a sweetener, and a preservative.

[0023] formulation The composition of the present invention can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compound to act quickly. Non-limiting examples of immediate release formulations include formulations that are easily soluble. A controlled release formulation can be a pharmaceutical formulation in which the release rate and release profile of the active agent are adapted to be able to meet physiological and chronotherapeutic requirements, or alternatively, formulated to provide the release of the active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations that include polymeric materials in which at least one active ingredient is dispersed), granules in a matrix, polymer mixtures, and granular agglomerates.

[0024] In some embodiments, the controlled release formulation is a delayed release form.Delayed release form can be formulated to delay the action of compound for a long period of time.Delayed release form can be formulated to delay the release of effective dose of one or more compounds for, for example, about 4 hours, about 8 hours, about 12 hours, about 16 hours or about 24 hours.

[0025] The controlled release formulation can be a sustained release form.The sustained release form can be formulated, for example, to sustain the action of the compound for a long period of time.The sustained release form can be formulated to provide an effective dose of any compound described herein (for example, provide a physiologically effective blood profile) for about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

[0026] Non-limiting examples of pharma- ceutically acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0027] dosage The compositions described herein can be given to supplement the meal consumed by the subject.The compositions described herein can be given as a meal replacement.The compositions described herein can be given immediately before or after a meal.The compositions described herein can be given within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours before or after a meal.

[0028] The compositions described herein can be in unit dosage form suitable for single administration of precise dosage. In unit dosage form, the formulation is divided into unit doses containing appropriate amounts of the composition. In some embodiments, the unit dose can be in the form of a package containing a separate amount of the formulation. In some embodiments, the formulation of the present disclosure can be presented in unit dosage form in a single-serving sachet. In some embodiments, the formulation of the present disclosure can be presented in a single-dose non-resealable container. In some embodiments, the formulation of the present disclosure can be presented in a resealable container, and the subject can obtain a single-dose dose of the formulation using a scoop or spoon designed to dispense a single dose. In some embodiments, the formulation of the present disclosure can be presented in a resealable container, and the subject can obtain a single-dose dose of the formulation using a scoop or spoon designed to dispense a half-dose (i.e., two scoops dispense a dose).

[0029] The compositions described herein can be present in a unit dose in the range of about 1 g to about 2 g, about 2 g to about 3 g, about 3 g to about 4 g, about 4 g to about 5 g, about 5 g to about 6 g, about 6 g to about 7 g, about 7 g to about 8 g, about 8 g to about 9 g, about 9 g to about 10 g, about 10 g to about 11 g, about 11 g to about 12 g, about 12 g to about 13 g, about 13 g to about 14 g, about 14 g to about 15 g, about 15 g to about 16 g, about 16 g to about 17 g, about 17 g to about 18 g, about 18 g to about 19 g, about 19 g to about 20 g, about 20 g to about 21 g, about 21 g to about 22 g, about 22 g to about 23 g, about 23 g to about 24 g, or about 24 g to about 25 g.

[0030] The compositions described herein can be present in a unit dose in an amount of about 1 g, about 2 g, about 3 g, about 4 g, about 5 g, about 6 g, about 7 g, about 8 g, about 9 g, about 10 g, about 11 g, about 12 g, about 13 g, about 14 g, about 15 g, about 16 g, about 17 g, about 18 g, about 19 g, about 20 g, about 21 g, about 22 g, about 23 g, about 24 g, or about 25 g. In some embodiments, the compositions described herein are present in a unit dose in an amount of about 10 g, 12 g, 15 g, 20 g, or 24 g.

[0031] In some embodiments, the compositions described herein are present in a unit dose in an amount of about 12 g. In some embodiments, the compositions described herein are present in a unit dose in a sachet in an amount of about 12 g. In some embodiments, the compositions described herein are present in a unit dose in an amount of about 15 g. In some embodiments, the compositions described herein are present in a unit dose in a sachet in an amount of about 15 g. In some embodiments, the compositions described herein are present in a unit dose in an amount of about 24 g. In some embodiments, the compositions described herein are present in a unit dose in a sachet in an amount of about 24 g.

[0032] In some embodiments, the dose of the composition of the present disclosure can be expressed in terms of the amount of drug divided by the mass of the subject, e.g., milligrams of drug per kilogram of body weight of the subject. In some embodiments, the composition is provided in an amount ranging from about 100 mg / kg to about 150 mg / kg, about 150 mg / kg to about 200 mg / kg, about 200 mg / kg to about 250 mg / kg, about 250 mg / kg to about 300 mg / kg, or about 300 mg / kg to about 350 mg / kg. In some embodiments, the composition is provided in an amount of about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, or about 350 mg / kg.

[0033] The compositions described herein can be provided to a subject to achieve an amount of protein per subject's body weight. In some embodiments, the compositions described herein can be provided to a subject to achieve a range of about 0.2 g protein / kg subject's body weight to about 0.4 g protein / kg subject's body weight, about 0.4 g protein / kg subject's body weight to about 0.6 g protein / kg subject's body weight, about 0.6 g protein / kg subject's body weight to about 0.8 g protein / kg subject's body weight, or about 0.8 g protein / kg subject's body weight to about 1 g protein / kg subject's body weight. In some embodiments, the compositions described herein can be provided to a subject to achieve a range of about 0.6 g protein / kg subject's body weight to about 0.8 g protein / kg subject's body weight.

[0034] The compositions described herein can be provided to a subject in one or multiple doses per day.In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve doses of the compositions described herein are provided to a subject in one day.In some embodiments, three doses of the compositions described herein are provided to a subject in one day.In some embodiments, six doses of the compositions described herein are provided to a subject in one day.In some embodiments, nine doses of the compositions described herein are provided to a subject in one day.

[0035] Method of administration The composition of the present disclosure can be administered to a subject, and the administration can be accompanied by a food-based diet that is low in or substantially devoid of at least one amino acid. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of one amino acid. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of serine. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of glycine. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of two amino acids or salts thereof. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of serine and glycine. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of three amino acids or salts thereof. In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of serine, glycine, and proline.In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of serine, glycine, and cysteine.In some embodiments, the administration of the composition of the present disclosure is accompanied by a food-based diet that is low in or substantially devoid of four amino acids or salts thereof.The salt of the amino acid disclosed herein can be a pharmaceutically acceptable salt.

[0036] The compositions of the present disclosure can be administered to a subject on a diet. In some embodiments, the compositions of the present disclosure are administered to a subject and the subject is on a low protein diet. In some embodiments, the compositions of the present disclosure are administered to a subject and the subject is on a low carbohydrate diet. In some embodiments, the compositions of the present disclosure are administered to a subject and the subject is on a high fat and low carbohydrate (e.g., ketogenic diet). In some embodiments, the compositions of the present disclosure are administered to a subject and the subject is on a vegetarian diet. In some embodiments, the compositions of the present disclosure are administered to a subject and the subject is on a vegan diet.

[0037] In some embodiments, the composition of the present disclosure is administered to a subject who is on a low-protein diet designed to be low in at least one non-essential amino acid.In some embodiments, the composition of the present disclosure is administered to a subject who is on a low-protein diet designed to be low in serine and glycine.In some embodiments, the composition of the present disclosure is administered to a subject who is on a low-protein diet of less than about 2 g / day, about 1.75 g / day, about 1.5 g / day, about 1.25 g / day, about 1 g / day, about 0.75 g / day, or about 0.5 g / day.In some embodiments, the composition of the present disclosure is administered to a subject who is on a low-protein diet of less than about 500 mg / day, about 450 mg / day, about 400 mg / day, about 350 mg / day, about 300 mg / day, about 250 mg / day, about 200 mg / day, about 150 mg / day, about 100 mg / day, or about 50 mg / day.

[0038] Multiple therapeutic agents can be administered in any order or simultaneously. In some embodiments, the composition of the present invention is administered in combination with, before, or after treatment with another therapeutic agent. If simultaneously, multiple therapeutic agents can be provided in a single unified form or in multiple forms, for example, as multiple separate pills. The agents can be packaged together or separately in a single package or multiple packages. One or all of the therapeutic agents can be given in multiple doses. If not simultaneously, the timing between multiple doses can vary up to about one month.

[0039] The therapeutic agent described herein can be administered before, during, or after the occurrence of disease or condition, and the timing of administering the composition containing the therapeutic agent can vary.For example, the composition can be used as a prophylactic agent and can be administered continuously to the subject with a condition or tendency to disease to reduce the possibility of the occurrence of disease or condition.The composition can be administered to the subject during the onset of symptoms or as soon as possible after onset.

[0040] The compositions disclosed herein can be administered as soon as practical after the onset of disease or condition is detected or suspected, and for the length of time required for disease treatment.In some embodiments, the length of time required for disease treatment is about 12 hours, about 24 hours, about 36 hours, or about 48 hours.In some embodiments, the length of time required for disease treatment is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days. In some embodiments, the length of time required to treat the disease is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, or about 20 weeks. In some embodiments, the length of time required to treat the disease is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months.

[0041] In some embodiments, the length of time that the compound can be administered is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months , about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. The length of treatment can vary for each subject.

[0042] The compositions described herein can be in unit dosage form suitable for single administration of accurate dosage.In unit dosage form, the formulation is divided into unit doses containing appropriate amount of one or more compounds.The unit dose can be in the form of a package containing a separate amount of formulation.The aqueous suspension composition can be packaged in a single-dose non-reclosable container.Multiple-dose reclosable containers can be used, for example, with or without preservatives.

[0043] In some embodiments, the composition is administered to the subject throughout the day. In some embodiments, the composition is administered to the subject with a meal. In some embodiments, the composition is administered to the subject with a snack. In some embodiments, the composition is administered to the subject without a meal. In some embodiments, the composition is administered to the subject at equal intervals throughout the day. In some embodiments, the first portion is administered before breakfast, the second portion is administered with breakfast, the third portion is administered with lunch, the fourth and fifth portions are administered with dinner, and the sixth portion is administered before bedtime.

[0044] The compositions provided herein can be administered in combination with other therapies, such as chemotherapy, radiation, surgery, anti-inflammatory agents, immunotherapy, biological agents, and selected vitamins. The other agents can be administered before, after, or simultaneously with the pharmaceutical composition.

[0045] Methods of using the compositions disclosed herein The present disclosure provides a method for treating a subject.The compositions disclosed herein can be used in the treatment of any disease.In some embodiments, the compositions disclosed herein are used to treat cancer in a subject in need thereof.Modifying the diet and nutrients of a subject can obtain desirable health benefits and can be effective in the treatment of disease.

[0046] Based on the specific disease and / or need of patient, the present disclosure provides a method for generalized treatment recommendation for subject and a method for subject-specific treatment recommendation.The method for treatment can include one of the following steps: determining the level of nutrients in subject; detecting the presence or absence of disease in subject based on the determination; and recommending at least one generalized treatment or subject-specific treatment to the subject to improve the symptoms of disease.

[0047] In some embodiments, the compositions disclosed herein can be used to manage a disease or condition through dietary intervention. In some embodiments, the compositions disclosed herein can be used as part of a treatment plan for a particular disease or condition.

[0048] In some embodiments, the subject has cancer. Cancer is caused by the uncontrollable proliferation of neoplastic cells, leading to the invasion of adjacent and distant tissues, and ultimately resulting in death. Cancer cells often have underlying genetic or epigenetic abnormalities that affect both the coding and regulatory regions of the genome. Genetic abnormalities in cancer cells can change the structure, dynamic level, and expression level of proteins, which in turn alter the cellular metabolism of cancer cells. Changes in cell cycle can cause cancer cells to proliferate at a much faster rate than normal cells. Along with increased metabolic rate and proliferation, cancer tissue has a much higher nutrient requirement compared to normal tissue.

[0049] Cancer cells have nutrient trophism and have much higher nutrient requirements compared to normal cells. As an adaptation to meet the increased nutrient requirements, cancer cells can upregulate glucose and amino acid transporters on the cell membrane to obtain more nutrients from the circulation. Cancer cells can also rewire metabolic pathways to sustain a higher rate of ATP production or energy supply by enhancing glycolysis and glutaminolysis. Glucose and amino acids are highly required nutrients in cancer cells. Some cancer cell types and tumor tissues are known to be nutrient trophic for certain amino acids. Cancer nutrient trophism for various amino acids can make the cancer type vulnerable to amino acid starvation treatment.

[0050] When mammalian cells experience amino acid starvation, the cells mount a homeostatic response to the amino acid shortage. Amino acid starvation can trigger the common amino acid regulatory pathway, which involves shifting cellular resources and energy to the expression of membrane transporters, growth hormones, and metabolic enzymes for amino acid homeostasis. Upregulation of membrane transporters can promote amino acid uptake, and upregulation of metabolic enzymes can enhance amino acid synthesis. Cells can also recycle proteins and organelles by autophagy to regenerate non-essential amino acids. Through the common amino acid regulatory pathway and autophagy, cells attempt to maintain amino acid homeostasis. Tumor tissues can also overcome amino acid starvation by enhancing angiogenesis to obtain more nutrient supply.

[0051] When homeostasis cannot be achieved during severe amino acid starvation, cancer cells can inhibit protein synthesis, suppress proliferation, or undergo programmed cell death. The cell death mechanism of amino acid starvation can be caspase-dependent apoptosis, autophagic cell death, or ferroptotic cell death. Amino acid transporters, metabolic enzymes, autophagy-related proteins, and amino acid starvation can be used to control cancer proliferation.

[0052] The method disclosed herein can monitor nutrient consumption by a subject.Nutrient consumption can be measured by collecting biological samples from a subject.Biological samples can be, for example, whole blood, serum, plasma, mucous membrane, saliva, buccal swab, urine, stool, cells, tissues, bodily fluids, sweat, breath, lymphatic fluid, CNS fluid, and lesion exudate.A combination of biological samples can be used in the method disclosed herein.

[0053] The disclosed composition methods can slow the growth of cancer cell lines or kill cancer cells. Non-limiting examples of cancers that can be treated by the compounds of the present invention include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoma of unknown primary, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer cancer), gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gliomas, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer including non-small cell lung cancer and small cell lung cancer, lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer cancer), oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, skin cancer Merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancercancer), T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic neoplasm (gestational), carcinoma of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0054] kit The composition of the present invention can be packaged as a kit. In some embodiments, the kit includes written instructions for administration / use of the composition. The written material can be, for example, a label. The written material can suggest the conditions and methods of administration. The instructions provide the subject and the supervising physician with the best guidance to achieve optimal clinical outcomes from the administration of the treatment. The written material can be a label. In some embodiments, the label can be approved by a regulatory agency, for example, the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or other regulatory agencies.

[0055] Radiation therapy Radiation therapy, or radiotherapy, is a treatment that uses ionizing radiation as part of cancer treatment to control or kill malignant cells, and is usually delivered by linear accelerator.Ionizing radiation damages the DNA of cancerous tissue, resulting in cell death.Radiation therapy can be curative in many types of cancer when localized in one area of ​​the body.In some embodiments, the method of the present disclosure can be performed in combination with a second treatment, such as radiation therapy, and the composition of the present disclosure can be administered in combination with the second treatment, such as radiation therapy.In some embodiments, radiation therapy can be used with the method or composition of the present disclosure, since it can control cell proliferation.

[0056] In some embodiments, radiation therapy can be used in combination with the disclosed method or composition to prevent or reduce the possibility of tumor recurrence after surgery to remove the primary malignant tumor. In some embodiments, radiation therapy and chemotherapy can be used in combination with the disclosed method or composition. In some embodiments, the disclosed method can be performed in combination with radiation therapy to treat cancer, and the disclosed composition can be administered in combination with radiation therapy to treat cancer. In some embodiments, the disclosed method can be performed in combination with radiation therapy to reduce cancer symptoms, and the disclosed composition can be administered in combination with radiation therapy to reduce cancer symptoms. In some embodiments, the disclosed method can be performed in combination with radiation therapy to slow cancer growth, and the disclosed composition can be administered in combination with radiation therapy to slow cancer growth.

