Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
  • A61N5/1001—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy using radiation sources introduced into or applied onto the body; brachytherapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4245—Oxadiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
  • A61N5/1042—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy with spatial modulation of the radiation beam within the treatment head
  • A61N5/1045—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy with spatial modulation of the radiation beam within the treatment head using a multi-leaf collimator, e.g. for intensity modulated radiation therapy or IMRT
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/175—Amino acids

Definitions

• the invention provides a method of treating a cancer in a subject in need thereof, the method 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, for a first amount of time; b) a radiation therapy for a second amount of time; and c) after the first amount of time and the second amount of time, waiting a third amount of time, wherein the subject is not administered the pharmaceutical composition or the radiotherapy during the third amount of time.
• the invention provides a method of treating a cancer in a subject in need thereof, the method 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.
• a pharmaceutical composition wherein the pharmaceutical composition is substantially devoid of at least two amino acids
• IDO1 indoleamine 2,3 -dioxygenase 1
• the invention provides a method of treating a cancer in a subject in need thereof, the method 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.
• FIG. 1 depicts the serine synthesis pathway.
• FIG. 2 depicts growth curves of the colon cancer cell lines grown in complete medium (CM) or equivalent medium lacking serine and glycine (-SG) and treated or not with 10 pM PH755. Data are presented as mean ⁇ SEM of triplicate cultures and are representative of at least two independent experiments (*p ⁇ 0.05, ** p ⁇ 0.01, ***p ⁇ 0.001, 0.0001; two-way ANOVA with Tukey’s post hoc test).
• FIG. 3 depicts growth curves of the indicated cell lines grown in complete medium (CM) or equivalent medium lacking serine and glycine (-SG) and treated or not with 10 pM PH755. Data are presented as mean ⁇ SEM of triplicate cultures and are representative of at least two independent experiments (* p ⁇ 0.05, ** p ⁇ 0.01, ***p ⁇ 0.001, 0.0001; two-way
• FIG. 4 shows the percentage of BrdU positive cells in HCT116 and DLD-1 cells grown in CM or -SG medium +/- lOpM PH755 for 48 hours followed by a 5 hours incubation with 10 pM BrdU.
• Data represents mean ⁇ SEM of 3 independent experiments (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, one-way ANOVA with Tukey’s post hoc test).
• FIG. 5 shows the gating strategy to determine the percentage of BrdU positive cells (left panel) and the percentage of cells undergoing different phases of the cell cycle (right panel), taking as an example HCT116 and DLD-1 cells grown in CM and incubated for 30 minutes with lOpM BrdU.
• FIG. 6 shows intracellular serine and glycine levels in HT-29, HCT116, DLD-1, and MDA-MB-468 cells grown in CM or -SG medium +/- 10 pM PH755 were 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). [0013] FIG. 7 shows the percentage of SubGl cells in HCT116 and DLD-1 cells grown in CM or -SG medium +/- 10 pM PH755 for 48 hours. Data represents mean ⁇ SEM of 5 independent experiments (** p ⁇ 0.01, *** p ⁇ 0.001, one-way ANOVA with Tukey’s post hoc test).
• FIG. 8 shows cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 2 days (HCT116) or 3 days (DLD-1).
• Western blots show the expression of cleaved Caspase-3 and Caspase-3.
• Membrane was reprobed with vinculin as a loading control. Data are representative of three independent experiments.
• FIG. 9 shows the intracellular serine level in HT-29, HCT116, DLD-1, and MDA- MB-468 cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[ 13 C]-glucose was measured by LC-MS. Metabolite percentages are represented 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).
• FIG. 10 shows intracellular glycine level in HT-29, HCT116, DLD-1 and MDA-MB- 468 cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[ 13 C]-glucose was measured by LC-MS. Metabolite percentages are represented 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).
• FIG. 11 HT-29 and DLD-1 cells infected with Cas9/PHGDH single guide RNA (sgRNA) were cultured in CM or -SG medium for 24 hours. Western blot shows efficient PHGDH depletion in these cells. Membrane was reprobed with vinculin as a loading control.
• FIG. 12 shows growth curves of HT-29 and DLD-1 cells infected with Cas9/PHGDH sgRNA (PHGDH) grown in CM or in -SG medium.
• FIG. 13 shows intestinal tumor organoids derived from Vill-creER;Apcfl/fl (Ape) and Vill-creER;Apcfl/fl;KrasG12D/+ (Ape Kras) mice grown in CM or -SG medium supplemented or not with 10 pM PH755.
• Left panel Representative pictures of the organoids are shown before (day 0) and 2 days after medium change.
• FIG. 14 shows intestinal organoids with Ape truncation (Apc5) or derived from Villin- CreER;Apcfl/fl;KrasG12D/+ mice (Ape Kras 2) grown for 4 days in tumor organoid medium with (CM) or without (-SG) serine and glycine supplemented or not with 10 pM PH755. Representative pictures of the organoids from at least 2 independent experiments are shown before (day 0), 2 days and 4 days after medium change. Scale bar represents 200 pm.
• FIG. 15 shows normal organoids derived from the proximal part of healthy small intestine from a Villin-CreERT2 mouse grown in normal organoid medium (containing Wnt- 3a) with (CM) or without (-SG) serine and glycine supplemented or not with 10 pM PH755. Representative pictures of the organoids from 3 independent experiments are shown 3 days after medium change. Scale bar represents 200 pm.
• FIG. 16 shows four patient-derived colorectal organoids grown in human organoid medium with (CM) or without (-SG) serine and glycine supplemented or not with 10 pM PH755. Representative pictures of the organoids from at least 2 independent experiments are shown 10 to 12 days after medium change. Scale bar represents 200 pm.
• FIG. 17 depicts a scheme representing the fate of uniformly carbon labelled glucose (m+6) into purine and glutathione synthesis.
• Glucose is converted through the pentose phosphate pathway into ribose-5-phosphate, a five-carbon sugar (m+5), that will be added to purine bases to form purine nucleosides.
• Purine rings also contain two one-carbon units and an intact glycine that can both come from serine metabolism.
• Serine is synthesized from the glycolytic intermediate 3-PG, producing an m+3 isotopomer from uniformly labelled glucose.
• Serine (m+3) can generate labelled glycine (m+2) and labelled one-carbon units (m+1).
• ribose phosphate glycine
• glycine a group consisting of glutathione
• glutamate both can be m+2 labelled from glucose
• cysteine The main isotopomer (m+2) of glutathione is likely to be derived from m+2 glycine with the m+4 labelling reflecting incorporation of m+2 glycine and m+2 glutamate.
• FIG. 18 shows intracellular ATP levels in HT-29, HCT116, DLD-1 and MDA-MB- 468 cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[ 13 C]-glucose were measured by LCMS. Metabolite percentages are represented as mean + SEM of triplicate cultures and are representative of three independent experiments.
• FIG. 19 shows intracellular glutamate levels in cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[ 13 C]-glucose were measured by LC-MS. Metabolite percentages are represented as mean ⁇ SEM of triplicate wells and are representative of three independent experiments.
• FIG. 20 shows intracellular GSH and GTP levels in HT-29, HCT116, DLD-1 and MDA-MB-468 cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[ 13 C]- glucose were measured by LCMS. Metabolite percentages are represented as mean + SEM of triplicate cultures and are representative of three independent experiments. Statistics have been performed comparing the sum of m+6-9 % of metabolite pool for ATP and GTP and the sum of m+2-4 % of metabolite pool for GSH between experimental groups (* p ⁇ 0.05, ** p
• FIG. 21 shows intracellular ATP, GTP, and GSH levels in HT-29, HCT116 and DLD-1 cells grown in CM or -SG medium +/- 10 pM PH755 containing U-[13C]-glucose for 3 hours or 6 hours were measured by LC-MS. Metabolite percentages are represented as mean ⁇ SEM of triplicate cultures and are representative of two independent experiments (* p
• FIG. 22 shows total levels of ATP, GTP and GSH in cells grown in CM or -SG medium +/- 10 pM PH755 were 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).
• FIG. 23 depicts a proliferation assay of HT-29 and HCT116 cells grown in -SG medium or -SG medium + 10 pM PH755 supplemented or not with 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).
• FIG. 24 and FIG. 25 show HCT116 cells were grown in -SG medium or -SG medium + 10 pM PH755 supplemented or not with 1 mM sodium formate (For), 0.4 mM glycine (Gly) or both (For/Gly) in presence of U-[ 13 C]-glucose.
• ATP and GTP levels were measured by LC-MS. Metabolite percentages are represented as mean + SEM of triplicate cultures and are representative of two independent experiments. Serine level was 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).
• FIG. 26 shows HT-29, HCT116 and DLD-1 cells grown in -SG medium + 10 pM PH755 supplemented with 1 mM sodium formate and 0.4 mM glycine for 24 hours in presence of 13 C2 15 Ni-Glycine for the last hour.
• 13 C2 15 Ni-Serine intracellular level was measured by LC-MS after adding a pulse of unlabeled ImM serine in the extracellular medium (+ serine pulse) or not (- serine pulse) 1 minute before metabolite extraction. Data are presented as mean + SEM of triplicate wells and are representative of three independent experiments.
• FIG. 27, FIG. 28, and FIG. 29 show HT-29 and DLD-1 cells infected with Cas9/PHGDH sgRNA (PHGDH) were grown in CM or in -SG medium in presence of U- [ 13 C]-glucose.
• 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 t test, (b-c) one-way ANOVA with Tukey’s post hoc test).
• FIG. 30 shows growth curves of HT-29, HCT116 and DLD-1 cells transiently depleted of ATF-4 using short interfering RNA (siRNA) and cultured in -SG medium for 4 days. 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; two-way ANOVA with Sidak’s post hoc test).
• siRNA short interfering RNA
• FIG. 31 shows cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• Western blots show the expression of the three SSP enzymes PHGDH, PSAT and PSPH (membrane was reprobed with vinculin as a loading control) or the expression of the ATF-4 target ASNS (membrane was reprobed with vinculin as a loading control).
• Data are representative of at least two independent experiments.
• FIG. 32 shows HT-29 and DLD-1 cells infected with Cas9/PHGDH single guide RNA (sgRNA) were cultured in CM or -SG medium for 24 hours.
• Western blots show expression of PHGDH, PSAT and PSPH (membrane was reprobed with vinculin as a loading control) or expression of ATF-4 and ASNS (membrane was reprobed with vinculin as a loading control) in these cells.
• Data are representative of three independent experiments.
• FIG. 33 shows HT-29 and HCT116 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 4h, 8h, 12h, 16h, and 24h.
• Western blots show SSP enzymes expression in these cells.
• Each membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 34 shows HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• Western blots show Phospho-GCN2 (Thr899), GCN2, Phospho-eIF2a (Ser51) and eIF2a.
• Membranes were reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 35 shows HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• cells were treated with 10 pM MG- 132, a proteasome inhibitor, 6 hours before harvesting the cells.
• Western blots show the expression of ATF-4 and its targets ASNS and PSAT. Membrane was reprobed with vinculin as a loading control. Data are representative of three independent experiments.
• FIG. 36 shows HT-29, HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 6 hours or 24 hours. Relative gene expression of ATF4 and PHGDH were measured by qPCR and normalized to the cells grown in CM for 6 hours. 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, 0.0001; one-way
• FIG. 37 shows HT-29, HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 6 hours or 24 hours.
• Relative gene expression of ASNS, PSAT1 and PSPH were measured by qPCR and normalized to the cells grown in CM for 6 hours. 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; oneway ANOVA with Tukey’s post hoc test).
• FIG. 38 shows HT-29, HCT116 and DLD-1 cells grown in CM, -SG medium or -SG medium +10 pM PH755 supplemented or not with 1 mM sodium formate plus 0.4 mM glycine (For/Gly).
• Western blot shows the expression of the three SSP enzymes PHGDH, PSAT and PSPH or the expression of ATF-4 and its canonical target ASNS after a 24 hours incubation in these medium.
• Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 39 shows HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• Puromycin 90 pM was added in culture medium 10 minutes before harvesting the cells.
• cells were treated with lOpg/mL cycloheximide (CHX), a well-known protein synthesis inhibitor, 5 hours before harvesting the cells.
• Western blots show puromycylated peptides. Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 40 shows HCT116 and DLD-1 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• cells were treated with 10 pM MG- 132, a proteasome inhibitor, 6 hours before harvesting the cells.
• Western blots show the expression of c-MYC, HIFla and p53.
• Membrane was reprobed with vinculin as a loading control. Data are representative of three independent experiments.
• FIG. 41 shows DLD-1 and HT-29 cells grown in CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours.
• Western blots show Phospho-p70S6K (Thr389) and p70S6K.
• Membrane was reprobed with actin as a loading control. Data are representative of three independent experiments.
• FIG. 42 shows HT-29, HCT116, and DLD-1 cells grown in CM for 24 hours.
• cells were treated with the ER stress inducer, tunicamycin (5 pg/mL) or 10 pM PH755.
• Western blots show the expression of the ATF-4 targets ASNS, PHGDH, PSAT and PSPH.
• Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 43 shows HT-29, HCT116, and DLD-1 cells grown in CM, -SG medium, or -SG medium +10 pM PH755 supplemented or not with 1 mM sodium formate plus 0.4 mM glycine (For/Gly).
• Western blot shows the expression of the three SSP enzymes PHGDH, PSAT and PSPH or the expression of ATF-4 and its canonical target ASNS after a 24 hours incubation in these medium.
