SCREENING METHOD FOR THE IDENTIFICATION OF CANCER THERAPEUTICS

Abstract

The present invention pertains to a method for identifying anti-cancer compounds. The invention is based on the finding that a direct protein-protein interaction between 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) and (F-box protein 28) FBXO28 silences a ubiquitin E3 ligase activity of FBXO28 towards HIF1a. Interfering with this protein-protein interaction leads to a strong induction of HIF1a proteasomal degradation and cell death in tumors, and therefore, compounds screened according to the present invention harbour therapeutic potential for the treatment of proliferative diseases such as cancer. The invention provides a screening method for cancer therapeutics based on the interaction of PFKFB4 and FBXO28, as well medical applications thereof.

Claims

1. A method for the identification of a compound which is useful as a medicament for the treatment of cancer, the method comprising the steps of: (a) Providing a candidate compound, (b) Providing a protein complex comprising PFKFB4 and FBXO28, or fragments or derivatives thereof, wherein PFKFB4 and FBXO28, or the fragments or derivatives thereof, are in direct protein-to-protein interaction with each other (such as binding each other), (c) Contacting said candidate compound with the protein complex comprising PFKFB4 and FBXO28, or fragments or derivatives thereof, and (d) Determining whether contacting in (c) results in a change of protein-protein interaction between PFKFB4 and FBXO28, or the fragments or derivatives thereof, optionally by comparison to a control, wherein in the event of a reduction of protein-protein interaction between PFKFB4 and FBXO28, or the fragments or derivatives thereof, as determined in step (d), the candidate compound is useful as a medicament for the treatment of cancer.

2. The method according to claim 1, wherein the complex is provided within a biological assay cell, or is provided in a cell-free system.

3. The method according to claim 1, wherein the candidate compound is selected from a small molecular compound (small molecule), a polypeptide, peptide, glycoprotein, a peptidomimetic, an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or a guide nucleic acid (gRNA or gDNA) and/or tracrRNA.

4. The method according to claim 1, wherein the determining in step (d) involves at least one of: (i) co-immuno precipitation of the interacting proteins, (ii) a Frster resonance energy transfer (FRET), (iii)yeast two hybrid assay, (iv)protein-protein covalent cross-linking, (v)mass spectroscopy, (vi) affinity chromatography, (vii) affinity blotting, (viii)two-hybrid reconstruction (ix)reporter gene assays (NanoBiT), (x) detection of HIF 1 a ubiquitylation, (xi) detection of HIF 1 a degradation, (xii)immunofluorescent based assays, (xiii)detection of assay cell viability.

5. The method according to claim 1, which is performed in a non-human animal system, ex-vivo, or in-vitro, preferably in a human cell line such as Human Embryonic Kidney cells (HEK).

6. The method according to claim 1, wherein the derivative or fragment of PFKFB4 is characterized by its ability to be in protein-protein interaction with a full length FBXO28 protein.

7. The method according to claim 1, wherein the derivative or fragment of FBXO28 is characterized by its ability to be in protein-protein interaction with a full length PFKFB4 protein.

8. The method according to claim 6, wherein the ability to be in protein-protein interaction is the ability of an interaction that mimics the native protein-protein interaction between PFKFB4 and FBXO28.

9. The method according to claim 1, wherein the cancer is a PFKFB4-expressing cancer, preferably a cancer associated with an elevated expression of PFKFB4, such as glioblastoma, breast, prostate or lung cancer.

10. A method for the production of pharmaceutical composition, the method comprising identifying a compound with a method according to claim 1, and formulating the compound as a pharmaceutical composition together with a pharmaceutically acceptable carrier and/or excipient.

11. Use of a compound identified according to the method of claim 1, for the production of a medicament for use in the treatment of cancer.

12. A method of treating a cancer in a subject, the method comprising the step of interrupting in cell associated with the cancer in the subject the protein-protein interaction between PFKFB4 and FBXO28.

13. The method according to claim 12, wherein the cancer is a cancer characterized by the expression of PFKFB4 and FBXO28.

14. The method according to claim 12, wherein the subject is a mammal, preferably a human patient suffering from cancer and in need of a treatment.

15. The method according to claim 12, wherein the method comprising the administration to the subject of a therapeutically effective amount of a compound which specifically reduces the protein-protein interaction between PFKFB4 and FBXO28 in a cell associated with the cancer.

16. A method of treating a cancer in a subject, the method comprising the step of interrupting in cell associated with the cancer in the subject the protein-protein interaction between PFKFB4 and FBXO28; wherein the compound is a compound as identified according to a method of claim 1.