[0057] In some embodiments, the radiation therapy is external beam radiation therapy. External beam radiation therapy uses a machine that delivers radiation locally to the cancer. In some embodiments, the radiation therapy is internal beam radiation therapy. In some embodiments, external beam radiation can be used to shrink tumors and treat pain, difficulty breathing, or loss of bowel or bladder control. In some embodiments, the external beam radiation therapy is three-dimensional conformal radiation therapy (3D-CRT). In some embodiments, the external beam radiation therapy is intensity-modulated radiation therapy (IMRT). In some embodiments, the external beam radiation therapy is proton therapy. In some embodiments, the external beam radiation therapy is image-guided radiation therapy (IGRT). In some embodiments, the external beam radiation therapy is stereotactic radiation therapy (SRT).

[0058] Internal radiation therapy is a procedure in which a radioactive source is placed in the subject's body. In some embodiments, the radioactive source is a liquid. In some embodiments, the radioactive source is a solid. In some embodiments, internal radiation therapy uses a permanent implant. In some embodiments, internal radiation therapy is a temporary internal radiation therapy, such as a needle, tube, or applicator. In some embodiments, a solid radiation source is used in brachytherapy. In some embodiments, a seed, ribbon, or capsule containing a radioactive source is placed in the subject's body. In some embodiments, the radiation therapy is brachytherapy, and a radioactive source is placed in or next to the area requiring treatment. In some embodiments, the radiation therapy is total body irradiation (TBI) in preparation for bone marrow transplantation.

[0059] In some embodiments, the radiation therapy is intraoperative radiation therapy (IORT). In some embodiments, the radiation therapy is total body radiation therapy. In some embodiments, the radiation therapy is radioimmunotherapy. In some embodiments, the radiation therapy uses a radiosensitizer or a radioprotector.

[0060] In some embodiments, brachytherapy is used to treat head, neck, breast, cervix, prostate, or eye cancer.In some embodiments, whole body radiation therapy, such as radioactive iodine, I-131, can be used to treat thyroid cancer.In some embodiments, targeted radionuclide therapy can be used to treat advanced prostate cancer or gastroenteropancreatic neuroendocrine tumors (GEP-NETs).

[0061] In some embodiments, the shaped radiation beam can be delivered from several angles to intersect the tumor while sparing normal tissue, hi some embodiments, the tumor absorbs a much larger amount of radiation than the surrounding healthy tissue.

[0062] In some embodiments, a subject or tumor can be treated with about 0.5 Gray (Gy), about 1 Gy, about 1.5 Gy, about 2 Gy, about 2.5 Gy, about 3 Gy, about 3.5 Gy, about 4 Gy, about 4.5 Gy, about 5 Gy, about 5.5 Gy, about 6 Gy, about 6.5 Gy, about 7 Gy, about 7.5 Gy, about 8 Gy, about 8.5 Gy, about 9 Gy, about 9.5 Gy, or about 10 Gy. In some embodiments, the subject or tumor can be treated with about 5 Gy, about 10 Gy, about 15 Gy, about 20 Gy, about 25 Gy, about 30 Gy, about 35 Gy, about 40 Gy, about 45 Gy, about 50 Gy, about 55 Gy, about 60 Gy, about 65 Gy, about 70 Gy, about 75 Gy, about 80 Gy, about 85 Gy, about 90 Gy, about 95 Gy, or about 100 Gy of radiation therapy. In some embodiments, the subject or tumor can be treated with about 5 Gy of radiation therapy. In some embodiments, the subject or tumor can be treated with about 10 Gy of radiation therapy. In some embodiments, the subject or tumor can be treated with about 20 Gy of radiation therapy.

[0063] In some embodiments, the subject or tumor can be treated with about 5 Gy to about 10 Gy; about 10 Gy to about 15 Gy; about 15 Gy to about 20 Gy; about 20 Gy to about 25 Gy; about 25 Gy to about 30 Gy; about 30 Gy to about 35 Gy; about 35 Gy to about 40 Gy; about 40 Gy to about 45 Gy; about 45 Gy to about 50 Gy; about 50 Gy to about 55 Gy; about 55 Gy to about 60 Gy; about 60 Gy to about 65 Gy; about 65 Gy to about 70 Gy; about 70 Gy to about 75 Gy; or about 75 Gy to about 80 Gy. In some embodiments, the subject or tumor can be treated with about 5 Gy to about 10 Gy. In some embodiments, the subject or tumor can be treated with about 20 Gy to about 40 Gy. In some embodiments, the subject or tumor can be treated with about 40 Gy to about 60 Gy.

[0064] In some embodiments, one cycle of radiation therapy can include subject or tumor being treated with radiation for several days. In some embodiments, radiation can be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, one cycle of radiation therapy can include subject or tumor being treated with radiation for 4 days. In some embodiments, one cycle of radiation therapy can include subject or tumor being treated with radiation for 5 days.

[0065] In some embodiments, one cycle of radiation can include administering 10 Gy over 5 days, e.g., 2 Gy per day for 5 days. In some embodiments, one cycle of radiation can include administering 15 Gy over 5 days, e.g., 3 Gy per day for 5 days. In some embodiments, one cycle of radiation can include administering 20 Gy over 5 days, e.g., 4 Gy per day for 5 days. In some embodiments, one cycle of radiation can include administering 25 Gy over 5 days, e.g., 5 Gy per day for 5 days.

[0066] In some embodiments, one cycle of radiation therapy can be repeated over a period of time.In some embodiments, one cycle of radiation therapy can be repeated every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks.

[0067] In some embodiments, the composition of the present disclosure can be administered simultaneously with the administration of radiation therapy.In some embodiments, the composition of the present disclosure can be administered simultaneously with radiation therapy for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.In some embodiments, the composition of the present disclosure can be administered simultaneously with the administration of radiation therapy for 5 days.In some embodiments, the composition of the present disclosure can be administered simultaneously with the administration of radiation therapy for 7 days.

[0068] In some embodiments, the composition of the present disclosure is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before the subject is treated with radiation therapy. In some embodiments, the composition of the present disclosure is administered 1 day before the subject is treated with radiation therapy. In some embodiments, the composition of the present disclosure is administered 2 days before the subject is treated with radiation therapy. In some embodiments, the composition of the present disclosure is administered 3 days before the subject is treated with radiation therapy. In some embodiments, the composition of the present disclosure is administered 4 days before the subject is treated with radiation therapy.

[0069] In some embodiments, a subject can be treated with the composition of the present disclosure and radiation therapy, and then discontinue treatment before starting a subsequent treatment cycle with the composition and radiation therapy. In some embodiments, the length of the treatment period and the off-treatment period are the same. In some embodiments, the length of the treatment period and the off-treatment period are different. In some embodiments, the length of the treatment period is longer than the off-treatment period. In some embodiments, the length of the treatment period is shorter than the off-treatment period.

[0070] In some embodiments, the length of the treatment period with the composition and radiation therapy is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, and the length of the treatment off period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the length of the treatment period is 5 days, and the length of the treatment off period is 2 days. In some embodiments, the length of the treatment period is 4 days, and the length of the treatment off period is 3 days. In some embodiments, the length of the treatment period is 3 days, and the length of the treatment off period is 4 days. In some embodiments, the length of the treatment period is 2 days, and the length of the treatment off period is 5 days.

[0071] In some embodiments, the cycle of treatment periods and treatment discontinuation periods is repeated every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks.

[0072] In some embodiments, the compositions and radiation therapy of the present disclosure are administered with a high fat diet. In some embodiments, the high fat diet is a diet that has more than about 50%, about 60%, about 70%, about 80%, or about 90% of daily calories from fat. In some embodiments, the compositions and radiation therapy of the present disclosure are administered with a low carbohydrate diet. In some embodiments, the low carbohydrate diet is a diet that has less than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of daily calories from carbohydrate. In some embodiments, the compositions and radiation therapy of the present disclosure are administered with a low protein diet. In some embodiments, the low protein diet is a diet that has less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of daily calories from total protein. In some embodiments, the low protein diet has a total protein content of less than about 50 g / day, about 40 g / day, about 30 g / day, about 20 g / day, or about 10 g / day. In some embodiments, the compositions of the present disclosure and radiation therapy are administered with a high fat, low carbohydrate, and low protein diet. In some embodiments, the compositions of the present disclosure are administered with a normal diet.

[0073] Combination therapy with immunotherapy In some embodiments, the amino acid starvation therapy of the present disclosure can be used in combination with a chemotherapy regimen. In some embodiments, the chemotherapy regimen is an immunotherapy. In some embodiments, the immunotherapy is an antibody therapy. In some embodiments, the antibody therapy is treatment with alemtuzumab, rituximab, ibritumomab tiuxetan, or ofatumumab. In some embodiments, the immunotherapy is an interferon. In some embodiments, the interferon is interferon alpha. In some embodiments, the immunotherapy is an interleukin, such as IL-2. In some embodiments, the immunotherapy is an interleukin inhibitor, such as an IRAK4 inhibitor.

[0074] In some embodiments, the immunotherapy is a cancer vaccine. In some embodiments, the cancer vaccine is a preventive vaccine. In some embodiments, the cancer vaccine is a treatment vaccine. In some embodiments, the cancer vaccine is an HPV vaccine, such as Gardisil™, Cervarix, Oncophage, or Sipuleucel-T. In some embodiments, the immunotherapy is gp100. In some embodiments, the immunotherapy is a dendritic cell-based vaccine, such as Ad.p53 DC. In some embodiments, the immunotherapy is a toll-like receptor modulator, such as TLR-7 or TLR-9. In some embodiments, the immunotherapy is a PD-1, PD-L1, PD-L2, or CTL4-A modulator, such as nivolumab. In some embodiments, the immunotherapy is an IDO inhibitor, such as indoximod. In some embodiments, the immunotherapy is an anti-PD-1 monoclonal antibody, such as MK3475 or nivolumab. In some embodiments, the immunotherapy is an anti-PD-L1 monoclonal antibody, such as MEDI-4736 or RG-7446. In some embodiments, the immunotherapy is an anti-PD-L2 monoclonal antibody. In some embodiments, the immunotherapy is an anti-CTL1-4 antibody, such as ipilumumab.

[0075] Cancer cells can change cellular metabolism to support the increased energetic and anabolic demands of cancer cell growth. Examples of altered metabolism include aerobic glycolysis (i.e., the Warburg effect) and a high reliance on non-essential amino acids. One-carbon metabolism encompasses a collection of metabolic pathways that allow cells to generate and use molecules that contain a single carbon. One-carbon units (i.e., methyl groups) are delivered for use and activated by tetrahydrofolate (THF) derived from dietary folic acid. Cells require one-carbon units to support nucleotide synthesis, methylation reactions, and reductive metabolism. Cancer cells rely on one-carbon pathways to support high growth rates, and one-carbon metabolism is critical to cancer cell growth.

[0076] THF-dependent one-carbon metabolism is an essential metabolic process that supports cell growth, providing carbon for the synthesis of nucleotides that are incorporated into DNA and RNA. Tryptophan is a theoretical source of one-carbon units through metabolism by indoleamine 2,3-dioxygenase 1 (IDO1). In cancer cells expressing IDO1, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis both in vitro and in vivo.

[0077] In cancer cell metabolism, serine is considered the major source of one-carbon units. Serine can be obtained either by de novo synthesis via the serine synthetic pathway (SSP) from the glycolytic intermediate 3-phosphoglycerate or by uptake from the extracellular environment. Some cancer cells show increased expression of SSP enzymes to meet the cellular serine demand, whereas others are primarily dependent on serine uptake. Serine hydromethyltransferases (SHMT1 and SHMT2) directly catalyze the conversion of serine to glycine and the release of one carbon that enters the THF cycle.

[0078] The amino acids glycine, histidine, and tryptophan are also potential one-carbon donors. Glycine can provide one-carbon units through the glycine cleavage system (GCS). Catabolism of histidine can also yield one-carbon units, making cancer cells more sensitive to antifolate treatment due to a reduction in the free THF pool.

[0079] As an essential amino acid, tryptophan is essential for protein synthesis, but it is also a precursor for the production of 5-hydroxytryptamine and kynurenine. In the kynurenine pathway, the first and rate-limiting step is the conversion of tryptophan to formyl-kynurenine. Three enzymes, IDO1, IDO2, and TDO, can catalyze this reaction. Both IDO2 and TDO have low expression levels and limited tissue specificity, and IDO1 is considered the predominant form. Formyl-kynurenine spontaneously forms kynurenine with the release of one molecule of formic acid. Formic acid can enter the one-carbon cycle by reacting directly with THF, and it is through this pathway that tryptophan can serve as a one-carbon donor.

[0080] IDO1 activity depletes tryptophan and increases kynurenine in the tumor microenvironment, causing various effects on immune cells. Tryptophan depletion reduces the activity of tumor-infiltrating T cells, and kynurenine, through aryl hydrocarbon receptor binding, reduces the proliferation of effector T cells and supports the differentiation of immunosuppressive T regulatory cells. The effect of the tumor microenvironment provides an immunologically permissive environment for tumor growth. The kynurenine pathway has several metabolic outputs, including reactive oxygen species (superoxide) levels, one-carbon metabolism, synthesis of NAD(P)+, synthesis of alanine, and carbon entry into the TCA cycle (via α-ketoadipic acid).

[0081] Disclosed herein is a method for treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; and b) an IDO1 inhibitor. In some embodiments, the at least two amino acids are serine and glycine.

[0082] In some embodiments, the IDO1 inhibitor is indoximod (D-1MT; NLG-8189), 4-phenylimidazole (4-PI), N3-benzyl-substituted 4-PI, ortho-hydroxy 4-PI, naboximide, or epacadostat. In some embodiments, the IDO1 inhibitor is epacadostat.

[0083] In some embodiments, the compositions and IDO1 inhibitors of the present disclosure can be used to treat cancer.In some embodiments, the cancer is pancreatic cancer.In some embodiments, the cancer is colon cancer.In some embodiments, the cancer is breast cancer.In some embodiments, the cancer is cervical cancer.In some embodiments, the cancer is lung cancer.

[0084] In some embodiments, the IDO1 inhibitor is administered 1, 2, 3, 4, or 5 times a day in combination with amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered once a day in combination with amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered twice a day in combination with amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered three times a day in combination with amino acid starvation therapy.

[0085] In some embodiments, the IDO1 inhibitor is administered in an amount of about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, or about 450 mg to about 500 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 50 mg to about 100 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 100 mg to about 150 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 250 mg to about 300 mg.

[0086] In some embodiments, the IDO1 inhibitor is administered in an amount of about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about 500 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 25 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 50 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 100 mg. In some embodiments, the IDO1 inhibitor is administered in an amount of about 300 mg.

[0087] In some embodiments, about 25 mg of epacadostat is administered to a subject in combination with serine and glycine starvation therapy. In some embodiments, about 50 mg of epacadostat is administered to a subject in combination with serine and glycine starvation therapy. In some embodiments, about 100 mg of epacadostat is administered to a subject in combination with serine and glycine starvation therapy. In some embodiments, about 300 mg of epacadostat is administered to a subject in combination with serine and glycine starvation therapy. EXAMPLES

[0088] Example 1: PHGDH inhibitors with serine and glycine depletion can block the proliferation of tumor cell lines Cells can either uptake exogenous serine or synthesize serine from the glycolytic intermediate 3-phosphoglycerate (3-PG) using the serine synthetic pathway (Figure 1). As a non-essential amino acid, serine can be taken up from the environment or synthesized de novo through the serine synthetic pathway (SSP). The SSP consists of a three-step enzymatic reaction that begins with the NAD+-dependent oxidation of the glycolytic intermediate 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP). This first reaction is catalyzed by phosphoglycerate dehydrogenase (PHGDH), an enzyme that can be targeted by the pharmacological compound PH755. 3-PHP, which is generated during the PHGDH reaction, is then converted to 3-phosphoserine (3-PS) by phosphoserine aminotransferase 1 (PSAT1) in a glutamate-dependent transamination reaction. Finally, phosphoserine phosphatase (PSPH) catalyzes the hydrolysis of 3-PS to generate serine. Serine is involved in many metabolic pathways, including nucleotide synthesis or glutathione, and is the major antioxidant in cells. Therefore, serine availability can be targeted by depleting it from the extracellular environment or by inhibiting SSP with PH755.