• Membrane was reprobed with vinculin as a loading control. Data are representative of two independent experiments.
• FIG. 48 shows plasma that was taken at time of 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.
• CTR control diet
• -SG an equivalent diet lacking serine and glycine
• Veh vehicle
• PH755 PH755
• CTR control diet
• -SG an equivalent diet lacking serine and glycine
• Veh vehicle
• FIG. 53 shows plasma that was taken at time of sacrifice from mice subcutaneously injected with DLD-1 cells, fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755.
• Data are presented as mean + SEM. (** p ⁇ 0.01, **** p ⁇ 0.0001, unpaired two-tailed Student t test).
• FIG. 54 shows plasma that was taken at time of sacrifice from mice subcutaneously injected with HCT116 cells fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755.
• CTR control diet
• Veh vehicle
• FIG. 55 shows CD-I nude mice that were subcutaneously injected with DLD-1 cells and fed a diet with (CTR) or without serine and glycine (-SG) two days after tumor cell injection. Two days after diet change, mice were dosed orally with vehicle (Veh) or PH755 once daily for 20 days. The starting dosage of PH755 was 100 mg/kg (for 7 days) and was subsequently lowered to 50 mg/kg (for 6 days) and increased again to 75 mg/kg (for 7 days). Tumor volumes were measured three times a week by caliper measurement. Data are presented as mean ⁇ SEM.
• FIG. 56 shows CD-I nude mice that were subcutaneously injected with HCT116 cells and fed a diet with (CTR) or without serine and glycine (-SG) ten days after tumor cell injection.
• CTR CCR
• -SG serine and glycine
• FIG. 57 provides representative immunohistochemistry pictures and quantification of active Caspase-3 positive cells in DLD-1 tumors harvested at end-points from mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (-SG) and treated with vehicle (Veh) or PH755.
• Data are presented as mean + SEM. (*P ⁇ 0.05; unpaired two-tailed Student t test with Welch’s correction). Scale bar represents 50 pm.
• TIC total ion count
• FIG. 62 and FIG. 63 show representative immunohistochemistry pictures and quantification of PHGDH staining and PS AT staining in DLD-1 tumors harvested at endpoints from mice fed a control diet (CTR) or an equivalent diet lacking serine and glycine (- SG) and treated with vehicle (Veh) or PH755.
• Data are presented as mean ⁇ SEM. (* p ⁇ 0.05, ***p ⁇ 0.001; one-way ANOVA with Tukey’s post hoc test). Scale bar represents 50 pm.
• FIG. 64 PANEL A-PANEL E show how combining dietary restriction of serine and glycine and PHGDH inhibition cooperate to lower tumor burden and improve survival in genetic models of intestinal cancer.
• FIG. 65 PANEL A-PANEL D show the metabolomic impact of radiation on pancreatic and colorectal cancer cells in vitro.
• FIG. 66 PANEL A-PANEL E show the effect of dietary amino acid restriction in response to targeted radiotherapy in vivo.
• FIG. 67 shows IDO1 expression in vivo.
• PANEL A is a schematic detailing the methods used to analyze IDO1 expression in genetically engineered mouse models (GEMM) of pancreatic ductal adenocarcinoma (PDAC).
• PANEL B shows the indicated proteins after analysis by immunoblotting.
• PANEL D shows KPC A cells, a line isolated from tumors of mixed-background Pdxl-cre;Kras G12D/+ ;Trp53 R172H/+ mice were treated either with mouse IFNy (Ing/ml) for 24h, or subcutaneously injected into the flank of CD 1 -nude mice to form tumors.
• PANEL E shows KPC cells were isolated from pure C567B16/J background Pdxl- cre;Kras G12D/+ ;Trp53 R172H/+ mice and either treated in culture with mouse IFNy (1 ng/mL) for 24h or subcutaneously injected into the flank of C567B16/J mice to form tumors.
• PANEL F shows the indicated cell lines were treated with human IFNy (Ing/ml) for 24h and cell lysates blotted for the indicated proteins.
• FIG. 68 shows data extracted from the MERAV database showing the relative abundance of IDO 1 mRNA from microarrays.
• FIG 69 shows that IDO expression was upregulated by ultra-low-attachment tissue culture plate (3D) growth of cells and proteins, and IFNy via JAK/STAT signaling.
• PANEL A shows a schematic detailing the kynurenine pathway through which tryptophan is metabolized.
• PANEL B shows the expression of proteins cultured under either normoxic (20% O2) or hypoxic (1% O2) conditions.
• PANEL C shows the expression of proteins treated with rotenone (IpM) or vehicle only control.
• PANEL D shows the expression of proteins cultured in media containing either glucose (Glc) (10 mM) or galactose (Gal) (10 mM).
• PANEL E shows proteins cultured in 2D or 3D conditions.
• PANEL G shows the results of CFPAC-1 cells cultured in 2D or 3D conditions for 24h and treated with epacadostat (IpM) or vehicle only control for 16h before media kynurenine was analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• PANEL H shows CFPAC-1 or HPAF-II cells cultured in either 2D or 3D conditions for 24h and then treated for 16h with JAKi (IpM) or vehicle only control (veh.) and/or human IFNy (Ing/ml). Cells were then lysed and indicated proteins analyzed by immunoblotting.
• CFPAC-1 and HPAF-II cells were grown in either normal tissue culture plates (2D) or ultra-low-attachment tissue culture plates (3D) for 24 hours, and cell lysates immunoblotted for the indicated proteins fter 16h treatment with MG132 (20 pM) or vehicle-only control (PANEL C); after treatment for the indicated times with bafilomycin Al (100 nM) or vehicle-only control or (PANEL D); or after 16h treatment with JAKi (at indicated concentrations), vehicle-only control or IFNy (1 ng/ml) (PANEL E).
• FIG. 71 shows that tryptophan-derived one carbon units are incorporated into serine and nucleotides in pancreatic cancer cells.
• FIG. 72 shows CFPAC-1 cells cultured in 2D or 3D for 24h, then treated for 24h with epacadostat (IpM) or vehicle only control in the presence of either unlabeled ( 12 C) or 13 Cn tryptophan and intracellular quantities of the indicated nucleotides were analyzed by LCMS (lex, triplicate wells, error bars are std. dev.)
• FIG. 73 shows that tryptophan-derived one carbon units are utilized for serine and nucleotide synthesis in PDAC tumors in vivo.
• FIG. 74 shows data from KPC cells from pure C57BL/J Pdxl- cre;Kras G12D/+ ;Trp53 R172H/+ mice expressing IDO1 or empty -vector control (EV).
• the KPC cells were injected subcutaneously into the flanks of C57BL/J mice; once tumors had formed the mice were given 800pL of 120mM 13 Cn tryptophan by intraperitoneal injection and left for 3h.
• FIG. 75 shows that cancer cells released tryptophan-derived formate, which was consumed by pancreatic stellate cells and incorporated into nucleotides.
• CFPAC-1 PANEL A
• HPAF-II PANEL B
• IFNy IFNy
• vehicle only control IFNy
• Media quantities of formate were analyzed by derivatization and GC- MS (lex, triplicate wells, error bars are std. dev.).
• PANEL C shows a schematic of the experimental approaches used in PANEL D-PANEL K.
• CFPAC-1 cells were treated with vehicle only control or human IFNy (Ing/ml) and epacadostat (epac., IpM) or vehicle only control in the presence of unlabeled ( 12 C) or 13 Cn tryptophan.
• Conditioned media was collected after 24h and ImPSC’s were cultured in this media, or in non-conditioned treatment-matched media.
• intracellular quantities of serine (PANEL D), ATP (PANEL E), ADP (PANEL F) and AMP (PANEL G) were analyzed by LCMS (fraction of major isotopologues relative to total are shown, lex, triplicate wells, error bars are std. dev.).
• ImPSC-GFP cells were cultured for 24h in 2D as a monoculture or in co-culture with CFPAC-1 cells. Cells were then treated with vehicle only control or human IFNy (Ing/ml) and epacadostat (1 pM) or vehicle only control in the presence of 13 Cn tryptophan for 24h. Cells were then trypsinised and sorted using FACS for GFP-positive cells and intracellular quantities of serine (PANEL H), ATP (PANEL I), ADP (PANEL J) and AMP (PANEL K) were analyzed by LCMS (fraction of major isotopologues relative to total are shown, lex, triplicate wells, error bars are std. dev.). PANEL L shows a proposed model for the use of tryptophan-derived formate in pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells.
• PDAC pancreatic ductal adenocarcino
• FIG. 76 shows intracellular uptake of 13 Ci formate in ATP, DP, AMP, and GTP in ImPSC #1, ImPSC #2, and ImPSC #3 cells.
• ImPSC #1, ImPSC #2 & ImPSC #3 cells were cultured for 24h in the presence of 13 Ci formate and intracellular quantities of ATP (PANEL A), ADP (PANEL B), AMP (PANEL C) and GTP (PANEL D), all possible destination for formate-derived one carbons were analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• CFPAC-1 cells were treated with IFNy (Ing/ml) and/or epacadostat (IpM) and/or vehicle only controls in the presence of unlabeled ( 12 C) or 13 Cn tryptophan.
• Conditioned media was collected after 24h and ImPSC#2 cells were cultured in this media, or in nonconditioned treatment-matched media.
• intracellular quantities of ATP (PANEL E), ADP (PANEL F) and serine (PANEL G) were analyzed by LCMS (fraction of major isotopologues relative to total are shown lex, triplicate wells, error bars are std. dev.).
• FIG. 77 LEFT PANEL shows cell proliferation over 5 days in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• FIG. 77 RIGHT PANEL shows fold changes in cell number at day 5 compared to day 0 in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• FIG. 78 shows the labelled fractions derived from carbon- 13 in cells of AMP, ADP, ATP, GDP, and GMP in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• Direct mechanisms of promoting resistance to the therapeutic approach of reducing the availability of serine and/or glycine include those that promote increased availability of serine e.g. by serine biosynthesis (at tumor or systemic level) via enhanced expression of the de novo serine synthesis pathway (SSP) enzymes, whose expression can also be promoted by certain oncogenic mutations.
• SSP de novo serine synthesis pathway
• Another route for increasing serine availability is the promotion of serine recycling e.g. by mechanisms such as authophagy.
• Indirect mechanisms of resistance can rely on metabolic adaptations beyond the metabolic pathways directly involved in serine synthesis, for example downregulating pathways (such as nucleotide synthesis) which consume serine.
• Combination with other therapeutic agents that target these direct or indirect mechanisms of resistance can improve the ability of serine and glycine starvation to inhibit, for example, tumor growth, tumor initiation, or metastasis.
• combination with therapeutic agents or interventions which increase the demands of a cancer cell or a tumor for serine and/or glycine can also sensitize the cancer cell or tumor to starvation of serine and/or glycine.
• compositions and method for the inhibition of Phosphoglycerate Dehydrogenase the first step in the SSP, in combination with compositions devoid of serine and/or glycine.
• PHGDH Phosphoglycerate Dehydrogenase
• In vitro inhibition of PHGDH combined with serine starvation can lead to a defect in global protein synthesis, which can block the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion.
• the combination of diet and inhibitor can show a therapeutic efficacy against tumors that are resistant to diet or drug alone, along with reduced one-carbon availability. Inhibition of PHGDH can augment the therapeutic efficacy of a serine depleted diet.
• Cancer cells can adapt their metabolism to support growth and survival, leading to various dependencies and vulnerabilities that could be targeted for therapy. While these metabolic alterations can be directed by numerous factors, including the genetic alterations in the tumor and the tumor environment or tissue of origin, serine metabolism in supporting cancer cell growth could also be important for these observed metabolic alterations. Serine and glycine (which is produced from serine by the SHMT1/2 reaction) contribute to a number of important processes, including protein, nucleotide, and lipid synthesis, the generation of antioxidant defense through glutathione and NADPH synthesis and the provision of one- carbon units for the folate cycle and methylation reactions.
• Serine and glycine which is produced from serine by the SHMT1/2 reaction
• serine can be taken up from the extracellular environment or synthesized de novo by cells using the serine synthesis pathway (SSP).
• SSP serine synthesis pathway
• Cancer cells can avidly consume serine and depend on an exogenous source 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 decreased PKM2 activity under serine depleted conditions can allow for the diversion of glycolytic intermediates into the SSP. This response is coordinated with an ATF-4 and histone methyltransferase G9A-dependent activation of the three enzymes of the SSP, which can allow most cancer cells to survive and continue to proliferate following serine starvation.
• the efficacy with which cancer cells can adapt to the loss of exogenous serine depends on several factors. Some cancers acquire an 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 an increase in SSP enzyme expression, also allowing cells to become resistant to depletion of exogenous serine. Conversely, although the p53 tumor suppressor protein can inhibit PHGDH expression, loss of p53 also makes cells more vulnerable to increased ROS that accompanies the switch to de novo serine synthesis, resulting in a decreased survival in serine free medium.
• oncogenes such as KRAS, MYC, MDM2, and NRF210
• loss of p53 also makes cells more vulnerable to increased ROS that accompanies the switch to de novo serine synthesis, resulting in a decreased survival in serine free medium
• a composition disclosed herein can lack serine.
• a composition disclosed herein can lack glycine.
• a composition disclosed herein can lack serine and glycine.
• a composition disclosed herein can be administered in combination with a PHGDH inhibitor, PSAT1 inhibitor, or a PSPH inhibitor.
• a composition of the disclosure comprises at least ten amino acids or salts thereof.