Description

[0053] FIG. 1: TMA and Western blots

[0054] FIG. 2: (A) Luminescent image of animals stably ex-pressing the luciferase gene and treated with doxycycline for 6 weeks. (B) Kaplan Meier curves of the animals treated with doxycycline expressing shNT (black) or shPFKFB4 (grey) and non-treated animals (dashed). (C) Tumor size as determined by luminescent imaging, expressed in total flux (photons/second). (D) Scanned H and E stained sections of mouse brains from the non-treated and the shNT expressing groups using the Leica ImageScope. Whole brain, to-fold and 4o-fold magnification are presented.

[0055] FIG. 3: PFKFB4 is involved in HIF transcriptional function

[0056] FIG. 4: Knockdown of PFKFB4 impairs HIF stability

[0057] FIG. 5: FBXO28 interacts with PFKFB4

[0058] FIG. 6: FBXO28 is required for HIF1a stability

[0059] FIG. 7: shows a NanoBiT assay for screening candidate compounds.

[0060] And in the sequences:

TABLE-US-00001 SEQIDNO:1 MASPRELTQNPLKKIWMPYSNGRPALHACQRGVCMTNCPTLIVMVGLPA RGKTYISKKLTRYLNWIGVPTREFNVGQYRRDVVKTYKSFEFFLPDNEE GLKIRKQCALAALRDVRRFLSEEGGHVAVFDATNTTRERRATIFNFGEQ NGYKTFFVESICVDPEVIAANIVQVKLGSPDYVNRDSDEATEDFMRRIE CYENSYESLDEDLDRDLSYIKIMDVGQSYVVNRVADHIQSRIVYYLMNI HVTPRSIYLCRHGESELNLKGRIGGDPGLSPRGREFAKSLAQFISDQNI KDLKVWTSQMKRTIQTAEALGVPYEQWKVLNEIDAGVCEEMTYEEIQDN YPLEFALRDQDKYRYRYPKGESYEDLVQRLEPVIMELERQENVLVICHQ AVMRCLLAYFLDKAAEQLPYLKCPLHTVLKLTPVAYGCKVESIFLNVAA VNTHRDRPQNVDISRPPEEALVTVPAHQ SEQIDNO:2 MAAAAEERMAEEGGGGQGDGGSSLASGSTQRQPPPPAPQHPQPGSQALP APALAPDQLPQNNTLVALPIVAIENILSFMSYDEISQLRLVCKRMDLVC QRMLNQGFLKVERYHNLCQKQVKAQLPRRESERRNHSLARHADILAAVE TRLSLLNMTFMKYVDSNLCCFIPGKVIDEIYRVLRYVNSTRAPQRAHEV LQELRDISSMAMEYFDEKIVPILKRKLPGSDVSGRLMGSPPVPGPSAAL TTMQLFSKQNPSRQEVTKLQQQVKTNGAGVTVLRREISELRTKVQEQQK QLQDQDQKLLEQTQIIGEQNARLAELERKLREVMESAVGNSSGSGQNEE SPRKRKKATEAIDSLRKSKRLRNRK

EXAMPLES

Example 1

Tumour-Specific Expression of PFKFB4

[0061] Recent studies have showed the importance of the key glycolysis gene PFKFB4 for the survival of different tumor cells in vitro and in vivo, highlighting its potential as therapeutic target. To investigate the endogenous protein expression level within different cancer entities, the inventors have developed a new PFKFB4 antibody that is suitable for Western blot and immunohistochemistry, allowing high-throughput staining of Tissue Microarrays (TMAs). As displayed in FIG. 1A, prostate tumor protein samples showed a significantly higher level of PFKFB4 expression than normal samples. Similarly, tumor and normal paired samples from lung cancer patients showed a marked difference of PFKFB4 expression level (FIG. 1A). As depicted in FIG. 1B, the level of PFKFB4 protein expression is ranging from not expressed to high expressed, normal tissues being mostly negative for PFKFB4. Interestingly, the level of expression correlated with the grading of the different tumor, irrespective of its tissue origin, suggesting that PFKFB4 could be involved in the maintenance and growth of tumors.

Example 2

Knockdown of PFKFB4 Impairs GSC Viability in Vivo

[0062] In order to verify the effect of PFKFB4 silencing on the tumor growth in vivo, we used a xenograft mouse model. For this purpose, we generated inducible expression constructs encoding an shRNA targeting PFKFB4 and a non-target shRNA as negative control. Stably transduced GSCs (NCH421k_TetONshPFKFB4 and NCH421k_TetONshNT) were treated for three days with doxycycline and the knockdown of PFKFB4 was characterized at the protein level. In order to allow the monitoring of the tumor growth in vivo, the inducible GSC lines were stably transduced with a luciferase-expressing construct.