[0089] To assess the relative contribution of each of these pathways to the proliferation of cells in culture, we measured the proliferation of a series of colorectal cancer cell lines grown in complete medium (CM), in medium lacking serine and glycine (-SG), in CM with PH755 (a PHGDH inhibitor) or a combination of -SG+PH755. Responses to serine and glycine starvation differed among the cell lines, ranging from RKO, HT-29, and SW48 cells, which showed significant dependence on exogenous serine and glycine for proliferation, to DLD-1, LoVo, CACO-2, and MDA-MB-468 cells (breast cancer lines previously shown to harbor PHGDH amplification), which were unaffected by the lack of serine and glycine in the medium (Figures 2 and 3). Compared with cell lines carrying BRAF mutations (RKO, HT-29, SW1417, CL-34), colorectal cancer cell lines carrying KRAS mutations (HCT-15, HCT116, DLD-1, LoVo, SW480) tended to be more resistant to serine and glycine deprivation, but SW620 (KRAS mutant) and VACO5 (BRAF mutant) were exceptions to this trend (Figures 2 and 3). Genetic mutations, such as PHGDH amplification in MDA-MB-468 cells, also contribute to the dependency of cancer cells on exogenous serine supply. Treatment of cells with PH755 in complete medium had no clear effect on the proliferation rate of cells, indicating that at this concentration, the inhibitor has no nonspecific inhibitory effect on cell proliferation. However, the combination of -SG medium with PH755 completely inhibited the proliferation of all cell lines tested (Figures 2 and 3).

[0090] Example 2: PHGDH inhibition combined with serine and glycine depletion limits DNA synthesis, survival and organoid growth This lack of proliferation observed in Example 1 was accompanied by a significant reduction in BrdU incorporation into newly synthesized DNA after 48 hours of incubation with -SG medium + PH755 compared to either treatment alone (Figures 4 and 5). The reduction in cells in S phase in the dual treatment condition was accompanied by an accumulation of cells in G2 / M phase (Figures 5 and 6) and an increase in the percentage of SubG1 cells, indicating increased cell death (Figure 7). The appearance of cleaved caspase 3 confirmed the induction of apoptosis in cells cultured in -SG medium and treated with PH755 (Figure 8). Using uniformly labeled glucose, cells grown in the presence of exogenous serine diverted little glucose into serine and glycine synthesis, as reflected by negligible accumulation of m+3 serine and m+2 glycine, with or without PH755 (Figures 9 and 10). Notably, these cells maintained much higher total intracellular serine and glycine levels than cells grown in -SG medium (Figure 6). Upon starvation of serine and glycine, all cell lines showed a clear increase in de novo serine synthesis, as indicated by the accumulation of m+3-labeled serine and m+2-labeled glycine (Figures 9 and 10). This response was weaker in HT-29 cells, consistent with their reduced ability to grow in the absence of exogenous serine. On the other hand, treatment of cells with PH755 completely blocked de novo serine and glycine synthesis both in complete medium and under serine and glycine starvation (Figures 9 and 10), demonstrating the efficiency of this inhibitor in blocking PHGDH activity and SSP.

[0091] To further validate the specificity of PH755, the effect of genetic deletion of PHGDH was tested. DLD-1 cells showed a strong induction of PHGDH expression in response to serine and glycine starvation, which was much less intense in HT-29 cells, consistent with the relative proliferation capacity of these cells in the absence of exogenous serine and glycine (Figures 11 and 12). The proliferation of these cells after CRISPR-mediated deletion of PHGDH (Figures 11 and 12) closely resembled that observed after PH755 treatment (Figure 2), supporting the function of PH755 as an inhibitor of PHGDH.

[0092] Cells grown in 2D on plastic may exhibit different metabolic demands compared to cells grown under more physiologically relevant conditions, and Vil1-cre ER ;Apc fl / fl (Apc) or Vil1-cre ER ;Apc fl / fl Kras G12D / + The effects of serine and glycine starvation and PH755 treatment on intestinal tumor organoids derived from (ApcKras) mice (Figures 13 and 14) were examined. Organoids derived from Apc mutant tumors showed some sensitivity to serine and glycine starvation, which was not evident in Apc / Kras mutant organoids. Consistent with the observations in 2D cell lines, treatment with PH755 alone did not affect the growth of Apc or Apc / Kras organoids. However, the combination of serine and glycine starvation with PH755 treatment efficiently inhibited the growth of both Apc and Apc / Kras organoids (Figures 13 and 14). Of note, this effect was not limited to cancer-derived intestinal organoids, as a substantial reduction in growth was also observed in normal small intestinal organoids treated with the combined treatment (Figure 15). To validate the effect of the dual treatment in human cells, colorectal cancer organoids from four patients with different KRAS status (C-001: WT, C-004: deletion, R-006: Gly12Asp, and R-008: Gly13Asp) were tested. Although -SG or PH755 treatment alone had no significant effect on proliferation, in both cases the combination of drug and inhibitor significantly reduced organoid growth, regardless of KRAS status (Figure 16).

[0093] Example 3: PHGDH inhibition in combination with serine and glycine depletion inhibits purine and GSH synthesis Serine participates in numerous metabolic pathways, including supplying one-carbon units in purine synthesis and glycine, and maintaining redox homeostasis through glutathione production. The contribution of de novo synthesized serine to these pathways can be assessed by tracking the fate of uniformly carbon-labeled glucose (Figure 17). Cells grown in serine and glycine showed little evidence of the use of de novo synthesized serine in ATP or GTP synthesis (Figure 18); rather, the majority of the label derived from ribose (m+5) was synthesized through the pentose phosphate pathway (Figure 17). Under serine and glycine starvation, the cells best able to adapt to these conditions (HCT116, DLD1, and MDA-MB-468) accumulated m+6 to m+9 labeled purines, consistent with the incorporation of labeled serine that arose through the SSP (Figure 18). Serine and glycine starvation by PH755 treatment efficiently inhibited the synthesis of ATP and GTP (Figure 18). Glutathione can be labeled from glucose-derived glycine (m+2) or glutamate (m+2) (Figure 17), but under these conditions, the generation of m+2 glutamate was not affected by PH755 treatment in most of the cell lines tested (Figure 19). The increased proportion of m+2 and m+4 labeled glutathione detected in response to removal of exogenous serine and glycine may reflect increased SSP activity and generation of labeled glycine, a reaction that was blocked by treatment with PH755 (Figure 20). Importantly, the inability of dual-treated cells to synthesize purines and glutathione de novo was evident as early as 3 or 6 hours after treatment, demonstrating that this response may be a primary effect of the combination treatment (Figure 21). Notably, total purine and GSH levels were not decreased in dual-treated cells compared to cells grown in -SG medium, likely reflecting a lack of consumption of these metabolites when growth was inhibited (Figure 22). These results demonstrate that metabolic pathways that are serine-dependent and critical for cancer cell growth are efficiently inhibited by the combination of serine and glycine starvation and PHGDH inhibitors.

[0094] Example 4: Metabolic rescue of co-treated, -SG / PHGDHi treated cells Cells starved of serine and glycine and treated with PH755 all showed strong growth inhibition (Figure 2, Figure 13, Figure 3, Figure 17, Figure 18, and Figure 20). Although supplementation of the dual-treated cells with either formate (to replenish the one-carbon cycle) or glycine alone did not restore growth, addition of formate and glycine efficiently rescued growth (Figure 23). This growth rescue was accompanied by restoration of ATP and GTP synthesis (Figure 24), and partial restoration of the pool of unlabeled serine (Figure 25). Using labeled glycine, it was shown that this pool of serine results from the one-carbon unit supplied by glycine and formate, a response that becomes more evident after the addition of unlabeled serine, stimulating the accumulation of labeled serine (Figure 26). These results indicate that the inhibition of growth is a direct effect of inhibition of de novo serine synthesis by PH755 and not a response to any off-target toxicity. The specificity of the metabolic defect induced by PH755 was further supported by the similarity of the response to gene deletion of PHGDH (Figures 27, 28, and 29).

[0095] Example 5: PHGDHi / -SG treatment impairs general ATF-4 response Although the action of PH755 was consistent with specific inhibition of PHGDH, analysis of the expression of serine synthesis pathway enzymes in response to PH755 treatment revealed an unexpected response in some of the cell lines. Serine and glycine starvation can lead to activation of ATF-4, which can mediate a general survival response to metabolic stress. Importantly, serine starvation leads to ATF-4-dependent induction of SSP enzymes, thus contributing to the ability of cells to adapt to reduced exogenous serine levels.

[0096] Depletion of ATF-4 caused the cells to lose the ability to adapt and grow under serine and glycine starvation (Figure 30). As expected, serine and glycine starvation led to induction of the expression of all three SSP enzymes in all cell lines tested, although this was less intense in MDA-MB-468 cells, which constitutively overexpress these enzymes (Figure 31). However, in four colon cancer lines (HT-29, HCT116, CACO2, and DLD-1), further treatment of serine and glycine starved cells with PH755 gradually attenuated this increase in SSP enzyme expression (Figure 31), whereas this was not observed in SW48 cells. Similar responses after serine and glycine starvation and PHGDH deletion confirmed that this was a response to the reduction in PHGDH activity (Figure 32). The reduction in activation of SSP enzymes correlated with the growth inhibition seen after serine and glycine starvation and PH755 treatment, with the failure of dual-treated cells to induce SSP enzymes becoming evident within 4-8 hours of serine and glycine starvation (Figure 33), suggesting that this is a direct response to PHGDH inhibition rather than an indirect response to growth arrest. The loss of ability to induce SSP enzyme expression in response to serine and glycine starvation was accompanied by a general loss of ability to activate the ATF-4 response, as measured by the lack of induction of the canonical ATF-4 target, ASNS (Figures 31 and 32). These results suggest that cells can respond to the combination of serine starvation and SSP inhibition, but not to either intervention treatment alone.

[0097] Example 6: PHGDHi / -SG treatment inhibits global protein synthesis To investigate how PHGDH activity affects the ATF-4 response induced after serine and glycine deprivation, we examined the level of activation of upstream regulators responsible for ATF-4 induction. In response to amino acid starvation, accumulation of uncharged tRNA leads to activation and autophosphorylation of the kinase General Control Nonderepressible 2 (GCN2). GCN2 then phosphorylates eukaryotic translation initiation factor 2α (eIF2α) at ​​serine 51, resulting in a general downregulation of global translation but selectively inducing the translation of ATF-434. Serine and glycine deprivation induced phosphorylation of GCN2 and its target eIF2α in HCT116 and DLD-1 cells (Figure 34). Interestingly, this induction of GCN2 and eIF2α phosphorylation was maintained or even more pronounced in cells co-treated with PH755 (Figure 34), demonstrating that the lack of ATF-4 upregulation in dual-treated cells was not due to lack of activation of its upstream regulators. ATF-4 protein levels can also be controlled by ubiquitin-dependent proteasomal degradation. However, although treatment of cells with the proteasome inhibitor MG-132 led to a strong accumulation of ATF-4 in cells grown in complete medium, it did not restore ATF-4 protein levels or expression of the target gene products ASNS and PSAT in cells grown in -SG medium + PH755 (Figure 35).

[0098] Furthermore, gene expression analysis showed that ATF4 was induced at the transcriptional level up to 24 hours after serine and glycine starvation, despite the presence of PHGDH inhibitors (Figure 36). These data indicate that the lack of ATF-4 induction in dual-treated cells was not due to increased protein degradation or decreased transcription. As expected, transcription of ATF-4 target genes, ASNS and SSP enzymes, was strongly upregulated in serine- and glycine-starved cells (Figures 36 and 37). Interestingly, transcription of ATF-4 target genes mirrored the degree of ATF-4 induction in various cell lines grown in -SG medium + PH755. DLD-1 cells, which maintained some induction of ATF-4 under these conditions (Figure 38), retained the ability to induce expression of PHGDH, ASNS, PSAT, and PSPH (Figures 36 and 37), while HT-29 and HCT116 cells, which showed a more blunted induction of ATF-4 (Figure 38), also showed a more profound loss of ability to transcriptionally activate these ATF-4 target genes (Figures 36 and 37).

[0099] PH755 treatment did not primarily affect transcription of ATF-4 or ATF-4 target genes, but did affect the subsequent expression of each of these proteins. To determine whether this reflected a general inhibition of translation resulting from the dramatic reduction in serine and glycine availability observed in these conditions, we analyzed the incorporation of puromycin, a tyrosyl-tRNA mimic, into newly synthesized polypeptides in cells grown in CM or -SG medium + PH755. Interestingly, a modest decrease in the amount of puromycin-labeled peptides was observed in response to serine / glycine depletion, and this decrease was more pronounced in the presence of PHGDH inhibitor (Figure 39). Consistent with the general inhibition of translation in dual-treated cells, the ability of proteasome inhibition to promote the accumulation of short-lived proteins such as c-MYC, HIF1α, and p53 was fully blocked in -SG + PH755-treated cells (Figure 40). Other conditions that induce a general inhibition of protein synthesis (e.g., cycloheximide or puromycin treatment), mTORC1 is hyperactivated in double-treated cells, as indicated by the accumulation of phosphorylated S6K (Figure 41). Thus, the lack of serine and glycine availability caused by inhibition of both the extracellular and intracellular supply of these amino acids (Figure 11) prevents normal translation and prevents the induction of ATF-4-mediated defense responses. In support of this model, the effect of PHGDH inhibition on the ATF-4 response was specific to serine and glycine deficiency, since treatment with PH755 did not prevent the induction of ATF-4 targets in response to ER stress (Figure 42). Furthermore, supplementation of double-treated cells with formate and glycine, a treatment that restored some levels of serine availability (Figure 25), fully rescued the ATF-4 response (Figure 43). Thus, in the absence of extracellular serine, PHGDH activity becomes essential to maintain general protein synthesis, allowing the induction of a protective ATF-4 response.

[0100] Example 7: The combination of a serine / glycine-free diet and a PHGDH inhibitor is well tolerated in vivo In vitro data show that the growth-suppressing response to serine and glycine starvation is significantly enhanced by treatment of cells with PHGDH inhibitors. Therefore, the in vivo efficacy of this approach was tested. To evaluate the tolerability of dietary serine / glycine restriction with PH755 treatment, the response to various treatments in a cohort of tumor-free, immunocompetent C57BL / 6J mice was tested. Mice shifted to the -SG diet showed a slight loss of body weight that stabilized over the study period (Figure 44). Treatment with PH755 alone did not result in any detectable adverse reactions in these mice, and they did not lose weight compared to control mice (Figure 44). However, mice co-treated with PH755 and the -SG diet showed a significant loss of body weight compared to either treatment alone, even though they remained active and appeared healthy (Figure 44). Weight loss was highly responsive to PH755 dose, and dose adjustment (75-50 mg / kg) was successful in limiting weight loss to less than 20% over the study period.

[0101] Serine is important in brain development and function, and PHGDH deficiency in humans can result in neurological abnormalities such as microcephaly, psychomotor retardation, and epileptic seizures. The effects of the -SG diet and PHGDH inhibitor on brain morphology of a cohort of C57BL / 6J mice after 20 days of treatment were evaluated. Microscopic examination of coronal sections from the brains of the four groups of mice at the level of the piriform cortex, caudal diencephalon, caudal midbrain, and rostral cerebellum did not reveal any histopathological lesions in any of the sections examined. In fact, hematoxylin & eosin stained sections show normal histological features with no evidence of degeneration, necrosis, or inflammation (Figure 45 and Figure 46). Furthermore, brain weights remained unchanged in all groups of mice (Figure 45). Other signs of toxicity of the dual treatment in these normal mice were looked for. Measurement of plasma AST and ALT activity at the end point did not show any significant elevation of these markers of liver toxicity in mice from the -SG diet and PH755 treated group (Figure 47), and plasma urea and creatinine levels remained normal in dually treated mice, suggesting that there was no kidney damage (Figure 48). The only obvious adverse effect of dual treatment in mice was weight loss, and in vitro studies with normal mouse organoids derived from the small intestine revealed that the combined treatment altered their growth capacity (Figure 18). Although serine and glycine starvation or PHGDH inhibition alone had no detectable effect on gut morphology, a significant reduction in the length of the intestinal villi was confirmed in animals co-treated with -SG diet and PH755 (Figure 49), consistent with the significant weight loss observed in these mice. However, these mice did not show obvious abnormalities in crypt proliferation, as assessed by Ki-67 staining (Figure 50), suggesting that crypt homeostasis was relatively unperturbed. These results are consistent with the observation that reducing the dose of the PHGDH inhibitor halted further weight loss, suggesting that short-term combination treatment does not cause long-term damage.

