CANCER TREATMENT BY GLYCEROL-3-PHOSPHATE DEHYDROGENASE 1 INHIBITION

Abstract

The present invention relates to an inhibitor of glycerol-3-phosphate dehydrogenase 1 (GPD1 inhibitor) for use in treatment and/or prevention of cancer. The present invention further relates to a kit comprising a GPD1 inhibitor comprised in a housing; to a method for identifying a subject suffering from cancer susceptible to cancer treatment by administration of a GPD1 inhibitor comprising a) determining in a sample of said subject the amount of a GPD1 gene product, b) comparing said amount determined in step a) to a reference, and c) based on the result of step b), identifying a subject susceptible to cancer treatment by administration of a GPD1 inhibitor; and to further means and methods related thereto.

Claims

1. A method for treating and/or preventing cancer in a subject, comprising administering to said subject an inhibitor of glycerol-3-phosphate dehydrogenase 1 (GPD1 inhibitor).

2. The method of claim 1, wherein said GPD1 inhibitor is a direct GPD1 inhibitor, preferably is a polypeptide or polynucleotide specifically binding and inhibiting GPD1.

3. The method of claim 1, wherein said GPD1 inhibitor is selected from the group consisting of an antibody, an aptamer, an anticalin, and a Designed Ankyrin Repeat Protein (DARPin).

4. The method of claim 1, wherein said GPD1 inhibitor is an indirect GPD1 inhibitor.

5. The method of claim 4, wherein said GPD1 inhibitor is selected from the group consisting of a shRNA, a siRNA, a miRNA agent, an antisense molecule, an antisense oligonucleotide, a ribozyme, and a pair of CRISPR/Cas oligonucleotides.

6. The method of claim 1, wherein said GPD1 inhibitor is ()-epicatechin, ()-epicatechin-3-gallate, ()-epigallocatechin, ()-epigallocatechin-3-gallate, preferably is ()-epigallocatechin-3-gallate.

7. The method of claim 1, wherein said cancer is bladder cancer or brain cancer, wherein said brain cancer preferably is a glioma, more preferably an astrocytoma, an oligodendroglioma or an oligoastrocytoma, most preferably a glioblastoma multiforme.

8. The method of claim 1, wherein said treating and/or preventing cancer further comprises administration of a cancer therapeutic agent, preferably comprises administration of chemotherapy.

9. (canceled)

10. (canceled)

11. A method for identifying a subject suffering from cancer susceptible to cancer treatment by administration of a GPD1 inhibitor comprising a) determining in a sample of said subject the amount of a GPD1 gene product, b) comparing said amount determined in step a) to a reference, and c) based on the result of step b), identifying a subject susceptible to cancer treatment by administration of a GPD1 inhibitor.

12. The method of claim 11, wherein said subject is a subject suffering from brain cancer, wherein said brain cancer preferably is a glioma, more preferably an astrocytoma, an oligodendroglioma or an oligoastrocytoma, most preferably a glioblastoma multiforme.

13. The method of 11, wherein said GPD1 inhibitor comprises at least one nucleic acid comprising at least one sequence selected from SEQ ID Nos: 1 to 4.

14. (canceled)

15. A method for identifying a compound for treating and/or preventing cancer, comprising a) contacting a reaction mixture comprising a GPD1 polypeptide, glycerol-3-phosphate, and nicotine-adenine-dinucleotide (NAD) with a compound suspected to have the activity of being a direct GPD1 inhibitor, b) further contacting said reaction mixture of a) with a luciferase and a luciferase substrate, c) determining (i) NADH absorbance and/or (ii) the luciferase-mediated luminescence in the reaction mixture, and (d) identifying a compound for treating and/or preventing cancer based on the result of the determination step of c).

16. The method of claim 4, wherein said indirect GPD1 inhibitor is a polypeptide comprising a lysosome-degradation sequence.

17. The method of claim 4, wherein said indirect GPD1 inhibitor is a chaperone-mediated autophagy-targeting motif (CTM) or is a polynucleotide.

18. The method of claim 4, wherein said GPD1 inhibitor comprises at least one nucleic acid comprising at least one sequence selected from SEQ ID NOs: 1 to 4.

Description

FIGURE LEGENDS

[0088] FIG. 1: GPD1 expression in brain tumor stem cells (BTSCs) and in highly tumorigenic human glioblastoma multiforme (GBM) cell lines; anti-GPD1 Western blot on SDS-PAGE separated cellular proteins of the indicated cells. NSC: neural stem cells; Turn: BTSCs; LN and U87MG: GBM cell lines.