• a composition of the disclosure comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids or a salt thereof.
• a composition of the disclosure comprises 10 amino acids or a salt thereof.
• a composition of the disclosure comprises 14 amino acids or a salt thereof.
• a composition of the disclosure comprises 18 amino acids or a salt thereof.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition disclosed herein is devoid of serine and glycine.
• a composition disclosed herein is devoid of serine.
• a composition disclosed herein is devoid of glycine.
• a composition of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 7, 8, or 9 essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 8 essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 9 essential amino acids or salts thereof.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition disclosed herein is devoid of serine and glycine. In some embodiments, a composition disclosed herein is devoid of serine. In some embodiments, a composition disclosed herein is devoid of glycine.
• a composition of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 7, 8, 9, 10, or 11 non-essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 7 non-essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 8 non- essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 9 non-essential amino acids or salts thereof.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt. In some embodiments, a composition disclosed herein is devoid of serine and glycine. In some embodiments, a composition disclosed herein is devoid of serine. In some embodiments, a composition disclosed herein is devoid of glycine.
• a composition of the disclosure can comprise essential amino acids or salts thereof and non-essential amino acids or salts thereof.
• a composition of the disclosure comprises 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.
• a composition of the disclosure comprises 7, 8, or 9 essential amino acids or salts thereof and 6, 7, 8, or 9 non-essential amino acids or salts thereof.
• a composition of the disclosure comprises 8 or 9 essential amino acids or salts thereof and 8 or 9 non- essential amino acids or salts thereof.
• a composition of the disclosure comprises 9 essential amino acids or salts thereof and 7 non-essential amino acids or salts thereof.
• a composition of the disclosure comprises 9 essential amino acids or salts thereof and 8 non-essential amino acids or salts thereof. In some embodiments, a composition of the disclosure comprises 9 essential amino acids or salts thereof and 9 non- essential amino acids or salts thereof.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition disclosed herein is devoid of serine and glycine. In some embodiments, a composition disclosed herein is devoid of serine. In some embodiments, a composition disclosed herein is devoid of glycine.
• a composition of the disclosure comprises histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, arginine, glutamine, alanine, aspartic acid, asparagine, glutamic acid or proline.
• a composition of the 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.
• a composition comprises histidine or a salt thereof, such as L- histidine or L-histidine hydrochloride.
• a composition of the disclosure comprises isoleucine or a salt thereof, such as L-isoleucine, L-isoleucine methyl ester hydrochloride, or L-isoleucine ethyl ester hydrochloride.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure comprises leucine or a salt thereof, such as L-leucine, L-leucine methyl ester hydrochloride, or L-leucine ethyl ester hydrochloride.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure comprises lysine or a salt thereof, such as L-lysine, L-lysine hydrochloride, or L- lysine dihydrochloride.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure comprises methionine or a salt thereof, such as L-methionine, L-methionine methyl ester hydrochloride, or L- methionine hydrochloride.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the 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.
• a composition discloses cystine or a salt thereof, such as L-cystine.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure comprises phenylalanine or a salt thereof, such as L-phenylalanine, DL-phenylalanine, or L- phenylalanine methyl ester hydrochloride.
• a composition of the disclosure comprises tyrosine or a salt thereof, such as L- tyrosine or L-tyrosine hydrochloride.
• a composition of the disclosure comprises threonine or a salt thereof, such as L-threonine or L-threonine methyl ester hydrochloride.
• a composition of the disclosure comprises L- tryptophan.
• a composition of the disclosure comprises valine or a salt thereof, such as L- valine, L-valine methyl ester hydrochloride, or L-valine ethyl ester hydrochloride.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure comprises arginine or a salt thereof, such as L-arginine or L-arginine hydrochloride.
• a composition of the disclosure comprises glutamine or a salt thereof, such as L-glutamine or L-glutamine hydrochloride.
• a composition of the disclosure comprises alanine or a salt thereof, such as L-alanine or P-alanine.
• a composition of the disclosure comprises aspartic acid or a salt thereof, such as L-aspartic acid, D-aspartic acid, L- or D-aspartic acid potassium salt, L- or D-aspartic acid hydrochloride salt; L- or D- aspartic acid magnesium salt, or L- or D-aspartic acid calcium salt.
• a composition of the disclosure comprises L-asparagine.
• a composition of the disclosure comprises glutamic acid or a salt thereof, such as L-glutamic acid or L- glutamic acid hydrochloride.
• a composition of the 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.
• a salt of an amino acid disclosed herein can be a pharmaceutically acceptable salt.
• a composition of the disclosure can comprise at least one pharmaceutical excipient, such as an anti-adherent, a binder, coating, colorant, disintegrant, flavorant, preservative, sorbent, sweetener, or vehicle.
• a composition of the disclosure comprises a colorant and a flavorant.
• a composition of the disclosure comprises a colorant, flavorant, and sweetener.
• a composition of the disclosure comprises a flavorant, sweetener, and a preservative.
• a composition of the invention can be, for example, an immediate release form or a controlled release formulation.
• An immediate release formulation can be formulated to allow the compounds to act rapidly.
• Non-limiting examples of immediate release formulations include readily dissolvable formulations.
• a controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of an active agent at a programmed rate.
• Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents e.g., gelforming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.
• hydrogels e.g., of synthetic or natural origin
• other gelling agents e.g., gelforming dietary fibers
• matrix-based formulations e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through
• a controlled release formulation is a delayed release form.
• a delayed release form can be formulated to delay a compound’s action for an extended period of time.
• a delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours.
• a controlled release formulation can be a sustained release form.
• a sustained release form can be formulated to sustain, for example, the compound’s action over an extended period of time.
• a sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically-effective blood profile) over about 4, about 8, about 12, about 16, or about 24 hours.
• Non-limiting examples of pharmaceutically-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, H.A.
• a composition described herein can be given to supplement a meal consumed by a subject.
• a composition described herein can be given as a meal replacement.
• a composition described herein can be given immediately before or immediately after a meal.
• a composition described here 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 one hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours before or after a meal.
• a composition described herein can be in unit dosage forms suitable for single administration of precise dosages.
• the formulation is divided into unit doses containing appropriate quantities of the composition.
• the unit dosage can be in the form of a package containing discrete quantities of the formulation.
• formulations of the disclosure can be presented in unit dosage form in single-serving sachet.
• formulations of the disclosure can be presented in a single-dose non-reclosable container.
• a formulation of the disclosure can be presented in a reclosable container, and the subject can obtain a single-dose serving of the formulation using a scoop or spoon designed to distribute a single-dose serving.
• a formulation of the disclosure can be presented in a reclosable container, and the subject can obtain a single-dose serving of the formulation using a scoop or spoon designed to distribute a half-dose serving (i.e., two scoops to distribute one serving).
• a composition described herein can be present in a unit dose serving in a range from about 1 g to about 2 g, from about 2 g to about 3 g, from about 3 g to about 4 g, from about 4 g to about 5 g, from about 5 g to about 6 g, from about 6 g to about 7 g, from about 7 g to about 8 g, from about 8 g to about 9 g, from about 9 g to about 10 g, from about 10 g to about 11 g, from about 11 g to about 12 g, from about 12 g to about 13 g, from about 13 g to about 14 g, from about 14 g to about 15 g, from about 15 g to about 16 g, from about 16 g to about
• a composition described herein can be present in a unit dose serving 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.
• a composition described herein is present in a unit dose serving in an amount of about 10 g, 12 g, 15 g, 20 g, or 24 g.
• a composition described herein is present in a unit dose serving in an amount of about 12 g. In some embodiments, a composition described herein is present in a unit dose serving in a sachet in an amount of about 12 g. In some embodiments, a composition described herein is present in a unit dose serving in an amount of about 15 g. In some embodiments, a composition described herein is present in a unit dose serving in a sachet in an amount of about 15 g. In some embodiments, a composition described herein is present in a unit dose serving in an amount of about 24 g. In some embodiments, a composition described herein is present in a unit dose serving in a sachet in an amount of about 24 g.
• a dose of a composition of the disclosure can be expressed in terms of an amount of the drug divided by the mass of the subject, for example, milligrams of drug per kilograms of subject body mass.
• a 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.
• a 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.
• a composition described herein can be provided to a subject to achieve an amount of protein per body weight of the subject.
• a composition described herein can be provided to a subject to achieve a range from about 0.2 g protein/kg to about 0.4 g protein/kg, about 0.4 g protein/kg to about 0.6 g protein/kg, about 0.6 g protein/kg to about 0.8 g protein/kg, or about 0.8 g protein/kg to about 1 g protein/kg of body weight of the subject.
• a composition described herein can be provided to a subject to achieve a range from about 0.6 g protein/kg to about 0.8 g protein/kg of body weight of the subject.
• a composition described herein can be provided to a subject in one or more servings per day.
• 1 serving, 2 servings, 3 servings, 4 servings, 5 servings, 6 servings, 7 servings, 8 servings, 9 servings, 10 servings, 11 servings, or 12 servings of a composition described herein is provided to a subject in one day.
• 3 servings of a composition described herein is provided to a subject in one day.
• 6 servings of a composition described herein is provided to a subject in one day.
• 9 servings of a composition described herein is provided to a subject in one day.
• a composition of the disclosure can be administered to a subject, and the administration can be accompanied by a food-based diet low in or substantially devoid of at least one amino acid. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of one amino acid. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of serine. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of glycine. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of two amino acids or salts thereof.
• administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of serine and glycine. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of three amino acids or salts thereof. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of serine, glycine, and proline. In some embodiments, administration of a composition of the disclosure is accompanied by a food-based diet low in or substantially devoid of serine, glycine, and cysteine.
• a composition of the disclosure can be administered to a subject that is on a diet.
• a composition of the disclosure is administered to the subject, and the subject is on a diet that is low in protein.
• a composition of the disclosure is administered to the subject, and the subject is on a low carbohydrate diet.
• a composition of the disclosure is administered to the subject, and the subject is on a high-fat, and low-carbohydrate (e.g. ketogenic type diet).
• a composition of the disclosure is administered to the subject, and the subject is on a vegetarian diet.
• a composition of the disclosure is administered to the subject, and the subject is on a vegan diet.
• a composition of the disclosure is administered to a subject that is on a low protein diet designed to be low in at least one non-essential amino acid. In some embodiments, a composition of the disclosure is administered to a subject that is on a low protein diet designed to be low in serine and glycine. In some embodiments, a composition of the disclosure is administered to a subject that is on a low protein diet with 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.
• a composition of the disclosure is administered to a subject that is on a low protein diet with 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.
• Multiple therapeutic agents can be administered in any order or simultaneously.
• a composition of the invention is administered in combination with, before, or after treatment with another therapeutic agent.
• the 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 packed together or separately, in a single package or in a plurality of packages.
• One or all of the therapeutic agents can be given in multiple doses. If not simultaneous, the timing between the multiple doses can vary to as much as about a month.
• compositions described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent can vary.
• the compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition.
• the compositions can be administered to a subject during or as soon as possible after the onset of the symptoms.
• a composition disclosed herein can be administered as soon as is practical after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease.
• the length of time necessary for the treatment of disease is about 12 hours, about 24 hours, about 36 hours, or about 48 hours.
• the length of time necessary for the treatment of disease 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.
• the length of time necessary for the treatment of 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.
• the length of time necessary for the treatment of 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.
• the length of time a compound can be administered can be 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
• a composition described herein can be in unit dosage forms suitable for single administration of precise dosages.
• the formulation is divided into unit doses containing appropriate quantities of one or more compounds.
• the unit dosage can be in the form of a package containing discrete quantities of the formulation.
• Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative.
• a composition is administered to a subject throughout a day. In some embodiments, a composition is administered to a subject with a meal. In some embodiments, a composition is administered to a subject with a snack. In some embodiments, a composition is administered to a subject without a meal. In some embodiments, a composition is administered to a subject through the day in equal intervals. In some embodiments, a first serving is administered before breakfast, a second serving is administered with breakfast, a third serving is administered with lunch, a fourth and fifth serving is administered with dinner, and a sixth serving is administered before bed.
• a composition provided herein can be administered in conjunction with other therapies, for example, chemotherapy, radiation, surgery, anti-inflammatory agents, immunotherapy, biologicals, and selected vitamins.
• the other agents can be administered prior to, after, or concomitantly with the pharmaceutical compositions.
• composition disclosed herein can be used in the treatment of any disease.
• a composition disclosed herein is used to treat cancer in a subject in need thereof. Altering the diet and nutrient of a subject can have desired health benefits and can be efficacious in the treatment of disease.
• a composition disclosed herein can be used to manage a disease or condition by a dietary intervention. In some embodiments, a composition disclosed herein can be used as part of a treatment plan for a particular disease or condition.
• the subject has cancer.
• Cancer is caused by uncontrollable growth of neoplastic cells, leading to invasion of adjacent and distant tissues resulting in death.
• Cancer cells often have underlying genetic or epigenetic abnormalities that affect both coding and regulatory regions of the genome. Genetic abnormalities in cancer cells can change protein structures, dynamic and expression levels, which in turn alter the cellular metabolism of the cancer cells. Changes in cell cycles can make cancer cells proliferate at a much higher speed than normal cells. With the increased metabolic rate and proliferation, cancer tissues have much higher nutrient demands compared to normal tissues.