[0102] Example 8: Combination of serine / glycine free diet and PHGDH prevents tumor growth in vivo To investigate the antitumor effects of the combination therapy, a xenograft model with two of the colon cancer cell lines that have been tested in vitro, DLD-1 and HCT116, was used. After subcutaneous injection of the cells, when tumors began to appear, mice were transitioned to -SG or control diets and treated with PH755 2-4 days later. As seen in non-tumor-bearing mice, dual-treated DLD1 tumor-bearing mice showed greater weight loss compared to either treatment alone, but careful adjustment of the dose of PH755 used in conjunction with the -SG diet can limit weight loss in these mice to less than 20% over the duration of the experiment (Figure 51).

[0103] This enhanced weight loss was avoided by increasing the time between diet change and PH755 treatment from 2 to 4 days in the HCT116 study (Figures 51 and 52). Analysis of circulating amino acid levels at the study endpoint confirmed previous observations that the -SG diet led to a decrease in plasma serine and glycine levels (Figures 53 and 54). Treatment of mice with PH755 had a more moderate effect on circulating serine and glycine, whereas the combination of the -SG diet with PH755 most efficiently reduced plasma serine and glycine levels, reaching absolute concentrations as low as 58 μM serine (vs. 267.7 μM in control mice and 99.9 μM in mice fed only the -SG diet) and 102.3 μM glycine (vs. 367.6 μM in control mice and 143.6 μM in mice fed only the -SG diet) (Figures 53 and 54). The growth of tumors arising from DLD-1 cells was not affected by dietary intervention treatment or PH755 treatment alone (Figure 55), consistent with the lack of effect of either of these treatments on the proliferation of these cells in vitro (Figure 2). However, the combination of diet and PH755 strongly suppressed the growth of these tumors (Figure 55). The growth of HCT116 xenograft tumors had some sensitivity to dietary serine and glycine restriction and also showed a trend toward reduction in mice treated with PH755 (Figure 56), consistent with previous reports showing the effect of PH755 on HCT116 tumor growth24. Meanwhile, the combined treatment of diet and PH755 almost completely blocked the growth of these tumors (Figure 56).

[0104] Interestingly, the strong growth inhibition observed in the dually treated tumors was accompanied by increased cell death, as reflected by an increased number of active caspase 3 positive cells in DLD-1 tumors treated with the combination therapy (Figure 57). Analysis of serine and glycine levels in tumors from these mice closely resembled the results from plasma, showing that either PH755 treatment or the -SG diet reduced intratumoral serine and glycine levels (Figures 58 and 59), but in both cases the -SG diet was more effective at reducing intratumoral serine and glycine levels than treatment with the PHGDH inhibitor. HCT116 tumors showed a slight further reduction in serine and glycine in mice treated with the diet and drug combination (Figure 59), whereas in DLD1 tumors the reduction in serine in response to the -SG diet was not further affected by additional PH755 treatment (Figure 58). Nevertheless, the further reduction in intratumoral glycine in dual-treated mice suggests that flux through SSP is lower in dual-treated tumors, and the maintenance of low steady-state levels of serine may reflect reduced growth (and serine consumption) under these conditions (Figure 58).

[0105] The in vitro data showed that complete inhibition of serine availability by serine starvation and PHGDH inhibition led to defects in one-carbon metabolism and a general inhibition of translation, which correlated with the inability to induce the ATF-4 response. To examine these responses to dietary serine / glycine starvation and PHGDH inhibition in vivo, purine levels in tumors were examined. As noted in vitro (Figure 25), no differences in total ATP or GTP levels were observed in tumors from dually treated mice (Figure 60), which may reflect reduced proliferation of dually treated tumor cells. In the methionine cycle, regeneration of SAM from SAH requires one-carbon units. Interestingly, a clear reduction in the SAM / SAH ratio was observed in tumors from -SG diet mice, which was further reduced in mice on -SG diet + PH755 (Figure 61). These results are consistent with a lack of one-carbon availability in mice on the -SG diet, which is exacerbated in dually treated mice.

[0106] To examine ATF-4 response, we measured the expression of two ATF-4 targets, PHGDH and PSAT1 (Figure 62 and Figure 63), in DLD-1 tumors. Immunohistochemical analysis of these tumors revealed that feeding mice with -SG diet led to a clear induction of PSAT1, and to a lesser extent PHGDH, in tumors, indicating induction of ATF-4 response in vivo. In contrast, treatment of mice with PHGDH inhibitor alone did not lead to any change in PHGDH or PSAT1 expression, suggesting that dietary restriction of serine and glycine alone was effective in depleting serine and glycine intratumoral levels sufficient to lead to ATF-4 response in vivo. Induction of PSAT-1 and PHGDH was equal or even more pronounced in the dual-treated tumors compared to tumors from mice fed the -SG diet alone, indicating that in vivo, the combined treatment did not reduce available serine enough to impair the ability of tumor cells to induce an ATF-4 response (Figures 62 and 63). Collectively, these data indicate that suppression of tumor growth correlates with defects in serine metabolism rather than inhibition of translation.

[0107] Example 9: Dietary restriction of serine and glycine combined with PHGDH inhibition synergistically reduces tumor burden and improves survival in a genetic model of intestinal cancer Figure 64A: ApcMin / + mice were transitioned to a CTR diet (red line) or -SG diet (black line) at day 80, followed by treatment with 100 mg / kg PH755 daily for 9 days at day 84. After treatment was stopped, mice were maintained on either the CTR or -SG diet until clinical endpoints were reached. These data are shown in comparison to data showing survival of ApcMin / + mice on control (dotted line) or -SG diet (dotted line). Survival rates were calculated from the change in diet. CTR: n=37; -SG n=35; CTR+PH755: n=12; -SG+PH755 n=12 (ns: no significant difference, * P < 0.05; *** P<0.001; Mantel-Cox test).

[0108] FIG. 64B. Total adenoma area was measured at clinical endpoints in small intestines from ApcMin / + mice treated with PH755 and fed either a CTR or -SG diet. Data are presented as mean ± SEM. CTR+PH755 n=12; -SG+PH755 n=9. **** P<0.0001, unpaired two-tailed Student's t test).

[0109] FIG. 64C. Plasma (left panel) or intestinal tumors (right panel) were collected at sacrifice from ApcMin / + mice treated with PH755 and fed either the CTR or -SG diet. LC-MS analysis was performed to assess serine and glycine content. Data are presented as mean ± SEM. Plasma: CTR+PH755 n=12; -SG+PH755 n=10. Tumor: CTR+PH755 n=10; -SG+PH755 n=8. ( ** p < 0.01; *** P < 0.001, unpaired two-tailed Student's t test).

[0110] Figure 64D. Villin-CreER;Apcfl / +;LSL-KrasG12D / + mice were induced with tamoxifen at 6-8 weeks of age and then either remained on normal chow or switched to -SG diet 2 weeks after induction. 14 days after switching to -SG diet, mice were either treated with 100mg / kg PH755 for 7 days and then switched to 50mg / kg for 2-12 days or treated with 50mg / kg PH755 for 30 days. After drug treatment was stopped, mice were then maintained on each diet until a humane endpoint was reached. Survival rates were calculated from the time of induction. CTR+Veh: n=10; CTR+PH755 n=10; -SG+PH755 n=14. (ns: no significant difference, * P < 0.05; ** P<0.01; Mantel-Cox test).

[0111] FIG. 64E. The total number of adenomas was scored from H&E staining of colon per roll in Villin-CreER;Apcfl / +;LSLKrasG12D / + mice fed a chow or -SG diet and treated with vehicle or PH755. Data are presented as mean±SEM. CTR+Veh: n=8; CTR+PH755 n=8; -SG+PH755 n=12. ** p < 0.01; **** P < 0.0001, unpaired two-tailed Student's t test).

[0112] Example 10: Methods used in previous examples cell culture All cell lines underwent routine quality controls, which included mycoplasma detection, STR profiling for validation, and species identification. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. HT-29, SW48, SW480, SW620, CACO2, HCT116, RKO, VACO5, and MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS; DLD-1, HCT-15, and SW1417 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, and LoVo and CL-34 cells were cultured in DMEM / F-12 (Gibco, 11320) supplemented with 10% FBS.

[0113] Serine and glycine deficiency For all serine and glycine starvation experiments, cells were cultured in MEM supplemented with 10% dialyzed FBS, 1% penicillin-streptomycin, D-glucose (5 mM), sodium pyruvate (65 μM), 1× MEM vitamin solution (Gibco, 11120), L-glutamine (2 mM), L-proline (0.15 mM), L-alanine (0.15 mM), L-aspartic acid (0.15 mM), L-glutamic acid (0.15 mM), and L-asparagine (0.34 mM) (-SG medium). Complete medium (CM) corresponds to the aforementioned medium supplemented with 0.4 mM L-serine and 0.4 mM L-glycine.

[0114] Growth curve Cells (2 × 10 depending on cell line) 4 ~3×10 4Cells / well) were plated in 24-well plates in their regular medium. The next day, after washing with PBS, cells were transferred to -SG medium or CM and treated with 10 μM PH755 diluted in DMSO or DMSO alone. For the counting step, cells were trypsinized, suspended in PBS-EDTA, and counted with a CASY model TT Cell Counter. The relative cell number at each time point was calculated based on the number of cells measured before the medium change. For growth curve experiments with formic acid and glycine supplementation, HT-29, HCT116, and DLD-1 cells were seeded (2 × 10 4 Cells / well). Sodium formate (1 mM) and / or glycine (0.4 mM) were diluted in -SG medium + 10 μM PH755 and the medium was refreshed every 2 days.

[0115] Organoids Crypts were isolated from adenomatous small intestinal tissues from Vil1-creER;Apcfl / fl and Vil1-creER;Apcfl / fl;KrasG12D / + mice. Generation of Apc5 organoids carrying the Apc truncating mutation using CRISPR / Cas9 technology and isolation of normal organoids from the proximal part of healthy small intestine from Villin-CreERT2 mice were performed. Cancer organoids from mice were cultured in tumor organoid medium (CM) consisting of Advanced DMEM / F12 supplemented with 1% penicillin-streptomycin solution, 0.1% BSA, 2 mM L-glutamine, 10 mM Hepes, 50 ng / mL EGF, 100 ng / mL Noggin, 500 ng / mL Spongin, 1x N-2 supplement, and 1x B-27 supplement (ThermoFisher 17504044). -SG medium corresponds to the aforementioned medium without serine and glycine. Normal organoids from mice were grown in a modification of tumor organoid medium supplemented with normal organoid medium, 100 ng / mL Wnt-3a, 1 mM N-acetyl-L-cysteine, 10 μM Y 27632, and 4 mM nicotinamide.

[0116] Human organoids were grown in a second modification of tumor organoid medium supplemented with human organoid medium, 10 nM FGF-basic, 100 ng / mL Wnt-3a, 1 μM prostaglandin E2, 4 mM nicotinamide, 20 ng / mL HGF, 10 nM FGF-10, 10 nM gastrin I, 10 μM Y-27632, 0.5 μM A 83-01, and 5 μM SB 202190.

[0117] For the splitting process, organoids were collected through mechanical pipetting with TrypLE, incubated at 37°C for 10 min, diluted to 3 volumes with ice-cold 1x HBSS, and spun down at 270g for 5 min at 4°C. The pellet was then resuspended in growth factor-reduced Matrigel and plated in a 24-well plate. Matrigel was then incubated at 37°C for 15 min, and 1 mL of CM as described above was added. The next day, organoids were washed with PBS, and the medium was replaced with CM or -SG medium supplemented or not with 10 μM PH755, and allowed to grow. Photographs were taken periodically under a light microscope, and organoid diameters were measured using ImageJ software.

[0118] Generation of PHGDH KO cells The following guide RNAs: PHGDH was targeted using the pLentiCRISPRv2 vector containing TIFF2024504260000002.tif4128. HEK293T cells were transfected with this lentiviral plasmid together with psPAX2 and VSV.G using jetPRIME reagent (Polyplus transfection). After 24 h of incubation, the medium was changed, and after 48 h, the medium containing viral particles was filtered (0.45 mm) and mixed with polybrene (4 μg / ml, Sigma-Aldrich). The medium containing the lentivirus was incubated with the target cells for 24 h. HT-29 and DLD-1 cells were then selected with puromycin for 3 weeks and analyzed for the reduction of PHGDH expression.

[0119] ATF-4 siRNA transfection Cells were transfected with siRNA using Lullaby transfection reagent.

[0120] BrdU / 7-AAD staining HCT116 and DLD-1 cells were grown in -SG medium or CM for 48 h and treated with 10 μM PH755 diluted in DMSO or DMSO alone. To determine the percentage of bromodeoxyuridine (BrdU) positive cells, 10 μM BrdU was then added to the culture medium for an additional 5 h, and for cell cycle analysis, 10 μM BrdU was added for only 30 min. Cells were then collected, fixed, and stained with APC anti-BrdU antibody (and 7-AAD for cell cycle analysis) using the APC BrdU Flow kit. Fluorescence was acquired by FACSdiva on a Fortessa flow cytometer, and analysis was performed using FlowJo (version 10.5.2).

[0121] Western blot Protein lysates were processed in RIPA buffer supplemented with phosphatase inhibitor cocktail and complete protease inhibitors. Lysates were separated using precast NuPAGE 4-12% Bis-Tris protein gels and transferred to nitrocellulose membranes. After incubation with primary antibodies, proteins were detected using appropriate secondary antibodies. Western blots were scanned using an Odyssey CLx or visualized using an ECL chemiluminescence detection kit.

[0122] Primary antibodies used were as follows: PHGDH (13428), ATF-4 (11815), phospho-eIF2α (Ser51) (3398), phospho-p70S6 kinase (Thr389) (9234), p70S6 kinase (9202), c-Myc (5605), HIF-1a (14179), caspase 3 (9662), cleaved caspase 3 (Asp175) (9661), β-actin (4970); GCN2 (sc-374609), eIF2α (sc-133132), p53 (sc-126), vinculin (sc-73614); PSAT (ab96136), PSPH (ab96414), phospho-GCN2 (Thr899) (ab75836); Atlas ASNS (HPA029318); puromycin (MABE343) from Antibodies. All primary antibodies were diluted at 1:1000 dilution.

[0123] Protein synthesis and degradation Cells were grown for 24 h in -SG medium or CM and treated with 10 μM PH755 diluted in DMSO or DMSO alone. To assess protein synthesis, puromycin (final concentration: 90 μM) was added to each well 10 min before cells were harvested for Western blot analysis, except for negative control wells. Where indicated, cells grown in CM medium were treated with cycloheximide (10 μg / mL) for the last 5 h, providing a control for translation inhibition. Incorporation of puromycin into newly synthesized proteins was assessed by Western blot using an anti-puromycin antibody. To assess accumulation of short-lived proteins in response to proteasome inhibition, cells grown for 24 h in -SG medium or CM with or without 10 μM PH755 were treated with the proteasome inhibitor MG-132 (10 μM) for the last 6 h before cells were harvested for Western blot analysis.

[0124] qPCR HT-29, HCT116, and DLD-1 cells were grown in -SG medium or CM for 6 or 24 hours and treated with 10 μM PH755 diluted in DMSO or DMSO alone. Total RNA was extracted using the RNeasy Mini kit, which performs on-column digestion of DNA, and reverse transcribed using the High-Capacity cDNA Reverse Transcription kit. qPCR was performed using PrimeTime Gene Expression Master Mix with the primers listed in Table 1 below.

[0125] [Table 1]

[0126] A QuantStudio 7 Flex Real-Time PCR System was used for all reactions. Gene expression was normalized to the ACTB (b-actin) housekeeper gene, analyzed according to the Pfaffl method, and expressed as relative units compared to cells grown in CM for 6 h.