[0089] FIG. 2: GPD1 KO tumor stem cells fail to form neurospheres. Phase contrast photographs of wildtype brain tumor stem cells (WT BTSCs, upper row) and of mouse brain tumor stem cells (mBTSCs) in which GPD1 expression was knocked out (lower row). Cells were seeded at four different densities and photographed at two different time points after seeding as indicated.

[0090] FIG. 3: Survival following GPD1-KD

Survival study of tumor-bearing mice w/o GPD1-KD. The tumors were induced at birth. At the age of four weeks tamoxifen was injected for ten days to permanently activate the GPD1 miRNA and the symptom free time was measured. Mice with a GPD1-KD survived significantly longer than mice with a WT tumor

[0091] FIG. 4: Loss of GPD1 reduces the G3P levels to that of normal neural stem cells

Intracellular levels of glycerol-3-phosphate (G3P) per 10.sup.6 cells in A) U87MG human glioblastoma cells; and in B) murine brain tumor stem cells (mBTSCs) and neural stem cell (Stem cells) lines; mKO1a and mKO1b indicate different guide RNAs used in BTSCs.

[0092] FIG. 5: Effects of GPD1 KO on lipid storage. Murine brain tumor stem cells (mBTSCs) and neural stem cell (Stem cells) lines were stained with Oil Red O; mKO#1 and mKO#2 indicate different guide RNAs used in BTSCs. Arrows point to exemplary lipid droplets.

[0093] FIG. 6: Sensitivity of GPD1 KO cells to Chemotherapy. % apoptotic cells after 72 h treatment with Temozolomide.

[0094] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: EXPRESSION OF GPD1 IN TUMOR CELLS AND IN TUMOR STEM CELLS

[0095] High GPD1 protein levels could be detected in mouse BTSCs; High GPD1 protein levels also correlated with high tumorigenicity of human glioma cell lines, in particular LN-229 and U87G (FIG. 1).

EXAMPLE 2: GPD1 EXPRESSION CO-LOCALIZES WITH STEM CELL MARKER TLX

[0096] In primary mouse brain tumors, GPD1 positive cells were found to form clusters at the tumor edge and these clusters did not overlap with PCNA staining. GPD1 expression was found in T1x-GFP expressing BTSCs, which are PCNA negative.

EXAMPLE 3: GPD1 KO PROMOTES DIFFERENTIATION

[0097] GPD1 was knocked out in primary mBTSCs and cells were allowed to differentiate in vitro. GPD1 KO BTSCs expressed significantly more neuronal cell markers (9%), compared to WT tumors (1%); also, WT BTSCs were more likely to differentiate into oligodendrocytes (consistent with GBM) compared to GPD1 KO cells (7% vs. <1%).

EXAMPLE 4: GPD1 KO TUMOR STEM CELLS FAIL TO FORM NEUROSPHERES

[0098] Stem cells isolated from tumors were cultured as neurospheres in vitro (FIG. 2). GPD1 KO tumor stem cells formed less and smaller neurospheres and instead grew as attached cells.

EXAMPLE 5: INDUCIBLE KNOCKDOWN OF GPD1 IN PRIMARY BRAIN TUMORS PROLONGS SURVIVAL IN VIVO

[0099] Animals with existing primary brain tumors were treated with Tamoxifen (QDx10) to induce the expression of a miRNA against GPD1. Animals with GPD1 knockdown showed significant increase in survival.

EXAMPLE 6: LOSS OF GPD1 REDUCES THE G3P LEVELS TO THAT OF NORMAL NEURAL STEM CELLS

[0100] Intracellular G3P was measured using a commercially available kit.

EXAMPLE 7: EFFECTS OF GPD1 KO ON LIPID STORAGE (OILREDO STAINING)

[0101] GPD1 KO phenotype shows lipid storage comparable to normal neural stem cells (FIG. 5). In contrast, mBTSCs showed increased lipid storage.

EXAMPLE 8: SENSITIVITY OF GPD1 KO CELLS TO CHEMOTHERAPY

[0102] U87MG, mBTSCs or the corresponding GPD1 KO cells were treated with 250 M Temozolomide for 72 hours. An apoptosis assay showed that GPD1 KO cells are more sensitive to chemotherapy than the BTSC or human U87MG cell line (FIG. 6).