• Cancer cells have nutrient auxotrophy and have a much higher nutrient demand compared to normal cells. As an adaptation to fulfill the increased nutritional demand, cancer cells can upregulate the glucose and amino acid transporters on the cell membrane to obtain more nutrients from circulation. Cancer cells can also rewire metabolic pathways by enhancing glycolysis and glutaminolysis to sustain a higher rate of ATP production or energy supply. Glucose and amino acids are highly demanded nutrients in cancer cells. Some cancer cell types and tumor tissues are known to be auxotrophic to specific amino acids. Cancers’ auxotrophy to different amino acids can render the cancer types vulnerable to amino acid starvation treatments.
• cancer cells can inhibit protein synthesis, suppress growth, or undergo programmed cell death.
• the cell death mechanisms of amino acid starvation can be caspase-dependent apoptosis, autophagic cell death, or ferroptotic cell death.
• Amino acid transporters, metabolic enzymes, autophagy- associated proteins, and amino acid starvation can be used to control cancer growth.
• a method disclosed herein can monitor nutrient consumption by a subject.
• the nutrient consumption can be measured by taking a biological sample from a subject.
• the biological sample can be for example, whole blood, serum, plasma, mucosa, saliva, cheek swab, urine, stool, cells, tissue, bodily fluid, sweat, breath, lymph fluid, CNS fluid, and lesion exudates.
• a combination of biological samples can be used with the methods of the disclosure.
• a method of composition of the disclosure can slow the proliferation of cancer cell lines, or kill cancer cells.
• Non-limiting examples of cancer that can be treated by a compound of the invention include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders
• compositions of the invention can be packaged as a kit.
• a kit includes written instructions on the administration/use of the composition.
• the written material can be, for example, a label.
• the written material can suggest conditions methods of administration.
• the instructions provide the subject and the supervising physician with the best guidance for achieving the optimal clinical outcome from the administration of the therapy.
• the written material can be a label.
• 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.
• FDA U.S. Food and Drug Administration
• EMA European Medicines Agency
• Radiation therapy is a therapy using ionizing radiation as a part of cancer treatment to control or kill malignant cells and is normally delivered by a linear accelerator. Ionizing radiation damages the DNA of cancerous tissue, resulting in cellular death. Radiation therapy can be curative in a number of types of cancer if localized to one area of the body.
• the methods and compositions of the disclosure can be administered in combination with a second therapy, for example, radiotherapy.
• radiotherapy can be used with a method or composition of the disclosure because radiotherapy can control cell growth.
• radiotherapy can be used in combination with a method or composition of the disclosure to prevent or reduce the likelihood of tumor recurrence after surgery to remove a primary malignant tumor.
• radiotherapy and chemotherapy can be used in combination with a method or composition of the disclosure.
• the methods and compositions of the disclosure can be administered in combination with radiotherapy to treat a cancer.
• the methods and compositions of the disclosure can be administered in combination with radiotherapy to reduce symptoms of a cancer.
• the methods and compositions of the disclosure can be administered in combination with radiotherapy to slow the growth of a cancer.
• the radiotherapy is external beam radiation therapy.
• External beam radiation therapy uses a machine that locally aims radiation at a cancer.
• the radiotherapy is internal beam radiation therapy.
• external beam radiation can be used to shrink tumors to treat pain, trouble breathing, or loss of bowel or bladder control.
• the external-beam radiation therapy is three-dimensional conformal radiation therapy (3D-CRT).
• the external-beam radiation therapy is intensity modulated radiation therapy (IMRT).
• IMRT intensity modulated radiation therapy
• the external -beam radiation therapy is proton beam therapy.
• the external -beam radiation therapy is image-guided radiation therapy (IGRT).
• the external-beam radiation therapy is stereotactic radiation therapy (SRT).
• Internal radiation therapy is a treatment that places a source of radiation in the subject’s body.
• the source of radiation is a liquid.
• the source of radiation is a solid.
• the internal radiotherapy uses a permanent implant.
• the internal radiotherapy is a temporary internal radiotherapy, for example, a needle, tube, or applicator.
• the solid source of radiation is used in brachytherapy.
• seeds, ribbons, or capsules containing a radiation source are placed in a subject’s body.
• the radiotherapy is brachytherapy, where a radioactive source is placed inside or next to an area requiring treatment.
• the radiotherapy is total body irradiation (TBI) in preparation for a bone marrow transplant.
• TBI total body irradiation
• the radiotherapy is intraoperative radiation therapy (IORT). In some embodiments, the radiotherapy is systemic radiation therapy. In some embodiments, the radiotherapy is radioimmunotherapy. In some embodiments, the radiotherapy uses a radiosensitizer or a radioprotector.
• IORT intraoperative radiation therapy
• the radiotherapy is systemic radiation therapy. In some embodiments, the radiotherapy is radioimmunotherapy. In some embodiments, the radiotherapy uses a radiosensitizer or a radioprotector.
• brachytherapy is used to treat a cancer of the head, neck, breast, cervix, prostate, or eye.
• a systemic radiation therapy such as radioactive iodine, or 1-131, can be used to treat thyroid cancer.
• targeted radionuclide therapy can be used to treat advanced prostate cancer or a gastroenteropancreatic neuroendocrine tumor (GEP-NET).
• shaped radiation beams can be aimed from several angles of exposure to intersect at the tumor while sparing normal tissue.
• a tumor absorbs a much larger dose of radiation than does a surrounding healthy tissue.
• 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.
• Gy Gray
• 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.
• a 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.
• a subject or tumor can be treated with about 5 Gy of radiation therapy.
• a subject or tumor can be treated with about 10 Gy of radiation therapy.
• a subject or tumor can be treated with about 20 Gy of radiation therapy.
• a subject or tumor can be treated with from 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.
• a subject or tumor can be treated with from about 5 Gy to about 10 Gy.
• a subject or tumor can be treated with from about 20 Gy to about 40 Gy.
• a subject or tumor can be treated with from about 40 Gy to about 60 Gy.
• one cycle of radiation therapy can comprise the subject or tumor being treated with radiation over a number of days.
• the radiation can be occur over 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.
• one cycle of radiation therapy can comprise the subject or tumor being treated with radiation over 4 days.
• one cycle of radiation therapy can comprise the subject or tumor being treated with radiation over 5 days.
• one cycle of radiation can comprise administering 10 Gy over 5 days, for example, 2 Gy a day for 5 days. In some embodiments, one cycle of radiation can comprise administering 15 Gy over 5 days, for example, 3 Gy a day for 5 days. In some embodiments, one cycle of radiation can comprise administering 20 Gy over 5 days, for example, 4 Gy a day for 5 days. In some embodiments, one cycle of radiation can comprise administering 25 Gy over 5 days, for example, 5 Gy a day for 5 days.
• one cycle of radiation therapy can be repeated over a period of time.
• a cycle of radiation therapy can be repeated for 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.
• a composition of the disclosure can be administered simultaneously with administration of a radiotherapy.
• a composition of the disclosure can be administered simultaneously with a radiotherapy 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.
• a composition of the disclosure can be administered simultaneously with administration of a radiotherapy for 5 days.
• a composition of the disclosure can be administered simultaneously with administration of a radiotherapy for 7 days.
• the composition of the disclosure is administered 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 before a subject is treated with radiotherapy.
• the composition of the disclosure is administered 1 day before a subject is treated with radiotherapy.
• the composition of the disclosure is administered 2 days before a subject is treated with radiotherapy.
• the composition of the disclosure is administered 3 days before a subject is treated with radiotherapy.
• the composition of the disclosure is administered 4 days before a subject is treated with radiotherapy.
• a subject can be treated with a composition of the disclosure and radiotherapy, then go off treatment before beginning a subsequent treatment cycle with the composition and radiotherapy.
• the length of the treatment period and off-treatment period are identical. In some embodiments, the length of the treatment period and 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.
• the length of a treatment period with a composition and radio 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
• the length of off-treatment 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.
• the length of the treatment period is 5 days, and the length of the off-treatment period is 2 days.
• the length of the treatment period is 4 days, and the length of the off-treatment period is 3 days.
• the length of the treatment period is 3 days, and the length of the off-treatment period is 4 days. In some embodiments, the length of the treatment period is 2 days, and the length of the off-treatment period is 5 days. [0141] In some embodiments, a cycle of a treatment period and an off-treatment period is repeated for 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.
• a composition of the disclosure and radiotherapy are administered with a high fat diet.
• the high fat diet is a diet that has greater than about 50%, about 60%, about 70%, about 80%, or about 90% daily calories from fat .
• a composition of the disclosure and radiotherapy are administered with a low carbohydrate diet.
• the low carbohydrate diet is a diet with less than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% daily calories from carbohydrates.
• a composition of the disclosure and radiotherapy are administered with a low protein diet.
• the low protein diet is a diet with 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 whole protein.
• the low protein diet has a whole protein amount of less than about 50 g/day, about 40 g/day, about 30 g/day, about 20 g/day, or about 10 g/day.
• a composition of the disclosure and radiotherapy are administered with a high fat, low carbohydrate, and low protein diet.
• a composition of the disclosure is administered with a normal diet.
• an amino acid starvation therapy of the disclosure can be used in combination with a chemotherapeutic regimen.
• the chemotherapeutic regimen is an immunotherapy.
• the immunotherapy is an antibody therapy.
• the antibody therapy is treatment with alemtuzumab, rituximab, ibritumomab tiuxetan, or ofatumumab.
• the immunotherapy is an interferon.
• the interferon is interferon a.
• the immunotherapy is an interleukin, for example, IL-2.
• the immunotherapy is an interleukin inhibitor, for example, an IRAK4 inhibitor.
• the immunotherapy is a cancer vaccine.
• the cancer vaccine is a prophylactic vaccine.
• the cancer vaccine is a treatment vaccine.
• the cancer vaccine is an HPV vaccine, for example, Gardisil TM, Cervarix, Oncophage, or Sipuleucel-T.
• the immunotherapy is gplOO.
• the immunotherapy is a dendridic cell-based vaccine, for example, Ad.p53 DC.
• the immunotherapy is a toll-like receptor modulator, for example, TLR-7 or TLR-9.
• the immunotherapy is a PD-1, PD-L1, PD-L2, or CTL4-A modulator, for example, nivolumab.
• the immunotherapy is an IDO inhibitor, for example, indoximod.
• the immunotherapy is an anti-PD-1 monoclonal antibody, for example, MK3475 or nivolumab.
• the immunotherapy is an anti-PD-Ll monoclonal antibody, for example, MEDI-4736 or RG-7446.
• the immunotherapy is an anti-PD-L2 monoclonal antibody.
• the immunotherapy is an anti-CTLl-4 antibody, for example, ipilumumab.
• Cancer cells can change cellular metabolism to support elevated energetic and anabolic demands of proliferation of cancer cells. Examples of altered metabolism include aerobic glycolysis (i.e., Warburg effect) and high dependency on non-essential amino acids.
• One-carbon metabolism encompasses a collection of metabolic pathways that allow cells to generate and use molecules containing single carbons.
• One-carbon units i.e., methyl groups
• THF tetrahydrofolates
• Cells require one-carbon units to support nucleotide synthesis, methylation reactions and reductive metabolism. Cancer cells are dependent on the one-carbon pathways for supporting high proliferative rates, and one-carbon metabolism is crucial for cancer cell proliferation.
• THF-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA.
• Tryptophan is a theoretical source of one-carbon units through metabolism by indoleamine 2,3-dioxygenase 1 (IDO1).
• IDO1 expressing cancer cells tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis both in vitro and in vivo.
• serine In cancer cell metabolism, serine is considered the predominant source of one-carbon units. Serine is obtained either by de novo synthesis from the glycolytic intermediate 3- phosphoglycerate via the serine synthesis pathway (SSP), or by uptake from the extracellular environment. Some cancer cells display increased SSP enzyme expression in order to meet cellular serine demands, whereas others rely predominantly on serine uptake. Serine hydroymethyltransferases (SHMT1 and SHMT2) directly catalyze the conversion of serine into glycine and the release of a one-carbon, which enters the THF cycle.
• SSP serine synthesis pathway
• 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). Histidine catabolism can also yield one-carbon units and can further sensitize cancer cells to anti-folate treatment due to a decrease in free THF pools.
• Glycine can provide one-carbon units through the glycine cleavage system (GCS). Histidine catabolism can also yield one-carbon units and can further sensitize cancer cells to anti-folate treatment due to a decrease in free THF pools.
• tryptophan is critical for protein synthesis, but is also a precursor for 5-hydroxytryptamine and kynurenine production. In the kynurenine pathway, the initial and rate-limiting step is the conversion of tryptophan to formyl-kynurenine.
• IDO1, IDO2, and TDO Three enzymes are capable of catalyzing this reaction: IDO1, IDO2, and TDO. 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 a molecule of formate. Formate can enter the one-carbon cycle by directly reacting with THF and it is via this pathway that tryptophan can serve as a one-carbon donor.
• IDO1 activity depletes tryptophan and increases kynurenine in the tumor microenvironment, causing a range of effects on immune cells. Tryptophan depletion decreases tumor infiltrating T-cell activity, and kynurenine decreases effector T-cell proliferation and supports the differentiation of immunosuppressive T-regulatory cells through binding of the aryl hydrocarbon receptor.
• the tumor micro-environmental effects provide 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 entry of carbons (via a-ketoadipate) into the TCA cycle.
• a method of treating a 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.
• the at least two amino acids is serine and glycine.