[0127] Liquid chromatography-mass spectrometry HT-29 cells (2.4×10 5 ), HCT116 cells (1.8×10 5 ), DLD-1 cells (1.8×10 5 cells), and MDA-MB-468 cells (2.4 × 10 5 (cells) were plated in 6-well plates in their normal medium. Duplicate plates were used for cell counting to normalize the LC-MS analysis based on cell number. After 16 h, cells were washed with PBS and shifted to CM or -SG medium supplemented or not with 10 μM PH755 for 24 h. Six hours before metabolite extraction, glucose was added to 10 mM U-[ 13 The medium was replaced with CM or -SG medium containing 10 mM U-[C]-glucose + / - 10 μM PH755. In short-term experiments, glucose was added at 10 mM U-[C]-glucose for only 3 or 6 h prior to metabolite extraction. 13For the measurement of glycine to serine conversion during rescue experiments, cells were grown for 24 h in -SG medium supplemented with 10 μM PH755, 1 mM sodium formate, and 0.4 mM glycine. The medium was then replaced with 0.4 mM glycine for 1 h before metabolite extraction. 13 C2 15 The medium was replaced with adapted medium containing N1-glycine.

[0128] In half of the samples, a 1 mM unlabeled serine stimulus was added to the medium 1 min prior to metabolite extraction to allow the accumulation of labeled serine. Cells were then washed with PBS, and metabolites were extracted using ice-cold extraction buffer composed of methanol, acetonitrile, and HO in the following ratio: 50:30:20. For LC-MS analysis on tumor samples, tissue was homogenized (20–40 mg of tissue per mL of extraction buffer as described above) using a Precellys 24 homogenizer. Samples were spun (16,000 g / 10 min / 0°C), the supernatant was collected, and centrifuged again (16,000 g / 10 min / 0°C). The supernatant was then collected for LC-MS analysis.

[0129] For LC-MS analysis of mouse plasma, the plasma was diluted 20-50 times with the same extraction buffer, vortexed for 30 seconds, and centrifuged (16,000 g / 10 min / 0°C). The supernatant was then collected for analysis. The absolute levels of serine and glycine in plasma were determined by diluting the plasma with 100% glycine and 100% glycine. 13 C3 15 N2-serine and 13 C2 15 The concentration was determined using an 8-point calibration curve (2.5-800 μM) with N1-glycine. LC-MS analysis was performed.

[0130] In vivo experiments Mice (3–5 / cage) were allowed free access to food and water and maintained on a 12-h day-night cycle starting at 07:00 and ending at 19:00. The room was maintained at 21°C with 55% humidity. Mice were acclimated for at least 1 week prior to the experiment. They were then randomly assigned to experimental groups. The experimental diets used in this study (control diet and -SG diet) were described as "Diet 1-Control" and "Diet 1-SG Free". Briefly, the control diet contained all essential amino acids, as well as serine, glycine, glutamine, arginine, cystine, and tyrosine. The -SG diet was the same as the control diet, except that serine and glycine were removed, which was compensated for by proportionally increased levels of the other amino acids to reach the same total amino acid content.

[0131] Xenograft experiments CD-1 female nude mice (7–9 weeks old) were inoculated with 100 μl of HCT116 cells (2 × 106 cells) or 100 μl of DLD-1 cells (4 × 10 6 Each mouse received a unilateral subcutaneous injection of 100 mM NaCl (100 mM TNF-α, 100 mM HEPATIC CELLS) of 10 mM DMSO.

[0132] Ten days (HCT116 xenograft experiment) or two days (DLD-1 xenograft experiment) after tumor injection, mice were maintained on experimental diet (control or -SG). Four days (HCT116 xenograft experiment) or two days (DLD-1 xenograft experiment) after diet change, mice were treated once daily by oral gavage with vehicle (0.5% methylcellulose, 0.5% Tween-80) or PH755 prepared in vehicle. The starting dose of PH755 was 100 mg / kg and was subsequently reduced to 75 mg / kg or 50 mg / kg as indicated in the figure legends. Subcutaneous growth was measured by calipers two to three times a week and calculated according to the following formula: (length × width) 2 Tumor volume was calculated using the formula: 1) / 2.

[0133] C57BL / 6J experiment C57BL / 6J male mice (14 weeks old) were placed on the experimental diet (control or -SG) 2 days before treatment with PH755 or its vehicle was initiated. Mice were treated with PH755 or its vehicle by oral gavage once daily for 20 days. The starting dose of PH755 was 75 mg / kg, which was then reduced to 50 mg / kg to maintain weight loss below 20% of initial body weight.

[0134] Immunohistochemistry All tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. For PHGDH and PSAT1 staining, slides were deparaffinized with xylene and rehydrated with a graded series of industrial methanol-denatured alcohol solutions and distilled water. Antigen retrieval was performed in a microwave with 0.1 M citrate buffer at pH 6 for 23 min. Endogenous peroxidase blocking was performed with 1.6% H2O2 for 10 min at room temperature, and protein blocking was performed with 2.5% normal horse serum (MP-7401, Vector) overnight at 4°C. Primary antibodies were diluted in 1% BSA at 1:1000 for PHGDH antibody (HPA021241) and 1:500 for PSAT1 antibody (PA5-22124) and incubated for 1 h at room temperature. HRP horse anti-rabbit IgG polymer (MP-7401, Vector) was incubated for 30 min at room temperature. 3,3-Diaminobenzidine (DAB) chromogen (SK-4100, Vector) was incubated for 10 min at room temperature. Slides were counterstained with Harris hematoxylin, dehydrated, cleared, and mounted on a Sakura Tissue-Tek Prisma R automated stainer. For quantification of PHGDH and PSAT1 staining intensity, a minimum of three fields per tumor were quantified with ImageJ. Active caspase 3 immunohistochemistry was performed on a Discovery Ultra Ventana platform.

[0135] Antigen retrieval was obtained by Cell Conditioning 1 (CC1) from Ventana Medical Systems. Primary antibody (AF835) was diluted 1:1250 and incubated for 60 min. For active caspase 3 staining, a minimum of 3 fields per tumor were quantified with the positive cell detection algorithm from QuPath (version 0.1.2). All slides were scanned with a ZEISS Axio Scan Z1 slide scanner and images were generated with ZEISS ZEN 2.6 (blue edition) software. For intestinal rolls, immunohistochemistry was performed with a Bond Rx Autostainer Leica Bond Intense R staining kit. Slides were deparaffinized with Bond Dewax for 30 min at 72°C and antigen retrieval was achieved with ER2 for 20 min at 100°C. Primary antibody (Ki67 SP6, ab16667) was diluted 1 / 100 and incubated for 35 min. For villus length measurements, villi (at least 15 per mouse) from the same region of the small intestine were measured from the crypt / villus junction to the villus tip using ImageJ.

[0136] Brain sampling and pathology C57BL / 6J mice were culled using carbon dioxide asphyxiation to avoid physical trauma to the brain. Mice were immediately dissected, and hairy skin and soft tissues were removed from the skull surface. An incision was made from the parietal to the frontal suture, allowing rapid infiltration of fixative into the brain parenchyma. Heads were immersed in 250 mL of 10% neutral buffered formalin and fixed for 2 weeks. After complete fixation, brains were removed from the skull and prepared using mouse brain matrix (BSMYS001-1). Four coronal sections were obtained at the level of the piriform cortex, caudal diencephalon, caudal midbrain, and rostral cerebellum. Tissue samples were routinely processed for paraffin embedding, sectioned at 4 μm, and stained with hematoxylin and eosin.

[0137] Histopathological examination of the brain was performed by a board-certified veterinary pathologist.

[0138] Blood Biochemistry Marker Assays Plasma ALT and AST activities were measured using an alanine transaminase activity assay kit (and an AST activity assay kit), respectively.

[0139] statistical analysis All data are expressed as mean ± SEM and each statistical analysis is detailed in the figure legend. Data were collected in Excel (version 16.16.26) and all statistical analyses were performed using GraphPad Prism 8 (version 8.3.1) software. Unpaired Student's t-tests were performed to compare two groups with each other. Welch's correction was applied when the variances between the two groups were unequal.

[0140] For comparisons of more than two groups, one-way ANOVA with Tukey's multiple comparison test was used to determine statistical significance. For analysis of tumor volume and body weight, two-way ANOVA + Tukey's post hoc test was performed. A p value below 0.05 was considered statistically significant.

[0141] Significance is indicated as follows: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns: not significant. All measurements were taken from separate samples. Tumor sizes were based on standard protocols in the field, and metabolic samples were randomly assigned prior to analysis. Mice were randomly assigned to treatments, and the identity of each mouse was made anonymous when measurements were collected.

[0142] Example 11: Metabolomic effects of radiation on pancreatic and colorectal cancer cells in vitro Figure 65, Panels A-D, show the metabolomic effects of radiation on pancreatic and colorectal cancer cells in vitro. In Panel A, primary mouse pancreatic cancer (KPC: Pdx1-cre; Kras G12D / + ;Trp53 R172H / -) and human colorectal cancer (HCT116) cells were exposed to 5–10 Gray (Gy) of radiation. After 24 h, metabolites were extracted and analyzed by LCMS using a Thermo Exactive Orbitrap mass spectrometer coupled with a pHILIC chromatography column. An unsupervised principal component analysis was performed with data from all identified metabolites. Panel B shows a volcano plot (control vs. 10 Gy irradiated) depicting the distribution of identified metabolites in terms of fold change and P-value in KPC and HCT116 cells. Panel C shows significantly changed metabolites identified when unbiased metabolomics was subjected to metabolic pathway analysis. Dominant pathway hits are shown. Panel D shows KPC (top panel) and HCT116 (bottom panel) cells grown in either complete medium (Ctr) or medium lacking serine and glycine (-SG) and irradiated with 10 or 5 Gray radiation (IR), respectively. Cell numbers over time (hours) are shown. Data are the mean of triplicate wells and error bars are SD.

[0143] Example 12: Effect of dietary amino acid restriction on response to targeted radiotherapy in vivo Figure 66, Panels A-E, show the effect of dietary amino acid restriction in response to targeted radiation therapy in vivo. Panel A is a schematic illustrating the experimental set-up of a pilot experiment testing the effect of dietary restriction of serine and glycine on the response of KPC tumors to radiation. Groups of C57BI6 (n=4) mice were fed 2×10 6Primary C57BI6 KPC cells were injected (subcutaneously, bilaterally). Once tumors had formed, mice were placed on a control or serine- and glycine-free diet. After 4 days on such a diet, mice were anesthetized and positioned in the Xstrahl SARRP. Each tumor was irradiated with a 2 mm focused beam of x-ray radiation (20 Gy) using high-resolution cone-beam CT imaging. Panels B and C show the results of an unsupervised principal component analysis performed on tumor tissue to evaluate the metabolic impact of radiation therapy alone. The most significantly altered metabolites in vivo were clustered in the same metabolic pathways as those identified in vitro. Panel D shows a representative tumor cross section stained for cleaved caspase 3 by immunohistochemistry. The top panel shows the stained section (cleaved caspase 3 stained brown) and the bottom panel shows the false color mapping of the histological scores generated by Halo image analysis software. Panel E shows quantification of Ki67 and cleaved caspase 3 staining quantified using Halo image analysis software. n=8 tumors / group, bars are SEM. Data show that cells obtained from mice on a serine- and glycine-free diet in combination with radiation therapy showed reduced proliferation and apoptosis compared to other experimental conditions.

[0144] Example 13: Sachet formulation lacking serine, glycine, and proline Prepare a sachet containing a formulation that lacks serine, glycine, and proline, which contains 0.8 g / kg / day of amino acids. Table 2 shows the components and amounts of the composition. The amino acid sachet is administered to the subject in conjunction with a low-protein and low-carbohydrate diet. The low-protein and low-carbohydrate diet provides the following daily dietary intake: 1) 1711 kcal / day (1923 kcal / day from the sachet); 2) about 10 g protein / day; 3) about 420 mg proline / day; 4) about 410 mg serine / day; 5) about 230 glycine / day; 6) a diet in which about 9% of the kilocalories of food alone are carbohydrates, 2% are protein, and 89% are fat.

[0145] [Table 2]

[0146] Example 14: Use of Radiation Therapy to Treat Cancer A first subject with cancer is treated with short-term radiation therapy to treat the cancer. Two days before the start of radiation therapy treatment (i.e., day -2), the first subject is placed on a diet substantially devoid of serine and glycine. An amino acid depleted diet is administered starting two days before treatment and ending four days after the second treatment (i.e., from day -2 to day 8), for a total of 10 days. The first subject is treated with 5 Gy per day for 5 days. The first subject returns to a normal diet after day 8, or 4 days after radiation treatment. If the first subject is treated with chemotherapy after radiation therapy, the first subject is placed on a cyclical diet during chemotherapy. The cyclical diet is a diet in which the first subject alternates between 5 days of an amino acid depleted diet (e.g., Monday-Friday) followed by 2 days of a normal diet (e.g., Saturday, Sunday) during the chemotherapy treatment period. Table 3 shows a short-term radiation therapy for treating cancer.

[0147] [Table 3]

[0148] A second subject with cancer is treated with long-term radiotherapy to treat the cancer. Two days before the start of radiotherapy treatment (i.e., day -2), the second subject is placed on a diet that is substantially devoid of serine and glycine. The amino acid depleted diet is administered for a total of seven days, beginning two days before treatment and continuing throughout treatment (i.e., from day -2 to day 4). The second subject is treated with 2 Gy per day for five days. The second subject returns to a normal diet for two days before the start of an additional round of radiation treatment. Subsequent radiation therapy cycles administer five days of amino acid depleted diet, followed by two days of normal diet, with five days of 2 Gy radiation. This cycle is repeated as necessary.

[0149] If the second subject is treated by chemotherapy after radiation therapy, the second subject is put on a cyclic diet during chemotherapy. The cyclic diet is to put the second subject on an alternating 5-day amino acid depletion diet (e.g., Monday-Friday) followed by 2-day normal diet (e.g., Saturday, Sunday) during the chemotherapy treatment period. Table 4 shows the long-term radiation therapy for treating cancer.

[0150] [Table 4] TIFF2024504260000007.tif97160

[0151] Example 15: IDO1-driven tryptophan metabolism is a source of one-carbon units for pancreatic tumor and stellate cells Using in vitro and in vivo pancreatic cancer models, we showed that IDO1 expression was highly context-dependent and influenced by adhesion-dependent growth as well as the canonical activator IFNγ. We also showed that cancer cells liberate tryptophan-derived formate, which can be taken up and utilized by pancreatic stellate cells to support purine nucleotide synthesis.

[0152] The metabolic consequences of IDO1-driven tryptophan metabolism were evaluated in the context of pancreatic ductal adenocarcinoma (PDAC). PDAC tumors are highly aggressive and associated with poor clinical outcomes. Characteristically, PDAC tumors exhibit low vascularization, metabolic derangements, and contain a large proportion of complex stroma. Noncancerous stromal stellate cells can support tumor cell metabolism by providing nutrients such as alanine. Unlike other tumor models, PDAC-bearing mice are nonresponsive to serine restriction.

[0153] Analysis of published data showed that multiple tumor types, including pancreatic cancer, had high IDO1-expressing subsets. IDO1 was expressed in a genetically engineered mouse model of PDAC. Results showed that IDO1 expression was not well expressed in standard in vitro cell culture conditions, but could be induced by the canonical activator IFNγ, which controls IDO1 via JAK / STAT signaling, or by culture in low-adhesion conditions. Results also showed that when IDO1 was expressed by cancer cells, it promoted the generation of one-carbon units from tryptophan used in de novo purine nucleotide synthesis. Furthermore, tryptophan-derived formate was liberated by the cancer cells. Pancreatic stellate cells, a key component of the tumor stroma, captured exogenously derived formate and redirected it into de novo nucleotide synthesis.

[0154] Experimental Method Cell Culture: All cell lines used in the study were cultured at 37°C with 5% CO2 in a humidified incubator. Cell lines were established using Promega GenePrint 10 and tested for mycoplasma using Mycoalert (Lonza). AsPC-1 (female), BxPC-3 (female), CFPAC-1 (male), HPAF-II (male), Panc 10.05 (male) & SW 1990 (male) cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin-streptomycin, 0.2% amphotericin B, and glutamine (2 mM). Mouse ImPSC and KPC cell lines were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 0.2% amphotericin B, and glutamine (2 mM). Pdx1-cre;LSL-Kras with either a mixed or pure C57BL / J background G12D / + ;LSL-Trp53 R172H / + KPC lines were isolated from mouse tumors. KPC-IDO1 & KPC-EV cell lines were generated from pure C57BL / J KPC cells using the PiggyBac transposon system. ImPSC #2 and #3 lines were transfected with Pdgfra tm11(EGFP)Sor It was isolated from a mouse.

