WO2021013794A1 - Transcriptomics for selecting druggable targets for combination therapy in cancer - Google Patents

Transcriptomics for selecting druggable targets for combination therapy in cancer Download PDF

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WO2021013794A1
WO2021013794A1 PCT/EP2020/070448 EP2020070448W WO2021013794A1 WO 2021013794 A1 WO2021013794 A1 WO 2021013794A1 EP 2020070448 W EP2020070448 W EP 2020070448W WO 2021013794 A1 WO2021013794 A1 WO 2021013794A1
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inhibitor
compound
pi3k
signalling
cancer
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David Stanley BAILEY
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Bailey David Stanley
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to combination therapy for cancer and particularly to methods for identifying new anti-cancer combinations.
  • the invention also relates to the combination of an inhibitor of phosphoinositide 3-kinase (PI3K) signalling with an inhibitor of Janus kinase (JAK) activity or function.
  • PI3K phosphoinositide 3-kinase
  • JAK Janus kinase
  • Glioblastoma multiforme is the most common and aggressive malignant glioma, with patients having a median survival of just over one year (Batash et al., 2017).
  • First line therapy remains empiric and consists of surgical resection followed by radiation with concurrent and adjuvant temozolomide, a DNA damaging agent (Stupp et al., 2005).
  • Clinical trials of inhibitors targeting the pathways frequently mutated in GBM have had disappointing results for a variety of reasons, including drug resistance and inclusion of molecularly heterogeneous patients (Cloughesy et al., 2014; Mendelsohn, 2013).
  • Current chemotherapies, together with surgery and radiotherapy, provide only minor patient benefit, and there is a considerable need for development of effective new therapies.
  • PI3K/Akt pathway which appears particularly important in glioblastoma proliferation but also plays a central role in the regulation of tumour cell survival, motility, angiogenesis and metabolism (Zhao et al., 2017). This has led to many attempts to target the PI3K/Akt pathway as a potential treatment option for glioblastoma (Cancer Genome Atlas Research Network,
  • LY-294002 is a non-selective pan- PI3K inhibitor, interacting with PI3K class I, III and PI4K proteins, among other unrelated proteins (Gharbi et al., 2007). LY-294002 has previously been shown to enhance the cytotoxicity of temozolomide in U87MG glioma cells by down-regulating genes involved in the PI3K/Akt pathway (Chen et al., 2012).
  • LY-294002 In drug-resistant leukemia cells, LY-294002 also blocks the cyclin-dependent kinases (CDKs), as well as PKC and other PI3K pathway components, in a manner similar to other drugs such as Flavopiridol, Roscovitine, Wortmannin ( Figure 1 B) and UCN-01 , leading to augmentation of apoptosis (Cory et al., 2005).
  • CDKs cyclin-dependent kinases
  • PKC PI3K pathway components
  • Chemical inhibitors of the PI3K pathway have been instrumental in understanding the role of PI3K enzymes in signal transduction and validating them as therapeutic targets, with the early pan-specific PI3K inhibitors Wortmannin and LY294002 being used extensively to probe the activity of the PI3K/Akt pathway in cancer.
  • LY-294002 nor Wortmannin are PI3K-selective inhibitors, showing additional Casein Kinase 2 (CK2) and Myosin Light-chain Kinase (MLCK) activities, respectively (Kong and Yamori, 2008).
  • CK2 Casein Kinase 2
  • MLCK Myosin Light-chain Kinase
  • the CMap method considers the most highly differentially expressed genes as query signatures and compares them to the reference signatures of drugs tested in a wide variety of biological systems, to date most particularly human cancer cell lines. Using this method, both pre-existing and novel signatures indicative of a compound’s functional effects, molecular modes of action, or toxicity can be defined (Alexander-Dann et al., 2018; lorio et al., 2010).
  • the present invention provides inhibitors of the phosphoinositide 3-kinase (PI3K) signalling pathway for use in combination with inhibitors of Janus kinase (JAK) activity or function. These find use, for example, in methods of treating or preventing a cancer.
  • PI3K phosphoinositide 3-kinase
  • JAK Janus kinase
  • the invention provides a method of treating or preventing cancer, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of an inhibitor of phosphoinositide 3-kinase (PI3K) signalling simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
  • PI3K phosphoinositide 3-kinase
  • JK Janus kinase
  • the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling for a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the inhibitor of PI3K signalling simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
  • the invention provides an inhibitor of Janus kinase (JAK) activity or function for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the inhibitor of PI3K signalling simultaneously or sequentially with an inhibitor of phosphoinositide 3-kinase (PI3K) signalling.
  • the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling for use in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament is administered simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
  • PI3K phosphoinositide 3-kinase
  • the invention provides an inhibitor of Janus kinase (JAK) activity or function for use in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament is administered simultaneously or sequentially with an inhibitor of phosphoinositide 3-kinase (PI3K) signalling
  • JK Janus kinase
  • PI3K phosphoinositide 3-kinase
  • the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and an inhibitor of Janus kinase (JAK) activity or function for use in a method of treating or preventing cancer in a subject.