• the IDO1 inhibitor is indoximod (D-1MT; NLG-8189), 4- phenylimidazole (4-PI), N3-benzyl substituted 4-PI, ortho-hydroxy 4-PI, navoximod, or epacadostat. In some embodiments, the IDO1 inhibitor is epacadostat.
• a composition of the disclosure and an IDO1 inhibitor can be used to treat a cancer.
• the cancer is pancreatic cancer.
• the cancer is colon cancer.
• the cancer is breast cancer.
• the cancer is cervical cancer.
• the cancer is lung cancer.
• the IDO1 inhibitor is administered 1, 2, 3, 4, or 5 times daily in combination with an amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered once daily in combination with an amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered twice daily in combination with an amino acid starvation therapy. In some embodiments, the IDO1 inhibitor is administered three times daily in combination with an amino acid starvation therapy.
• the IDO1 inhibitor is administered in an amount of from about 10 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, or about 450 mg to about 500 mg.
• the IDO1 inhibitor is administered in an amount of from about 50 mg to about 100 mg.
• the IDO1 inhibitor is administered in an amount of from about 100 mg to about 150 mg.
• the IDO1 inhibitor is administered in an amount of from about 250 mg to about 300 mg.
• 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.
• the IDO1 inhibitor is administered in an amount of about 25 mg.
• the IDO1 inhibitor is administered in an amount of about 50 mg.
• the IDO1 inhibitor is administered in an amount of about 100 mg.
• the IDO1 inhibitor is administered in an amount of about 300 mg.
• 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.
• EXAMPLE 1 PHGDH inhibitor along with lack of serine and glycine can impede growth of tumor cell lines.
• Cells can take up exogenous serine or synthesize serine from the glycolytic intermediate 3 -phosphoglycerate (3-PG), using the serine synthesis pathway (FIG. 1).
• serine can be taken up from the environment or newly synthesized through the serine synthesis pathway (SSP).
• SSP serine synthesis pathway
• the SSP consists of a three-step enzymatic reaction starting 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.
• PSGDH phosphoglycerate dehydrogenase
• the 3-PHP produced during the PHGDH reaction is then converted into 3 -phosphoserine (3-PS) by phosphoserine aminotransferase 1 (PSAT1) in a glutamate-dependent transamination reaction.
• PSAT1 phosphoserine aminotransferase 1
• PSPH phosphoserine phosphatase
• Serine is involved in numerous metabolic pathways including nucleotide synthesis or glutathione synthesis, a major antioxidant for the cells. Serine availability can thus be targeted by depleting it from the extracellular environment or by inhibition of the SSP using PH755.
• CM complete medium
• -SG medium lacking serine and glycine
• PH755 a PHGDH inhibitor
• EXAMPLE 2 PHGDH inhibition combined with lack of serine and glycine limits DNA synthesis, survival & organoid growth.
• EXAMPLE 1 Accompanying this lack of proliferation seen in EXAMPLE 1 was a strong reduction of BrdU incorporation into newly synthesized DNA after 48 hours incubation with -SG medium plus PH755, compared to either treatment alone (FIG. 4 and FIG. 5).
• the decrease in cells undergoing S-phase was accompanied by an accumulation of cells in G2/M phase (FIG. 5 and FIG. 6) and an increase in the proportion of SubGl cells in the double-treated condition, indicating an increase in cell death (FIG. 7).
• cleaved-caspase 3 confirmed the induction of apoptosis in cells cultured in -SG medium and treated with PH755 (FIG. 8).
• Using uniformly labelled glucose cells grown in the presence of exogenous serine diverted little glucose into serine and glycine synthesis, as reflected by the negligible accumulation of m+3 serine and m+2 glycine (FIG. 9 and FIG. 10), regardless of the presence or absence of PH755. Of note, these cells maintained much higher overall intracellular serine and glycine levels than cells grown in the -SG medium (FIG. 6).
• EXAMPLE 3 PHGDH inhibition combined with lack of serine and glycine inhibits purine and GSH synthesis.
• Serine is involved in numerous metabolic pathways, including the provision of one- carbon units and glycine for purine synthesis and the maintenance of redox homeostasis through glutathione production.
• the contribution of de novo synthesized serine to these pathways can be assessed by following the fate of uniformly carbon-labeled glucose (FIG. 17).
• Cells grown in serine and glycine showed little evidence of the use of de novo synthesized serine for ATP or GTP synthesis (FIG. 18); rather, the majority of label (m+5) deriving from ribose was synthesized through the pentose phosphate pathway (FIG. 17).
• EXAMPLE 4 Metabolic rescue of cells co-treated -SG/PHGDHi treated cells.
• All cells deprived of serine and glycine and treated with PH755 showed a strong growth inhibition (FIG. 2, FIG. 13, FIG. 3, FIG. 17, FIG. 18, and FIG. 20).
• formate to replenish the one-carbon cycle
• glycine alone did not restore growth
• addition of formate and glycine effectively rescued proliferation (FIG. 23).
• This proliferation rescue was accompanied by the recovery of ATP and GTP synthesis (FIG. 24), and the partial restoration of the pool of unlabeled serine (FIG. 25).
• EXAMPLE 5 PHGDHi/ -SG treatment impairs the general ATF-4 response.
• PH755 While the effect of PH755 was consistent with a specific inhibition of PHGDH, analysis of the expression of the 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 the activation of ATF-4, which can mediate a general survival response to metabolic stress. Importantly, serine starvation leads to an ATF-4 dependent induction of expression of the SSP enzymes, so contributing to the ability of the cells to adapt to a reduction in exogenous serine levels.
• EXAMPLE 6 PHGDHi/ -SG treatment inhibits global protein synthesis.
• PH755 treatment did not primarily affect the transcription of ATF-4 or ATF-4 target genes but impacted the subsequent expression of each of these proteins. To determine whether this reflected a general inhibition of translation resulting from the dramatic decrease of serine and glycine availability seen in this condition, the incorporation of puromycin, a tyrosyl-tRNA mimetic, into newly synthesized polypeptides in cells grown in CM or -SG medium plus PH755 was analyzed. Interestingly, while a modest decrease in the amount of puromycin-labelled peptides in response to serine/glycine withdrawal was observed, this reduction was much more pronounced in presence of the PHGDH inhibitor (FIG. 39).
• EXAMPLE 7 Combining a serine/glycine-free diet and a PHGDH inhibitor is well tolerated in vivo.
• mice cotreated with PH755 and the -SG diet showed greater weight loss compared to either treatment alone (FIG. 44), despite remaining active and appearing healthy.
• the weight loss was highly responsive to the dose of PH755, and modulation of the dose (from 75 to 50 mg/kg) was successful in limiting weight loss to less than 20% over the course of the study.
• Serine is important in brain development and function and PHGDH deficiency in humans can lead to neurological defects such as microcephaly, psychomotor retardation, and seizures.
• the impact of -SG diet and PHGDH inhibitor treatment on the brain morphology of a cohort of C57BL/6J mice after 20 days of treatment was assessed.
• mice Microscopic examination of coronal sections from the brains of the 4 groups of mice at the level of the pyriform cortex, caudal diencephalon, caudal mesencephalon, and rostral cerebellum did not reveal any histopathological lesions in any of the sections examined. Indeed, hematoxylin & eosin stained sections exhibit normal histological features with no evidence of degeneration, necrosis or inflammation (FIG. 45 and FIG. 46). Furthermore, the brain weight remained unchanged in all groups of mice (FIG. 45). Other signs of toxicity of the double treatment in these normal mice were looked for.
• EXAMPLE 8 Combining a serine/glycine-free diet and a PHGDH impedes tumor growth in vivo.
• mice were transferred to a -SG or control diet when tumors started to become evident and treated with PH755 two-four days later.
• the double-treated DLD1 tumor-bearing mice showed more weight loss compared to either treatment alone but a careful modulation of the dose of PH755 used in association with the -SG diet was able to limit the weight loss in these mice to less than 20% over the course of the experiment (FIG. 51).
• HCT116 tumors showed a modest further drop in serine and glycine in the combination diet and drug treated mice (FIG. 59) but in DLD1 tumors, the reduction in serine in response to the -SG diet was not further affected by additional PH755 treatment (FIG. 58). Nevertheless, a further reduction in intra-tumoral glycine in the double treated mice suggests that flux through the SSP is lower in the double treated tumors and that the maintenance of the low steady state levels of serine may reflect the decrease in growth (and serine consumption) under these conditions (FIG. 58).
• EXAMPLE 9 Combining dietary restriction of serine and glycine and PHGDH inhibition cooperates to lower tumor burden and improve survival in genetic models of intestinal cancer.
• FIG. 64A ApcMin/+ mice were transferred to a CTR diet (red line) or a -SG diet (black line) at 80 days and were subsequently treated at 84 days with 100 mg/kg PH755 daily for 9 days. After stopping treatment, mice were maintained either on a CTR diet or a -SG diet until clinical end point was reached. These data are shown in comparison to data showing survival of ApcMin/+ mice on control (dotted line) or -SG diet (dotted line). Survival was calculated from change of diet.
• All cell lines underwent routine quality control, which included mycoplasma detection, STR profiling and species identification for validation.
• 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.
• CM complete medium
• Cells (2 x 10 4 to 3 x 10 4 cells/well depending on the cell lines) were plated in 24-well plates in their regular medium. The next day, after being washed with PBS, cells were transferred to -SG medium or CM and treated with 10 pM 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 . Relative cell number at each time point was calculated based on the number of cells measured before the medium change.
• HT-29, HCT116 and DLD-1 cells were seeded in 24-well plates (2 x 10 4 cells/well).
• Sodium formate (1 mM) and/or glycine (0.4 mM) were diluted in -SG medium + 10 pM PH755 and medium was refreshed every two days.
• Crypts were isolated from adenomatous small intestine tissue derived from Vill- creER;Apcfl/fl and Vill-creER;Apcfl/fl;KrasG12D/+ mice.
• the generation of the Apc5 organoid bearing an Ape truncating mutation using CRISPR/Cas9 technology and isolation of normal organoids derived from the proximal part of healthy small intestine from Villin- CreERT2 mouse were performed.
• CM tumor organoid medium
• CM tumor organoid medium
• the -SG medium corresponds to the previously described medium without serine and glycine.
• mice Normal organoids from mice were grown in normal organoid medium, a modification of tumor organoid medium that was supplemented with 100 ng/mL Wnt-3a, 1 mM N-Acetyl-L-cysteine , 10 pM Y 27632 and 4 mM Nicotinamide.
• Human organoids were grown in human organoid medium, a second modification of tumor organoid medium that was supplemented with 10 nM FGF-basic, 100 ng/mL Wnt-3a 1 pM Prostaglandin E2, 4 mM Nicotinamide, 20 ng/mL HGF, 10 nM FGF-10, 10 nM Gastrin I, 10 pM Y-27632, 0.5 pM A 83-01 and 5 pM SB 202190.
• organoids were harvested through mechanical pipetting using TrypLE, incubated for 10 minutes at 37 °C, diluted 3 times in volume in ice-cold IX HBSS, and spun down at 270g for 5 minutes at 4 °C. Pellet was then resuspended in growth factor reduced Matrigel and plated in 24-well plates. Matrigel was then incubated for 15 min at 37 °C and ImL of the CM 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 pM PH755 and allowed to grow. Pictures were regularly taken with a light microscope and organoid diameter was measured using ImageJ software.
• pLentiCRISPRv2 vector containing the following guide RNA was used to target PHGDH.
• HEK293T cells were transfected with this lentiviral plasmid together with psPAX2 and VSV.G using jetPRIME reagent (Polyplus transfection). After 24 hours incubation, medium was changed and 48 hours later, the viral particle containing-medium was filtered (0.45 mm) and mixed with polybrene (4 pg/ml, Sigma-Aldrich). The medium containing lentiviruses was incubated with the target cells for 24 hours. HT-29 and DLD- 1 cells were then selected with puromycin for 3 weeks and analyzed for loss of PHGDH expression.
• HCT116 and DLD-1 cells were grown for 48 hours in -SG medium or CM and treated with 10 pM PH755 diluted in DMSO or DMSO alone.
• 10 pM BrdU was then added to culture media for an additional 5 hours while for cell cycle analysis, 10 pM BrdU was added for only 30 minutes.
• Cells were then harvested, fixed and stained with APC anti-BrdU antibody (and 7- AAD for cell cycle analysis) using the APC BrdU Flow kit. Fluorescence was acquired with FACSdiva on a Fortessa flow Cytometer and the analysis performed using FlowJo (version 10.5.2).
• 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.
• HT-29, HCT116 and DLD-1 cells were grown for 6 hours or 24 hours in -SG medium or CM and treated with 10 pM PH755 diluted in DMSO or DMSO alone.
• Total RNA was extracted using RNeasy Mini kit performing 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 TABLE 1 below.
• the QuantStudio 7 Flex Real-Time PCR System was used for all reactions. Gene expression was normalized to ACTB (b-actin) housekeeper gene, analyzed according to Pfaffl method and expressed as relative units compared to the cells grown in CM for 6 hours.
• ACTB b-actin
• HT-29 cells (2.4 x 10 5 ), HCT116 cells (1.8 x 10 5 ), DLD-1 cells (1.8 x 10 5 ) and MDA- MB-468 cells (2.4 x io 5 ) were plated in 6-well plates in their regular medium. Duplicate plates were used for cell counting to normalize LC-MS analysis based on cell number. After 16 hours, cells were washed with PBS and transferred to CM or -SG medium supplemented or not with 10 pM PH755 for 24 hours. 6 hours before metabolite extraction, medium was replaced with CM or -SG medium +/- 10 pM PH755 with glucose substituted for 10 mM U- [ 13 C]-glucose.