[0063] Two groups of 8 and 14 animals were orthotopically transplanted with 100.000 NCH421k_Luc_TetONshNT and NCH421k_Luc_TetONshPFKFB4 cells respectively. The tumor growth expressed as luminescent signal was monitored twice a week. Apparition of the signal of sufficient size (ca. 200.000 flux/photon/second) determined the start of doxycycline treatment. Animals were randomly separated in three groups, namely shNT_dox treated (n=8), shPFKFB4_dox treated (n=7) and shPFKFB4_untreated (n=7). Pictures of the animals were taken twice a week (FIG. 2A). Animals expressing shPFKFB4 upon doxycycline treatment showed a significantly better survival than the control animals (non-treated animals and shNT expressing animals) (FIG. 2B).

[0064] Knockdown of PFKFB4 reduced significantly tumor size overtime as observed by bioluminescent intensity, ultimately leading to the loss of tumor cells about three weeks after treatment (FIG. 2C). Indeed, tumor could be observed in the brain of the sacrified sick animals (non-treated and doxycycline treated shNT) by H and E staining (FIG. 2D), while most of the shPFKFB4 doxycycline-treated mice were tumor-free. Residual tumors of shPFKFB4_dox treated animals still expressed PFKFB4, suggesting that the doxycycling induction of shPFKFB4 was not completely efficient in these tumors as confirmed by immunofluorescence.

Example 3

Impact of PFKFB4 Silencing on PDK1 Expression

[0065] As shown in vitro and in vivo, the impact of PFKFB4 silencing is particularly high for GSC viability. In order to determine its effect on the regulation of the expression of other genes, the gene expression of three different GSC lines (NCH421k, NCH441 and NCH644) transduced with pLKO_shPFKFB4 and pLKO_shNT was profiled. Interestingly, knockdown of PFKFB4 led to a decrease of the HIF1a gene signature, as determined by Gene Set Enrichment Analysis (GSEA) (FIG. 3A). Among the numerous known target of HIF1a the inventors identified, such as LDHA, CA9 and IGF2, the Pyruvate Dehydrogenase Kinase 1 (PDK1) showed the strongest reduction upon PFKFB4 knockdown, which was verified by qRT-PCR (FIG. 3B). PDKi is a key enzyme that regulates the fate of pyruvate within the glycolytic pathway by phosphorylating and thereby inhibiting the Pyruvate Dehydrogenase (PDH). In order to verify that the knockdown of PFKFB4 also affected the phosphorylation of PDKi's target, the inventors performed western blot analysis upon knockout of PFKFB4 using CRISPR. As depicted in FIG. 3C, the level of phosphorylated PDH decreased upon PFKFB4 knockdown.

[0066] Because the effect of PFKFB4 silencing on PDKi expression was significant, the endogenous expression of both genes was investigated in a cohort of glioblastoma patients (n=154), available from The Cancer Genome Atlas (TCGA). Notably, PDKi showed the highest expression correlation with PFKFB4 (R=0.67) (FIG. 3D), a phenomenon that seemed to be specific to that PFK2/FBP2 isoform. Indeed, none of the other isoforms that are expressed in glioblastoma samples showed any correlation with PDKi expression (FIG. 3D).

Example 4

PFKFB4 is Involved in the Regulation of HIF1a Protein Levels

[0067] As highlighted by the gene expression profiling of PFKFB4 knockdown GSC lines, PFKFB4 seems to be involved in the regulation of the expression of genes that are targets of the HIF1a transcription factor. In order to investigate the potential role of PFKFB4 on the regulation of HIF1a expression, qRT-PCR on PFKFB4 knockdown GSCs was performed. As shown in FIG. 4A, no decrease of HIF1a mRNA could be observed upon PFKFB4 knockdown. Indeed, it seems that upon silencing, HIF1a is upregulated at the mRNA level. However, the knockdown of PFKFB4 led to a strong decrease of HIF1a at the protein level (FIG. 4B), suggesting that PFKFB4 is involved post-transcriptionally in HIF1a regulation. This phenomenon was confirmed by knockout of PFKFB4 using two different CRISPR guide RNAs (FIG. 4C).

[0068] As GSCs are cultivated as neurospheres, which could lead to different level of oxygen and nutrient availability, thereby influencing the dependence on HIF1a transcription factor, the inventors performed a PFKFB4 knockout on adherent GSCs. Interestingly, even on these normoxic conditions, HIF1a is strongly expressed, suggesting that HIF1a protein levels are dependent on PFKFB4 expression, irrespective of the culture conditions (FIG. 4C). Furthermore, the overexpression of PFKFB4 in HEK293 cells led to the up-regulation of HIF1a, while its knockdown under hypoxic conditions decreased HIF1a protein levels (FIG. 4D).