  • PI3K phosphoinositide 3-kinase
  • JK Janus kinase
  • the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and an inhibitor of Janus kinase (JAK) activity or function for use in the manufacture of a medicament for treating or preventing cancer in a subject.
  • PI3K phosphoinositide 3-kinase
  • JK Janus kinase
  • the invention provides a composition comprising an inhibitor of phosphoinositide 3- kinase (PI3K) signalling, an inhibitor of Janus kinase (JAK) activity or function, and a pharmaceutically acceptable excipient.
  • PI3K phosphoinositide 3- kinase
  • JAK Janus kinase
  • the invention provides a kit comprising (i) an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and (ii) an inhibitor of Janus kinase (JAK) activity or function.
  • PI3K phosphoinositide 3-kinase
  • JK Janus kinase
  • the inhibitor of PI3K signalling interacts with and inhibits the activity or function of PI3Kpreferably a PI3K isoform selected from PIK3CA/p110a, PIK3CB/p1 10b and/or PIK3CD/p1 10d.
  • the inhibitor of PI3K signalling may additionally interact with and inhibit the activity or function of one or more of Casein Kinase 2 (CK2) and/or Myosin Light-chain Kinase (MLCK).
  • the inhibitor of PI3K signalling is 2-Morpholin-4- enylchromen-4-one (LY-294002).
  • the inhibitor of JAK activity or function is an inhibitor of JAK2 activity or function, preferably AZD1480.
  • the cancer is a cancer of the brain, for example a primary brain tumour, preferably a glioma, most preferably glioblastoma multiforme.
  • the inhibitor of PI3K signalling is LY-294002, or a salt, conjugate, prodrug, or derivative thereof, and the inhibitor of JAK activity or function is AZD1480, or a salt, conjugate, prodrug, or derivative thereof, and the cancer is glioblastoma multiforme.
  • the invention relates to methods useful in the development of combination therapies for cancer and, in particular, for glioblastoma multiforme.
  • the invention provides a method, preferably an in vitro method, of identifying druggable targets for combination therapy, comprising the steps of:
  • the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • the combination therapy is combination cancer therapy, more preferably a glioblastoma multiforme combination therapy.
  • the invention provides a method, preferably an in vitro method, of providing a combination therapy, said combination comprising a first and a second compound, the method comprising
  • the combination therapy is preferably a cancer combination therapy, preferably where the cancer is glioblastoma multiforme.
  • FIG. 1 Chemical structures of A. LY-294002; B. Wortmannin; C. Fucoxanthin; and D. Fucoxanthinol. Structures taken from PubChem and redrawn by ChemDraw
  • FIG. 3 Morphology of U87MG cells treated with Fucoxanthin or LY-294002.
  • Panels A, B U87MG cells without compound treatment (controls), visualised at 24h and 48h, respectively;
  • Panels C, D U87MG cells treated with Fucoxanthin, visualised at 24h and 48h respectively;
  • Panels E, F U87MG cells treated with LY-294002, visualised at 24h and 48h, respectively.
  • Treatments with LY-294002 and Fucoxanthin were performed at concentrations of 19.9 pM and 199.5 pM, respectively.
  • G is control
  • H Fucoxanthin
  • I LY-294002 48h after treatment.
  • Apoptotic cells were detected by Annexin V- FITC/PI staining and death cells were detected by PI. Viable cells are shown in the lower left quarter (Q3- LL), early apoptotic cells are shown in the lower right quarter (Q4-LR), late apoptotic cells are shown in the upper right quarter (Q2-UR) and necrotic or mechanically damaged cells are shown in the upper left quarter (Q1 -UL).
  • PCA Principal Components
  • Figure 5 Euclidean distance based heatmap and clustering of the samples. The samples were clustered based on treatment first and then by time. LY-294002 24h (L24), LY-294002 48h (L48) treatments, Fucoxanthin 24h (F24) and Fucoxanthin 48h (F48) treatments, Control 24h (C24), Control 48h (C48). Up- regulated genes are shown in red; down-regulated genes are shown in green. Only significantly differentially expressed genes are shown. The colors used are the same as those used in the PCA analysis.
  • FIG. 6 Venn Diagram showing the in-common and unique responses of the two treatments in gene expression space.
  • A Gene up-expression at 24 h and 48 h for LY294002 and Fucoxanthin treated U87MG cells.
  • B Gene down-expression at 24 h and 48 h for LY294002 and Fucoxanthin treated U87MG cells. Note that few up-regulated genes are shared by the two treatments, in contrast to much higher in down-regulated genes.
  • Figure 7. Comparison of gene expression signature in response to LY-294002 and Fucoxanthin at 24h and 48h.
  • Figure 8 Comparison of gene expression signature in response to LY-294002 and Fucoxanthin at 24h and 48h.
  • A Top 25 up-regulated genes in Fucoxanthin treatment at 24h (F24, left side) and at 48h (F48, right side) relative to no-treatment controls, and comparison to the expression signature seen in LY- 294002 at the same timepoints.