• cells were moved to the previously described medium with glucose substituted for 10 mM U-[ 13 C]-glucose for only 3 hours or 6 hours before metabolite extraction.
• glucose substituted for 10 mM U-[ 13 C]-glucose for only 3 hours or 6 hours before metabolite extraction.
• cells were grown for 24 hours in -SG medium supplemented with 10 pM PH755, 1 mM sodium formate and 0.4 mM glycine. This medium was then replaced with matched medium with glycine substituted for 0.4 mM 13 C2 15 Ni-glycine for 1 hour before metabolite extraction.
• mice (3 to 5 per cage) were allowed access to food and water ad libitum and were kept in a 12-hour day/night cycle starting at 7:00 until 19:00. Rooms were kept at 21 °C at 55% humidity. Mice were allowed to acclimatize for at least one 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 but was deprived of serine and glycine, which were compensated by a proportionally increased level of the other amino acids to reach the same total amino acid content.
• CD-I female nude mice (7-9 weeks old) received unilateral subcutaneous injections of lOOpl of HCT116 cells (2x106 cells) or 100 pl of DLD-1 cells (4xl0 6 cells) suspended in PBS.
• mice were placed on experimental diets (control or -SG) 10 days (for HCT116 xenograft experiment) or 2 days (for DLD-1 xenograft experiment) after tumor injections. 4 days (for HCT116 xenograft experiment) or 2 days (for DLD-1 xenograft experiment) after the diet change, mice were treated either with vehicle (0.5% methylcellulose, 0.5% Tween- 80) or PH755 prepared in vehicle once daily by oral gavage. The starting dosage of PH755 was 100 mg/kg and was subsequently lowered to 75 mg/kg or 50 mg/kg as indicated in the figure legends. Subcutaneous growth was measured two to three times a week by caliper and the following formula: (length x width 2 i was used to calculate tumor volume.
• mice C57BL/6J male mice (14 weeks old) were placed on experimental diets (control or - SG) two days before stating the treatment with PH755 or its vehicle. Mice were treated once daily by oral gavage with PH755 or its vehicle for 20 days. The starting dosage of PH755 was 75 mg/kg and was subsequently lowered to 50 mg/kg to maintain weigh loss below 20% of the initial body weight.
• Antigen retrieval was obtained with Cell Conditioning 1 (CC1) from Ventana Medical Systems. Primary antibody (AF835) was diluted at 1 : 1250 and incubated for 60 minutes. 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 the ZEISS Axio Scan.Zl slide scanner and images were generated through ZEISS ZEN 2.6 (blue edition) software. For gut rolls, immunohistochemistry was performed on Bond Rx Autostainer Leica Bond Intense R staining kit.
• C57BL/6J mice were culled using carbon dioxide asphyxiation to avoid physical trauma to the brain. Mice were immediately dissected, and haired skin and soft tissue were removed from the cranial surface. Incisions throughout the parietal and frontal sutures were performed to allow fast penetration of the fixative solution into the brain parenchyma. The head was immersed in 250 mL of 10% neutral buffered formalin and fixed for 2 weeks. After complete fixation, the brains were removed from the skull and trimmed using a mouse brain matrix (BSMYS001-1). Four coronal sections were obtained at the level of the pyriform cortex, caudal diencephalon, caudal mesencephalon and rostral cerebellum. Tissue samples were routinely processed for paraffin embedding, sectioned at 4 pm, and stained with hematoxylin and eosin.
• Plasma ALT and AST activities were measured using Alanine Transaminase Activity Assay Kit (and AST Activity Assay Kit respectively.
• Statistical analyses were performed using Alanine Transaminase Activity Assay Kit (and AST Activity Assay Kit respectively.
• EXAMPLE 11 Metabolomic impact of radiation on pancreatic and colorectal cancer cells in vitro.
• FIG. 65 PANEL A-PANEL D show the metabolomic impact of radiation on pancreatic and colorectal cancer cells in vitro.
• PANEL A primary murine pancreatic cancer (KPC: Pdxl-cre; Kras G12D/+ ; Trp53 R172H/ ") and human colorectal cancer (HCT116) cells were exposed to 5-10 Gray (Gy) radiation.
• KPC Pdxl-cre; Kras G12D/+ ; Trp53 R172H/
• HCT116 human colorectal cancer
• metabolites were extracted and analyzed by LCMS using a Thermo Exactive Orbitrap Mass Spectrometer coupled to a pHILIC chromatography column. Unsupervised principle component analysis was performed using data from all identified metabolites.
• PANEL B shows volcano plots showing distribution of identified metabolites (Control vs.
• PANEL C shows significantly altered metabolites identified during unbiased metabolomics were subjected to metabolic pathway analysis. The dominant pathway hits are shown.
• PANEL D shows that KPC (Top panel) and HCT116 (Bottom panel) cells were either grown in complete medium (Ctr) or medium lacking serine and glycine (-SG) and irradiated with 10 or 5 Gray radiation (IR), respectively. Cell number over time (hours) is shown. Data are averages of triplicate wells, and error bars ar SD.
• FIG. 66 PANEL A-PANEL E show the effect of dietary amino acid restriction in response to targeted radiotherapy in vivo.
• PANEL A is a cartoon illustrating the experimental setup of a pilot experiment testing the impact of dietary restriction of serine and glycine on the response of KPC tumors to radiation.
• mice were anesthetized and positioned in an Xstrahl SARRP.
• high resolution cone-beam CT imaging a 2 mm focused beam of x-ray radiation (20 Gy) was delivered to each tumor.
• PANEL B and PANEL C show the results of an unsupervised principle component analysis performed on tumor tissue to assess the metabolic impact of radiotherapy alone. The most significantly altered metabolites in vivo converged on the same metabolic pathways as identified in vitro.
• PANEL D shows representative tumor cross sections stained for cleaved caspase-3 by immunohistochemistry. The upper panels show stained sections (cleaved caspase-3 stained brown), lower panels show false color mapping of histological scores generated by Halo image analysis software.
• EXAMPLE 13 Sachet formulation devoid of serine, glycine, and proline.
• a sachet containing a formulation devoid of serine, glycine, and proline is prepared and 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 a subject in conjunction with a low protein and low carbohydrate diet.
• the low protein and low carbohydrate diet results in a daily dietary intake of: 1) 1711 kcals/day (1923 kcals/day with sachets); 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 that is about 9% carbohydrates, 2% protein, and 89% fat of food-only kcals.
• EXAMPLE 14 Use of radiotherapy to treat a cancer
• a first subject with a cancer is treated with a short course of radiotherapy to treat the cancer.
• the first subject is placed on a diet substantially devoid of serine and glycine two days before starting radiotherapy treatment (i.e., day -2).
• the amino acid-depleted diet is administered for a total of 10 days, starting 2 days before treatment through 4 days posttreatment (i.e., day -2 through day 8).
• the first subject is treated with 5 Gy a day for 5 days.
• the first subject returns to a normal, habitual diet after day 8, or 4 days post-radiation treatment.
• the first subject is placed on a cycled diet throughout the chemotherapy.
• the cycle diet places the first subject on an alternating 5 day amino acid-depleted diet (e.g., Monday -Friday) followed by a 2 day habitual diet (e.g., Saturday, Sunday) throughout the chemotherapy treatment period.
• TABLE 3 shows a short course radiotherapy to treat a cancer.
• a second subject with a cancer is treated with a long course of radiotherapy to treat the cancer.
• the second subject is placed on a diet substantially devoid of serine and glycine two days before starting radiotherapy treatment (i.e., day -2).
• the amino acid-depleted diet is administered for a total of 7 days, starting 2 days before treatment through the course of treatment (i.e., day -2 through day 4).
• the second subject is treated with 2 Gy a day for 5 days.
• the second subject returns to a normal, habitual diet for two days before starting an additional round of radiotherapy.
• Subsequent radiation therapy cycles administer 5 days of an amino-acid depleted diet with 2 Gy of radiation for 5 days, followed by 2 days of a habitual diet. The cycle is repeated as needed.
• the second subject is treated with chemotherapy after the radiotherapy, the second subject is placed on a cycled diet throughout the chemotherapy.
• the cycle diet places the second subject on an alternating 5 day amino acid-depleted diet (e.g., Monday -Friday) followed by a 2 day habitual diet (e.g., Saturday, Sunday) throughout the chemotherapy treatment period.
• TABLE 4 shows a long course radiotherapy treatment to treat a cancer.
• EXAMPLE 15 IDOl-driven tryptophan metabolism is a source of one-carbon units for pancreatic tumor and stellate cells.
• pancreatic cancer models were used to show that IDO1 expression was highly context dependent, influenced by attachment independent growth as well as canonical activator fFNy. Cancer cells were also shown to release tryptophan-derived formate, which can be taken up and utilized by pancreatic stellate cells to support purine nucleotide synthesis.
• PDAC pancreatic ductal adenocarcinoma
• IDO1 was expressed in genetically engineered mouse models for PDAC. The resulted showed that IDO1 expression was not well represented in standard in vitro cell culture conditions, but could be induced by the canonical activator IFNy, or by culture in low attachment conditions, which regulate IDO1 via JAK/STAT signaling. The results also showed that when IDO1 was expressed by cancer cells, IDO1 promoted the generation of one-carbon units from tryptophan that are used in de novo purine nucleotide synthesis. Further, tryptophan-derived formate was released by cancer cells. Pancreatic stellate cells (a key component of the tumor stroma) captured the exogenously derived formate and channeled the formate into de novo nucleotide synthesis.
• Cell culture All cell lines used in the study were cultured at 37 °C in 5% CO2 in a humidified incubator. Cell lines were authenticated using Promega GenePrint 10 and tested for mycoplasma using Mycoalert (Lonza). AsPC-1 (female), BxPC-3 (female), CFPAC-1 (male), HPAF-II (male), Pane 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).
• KPC lines were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 0.2% amphotericin B and glutamine (2 mM).
• KPC lines were isolated from the tumors of Pdxl-cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ mice either with a mixed or pure C57BL/J background.
• KPC-IDO1 & KPC-EV cell lines were made from pure C57BL/J KPC cells using the PiggyBac transposon system.
• ImPSC #2 and #3 lines were isolated from Pdgfra tmll(EGFP)Sor mice
• mice Mus musculus cohorts were housed in a barrier facility proactive in environmental enrichment and maintained on a normal chow diet. Mixed male and female populations were used for each genotype. Cohorts were on a mixed strain background but all cohorts consisted of litter-matched controls and were killed at a humane clinical end point. For allograft of mixed background KPC cells, Crl:CDl-Foxnl nu (CDl-Nude) female mice were used (7 weeks old). For allograft of pure C57BL/J KPC cells, C57BL/J female mice were used (7 weeks old).
• Stellate cells were harvested from the interface of the Nycodenz solution at the bottom and the aqueous solution at the top. The PSCs isolated were then washed with GBSS and resuspended in DMEM with 10% characterized FBS (HyClone), 100 U/mL penicillin and 100 pg/mL streptomycin. The cells were immortalized with pRetro. Super. shARF retroviral plasmid and selected with blasticidin (4pM).
• ImPSC #2 and #3 lines were isolated using a very similar protocol as ImPSC #1 with some minor differences detailed below.
• Pancreas tissue was extracted from Pdgfra tmll(EGFP)Sor mice, minced with a scalpel and digested with 0.1% DNase I and 0.05% collagenase P in GBSS for 30 mins at 37 °C. The solution was then passed through a 100 pm 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 x g for 20mins at 4 °C. Stellate cells were harvested from the interface of the Histodenz solution at the bottom and the aqueous solution at the top.
• the PSCs were washed in PBS containing 3% FBS and resuspended in DMEM containing 10% FBS, 1% penicillin- streptomycin, 0.2% amphotericin B and glutamine (2 mM). After the culture was established, fibroblasts expressing GFP were isolated via FACS and immortalized spontaneously.
• ImPSC #1 cells stably expressing GFP were generated by the PiggyBac transposon system. Briefly, 5 x 10 4 ImPSC #1 cells were seeded in a 6-well plate. 24h after seeding, cells were transfected using Lipofectamine 3000with 1.5pg Super piggyBac Transposase expression vector and 0.6 pg PB-GFP PB-CMV-MCS-EF1- GreenPuro. 24h after transfection, cells were selected in 5pg/mL puromycin for 48h, until puromycin sensitive control cells treated in parallel were dead.
• KPC-EV and KPC-IDO1 cell lines Pure C57BL/J KPC cells stably expressing IDO1-RFP or RFP only (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 Xbal and EcoRI. Successful cloning was confirmed by full sequencing of the insert. 2.5 x 10 5 pure C57BL/J KPC cells were seeded in a 6-well plate.
• Conditions medium experiments 8.7 x 10 6 HPAF-II or CFPAC-1 cells were seeded in 10cm dishes in their normal growth media. Experimental media for conditioning were formulated lacking tryptophan and supplemented with the stated concentrations of 13 Cn- tryptophan. After 48 h in culture, cells were washed in PBS and media for conditioning was added. After 48 h, conditioned medium was collected and passed through a 0.45 pm filter to remove cells. Conditioned medium was stored at -20 °C prior to use.
• the volume of lysis solvent was normalized to 2 x 10 6 cells per ml. Subsequent isolation of metabolites for LCMS was performed as below.