[0155] Mice: Cohorts of Mus musculus mice were housed in pre-enriched barrier facilities and maintained on a normal chow diet. A mixed population of males and females of each genotype was used. Cohorts were of mixed strain background, but all cohorts consisted of litter-matched controls and were sacrificed at a humane clinical endpoint. Allogeneic transplantation of mixed background KPC cells resulted in increased Crl:CD1-Foxn1 nu (CD1 nude) female mice were used (7 weeks old). For allogeneic transplantation of pure C57BL / J KPC cells, C57BL / J female mice were used (7 weeks old).

[0156] Extraction of ImPSC cell lines: Healthy pancreatic tissue extracted from C57BL / J mice was minced and digested with 0.1% DNase, 0.05% collagenase P, and 0.02% pronase in Gey's balanced salt solution (GBSS) at 37°C for 20 minutes. The tissue was then triturated until large fragments were no longer visible, passed through a 100 μm filter, and washed with GBSS. Cells were then pelleted and resuspended in 9.5 mL GBSS with 0.3% BSA and 8 mL Nycodenz solution. The cell suspension was layered beneath GBSS containing 0.3% BSA and centrifuged at 1400×g for 20 minutes at 4°C. Stellate cells were collected from the interface of the bottom Nycodenz solution and the top aqueous solution. The isolated PSCs were then washed with GBSS and resuspended in DMEM with 10% characterized FBS (HyClone), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were immortalized with the pRetro.Super.shARF retroviral plasmid and selected with blasticidin (4 μM).

[0157] ImPSC #2 and #3 lines were isolated using a protocol very similar to ImPSC #1 with some minor differences detailed below. Pancreatic tissue was purified using Pdgfra tm11(EGFP)SorPSCs were extracted from mice, minced with a scalpel, and digested with 0.1% DNase I and 0.05% collagenase P in GBSS for 30 min at 37°C. The solution was then passed through a 100 μm filter, washed with GBSS, pelleted, and resuspended in 6 mL GBSS containing 0.3% BSA. The cell suspension was then mixed with 8 mL Histodenz solution (43.75% in GBSS), layered beneath GBSS containing 0.3% BSA, and centrifuged at 1400×g for 20 min at 4°C. Stellate cells were collected from the interface of the bottom Histodenz solution and the top aqueous solution. PSCs were washed with PBS containing 3% FBS and resuspended in DMEM containing 10% FBS, 1% penicillin-streptomycin, 0.2% amphotericin B, and glutamine (2 mM). After culture establishment, fibroblasts expressing GFP were isolated via FACS and spontaneously immortalized.

[0158] ImPSC #1 cells stably expressing GFP (ImPSC-GFP cells) were generated by the PiggyBac transposon system. Briefly, 5 × 10 4 ImPSC #1 cells were seeded in 6-well plates. 24 hours after seeding, cells were transfected with 1.5 μg Super piggyBac transposase expression vector and 0.6 μg PB-GFP PB-CMV-MCS-EF1-GreenPuro using Lipofectamine 3000. 24 hours after transfection, cells were selected with 5 μg / mL puromycin for 48 hours until puromycin-sensitive control cells treated in parallel died.

[0159] Generation of KPC-EV and KPC-IDO1 cell lines: Pure C57BL / J KPC cells stably expressing IDO1-RFP or RFP alone (empty vector control) were generated using the piggyback system. Human IDO1 cDNA was cloned into the PB-RFP PB-CMV-MCS-EF1-RedPuro cDNA cloning and expression vector using XbaI and EcoRI. Successful cloning was confirmed by full sequencing of the insert. 2.5 × 10 5 Pure C57BL / J KPC cells were seeded in 6-well plates. 24 hours after seeding, cells were transfected with 1.5 μg Super piggyBac transposase expression vector and 0.6 μg of either PB-RFP PB-CMV-IDO1-EF1-RedPuro (IDO1 overexpression) or PB-RFP PB-CMV-MCS-EF1-RedPuro (empty vector control) using Lipofectamine 3000. 24 hours after transfection, cells were selected with 5 μg / mL puromycin for 48 hours until puromycin-sensitive control cells treated in parallel died. To identify high expressers of IDO1, cells were clonally expanded and expression was verified by immunoblotting.

[0160] Hypoxia experiments: Cells were seeded and grown under normal tissue culture conditions for 48 hours to approximately 80% confluence. They were then transferred to a humidified Whitley H35 hypoxia station controlled by a hypoxic gas mixer at 1% O2, 5% CO2, and 94% N2 at 37°C for 24 hours prior to lysis, following standard RIPA lysis protocols.

[0161] Adhesion-independent 3D growth experiments: 1 × 10 6 Cells were seeded on ultra-low attachment plates for 48 hours. For treatment, cells were harvested, centrifuged at 50×g for 5 minutes, and washed with PBS. Cells were then resuspended in 2 mL of treatment medium and returned to the ultra-low attachment plates for the indicated treatment times.

[0162] Conditioned medium experiment: 8.7 x 106 HPAF-II or CFPAC-1 cells were seeded in 10 cm dishes in their normal growth medium, lacking tryptophan and supplemented with the indicated concentrations of tryptophan. 13 C 11 -Conditioned experimental medium supplemented with tryptophan was prepared. After 48 hours of culture, cells were washed with PBS and conditioned medium was added. After 48 hours, conditioned medium was collected and passed through a 0.45 μm filter to remove cells. Conditioned medium was stored at -20°C before use.

[0163] Simultaneous culture experiment: 5 × 10 5 ImPSC-GFP cells were either alone or at 1 × 10 4 CFPAC-1 cells were seeded in 6-well plates with either tryptophan-deficient or 0.4 mM 13 C 11 Experimental medium supplemented with tryptophan was prepared. After 24 h of culture, cells were washed with PBS and medium containing human IFNγ (1 ng / mL) or vehicle only control and / or epacadostat (1 μM) or vehicle only control was added. After 24 h, cells were detached by trypsinization, washed with PBS, and plated at 1 × 10 in cold supplemented PBS (PBS + 3% FBS, 5 mM glucose, MEM amino acids, and MEM NEAA). 7 The cells were then resuspended to a concentration of 1000 cells / mL. The cell suspension was then passed through a 70 μm mesh to ensure a single cell suspension and subjected to fluorescence activated cell sorting (FACS) using an Aria sorter Z6001 to separate GFP positive cells from unlabeled cells. The resulting cell suspension was centrifuged at 300 × g for 5 min and the pellet was resuspended in ice-cold lysis medium. Using the cell count obtained from FACS, the volume of lysis medium was reduced to 2 × 10 6 Normalized to cells / ml. Subsequent isolation of metabolites by LCMS was performed as follows.

[0164] Western blotting: Proteins were extracted from whole cells by lysis in RIPA buffer supplemented with protease and phosphate inhibitor cocktail. For cells grown in ultra-low attachment plates, cells were collected by centrifugation at 50×g for 5 min, washed with PBS, and resuspended in 100 μL RIPA lysis buffer. Cells were left to lyse on ice for 10 min and then homogenized by pipetting. For adherent cells, cells were washed with PBS, lysed in situ in 200 μL RIPA lysis buffer on ice, collected using a cell scraper, and homogenized by pipetting. Tissue samples were flash frozen and stored at -80°C. Frozen samples were weighed prior to lysis to ensure a minimum sample size of 20 mg. Samples were homogenized in 2 mL RIPA lysis buffer using a TissueLyser II. Lysates were clarified by centrifugation at 12,000×g for 15 min at 4°C. Supernatants were collected and total protein content was quantified by BCA assay. Lysates were normalized by total protein content and prepared for Western blotting by addition of 4x Bolt™ LDS Sample Buffer (+355 mM β-mercaptoethanol) and heating to 95°C for 10 min. Lysates (25 μg) were resolved on Bolt™ 4-12% bis-Tris Plus precast gels using Bolt™ MOPS SDS Running Buffer and transferred to nitrocellulose membranes. If total protein staining was performed, it was done before blocking with Revert™ Total Protein Stain. Membranes were blocked for 1 h with Odyssey® Blocking Buffer (TBS) and incubated with primary antibodies overnight at 4°C. All primary antibodies were diluted to a concentration of 1:1000 in Odyssey® Blocking Buffer, except for actin, which was used at 1:10,000. Membranes were washed three times with TBS + 1% TWEEN® 20 and incubated with secondary antibody (1:10,000) for 1 hour at room temperature. Fluorescence intensity was captured and quantified using a LI-COR Odyssey® Fc Imaging system with Image Studio software (version 5.2).

[0165] In vivo model: LSL-Kras G12D / + , Pdx1-cre;LSL-Kras G12D / + ;Trp53 fl / + and Pdx1-cre;LSL-Kras G12D / + ;LSL-Trp53 R172H / + Mice were allowed to develop tumors, sacrificed at a humane clinical endpoint, and tumors were removed for analysis. Pancreatic tissue from healthy, non-cre-expressing littermates served as controls. For allograft experiments, pure C567Bl6 / J KPC cells were injected subcutaneously (1 × 10 per injection) in a unilateral manner. 6 KPC cells were transplanted into pure C567Bl6 / J female mice by unilateral subcutaneous injection (2 × 10 6 Cells / flank) Crl:CD1-Foxn1 nu (CD1 nude) female mice. Mice were implanted with 0.1% CI, 0.1–0.2% until the tumor reached clinical endpoint or tumor size reached 300 mm 3 Mice were monitored daily until they reached a median daily intake of 100 mg / kg / day. Mice were fasted for 3 hours and then ingested with 800 μL of 120 mM 13 C tryptophan intraperitoneally. Three hours after injection, the mice were sacrificed and the tumors were removed for analysis.

[0166] LCMS for steady-state metabolite measurements: Cells were seeded in 6-well plates in complete medium and grown to approximately 80% confluence. Cells were washed with PBS and the relevant experimental medium was added for the indicated times. Duplicate wells were used for cell counting: prior to metabolite extraction, the volume of dissolution medium was normalized using cell number (2D cells) or protein concentration (3D cells BCA assay) (1 × 10 6Cells / ml). For 2D grown cells, cells were quickly washed with PBS, then ice-cold lysis solvent (50% methanol, 30% acetonitrile, 20% water) was added and cells were detached on ice. For 3D grown cells, cells were transferred to 15 mL Falcon tubes and centrifuged at 50×g for 5 min. The supernatant was removed and the cell pellet was washed with PBS and centrifuged again. The supernatant was removed and the cell pellet was resuspended in ice-cold lysis solvent. The lysate was transferred to 1.5 ml tubes on ice, vortexed and then centrifuged at 18,000×g for 10 min at 4°C. The supernatant was collected and stored at -80°C for LCMS analysis. Tissue samples were flash frozen and stored at -80°C. Frozen samples were weighed prior to lysis. Samples were homogenized in 2 mL of ice-cold lysis solvent using a TissueLyser II. Lysates were then cleared of protein by centrifugation at 18,000×g for 10 min at 4° C. and then normalized to 10 mg / mL with lysis buffer based on the original tissue mass.

[0167] GCMS for formic acid analysis: 40 μL of sample was added to 20 μL of d2-formic acid (50 μM, internal standard), 50 μL pyridine, 10 μL NaOH (1N), and 5 μL benzyl alcohol. While vortexing, 20 μL of methyl chloroformate was added to this mixture for derivatization. Then, 100 μL methyl tertiary butyl ether and 200 μL H2O were added, followed by vortexing the sample for 10 s and centrifuging at maximum speed for 10 min. The nonpolar phase was then transferred to a GC vial and capped. Standards and blank samples (water) were prepared in the same way and analyzed alongside the experimental samples to subtract background and verify quantification. MassHunter Quantitative analysis software was used to analyze formic acid, 2-formic acid, and 13 The C formate peak area was extracted and processed. After correction for background signal, quantification was performed by comparing the peak areas of formate (m / z of 136) and C formate (m / z of 137) to the peak area of ​​d2-formate (m / z of 138).

[0168] Sample analysis was performed using an LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer. Metabolites were separated using a Sequant ZIC-pHILIC column (4.6 mm × 150 mm, 3.5 μm), with mobile phases mixed by A = 0.1% (v / v) formic acid in water and B = 0.1% (v / v) formic acid in acetonitrile. A gradient program was used starting with 20% A and increasing linearly to 80% in 30 min, followed by a wash (92% A for 5 min) and re-equilibration (20% A for 10 min) step. The total run time of the method was 45 min. The LC flow path was desolvated and ionized with a HESI probe. The Exactive mass spectrometer was operated in full scan mode with a mass range of 70–1,200 m / z at a resolution of 50,000 with polarity switching. The LCMS raw data were converted to mzML by using ProteoWizard and imported into MZMine 2.10 for peak extraction and sample alignment. All possible relevant metabolites were 13 C and 15 A home-made database containing N isotope m / z values ​​was used for the assignment of LCMS signals. Finally, peak areas were used for comparative quantification.

[0169] Carbon-13 labeling of metabolites: Tryptophan or serine-free and at the indicated concentrations. 13 C 11 -Tryptophan, 13 C3 15 N1-serine, or 13 Experimental media supplemented with C1-formic acid were prepared. The same basic protocol as for steady-state metabolite measurements was used. Metabolites were extracted as described above.

[0170] result PDAC cells express IDO1 in a context-dependent manner: Expression of IDO1 in pancreatic cancer cells was examined in vitro and in vivo. Using the mouse KPC model, IDO1 expression was assessed in different contexts (Figure 67): Pdx1-cre; LSL-Kras G12D / + ;Trp53 fl / +and Pdx1-cre;LSL-Kras G12D / + ;LSL-Trp53 R172H / + Direct analysis of pancreatic tumor tissue from mice showed that tumors had increased IDO1 expression versus normal pancreatic tissue, and that certain tumors expressed high levels of IDO1 (Figure 67 Panels B and C). Compared to GEMM tumor tissue, tumor-derived primary KPC cells cultured under normal in vitro conditions showed undetectable IDO1 (Figure 67 Panel D). Addition of the murine form of the cytokine IFNγ, a canonical activator of IDO1, increased IDO1 expression in vitro. Human IFNγ did not affect IDO1 expression in mouse cells (Figure 67 Panel D). To assess whether in vivo growth could restore IDO1 expression, KPC cells were injected as subcutaneous allografts into CD-1 nude mice. Evaluation of IDO1 expression in allograft tumor tissue revealed extremely low IDO1 expression (Figure 67 Panel D).

[0171] Given the ability of IFNγ to promote IDO1 expression and the known immunological role of IDO1, increased IDO1 expression in an immunocompetent host was tested using KPC cells. G12D / + ; and LSL-Trp53 R172H / + Using primary tumor cells extracted from mice, they were successfully engrafted into normal recipient C57BL / J mice. When the cells were injected as subcutaneous allografts into syngeneic immunocompetent mice, IDO1 expression was elevated in tumor tissue (compared to in vitro culture) in two of the three cell lines (Figure 67 Panel E).

[0172] We also examined the expression of IDO1 in a panel of human pancreatic cancer cells. Similar to KPC cells, IDO1 expression was very low or below the limit of detection under normal culture conditions (Figure 67 Panel F). Addition of IFNγ (human) consistently increased IDO1 expression. To globally evaluate IDO1 expression in human cancers, data was extracted from metabolic gene rapid visualizer. In the pancreas, IDO1 had a comparable range of expression in healthy tissues compared to in vitro grown cancer cell lines (Figure 68). However, pancreatic tumor tissues had multiple high or very high IDO1 expressing tumors. This trend was also observed in a variety of other tumors, notably colon, breast, and cervix. The dataset consistently showed that IDO1 expression was elevated in tumors compared to healthy tissues, but cancer cells grown under normal in vitro culture conditions did not necessarily exhibit tumor-associated levels of IDO1. Overall, these data indicated that IDO1 expression was upregulated during tumorigenesis in an immunocompetent context.