Example 5

Identification of a New E3 Ubiquitin Ligase of HIF1a

[0069] As both proteins do not directly interact with each, the inventors performed mass spectrometry of immunoprecipitated PFKFB4 samples to find binding partners of PFKFB4 that could be involved in the mechanisms stabilizing HIF1a.

[0070] Proteins showing more than two signature sequences are listed in FIG. 5A. The binding of FBXO28 to PFKFB4 was verified by coIP and by Yeast-Two-Hybrid (FIG. 5B). In addition, the cytoplasmic localization of both proteins was verified by immunofluorescence (FIG. 5C). FBXO28 is a member of the F-Box protein family and is thought to be part of the SCF complex formed by SKPi, cullin and F-box proteins, as shown by coIP (FIG. 5D), acting as ubiquitin ligases. Interestingly, unlike PFKFB4, FBXO28 mRNA expression is decreased in glioblastoma as compared to normal brain and patients with a lower expression have a better survival (FIG. 5E).

Example 6

PFKFB4 Binds to FBXO28 to Inhibit HIF1a Ubiquitylation and Degradation

[0071] To verify the role of PFKFB4 in the ubiquitylation of HIF1a, the inventors performed immunoblotting using ubiquitin antibody on immunoprecipitated HIF1a upon MG132 treatment. As highlighted in FIG. 6A, the ubiquitylation of HIF1a is decreased in cells overexpressing PFKFB4. Next, the importance of the link between FBXO28 and for HIF1a protein expression and GSCs survival PFKFB4 was identified. In that respect, PFKFB4 was knocked down solely or in combination with FBXO28 silencing in GSCs and FACS analysis was performed. The effect on HIF1a protein level was verified by Western blot. As shown in FIG. 6B, knockdown of PFKFB4 decreased the protein level of HIF1a while silencing of FBXO28 alone did not reduce it. However, silencing of both FBXO28 and PFKFB4 in the GSCs rescued the level of HIF1a. The rescue was also seen at the phenotypic level (FIG. 6C).

[0072] Taken together, these results emphasize the potential role of PFKFB4 to protect HIF1a from the SCF complex, enabling the expression of HIF target genes in glioblastoma stem-like cells.

Example 7

Development of a Screening Method to Identify Small-Compound in-Hibitor to Inhibit PFKFB4 Function

[0073] The NanoBiT assay from Promega is widely used to investigate protein-protein interactions. The NanoBiT Assay is described in detail in Dixon et al., (2016) NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells (ACS Chem. Biol., 11, 400-408; which is incorporated herein by reference in its entirety). The inventors adapted this method to develop a cellular screening assay in order to investigate the interaction of PFKFB4 with FBXO28. In that respect, both genes were cloned into pLVX vectors containing the four NanoBiT versions (pLVX1.1-N[TK/LgBiT], pLVX2.1-N[TK/SmBiT], pLVX1.1-C[TK/LgBiT] and pLVX2.1-C[TK/SmBiT]), allowing the small and large BiT tags to be at the N- or C-terminal of both proteins of interest. As negative control, a vector containing a HaloTag protein cloned to the small BiT was used in combination with the large BiT cloned to either FBXO28 or PFKFB4.

[0074] All vectors were transfected in HEK293 cells and the combinations were tested as depicted in FIG. 7A. The luminescent signal was detected with the Nano-Glo Luciferase Assay System from Promega. The interaction was considered positive if the signal was at least 10-fold of the respective negative control. The combination of the vector containing PFKFB4 tagged at the C-terminal with the Large BiT with the vector expressing FBXO28 tagged at the N-terminal with the small BiT (see FIG. 7A, 2.sup.nd column) gave the highest signal and was therefore selected for further validation.

[0075] In order to verify the specificity of the assay, the inventors transfected HEK293 cells with the combination showing the best results (C-ter[LgBiT]PFKFB4+N-ter[SmBiT]FBXO28) together with increasing concentration of a vector overexpressing untagged PFKFB4. As shown in FIG. 7B, the signal given by the interaction of tagged PFKFB4 and FBXO28 decreased upon addition of untagged PFKFB4 which competes with tagged PFKFB4 to form the interacting complex. This is a clear indication that a candidate interacting compound (here represented by the untagged PFKFB4) in a screening assay would yield signals indicative for the impairment of the interaction between both proteins.

[0076] Finally, by transfecting different truncated versions of tagged PFKFB4 together with tagged FBXO28, the inventors were able to determine that the interaction is reduced if the phosphatase domain is removed and therefore, that the site of interaction between both proteins is most likely located within the phosphatase domain of PFKFB4 (FIG. 7C).