• Lysates (25 pg) were resolved on BoltTM 4-12% bis-tris plus pre-cast gels using BoltTM MOPS SDS Running Buffer running buffer and transferred to nitrocellulose membranes. When total protein staining was performed, it was done prior to blocking using RevertTM Total Protein Stain. Membranes were blocked for 1 hour using Odyssey® Blocking Buffer (TBS) and incubated overnight at 4°C with primary antibodies. All primary antibodies were diluted in Odyssey® Blocking Buffer at a concentration of 1 : 1000, except actin, which was used at 1 : 10,000. Membranes were washed three times in TBS + 1% TWEEN® 20 and incubated with secondary antibodies (1 : 10,000) for Ih at room temperature. Fluorescence intensity was captured and quantified using a LL COR Odyssey® Fc Imaging System with Image Studio software (version 5.2).
• mice Mixed background KPC cells were implanted by unilateral subcutaneous injections (2 x 10 6 cells per flank) into Crl:CDl-Foxnl nu (CDl-Nude) female mice. Mice were monitored daily until they reached clinical end point or tumor size reached 300mm 3 . Mice were fasted for 3h and then received an intraperitoneal injection of 800 pL of 120mM 13 C Tryptophan. 3 h after injection, mice were killed and tumors removed for analysis.
• LCMS for steady state metabolite measurements Cells were seeded into 6-well plates in complete medium and allowed to grow to -80% confluence. Cells were washed with PBS and the relevant experimental media were added for the stated times. Duplicate wells were used for cell counting: cell counts (2D cells) or protein concentration (3D cells BCA assay) were used to normalize the volume of lysis solvent prior to metabolite extractions (1 x 10 6 cells per ml). For 2D grown cells, cells were washed quickly in PBS, then ice-cold lysis solvent (Methanol 50%, acetonitrile 30%, water 20%) was added and cells scraped on ice.
• ice-cold lysis solvent Methanol 50%, acetonitrile 30%, water 20%
• cells were transferred to 15 mL falcon tubes and centrifuged at 50 x g for 5 minutes. The supernatant was removed and the cell pellet was washed in PBS and centrifuged again. The supernatant was removed and the cell pellet resuspended in ice-cold lysis solvent. Lysates were transferred to 1.5ml tubes on ice, vortexed, then centrifuged at 18,000 x g at 4 °C for 10 mins. Supernatants were collected and stored at -80 °C for LCMS analysis. Tissue samples were snap-frozen and stored at -80°C. Frozen samples were weighed before lysis.
• GCMS for formate analysis 40 pL of sample was added to 20 pL of d2-formate (50 pM, internal standard), 50 pL pyridine, 10 pL NaOH (IN), and 5 pL benzyl alcohol. While vortexing, 20 pL of methyl chloroformate was added to this mixture for derivatization. 100 pL methyl tertiary butyl ether and 200 pL H2O were then added, and the samples subsequently vortexed for 10 s and centrifuged for 10 mins at maximum speed. The apolar phase was then transferred to a GC-vial and capped.
• Sample analysis was performed using an LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer.
• a gradient program starting at 20% of A and linearly increasing to 80% at 30 min was used followed by washing (92% of A for 5 mins) and re-equilibration (20% of A for lOmin) steps. The total run time of the method was 45 min.
• the LC stream was desolvated and ionized in the HESI probe.
• the Exactive mass spectrometer was operated in full scan mode over a mass range of 70-1,200 m/z at a resolution of 50,000 with polarity switching.
• the LCMS raw data was converted into mzML files by using ProteoWizard and imported to MZMine 2.10 for peak extraction and sample alignment.
• a house-made database including all possible 13 C and 15 N isotopic m/z values of the relevant metabolites was used for the assignment of LCMS signals. Finally the peak areas were used for comparative quantification.
• Carbon-13 labelling of metabolites Experimental media were formulated lacking tryptophan or serine and supplemental with the stated concentrations of 13 Cn-tryptophan, 13 C3 15 Ni-serine, or 13 Ci-formate. The same basic protocol was used as for steady state metabolite measurements. Metabolites were extracted as above.
• PDAC cells express IDO1 in a context-dependent manner: The expression of IDO1 in pancreatic cancer cells was investigated in vitro and in vivo. Utilizing murine KPC models, IDO1 expression was assessed in a range of contexts (FIG. 67): Direct analysis of pancreatic tumor tissue from Pdxl-cre;LSL-Kras G12D/+ ;Trp53 fl/+ and Pdxl-cre;LSL- Kras G12D/+ ;LSL-Trp53 R172H/+ mice showed that tumors had increased IDO1 expression versus normal pancreas tissue, and that certain tumors expressed high levels of IDO 1 (FIG. 67 PANEL B and PANEL C).
• tumor derived primary KPC cells cultured under normal in vitro conditions displayed undetectable IDO1 (FIG. 67 PANEL D).
• Addition of the murine form of cytokine IFNy - a canonical activator of IDO 1 - increased IDO1 expression in vitro.
• the human form of IFNy did not impact IDO1 expression in murine cells (FIG. 67 PANEL D).
• KPC cells were injected into CD-I nude mice as subcutaneous allografts. Assessment of IDO 1 expression in allograft tumor tissue revealed extremely low IDO1 expression (FIG. 67 PANEL D).
• IDO 1 The expression of IDO 1 in a panel of human pancreatic cancer cells was also investigated. Similar to KPC cells, IDO1 expression was very low or undetectable under normal culture conditions (FIG. 67 PANEL F). Addition of IFNy (human form) consistently increased IDO1 expression. To globally assess IDO1 expression in human cancers, data were extracted from the metabolic gene rapid visualizer. In the pancreas, IDO1 had a similar range of expression in healthy tissue compared to cancer cell lines grown in vitro (FIG. 68). However, pancreatic tumor tissue had multiple high or very high IDO 1 -expressing tumors. The trend was also observed in a variety of other tumors, particularly in the colon, breast, and cervix. The dataset showed consistently that IDO1 expression was elevated in tumor versus healthy tissue, but cancer cells grown under normal in vitro culture conditions did not necessarily display tumor relevant levels of IDO 1. Overall, these data showed that IDO1 expression was up-regulated during tumor formation in an immune competent setting.
• FIG. 67 shows IDO1 expression in vivo.
• PANEL A shows a schematic detailing the methods used to analyze IDO1 expression in genetically engineered mouse models (GEMM) of pancreatic ductal adenocarcinoma (PDAC). Tumors from Pdxl-cre;Kras G12D/+ ;Trp53 fl/+ and Pdxl-cre;Kras G12D/+ ;Trp53 R172H/+ mice and healthy pancreas tissue from non-cre- expressing isogenic control mice were lysed.
• PANEL B shows the indicated proteins after analysis by immunoblotting.
• PANEL D shows KPC A cells, a line isolated from tumors of mixed-background Pdxl-cre;Kras G12D/+ ;Trp53 R172H/+ mice were either treated with mouse IFNy (Ing/ml) for 24h, or subcutaneously injected into the flank of CD 1 -nude mice to form tumors. Cell and tumor lysates were subjected to immunoblotting for the indicated proteins.
• PANEL E shows KPC cells were isolated from pure C567B16/J background Pdxl-cre;Kras G12D/+ ;Trp53 R172H/+ mice and either treated in culture with mouse IFNy (Ing/ml) for 24h or subcutaneously injected 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 IFNy (1 ng/mL) for 24h and cell lysates blotted for the indicated proteins.
• FIG. 68 shows data extracted from the MERAV database showing the relative abundance of IDO 1 mRNA from microarrays.
• IDO1 expression can be regulated by attachment independent growth in vitro.
• IDO1 expression promotes the utilization of tryptophan via the kynurenine pathway. Given the diversity of potential metabolic interactions of this pathway (FIG. 69 PANEL A) the effect of immune independent stimuli on IDO1 expression was investigated. Mitochondrial metabolism is potentially linked to the kynureneine pathway in two ways: (1) mitochondrial production of superoxide and (2) entry of tryptophan derived carbons into the TCA cycle via a-ketoadipate. Exposure of PDAC cells to low oxygen or rotenone - both predicated to impact OXPHOS and potentially modulate superoxide levels - had little impact on IDO1 expression (FIG. 69 PANEL B and PANEL C).
• FIG 69 shows that IDO expression was upregulated by 3D growth and IFNy via JAK/STAT signaling.
• PANEL A shows a schematic detailing the kynurenine pathway through which tryptophan is metabolized. The indicated proteins were analyzed by immunoblotting in the indicated cell lines after 24 h 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 pM) 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 h, and cell lysates were immunoblotted for the indicated proteins.
• PANEL E shows proteins cultured in 2D or 3D conditions.
• PANEL G shows the results of CFPAC-1 cells cultured in 2D or 3D conditions for 24h and treated with epacadostat (IpM) or vehicle only control for 16h before media kynurenine was analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• PANEL H shows CFPAC- 1 or HPAF-II cells cultured in either 2D or 3D conditions for 24h and then treated for 16h with JAKi (IpM) or vehicle only control (veh.) and/or human IFNy (Ing/ml). Cells were then lysed and indicated proteins analyzed by immunoblotting.
• Indicated cell lines were grown in either 2D or 3D for 24 hours and lysates immunoblotted for indicated proteins (PANEL C) after 16h treatment with MG132 (20 pM) or vehicle-only control (PANEL D) after treatment for the indicated times with bafilomycin Al (100 nM) or vehicle-only control or (PANEL E) after 16h treatment with JAKi (at indicated concentrations), vehicle-only control or IFNy (1 ng/ml).
• Attachment independent (Al) growth stimulated IDO1 expression is regulated through JAK/STAT signaling: The molecular mechanism through which attachment independent (Al) growth up-regulates IDO1 expression was studied. Treatment with the proteasome inhibitor MG132 (FIG. 70 PANEL C) or with lysosomal inhibitor bafilomycin (FIG. 70 PANEL D) had no effect on IDO1 protein levels. The data showed that increased IDO1 levels observed during Al growth were not due to changes in IDO1 degradation via proteasomal or lysosomal systems.
• IFNy mediates changes in gene expression through activation of the JAK/STAT signaling cascade.
• the role of Al growth, independent of IFNy, on activation of the JAK/STAT pathway leading to increased IDO1 expression was investigated.
• Activation of STAT proteins was studied using a small molecule JAK inhibitor I (JAKi).
• JAKi JAK inhibitor I
• the results showed that STAT3 phosphorylation was increased upon Al growth (FIG. 69 PANEL H), indicating up-regulated JAK/STAT pathway activation. This appeared to be specific to STAT3, as no such increase was detected for STAT1 (FIG. 70 PANEL E).
• Up-regulation of IDO 1 protein levels in Al-grown cells was blocked by treatment with JAKi (FIG.
• LCMS Liquid chromatography mass spectrometry
• kynurenine from tryptophan a one-carbon unit is released as formate.
• a potential destination for this formate is to enter the THF cycle.
• the tryptophan-derived carbon could be used in a number of anabolic pathways, including purine nucleotide synthesis. Tryptophan-derived carbons were observed in purine nucleotides (FIG. 71), indicating that tryptophan is a legitimate source of one-carbon units for the THF cycle in PDAC cells.
• Another THF-dependent fate for one-carbons is de novo serine synthesis (by combination with glycine via SHMT1/2), and increased labelling of serine from labelled tryptophan was also observed.
• CFPAC-1 cells cultured in 2D or 3D for 24h then treated for 24h with epacadostat (IpM) or vehicle only control in the presence of either unlabeled ( 12 C) or 13 Cn tryptophan and intracellular quantities of the indicated nucleotides were analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• IpM epacadostat
• vehicle only control in the presence of either unlabeled ( 12 C) or 13 Cn tryptophan and intracellular quantities of the indicated nucleotides were analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• mice received a single intraperitoneal injection of 13 Cn-tryptophan solution and we assessed the incorporation of tryptophan-derived carbons using LCMS at a single time-point post injection.
• LCMS liquid phase shift
• FIG. 73 shows that tryptophan-derived one-carbon units are incorporated into nucleotides in in vivo pancreatic tumors.
• PANEL A shows a schematic detailing the experimental approaches for this figure.
• PANEL B KPC cells from pure C57BL/J Pdxl- cre;Kras G12D/+ ;Trp53 R172H/+ mice expressing IDO1 or empty -vector control (EV) were injected subcutaneously into the flanks of C57BL/J mice, once tumors had formed the mice were given 800pL of 120 mM 13 Cn tryptophan by intraperitoneal injection and left for 3h.
• EV empty -vector control
• FIG. 74 shows data from KPC cells from pure C57BL/J Pdxl- cre;Kras G12D/+ ;Trp53 R172H/+ mice expressing IDO1 or empty -vector control (EV) were injected subcutaneously into the flanks of C57BL/J mice, once tumors had formed the mice were given 800pL of 120mM 13 Cn tryptophan by intraperitoneal injection and left for 3h. Tumor tissue was excised and analyzed by immunoblotting for the indicated proteins.
• EV empty -vector control
• PDAC cells excrete tryptophan-derived formate Whether IDO 1 -expressing PDAC cells released formate produced from tryptophan was investigated. After culture with 13 Cn- tryptophan, labelled formate was identified by gas chromatography mass spectrometry in the spent medium from CFPAC-1 and HPAF-II cells expressing IDO1 (+IFNy) (FIG. 75 PANEL A and PANEL B) The release of tryptophan-derived formate was considerably higher than serine-derived formate in CFPAC-1 cells and equivalent in HPAF-II cells. These results were surprising because serine is generally viewed as the dominant one-carbon source in cancer cells, and because exogenous serine levels are higher than tryptophan.