[0173] Figure 67 shows IDO1 expression in vivo. Panel A shows a schematic diagram detailing the method used to analyze IDO1 expression in genetically engineered mouse models (GEMMs) of pancreatic ductal adenocarcinoma (PDAC). Pdx1-cre; Kras G12D / + ;Trp53 fl / + and Pdx1-cre;Kras G12D / + ;Trp53 R172H / + Tumors from mice and healthy pancreatic tissue from isogenic control mice not expressing cre were lysed. Panel B shows the indicated proteins after analysis by immunoblotting. Panel C shows the indicated proteins analyzed with the fluorescence intensity of IDO1 quantified relative to total protein (loading control) (healthy pancreas n=5, Pdx1-cre; Kras G12D / + ;Trp53 fl / + Tumors n=6, Pdx1-cre;Kras G12D / + ;Trp53 R172H / +(n=5, unpaired t-test, p-values ​​shown, error bars are standard deviations). Panel D shows KPC A cells, mixed background Pdx1-cre;Kras, treated with murine IFNγ (1 ng / ml) for 24 h or injected subcutaneously into the flank of CD1 nude mice to form tumors. G12D / + ;Trp53 R172H / + Cell lines isolated from mouse tumors are shown. Cell and tumor lysates were subjected to immunoblotting for the indicated proteins. Panel E shows a pure C567Bl6 / J background Pdx1-cre;Kras G12D / + ;Trp53 R172H / + KPC cells were isolated from mice and treated with mouse IFNγ (1 ng / ml) for 24 hours or injected subcutaneously into the flank of C567B16 / J mice to form tumors. Cell and tumor lysates were subjected to immunoblotting for the indicated proteins. Panel F shows the indicated cell lines were treated with human IFNγ (1 ng / mL) for 24 hours and cell lysates were blotted for the indicated proteins.

[0174] FIG. 68 shows data extracted from the MERAV database showing the relative abundance of IDO1 mRNA from microarrays.

[0175] IDO1 expression can be controlled by adhesion-dependent growth in vitro: IDO1 expression promotes tryptophan utilization via the kynurenine pathway. Given the diversity of potential metabolic interactions in this pathway (Figure 69 Panel A), the effect of immune-dependent stimulation on IDO1 expression was examined. Mitochondrial metabolism potentially links with the kynurenine pathway in two ways: (1) mitochondrial generation of superoxide and (2) influx of tryptophan-derived carbon into the TCA cycle via α-ketoadipic acid. Exposure of PDAC cells to hypoxia or rotenone, both of which are predicted to affect OXPHOS and potentially regulate superoxide levels, had little effect on IDO1 expression (Figure 69 Panel B and Panel C). Similarly, replacement of glucose (which promotes OXPHOS) with galactose did not regulate IDO1 expression (Figure 69 Panel D). Transitioning cells from 2D monolayer culture to adhesion-dependent growth (without any other adjustments to culture conditions) caused an increase in IDO1 expression in BxPC-3, CFPAC-1, and HPAF-II cells (Figure 69 Panels E, F; Figure 70 Panels A and B). This observation was consistent in CFPAC-1 cells, with a dramatic increase in kynurenine pathway activity as measured by kynurenine efflux, which was ablated by the IDO1 inhibitor epacadostat (Figure 69 Panel G).

[0176] Figure 69 shows that IDO expression was upregulated by IFNγ through 3D growth and JAK / STAT signaling. Panel A shows a schematic detailing the kynurenine pathway by which tryptophan is metabolized. The indicated proteins were analyzed by immunoblotting in the indicated cell lines after 24 hours of culture. Panel B shows proteins cultured under either normoxic (20% O2) or hypoxic (1% O2) conditions; Panel C shows proteins treated with rotenone (1 μM) or vehicle only control; and Panel D shows proteins cultured in media containing either glucose (Glc) (10 mM) or galactose (Gal) (10 mM). The indicated cell lines were cultured in 2D or 3D conditions for 24 hours and cell lysates were immunoblotted for the indicated proteins. Panel E shows proteins cultured in 2D or 3D conditions. Panel F shows quantified IDO1 / actin fluorescence intensity of CFPAC-1 in 2D and 3D conditions (n=4, paired t-test, p-values ​​shown, error bars are SEM). Panel G shows results of CFPAC-1 cells cultured in 2D or 3D conditions for 24 h and treated with epacadostat (1 μM) or vehicle-only control for 16 h before media kynurenine was analyzed by LCMS (1ex, triplicate wells, error bars are standard deviation). Panel H shows CFPAC-1 or HPAF-II cells cultured in either 2D or 3D conditions for 24 h and then treated with JAKi (1 μM) or vehicle-only control (veh.) and / or human IFNγ (1 ng / ml) for 16 h. Cells were then lysed and the indicated proteins were analyzed by immunoblotting.

[0177] In Figure 70, CFPAC-1 or HPAF-II cells were grown for 24 hours in normal tissue culture plates (2D) or ultra-low attachment tissue culture plates (3D), or cultured in 2D and treated with 1 ng / ml IFNγ. Lysates (Panel A) were blotted for the indicated proteins, and (Panel B) the fluorescence intensity of IDO1 relative to actin (loading control) was quantified (n=4, paired t-test, p-values ​​shown, error bars are SEM). The indicated cell lines were grown in either 2D or 3D for 24 hours and lysates were immunoblotted for the indicated proteins after (panel C) 16 hours of treatment with MG132 (20 μM) or vehicle only control, (panel D) treatment with bafilomycin A1 (100 nM) or vehicle only control for the indicated times, or (panel E) 16 hours of treatment with JAKi (at the indicated concentrations), vehicle only control, or IFNγ (1 ng / ml).

[0178] Adhesion-dependent (AI) growth stimulated IDO1 expression is regulated by JAK / STAT signaling: The molecular mechanism by which adhesion-dependent (AI) growth upregulates IDO1 expression was examined. Treatment with the proteasome inhibitor MG132 (Figure 70 panel C) or the lysosomal inhibitor bafilomycin (Figure 70 panel D) had no effect on IDO1 protein levels. The data indicated that the increase in IDO1 levels observed during AI growth was not due to changes in IDO1 degradation via the proteasome or lysosomal system.

[0179] IFNγ mediates changes in gene expression through activation of the JAK / STAT signaling cascade. The role of IFNγ-independent AI proliferation on activation of the JAK / STAT pathway leading to increased IDO1 expression was examined. Activation of STAT proteins (by phosphorylation) was examined using small molecule JAK inhibitor I (JAKi). Results showed that STAT3 phosphorylation was increased by AI proliferation (Figure 69 Panel H), suggesting upregulation of JAK / STAT pathway activation. This appeared to be specific for STAT3, as such an increase was not detected for STAT1 (Figure 70 Panel E). The upregulation of IDO1 protein levels in AI-proliferated cells was blocked by treatment with JAKi (Figure 69 Panel H). These data indicated that during AI proliferation, the JAK / STAT pathway was activated (similar to IFNγ treatment) and that the stimulation increased IDO1 expression.

[0180] Tryptophan provides a one-carbon unit for purine synthesis in vitro: during metabolism of tryptophan via the kynurenine pathway, a number of metabolites are formed that are known to have potentially important roles in cancer metabolism (Figure 69 Panel A). To examine the production of such metabolites from tryptophan in IDO1-expressing cancer cells, cells were cultured in vitro. 13 C 11 The cells were cultured with IFNγ in the presence of -tryptophan. Liquid chromatography mass spectrometry (LCMS) was used to track the incorporation of labeled carbon into kynurenine pathway metabolites and across them into nucleotide synthesis and the TCA cycle. 13 C 11-tryptophan was readily taken up and extensively labeled the cellular tryptophan pool for over 24 hours. A high percentage (approximately 95%) of labeling was observed with kynurenine (Figure 71). Downstream of kynurenine, a small amount of labeling was identified with alanine, however, this was a very small percentage (<1%) of the total alanine pool. Tryptophan can be metabolized to acetyl-coA via either alanine or α-ketoadipic acid. Some evidence of labeling from tryptophan in acetyl-coA was also detected (Figure 71). No evidence of labeling in TCA cycle components or NAD / H or NADP / H was observed, suggesting limited effects of tryptophan on these pathways in PDAC cells.

[0181] During the production of kynurenine from tryptophan, a one-carbon unit is liberated as formate. A potential destination of this formate is to enter the THF cycle. From here, tryptophan-derived carbon could be used in multiple protein anabolic pathways, including purine nucleotide synthesis. Tryptophan-derived carbon was observed in purine nucleotides (Figure 71), indicating that tryptophan is a legitimate source of one-carbon units in the THF cycle in PDAC cells. Another THF-dependent fate of the one-carbon is de novo serine synthesis (by combination with glycine via SHMT1 / 2), and increased labeling of serine from labeled tryptophan was also observed.

[0182] The degree of labeling seen with purines is remarkable, especially considering that these cells were grown with ample exogenous serine, the major one-carbon source (the medium contained 0.4 mM serine compared with 0.08 mM tryptophan). Approximately 30% of the ATP pool and approximately 40% of the GTP pool were labeled with carbon derived from tryptophan. Labeling with serine was at a much lower rate (approximately 3%), suggesting that purine labeling occurs directly via the THF cycle.

[0183] We confirmed that AI proliferation stimulates the incorporation of one-carbon units derived from tryptophan into purine nucleotides, and found increased rates of labeling in AMP, ADP, ATP, and GTP in AI-proliferated cells, albeit to a lesser extent than that seen in IFNγ-treated cells (Figure 72). Labeling was prevented by treatment with the IDO1 inhibitor epacadostat (Figure 72). Overall, these data clearly demonstrate that PDAC cells expressing IDO1 can utilize tryptophan as an important source of one-carbon units in purine nucleotide synthesis.

[0184] Figure 72 shows that the cells were cultured in 2D or 3D for 24 hours and then unlabeled ( 12 C) or 13 C 11 CFPAC-1 cells treated for 24 h with epacadostat (1 μM) or vehicle-only control in the presence of either tryptophan are shown, and the intracellular content of the indicated nucleotides was analyzed by LCMS (1ex, triplicate wells, error bars are standard deviations).

[0185] Tryptophan can contribute one-carbon units to purine synthesis in vivo: We investigated whether tryptophan can contribute one-carbon units to purine synthesis in PDAC tumors in vivo. Given the high variability of IDO1 expression in spontaneous GEMM and KPC allograft tumors, and to accurately recapitulate the situation in high IDO1-expressing tumors, KPC cells were engineered to constitutively express IDO1. Cells were implanted as subcutaneous allografts into syngeneic immunocompetent mice (Figure 73 Panel A). Once tumors were formed, mice were 13 C 11-Tryptophan solution was administered intraperitoneally once, and the inventors evaluated the incorporation of tryptophan-derived carbon using LCMS at a single time point after injection. The inventors found a significant increase in the labeling percentage of serine, ATP, ADP, GMP, and GDP in IDO1-expressing tumor tissues compared to the empty vector (EV) control (Figure 73 Panel B, Figure 74). These results indicate that IDO1-expressing tumors can indeed incorporate tryptophan-derived carbon into purine nucleotides in vivo.

[0186] Figure 73 shows that one-carbon units derived from tryptophan are incorporated into nucleotides in in vivo pancreatic tumors. Panel A shows a schematic detailing the experimental approach in this figure. Panel B shows pure C57BL / J Pdx1-cre;Kras expressing IDO1 or an empty vector control (EV). G12D / + ;Trp53 R172H / + KPC cells from mice were injected subcutaneously into the flanks of C57BL / J mice, and once tumors were formed, mice were administered 800 μL of 120 mM 13 C 11 Tryptophan was given and left for 3 hours. Tumor tissue was excised and analyzed by LCMS (fraction of major isotopologue compared to total shown, EV n=7, IDO1 n=7, unpaired t-test, p-values ​​shown, error bars are standard deviation).

[0187] FIG. 74 shows pure C57BL / J Pdx1-cre;Kras expressing IDO1 or empty vector control (EV) injected subcutaneously into the flank of C57BL / J mice. G12D / + ;Trp53 R172H / + Data from KPC cells from mice are shown. Once tumors were formed, mice were injected intraperitoneally with 800 μL of 120 mM 13 C 11 Tryptophan was administered for 3 hours, and tumor tissue was excised and analyzed by immunoblotting for the indicated proteins.

[0188] PDAC cells excrete formate derived from tryptophan: We investigated whether IDO1-expressing PDAC cells liberated formate generated from tryptophan. 13 C 11 Labeled formate was identified by gas chromatography-mass spectrometry in spent medium (+IFNγ) from IDO1-expressing CFPAC-1 and HPAF-II cells after incubation with -tryptophan (Figure 75 Panels A and B). The release of formate from tryptophan was significantly higher than that from serine in CFPAC-1 cells and was comparable in HPAF-II cells. These results were surprising since serine is generally considered to be the predominant one-carbon source in cancer cells and exogenous serine levels are higher than tryptophan.

[0189] Stellate cells take up tryptophan-derived formate and utilize it in purine synthesis: We investigated the ability of pancreatic stellate cells to take up tryptophan-derived formate released by PDAC cells and utilize it in the synthesis of purine nucleotides. To directly test the ability of pancreatic stellate cells to take up and utilize exogenous formate, we transformed immortalized mouse stellate cells (ImPSCs) into 13 The cells were cultured in medium supplemented with C1-formate. LCMS analysis revealed that stellate cells consumed extracellular formate and incorporated one carbon into purines. Purine synthesis utilized two THF-derived one-carbons, resulting in the elevation of the major isotopologue peaks at m+1 and m+2 (Figure 76 Panel B).

[0190] To assess whether the formate derived from tryptophan produced in PDAC cells could be used in a similar manner, two techniques were used: (1) transfer to conditioned medium and (2) direct co-culture (Figure 75 Panel C). 13 C 11-tryptophan-containing medium and conditioned medium was collected after 24 hours. Filtered medium was then transferred on top of the astrocytes prior to analysis of the astrocytes by LCMS. Purine nucleotide and serine labeling was detected in astrocytes fed with conditioned medium from IDO1-expressing (+IFNγ) PDAC cells (Figure 75 Panels D-G). Labeling was prevented when PDAC cells were treated with epacadostat during the conditioning step. Labeling was not affected when epacadostat was added after conditioning (i.e., while the astrocytes were cultured in conditioned medium), indicating that IDO1 activity in PDAC cells, but not in astrocytes, was crucial for astrocyte formate utilization. The same was observed when the experiment was repeated with ImPSC #2 cells (Figure 76 Panels E-G).

[0191] To further confirm this finding, a direct co-culture assay was performed. CFPAC-1 cells were co-cultured with ImPSC cells engineered to ectopically express GFP for 24 h. The co-culture medium was 13 C 11 -tryptophan and IFNγ. ImPSC-GFP cells were then separated from PDAC cells by FACS and subjected to LCMS analysis. Purine nucleotide labeling was evident in astrocytes co-cultured with PDAC cells in the presence of IFNγ (Figure 75 Panels H-K). With this method, the labeled fraction was generally smaller and no clear labeling at serine was identified. Nucleotide labeling was not observed in astrocytes cultured alone. Importantly, labeling was also reduced when IDO1 levels were low (-IFNγ) or IDO1 was inhibited by treatment with epacadostat (Figure 75 Panels H-K).

[0192] Figure 75 shows that cancer cells liberated formate from tryptophan, which was consumed by pancreatic stellate cells and incorporated into nucleotides. CFPAC-1 (panel A) or HPAF-II (panel B) cells were cultured in 3D for 4 days and then incubated with unlabeled ( 12 C). 13 C 11Tryptophan, or 13 C3 15 CFPAC-1 cells were treated for 24 h with IFNγ (1 ng / ml) or vehicle-only control in the presence of either N1 serine. The medium content of formic acid was analyzed by derivatization and GC-MS (1ex, triplicate wells, error bars are standard deviations). Panel C shows a schematic of the experimental approach used in panels D-K. CFPAC-1 cells were treated with unlabeled ( 12 C) or 13 C 11 ImPSCs were treated with vehicle only control or human IFNγ (1 ng / ml) and epacadostat (epac., 1 μM) or vehicle only control in the presence of tryptophan. Conditioned medium was collected after 24 hours and ImPSCs were cultured in this medium or in non-conditioned treatment matched medium. After 24 hours, intracellular contents of serine (panel D), ATP (panel E), ADP (panel F), and AMP (panel G) were analyzed by LCMS (fractions of major isotopologues relative to total shown, 1ex, triplicate wells, error bars are standard deviation). ImPSC-GFP cells were cultured for 24 hours in 2D as monoculture or in coculture with CFPAC-1 cells. Then, 13 C 11 Cells were treated with vehicle-only control or human IFNγ (1 ng / ml) and epacadostat (1 μM) or vehicle-only control in the presence of tryptophan for 24 hours. Cells were then trypsinized, sorted using FACS for GFP-positive cells, and the intracellular content of serine (panel H), ATP (panel I), ADP (panel J), and AMP (panel K) was analyzed by LCMS (fractions of major isotopologues relative to total shown, 1ex, triplicate wells, error bars are standard deviation). Panel L shows a proposed model for the use of tryptophan-derived formate in pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells.