• Stellate cells take up tryptophan-derived formate and utilize formate for purine synthesis: The ability of pancreatic stellate cells to take up tryptophan-derived formate released by PDAC cells and utilize it in synthesis of purine nucleotides was investigated. To directly test the ability of pancreatic stellate cells take up and utilize exogenous formate, immortalized mouse stellate cells (ImPSCs) were cultured in media supplemented with 13 Ci- formate. LCMS analysis revealed that stellate cells consumed extracellular formate and incorporated single carbon into purines. Purine synthesis utilized two THF-derived one carbons, giving rise to major isotopologue peaks of m+1 and m+2 (FIG.
• ImPSCs immortalized mouse stellate cells
• CFPAC-1 cells were co-cultured for 24 hours with ImPSC cells engineered to ectopically express GFP.
• the co-culture medium contained 13 Cn-tryptophan and IFNy.
• the ImPSC-GFP cells were then separated from the PDAC cells by FACS and subjected to LCMS analysis. Labelling of purine nucleotides was evident in stellate cells co-cultured with PDAC cells in the presence of IFNy (FIG. 75 PANEL H-PANEL K) With this method, the labelled fractions were generally smaller and clear labelling in serine was not observed. Nucleotide labelling was not seen in stellate cells cultured alone. Importantly, labelling was also lower when IDO1 levels were low (-IFNy) or IDO1 was inhibited by treatment with epacadostat (FIG. 75 PANEL H- PANEL K)
• FIG. 75 shows that cancer cells released tryptophan-derived formate, which was consumed by pancreatic stellate cells and incorporated into nucleotides.
• CFPAC-1 PANEL A
• HPAF-II PANEL B
• IFNy IFNy
• vehicle only control IFNy
• Media quantities of formate were analyzed by derivatization and GC- MS (lex, triplicate wells, error bars are std. dev.).
• PANEL C shows a schematic of the experimental approaches used in PANEL D-PANEL K.
• CFPAC-1 cells were treated with vehicle only control or human IFNy (Ing/ml) and epacadostat (epac., IpM) or vehicle only control in the presence of unlabeled ( 12 C) or 13 Cn tryptophan.
• Conditioned media was collected after 24h and ImPSC’ s were cultured in this media, or in non-conditioned treatment-matched media.
• intracellular quantities of serine (PANEL D), ATP (PANEL E), ADP (PANEL F) and AMP (PANEL G) were analyzed by LCMS (fraction of major isotopologues relative to total are shown, lex, triplicate wells, error bars are std. dev.).
• ImPSC-GFP cells were cultured for 24h in 2D as a monoculture or in co-culture with CFPAC-1 cells. Cells were then treated with vehicle only control or human IFNy (Ing/ml) and epacadostat (1 pM) or vehicle only control in the presence of 13 Cn tryptophan for 24h. Cells were then trypsinised and sorted using FACS for GFP-positive cells and intracellular quantities of serine (PANEL H), ATP (PANEL I), ADP (PANEL J) and AMP (PANEL K) were analyzed by LCMS (fraction of major isotopologues relative to total are shown, lex, triplicate wells, error bars are std. dev.). PANEL L shows a proposed model for the use of tryptophan-derived formate in pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells.
• PDAC pancreatic ductal adenocarcino
• FIG. 76 shows intracellular uptake of 13 Ci formate in ATP, DP, AMP, and GTP in ImPSC #1, ImPSC #2, and ImPSC #3 cells.
• ImPSC #1, ImPSC #2 & ImPSC #3 cells were cultured for 24h in the presence of 13 Ci formate and intracellular quantities of ATP (PANEL A), ADP (PANEL B), AMP (PANEL C) and GTP (PANEL D), all possible destination for formate-derived one carbons were analyzed by LCMS (lex, triplicate wells, error bars are std. dev.).
• CFPAC-1 cells were treated with IFNy (Ing/ml) and/or epacadostat (IpM) and/or vehicle only controls in the presence of unlabeled ( 12 C) or 13 Cn tryptophan.
• Conditioned media was collected after 24h and ImPSC#2 cells were cultured in this media, or in nonconditioned treatment-matched media.
• intracellular quantities of ATP (PANEL E), ADP (PANEL F) and serine (PANEL G) were analyzed by LCMS (fraction of major isotopologues relative to total are shown lex, triplicate wells, error bars are std. dev.).
• IDO1 activator IFNY Beyond the canonical IDO1 activator IFNY, the impact of metabolic perturbations caused by hypoxia, rotenone treatment or galactose on IDO1 expression was examined. None of the conditions changed IDO1 levels. A transfer of PDAC cells from standard monolayer culture to attachment independent conditions up-regulated IDO1 was observed, albeit to a lesser extent than IFNY. The mechanism of the effect was investigated, and attachment independent growth regulated IDO1 via the JAK/STAT signaling pathway was observed. [0281] Improved understanding of IDO 1 expression allowed a detailed analysis of IDO 1- dependent tryptophan metabolism in PDAC cells.
• the kynurenine pathway made a significant contribution to nucleotide synthesis in PDAC cells in vitro, contributing carbons to approximately 30% of the purine pool over 24 hours.
• IDO 1 -dependent tryptophan labelling was detected in tumor serine and purine pools following a single injection of 13C11- tryptophan.
• PDAC cells were tested for tryptophan-derived formate efflux. Certain PDAC cells released double the quantity of tryptophan-derived versus serine-derived formate, despite exogenous serine outweighing tryptophan 4: 1.
• a robust ability of stellate cells to capture tryptophan-derived formate produced by PDAC cells and incorporate the formate into nucleotide synthesis was also observed.
• EXAMPLE 16 Effect of epacadostat on cell proliferation and nucleotide synthesis.
• Epacadostat enhances the antiproliferative effect or serine starvation.
• KPC cells tumor cells derived from a pancreatic tumor of a Kras mut p53 mut genetically engineered mouse
• KPC cells tumor cells derived from a pancreatic tumor of a Kras mut p53 mut genetically engineered mouse
• Cells were washed with PBS and received either control medium containing all amino acids or matched medium lacking serine (-Serine) with or without IDO1 inhibitor epacadostat (1 pM).
• Cell number was recorded every 24 h for 5 days. A time zero plate was used to calculate the starting cell number.
• FIG. 77 LEFT PANEL shows cell proliferation over 5 days in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• RIGHT PANEL shows fold changes in cell number at day 5 compared to day 0 in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• Serine starvation was shown to increase the amount of tryptophan-derived carbon used in nucleotide synthesis in an IDO 1 -dependent matter.
• KPC cells tumor cells derived from a pancreatic tumor of a Kras mut p53 mut genetically engineered mouse
• Cells were fed medium containing carbon- 13 labelled tryptophan either with (+) or without (-) serine (plus all other amino acids), with or without the IDO1 inhibitor epacadostat (1 pM).
• metabolites were extracted from cells and analyzed by LCMS.
• the labelled fraction (derived from carbon-13) of purine nucleotides ATP, ADP, AMP, GDP, GTP are shown.
• FIG. 78 shows the labelled fractions derived from carbon- 13 in cells of AMP, ADP, ATP, GDP, and GMP in cells treated with: 1) control + vehicle; 2) -Serine + vehicle; 3) control + epacadostat (1 pM); or 4) -Serine + epacadostat (1 pM).
• Embodiment 1 A method of treating a cancer in a subject in need thereof, the method 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, for a first amount of time; b) a radiation therapy for a second amount of time; and c) after the first amount of time and the second amount of time, waiting a third amount of time, wherein the subject is not administered the pharmaceutical composition or the radiotherapy during the third amount of time.
• Embodiment 2 The method of embodiment 1, wherein the cancer is rectal cancer.
• Embodiment 3. The method of embodiment 1, wherein the cancer is breast cancer.
• Embodiment 4. The method of any one of embodiments 1-3, wherein the administration is oral.
• Embodiment 5 The method of any one of embodiments 1-4, wherein the radiation therapy is an external beam therapy.
• Embodiment 6 The method of embodiment 5, wherein the external beam therapy is three dimensional conformal radiation therapy (3D-CRT).
• 3D-CRT three dimensional conformal radiation therapy
• Embodiment 7 The method of embodiment 5, wherein the external beam therapy is intensity-modulated radiation therapy (IMRT).
• IMRT intensity-modulated radiation therapy
• Embodiment 8 The method of any one of embodiments 1-7, wherein the radiation therapy comprises administering about 5 Grays (Gy) to about 50 Gy of radiation to the subject.
• Embodiment 9 The method of any one of embodiments 1-8, wherein the radiation therapy comprises administering about 5 Gy of radiation to the subject.
• Embodiment 10 The method of any one of embodiments 1-8, wherein the radiation therapy comprises administering about 50 Gy of radiation to the subject.
• Embodiment 11 The method of any one of embodiments 1-4 or 8-10, wherein the radiation therapy is an internal beam therapy.
• Embodiment 12 The method of any one of embodiments 1-11, wherein the at least two amino acids is serine and glycine.
• Embodiment 13 The method of any one of embodiments 1-12, wherein the pharmaceutical composition is further substantially devoid of proline.
• Embodiment 14 The method of any one of embodiments 1-13, wherein the pharmaceutical composition is further substantially devoid of cysteine.
• Embodiment 15 The method of any one of embodiments 1-14, further comprising administering a high fat diet to the subject.
• Embodiment 16 The method of embodiment 15, wherein the high fat diet has greater than about 50% of daily calories from fat.
• Embodiment 17 The method of any one of embodiments 1-16, further comprising administering a low carbohydrate diet to the subject.
• Embodiment 18 The method of embodiment 17, wherein the low carbohydrate diet has less than about 50% of daily calories from carbohydrates.
• Embodiment 19 The method of any one of embodiments 1-18, further comprising administering a low protein diet to the subject.
• Embodiment 20 The method of embodiment 19, wherein the low protein diet has less than about 15% of daily calories from whole protein.
• Embodiment 21 The method of any one of embodiments 1-20, wherein the first amount of time and the second amount of time are equal.
• Embodiment 22 The method of any one of embodiments 1-21, wherein the first amount of time and the second amount of time are 5 days.
• Embodiment 23 The method of any one of embodiments 1-20, wherein the first amount of time and the second amount of time is greater than the third amount of time.
• Embodiment 24 The method of any one of embodiments 1-23, wherein the third amount of time is 2 days.
• Embodiment 25 The method of any one of embodiments 1-24, further comprising repeating steps a), b), and c).
• Embodiment 26 A method of treating a cancer in a subject in need thereof, the method 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) administering a therapeutically effective amount of an immunotherapy, wherein the immunotherapy is administered at least twice per day.
• Embodiment 27 The method of embodiment 26, wherein the cancer is pancreatic cancer.
• Embodiment 28 The method of embodiment 26, wherein the cancer is colon cancer.
• Embodiment 29 The method of embodiment 26, wherein the cancer is breast cancer.
• Embodiment 30 The method of embodiment 26, wherein the cancer is cervical cancer.
• Embodiment 31 The method of embodiment 26, wherein the cancer is lung cancer.
• Embodiment 32 The method of any one of embodiments 26-31, wherein the immunotherapy is an IDO1 inhibitor.
• Embodiment 33 The method of embodiment 32, wherein the IDO1 inhibitor is indoximod.
• Embodiment 34 The method of embodiment 32, wherein the IDO1 inhibitor is navoximod.
• Embodiment 35 The method of embodiment 32, wherein the IDO1 inhibitor is epacadostat.
• Embodiment 36 The method of any one of embodiments 26-35, wherein the at least two amino acids is serine and glycine.
• Embodiment 37 The method of any one of embodiments 26-36, 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 26-39, wherein the therapeutically effective amount of the immunotherapy is about 25 mg to about 500 mg.
• Embodiment 41 The method of any one of embodiments 26-40, wherein the therapeutically effective amount of the immunotherapy is about 25 mg.
• Embodiment 42 The method of any one of embodiments 26-40, wherein the therapeutically effective amount of the immunotherapy is about 50 mg.
• Embodiment 43 The method of any one of embodiments 26-40, wherein the therapeutically effective amount of the immunotherapy is about 100 mg.
• Embodiment 44 The method of any one of embodiments 26-40, wherein the therapeutically effective amount of the immunotherapy is about 300 mg.
• Embodiment 45 The method of any one of embodiments 26-44, wherein the immunotherapy is administered twice per day.
• Embodiment 46 The method of any one of embodiments 26-44, wherein the immunotherapy is administered three times per day.
• Embodiment 47 A method of treating a cancer in a subject in need thereof, the method 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.
• Embodiment 48 The method of embodiment 47, wherein the cancer is pancreatic cancer.
• Embodiment 49 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.
• Embodiment 52 The method of embodiment 47, wherein the cancer is lung cancer.
• Embodiment 53 The method of any one of embodiments 47-52, wherein the at least two amino acids is serine and glycine.
• Embodiment 54 The method of any one of embodiments 47-53, 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 47-56, wherein the therapeutically effective amount of epacadostat is about 25 mg to about 500 mg.
• Embodiment 58 The method of any one of embodiments 47-57, wherein the therapeutically effective amount of epacadostat is about 25 mg.
• Embodiment 59 The method of any one of embodiments 47-57, 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 47-57, wherein the therapeutically effective amount of epacadostat is about 300 mg.

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