[0193] FIG. 76 shows the changes in ATP, DP, AMP, and GTP in ImPSC #1, ImPSC #2, and ImPSC #3 cells. 13The cellular uptake of C1 formic acid was shown. ImPSC #1, ImPSC #2 & ImPSC #3 cells 13 CFPAC-1 cells were incubated for 24 h in the presence of C1 formate and the intracellular contents of all possible destinations of one carbon from formate, ATP (panel A), ADP (panel B), AMP (panel C), and GTP (panel D), were analyzed by LCMS (1ex, triplicate wells, error bars are standard deviation). 12 C) or 13 C 11 Treated with IFNγ (1 ng / ml) and / or epacadostat (1 μM) in the presence of tryptophan and / or vehicle-only control. Conditioned medium was collected after 24 hours and ImPSC#2 cells were cultured in this medium or in non-conditioned treatment matched medium. After 24 hours, the intracellular content of ATP (panel E), ADP (panel F), and serine (panel G) was analyzed by LCMS (1ex, triplicate wells showing fractions of major isotopologues relative to the total, error bars are standard deviation).

[0194] Enrichment in pancreatic cancer and relative expression of IDO1 in GEM models were determined. Tumors from KPC mice showed clear elevation of IDO1 compared to healthy tissue in vivo, but IDO1 expression was absent in cell culture. Expression data from a large set of human cancers further demonstrated that high IDO1 expression was found in multiple tumors, but was not observed in cell culture.

[0195] Beyond the canonical IDO1 activator IFNγ, we examined the effects of metabolic perturbations caused by hypoxia, rotenone treatment, or galactose on IDO1 expression. None of the conditions altered IDO1 levels. Transfer of PDAC cells from standard monolayer culture to adhesion-dependent conditions resulted in upregulation of IDO1, albeit to a lesser extent by IFNγ. The mechanism of action was examined and regulated IDO1 in adhesion-dependent growth via the JAK / STAT signaling pathway.

[0196] Improved understanding of IDO1 expression has allowed detailed analysis of IDO1-dependent tryptophan metabolism in PDAC cells. The kynurenine pathway makes a significant contribution to nucleotide synthesis in PDAC cells in vitro, contributing carbon to approximately 30% of the purine pool over a 24-hour period. IDO1-dependent tryptophan labeling was detected in tumor serine and purine pools after a single injection of 13C11-tryptophan. PDAC cells were tested for tryptophan-derived formate excretion. Certain PDAC cells liberated twice the content of tryptophan-derived formate relative to serine-derived formate, despite a 4:1 predominance of exogenous serine over tryptophan. We also observed a robust ability of stellate cells to capture tryptophan-derived formate produced by PDAC cells and incorporate the formate into nucleotide synthesis.

[0197] Overall, the results indicate that IDO1 can affect one-carbon metabolism in cancer and stromal cells. In IDO1-expressing tumors, we showed that tryptophan was a legitimate one-carbon source in the THF cycle. The data also provide a mechanistic explanation for why tryptophan may be the most highly depleted stromal nutrient in PDAC.

[0198] Example 16: Effects of epacadostat on cell proliferation and nucleotide synthesis Epacadostat enhances antiproliferative effects or serine starvation in KPC cells (Kras mut p53 mut Tumor cells (derived from pancreatic tumors in genetically modified mice) were seeded in 24-well plates and allowed to adhere overnight. Cells were washed with PBS and received either control medium containing all amino acids with or without the IDO1 inhibitor epacadostat (1 μM) or adapted medium lacking serine (-serine). Cell numbers were recorded every 24 hours for 5 days. The plate at time 0 was used to calculate the starting cell number.

[0199] Figure 77 left panel shows cell proliferation over 5 days in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM). Right panel shows fold change in cell number at day 5 compared to day 0 in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM).

[0200] Serine starvation was shown to increase the amount of tryptophan-derived carbon used in nucleotide synthesis in an IDO1-dependent manner. mut p53 mut Tumor cells (derived from pancreatic tumors in genetically engineered mice) were seeded in 6-well plates and allowed to adhere overnight. Cells were fed with medium containing carbon-13 labeled tryptophan with (+) or without (-) serine (+ all other amino acids) and with or without the IDO1 inhibitor epacadostat (1 μM). After 48 hours, metabolites were extracted from cells and analyzed by LCMS. The labeled fraction (derived from carbon-13) of purine nucleotides (ATP, ADP, AMP, GDP, GTP) is shown.

[0201] FIG. 78 shows the cellular carbon-13-derived labeled fractions of AMP, ADP, ATP, GDP, and GMP in cells treated with 1) control + vehicle; 2) -serine + vehicle; 3) control + epacadostat (1 μM); or 4) -serine + epacadostat (1 μM).

[0202] Aspects The following non-limiting embodiments provide illustrative examples of the present invention, but do not limit the scope of the invention. Embodiment 1. A method of treating cancer in a subject in need thereof comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition for a first period of time, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; b) radiation therapy for a second period of time; and c) after the first and second periods of time, waiting a third period of time, wherein the subject is not administered the pharmaceutical composition or radiation therapy during the third period of time. Embodiment 2. The method of embodiment 1, wherein said cancer is rectal cancer. Embodiment 3 The method of embodiment 1, wherein said cancer is breast cancer. Aspect 4. The method of any one of aspects 1-3, wherein said administration is oral. Aspect 5. The method of any one of aspects 1-4, wherein said radiation therapy is external beam therapy. The method of embodiment 5, wherein said external beam therapy is three-dimensional conformal radiotherapy (3D-CRT). Aspect 7. The method of aspect 5, wherein said external beam therapy is intensity modulated radiation therapy (IMRT). Aspect 8. The method of any one of Aspects 1-7, wherein said radiation therapy comprises administering to the subject about 5 Gray (Gy) to about 50 Gy of radiation. Embodiment 9. The method of any one of embodiments 1-8, wherein said radiation therapy comprises administering about 5 Gy of radiation to the subject. Embodiment 10. The method of any one of embodiments 1-8, wherein said radiation therapy comprises administering about 50 Gy of radiation to the subject. Aspect 11. The method of any one of aspects 1-4 or 8-10, wherein the radiation therapy is internal beam therapy. Embodiment 12 The method of any one of embodiments 1 to 11, wherein said at least two amino acids are serine and glycine. Embodiment 13 The method of any one of embodiments 1 to 12, wherein said pharmaceutical composition is further substantially devoid of proline. Embodiment 14 The method of any one of embodiments 1 to 13, wherein said pharmaceutical composition further substantially lacks cysteine. Embodiment 15 The method of any one of embodiments 1-14, further comprising administering to the subject a high fat diet. Embodiment 16 The method of embodiment 15, wherein said high fat diet has greater than about 50% of daily calories from fat. Embodiment 17 The method of any one of embodiments 1 to 16, further comprising administering to the subject a low carbohydrate diet. Embodiment 18 The method of embodiment 17, wherein said low carbohydrate diet has less than about 50% of daily calories from carbohydrates. Embodiment 19. The method of any one of embodiments 1 to 18, further comprising administering to the subject a low protein diet. Embodiment 20 The method of embodiment 19, wherein said low protein diet has less than about 15% of daily calories from total protein. Embodiment 21 The method of any one of embodiments 1-20, wherein the first time and the second time are equivalent. Embodiment 22 The method of any one of embodiments 1 to 21, wherein the first time and the second time are 5 days. Embodiment 23 The method of any one of embodiments 1-20, wherein the first time period and the second time period are longer than the third time period. Embodiment 24 The method of any one of embodiments 1 to 23, wherein the third time period is 2 days. Embodiment 25. The method of any one of embodiments 1 to 24, further comprising repeating steps a), b), and c). Embodiment 26. A method of treating cancer in a subject in need thereof, comprising: a) administering to said subject a therapeutically effective amount of a pharmaceutical composition, said pharmaceutical composition substantially lacking at least two amino acids; and b) administering a therapeutically effective amount of an immunotherapy, said immunotherapy being administered at least twice per day. The method of embodiment 26, wherein the cancer is pancreatic cancer. The method of embodiment 26, wherein the cancer is colon cancer. The method of embodiment 26, wherein the cancer is breast cancer. The method of embodiment 26, wherein the cancer is cervical cancer. The method of embodiment 26, wherein the cancer is lung cancer. Embodiment 32 The method of any one of embodiments 26 to 31, wherein the immunotherapy is an IDO1 inhibitor. The method of embodiment 32, wherein the IDO1 inhibitor is indoximod. The method of embodiment 32, wherein the IDO1 inhibitor is naboximod. The method of embodiment 32, wherein the IDO1 inhibitor is epacadostat. Embodiment 36 The method of any one of embodiments 26 to 35, wherein the at least two amino acids are serine and glycine. Embodiment 37 The method of any one of embodiments 26 to 36, wherein the pharmaceutical composition is substantially devoid of three amino acids. Embodiment 38. The method of embodiment 37, wherein the three amino acids are serine, glycine, and proline. Embodiment 39. The method of embodiment 37, wherein the three amino acids are serine, glycine, and cysteine. Embodiment 40. The method of any one of embodiments 26 to 39, wherein the therapeutically effective amount of said immunotherapy is from about 25 mg to about 500 mg. Embodiment 41 The method of any one of embodiments 26 to 40, wherein the therapeutically effective amount of said immunotherapy is about 25 mg. Embodiment 42 The method of any one of embodiments 26 to 40, wherein the therapeutically effective amount of said immunotherapy is about 50 mg. Embodiment 43 The method of any one of embodiments 26 to 40, wherein the therapeutically effective amount of said immunotherapy is about 100 mg. Embodiment 44 The method of any one of embodiments 26 to 40, wherein the therapeutically effective amount of said immunotherapy is about 300 mg. Embodiment 45 The method of any one of embodiments 26 to 44, wherein said immunotherapy is administered twice per day. Embodiment 46 The method of any one of embodiments 26 to 44, wherein the immunotherapy is administered three times per day. Embodiment 47. A method of treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition is substantially devoid of at least two amino acids; and b) a therapeutically effective amount of epacadostat. The method of embodiment 47, wherein the cancer is pancreatic cancer. The method of embodiment 47, wherein the cancer is colon cancer. Embodiment 50 The method of embodiment 47, wherein the cancer is breast cancer. Embodiment 51 The method of embodiment 47, wherein the cancer is cervical cancer. The method of embodiment 47, wherein the cancer is lung cancer. Embodiment 53. The method of any one of embodiments 47 to 52, wherein the at least two amino acids are serine and glycine. Embodiment 54 The method of any one of embodiments 47 to 53, wherein the pharmaceutical composition is substantially devoid of three amino acids. Embodiment 55. The method of embodiment 54, wherein the three amino acids are serine, glycine, and proline. Embodiment 56. The method of embodiment 54, wherein the three amino acids are serine, glycine, and cysteine. Embodiment 57. The method of any one of embodiments 47 to 56, wherein the therapeutically effective amount of epacadostat is from about 25 mg to about 500 mg. Embodiment 58 The method of any one of embodiments 47 to 57, wherein the therapeutically effective amount of epacadostat is about 25 mg. Embodiment 59 The method of any one of embodiments 47 to 57, wherein the therapeutically effective amount of epacadostat is about 50 mg. Embodiment 60 The method of any one of embodiments 47-57, wherein the therapeutically effective amount of epacadostat is about 100 mg. Embodiment 61 The method of any one of embodiments 47 to 57, wherein the therapeutically effective amount of epacadostat is about 300 mg.

Claims

1. 1. A pharmaceutical composition for use in a method of treating cancer in a subject in need thereof, the treatment comprising: a) administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition for a first period of time, wherein said pharmaceutical composition is substantially devoid of at least two amino acids, said at least two amino acids being serine and glycine; b) administering a second period of radiation therapy to the subject in need thereof; and c) waiting a third time period after said first and second time periods, wherein said subject in need thereof is not administered said pharmaceutical composition or administered said radiation therapy during said third time period; The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the cancer is rectal cancer.

3. The pharmaceutical composition of claim 1, wherein the cancer is breast cancer.

4. 2. The pharmaceutical composition of claim 1, wherein the administration is oral.

5. 2. The pharmaceutical composition of claim 1, wherein the radiation therapy is external beam therapy.

6. 6. The pharmaceutical composition of claim 5, wherein the external beam therapy is three-dimensional conformal radiotherapy (3D-CRT).

7. 6. The pharmaceutical composition of claim 5, wherein the external beam therapy is intensity modulated radiation therapy (IMRT).

8. 10. The pharmaceutical composition of claim 1, wherein said radiation therapy comprises administering to said subject in need thereof about 5 Gray (Gy) to about 50 Gy of radiation.

9. The pharmaceutical composition of claim 1 , wherein the radiation therapy is internal beam therapy.

10. The pharmaceutical composition of claim 1 , further comprising a substantial lack of proline.

11. 10. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering to the subject in need thereof a high fat diet, wherein the high fat diet has greater than about 50% of its daily calories from fat.

12. 2. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering a low carbohydrate diet to the subject in need thereof, the low carbohydrate diet having less than about 50% of its daily calories from carbohydrates.

13. 2. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering a low carbohydrate diet to the subject in need thereof, the low carbohydrate diet having less than about 10% of its daily calories from carbohydrates.

14. 2. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering to the subject in need thereof a low protein diet, wherein the low protein diet has less than about 15% of its daily calories from total protein.

15. 2. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering to the subject in need thereof a low protein diet, the low protein diet having a total protein content of less than about 40 g / day.

16. 2. The pharmaceutical composition of claim 1, wherein the first time period and the second time period are equivalent.

17. 2. The pharmaceutical composition of claim 1, wherein the first time and the second time are 5 days.

18. 2. The pharmaceutical composition of claim 1, wherein the first time period and the second time period are also longer than the third time period.

19. 20. The pharmaceutical composition of claim 18, wherein the third time is 2 days.

20. 2. The pharmaceutical composition of claim 1, wherein the treatment further comprises repeating steps a), b), and c).

21. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is further substantially devoid of cysteine.

22. 1. A pharmaceutical composition for use in a method of treating cancer in a subject in need thereof, the treatment comprising: a) administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition, wherein said pharmaceutical composition is substantially devoid of at least two amino acids, and wherein said at least two amino acids are serine and glycine; and b) administering a therapeutically effective amount of immunotherapy The pharmaceutical composition.

23. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is substantially devoid of three amino acids.

24. 24. The pharmaceutical composition of claim 23, wherein the three amino acids are serine, glycine, and proline.

25. 24. The pharmaceutical composition of claim 23, wherein the three amino acids are serine, glycine, and cysteine.

26. 23. The pharmaceutical composition of claim 22, wherein the immunotherapy is an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor.

27. 27. The pharmaceutical composition of claim 26, wherein the IDO1 inhibitor is indoximod, navoximod or epacadostat.

28. The pharmaceutical composition of claim 22, wherein the immunotherapy is an antibody therapy.

29. 29. The pharmaceutical composition of claim 28, wherein the antibody therapy is a programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), or cytotoxic T-lymphocyte antigen-4 (CTL4-A) modulator.

30. 29. The pharmaceutical composition of claim 28, wherein the antibody therapy is an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-PD-L2 monoclonal antibody, or an anti-CTL1-4 antibody.

31. 23. The pharmaceutical composition of claim 22, wherein the cancer is pancreatic cancer, colon cancer, breast cancer, cervical cancer or lung cancer.

32. 23. The pharmaceutical composition of claim 22, wherein the therapeutically effective amount of said immunotherapy is from about 25 mg to about 500 mg.

33. 23. The pharmaceutical composition of claim 22, wherein the therapeutically effective amount of the immunotherapy is about 25 mg, about 50 mg, about 100 mg, or about 300 mg.

34. 23. The pharmaceutical composition of claim 22, wherein the immunotherapy is administered at least twice per day.

35. 35. The pharmaceutical composition of claim 34, wherein the immunotherapy is administered two or three times per day.

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