  • B Top 25 down-regulated genes in fucoxanthin treatment at 24h (F24, left side) and at 48h (F48, right side) relative to no-treatment controls, and comparison to the expression signature seen in LY-294002 at the same timepoints.
  • FIG. 9 Volcano plot of the 4 treatments showing the top 25 down-regulated genes (left side, in green) and the top 25 up-regulated genes (right side, in red) accompanied by their level of expression, expressed as logarithm-based 2 fold changes (Log2FC, ⁇ -1 or > 1) and corrected p-value as logarithm- based 10 false discovery rate (Logl OFDR P-value, ⁇ 0.05).
  • D top 25 differentially expressed genes in U87MG responding to Fucoxanthin at 48h.
  • FIG. 10 PI3K/Akt signalling Pathway WikiPathway map representations.
  • A Down-regulated (in green) and up-regulated genes (in red) in response to individual treatments;
  • B L24, PI3K/Akt signalling pathway affected by LY-294002 at 24h;
  • C L48, PI3K/Akt signalling Pathway affected by LY-294002 at 48h;
  • D F24, PI3K/Akt signalling Pathway affected by Fucoxanthin at 24h;
  • E F48, PI3K/Akt signalling Pathway affected by Fucoxanthin 48h.
  • FIG. 1 Retinoblastoma gene in cancer pathway, WikiPathway map representations.
  • A Down- regulated (in green) and up-regulated genes (in red) in response to individual treatments;
  • B L24.
  • Yellow lines are perturbagens with significantly correlating gene signatures and blue lines perturbagens with anti-correlating signatures.
  • Blue squares are compounds from IOTA ' S GBM Drug Bank.
  • LY-294002 and Fucoxanthin have drug counterparts with similar gene signatures, including groups of antibiotics and anti-protozoal / antifungal agents as well as antipsychotics and antidepressants and compounds with described effects in GBM cell lines. They also share similar gene expression signatures with other PI3K inhibitors such as Quinostatin and Wortmannin, as indicated.
  • FIG. 14 Differential drug induced gene expression in the PI3K pathway. Shown are members of the PI3K pathway which are among the top 25 upregulated genes in LY-294002 (left) and Fucoxanthin (right) treated cells, at 24h and 48h. Note that JAK2 is up-regulated 24 h and 48 h following LY-294002 treatment. JAK2 is also up-regulated 24 h but not 48 h after Fucoxanthin treatment.
  • FIG. 15 Combination of LY-294002 with JAK2 inhibitors (A) ruxolitinib and (B) AZD1480.
  • Cells were exposed to LY294002 in concentrations ranging from 10 _6 M to 10 _3 M, alone or in combination with JAK inhibitor at 10, 25 or 50 pM.
  • Cell survival after 72h is given as a percentage of 1 .1 % DMSO control.
  • Also shown is an analysis of the synergy of the two combinations, from which it can be seen that the combination of AZD1480 and LY-294002 is synergistic (indicated in blue).
  • inhibitor relates to a compound or substance which reduces or suppresses the activity or function of a target.
  • a target may be one or more proteins or nucleic acids (such as mRNAs).
  • An inhibitor may have multiple targets, which may share structural homology (e.g. in the case of related proteins) or a shared function (e.g. in the case of a pathway inhibitor).
  • Inhibitors include a compound or substance which interacts with its target, for example a competitive or non-competitive/allosteric inhibitor.
  • a competitive inhibitor competes with a substrate for the active site, whist an allosteric inhibitor binds to a site other than the active site and prevents substrate binding for example by stabilising a conformation which abolishes or disrupts the active form of the target.
  • interactions are typically reversible, however an inhibitor may be an irreversible inhibitor, for example an inhibitor which covalently links to a target, blocking or disrupting the active form.
  • an inhibitor may be a transcriptional inhibitor, which reduces or abolishes the expression of a target.
  • a“phosphatidylinositol 3-kinase (PI3K) pathway inhibitor”, an“inhibitor of phosphatidylinositol 3-kinase (PI3K) signalling” or an“inhibitor of the phosphatidylinositol 3-kinase (PI3K) signalling pathway” are used interchangeably and refer to a compound which inhibits, downregulates or abolishes signalling through the PI3K signalling pathway. It is therefore an inhibitor which targets one or more components in the PI3K pathway and, as a consequence, downregulates or abolishes PI3K signalling.
  • the inhibitor’s action may comprise or consist of direct interaction with and inhibition of PI3K activity or function.
  • the PI3K pathway is an intracellular signal transduction pathway that functions to stimulate cell to proliferation and growth, and simultaneously inhibit cell apoptosis.
  • the pathway involves many members, however key proteins involved include receptor tyrosine kinases (RTKs), phosphatidylinositol 3-kinases (PI3Ks), phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-bisphosphate (PIP3) and AKT/protein kinase B.
  • RTKs are cell surface receptors for multiple growth factors, cytokines and hormones. Ligands binding to RTKs promote activation of PI3Ks.
  • PI3K family kinases are capable of phosphorylating the 3’-hydroxyl group of the inositol ring of phosphatidylinositol.
  • Phosphatidylinositol containing PIP2 and PIP3 are minor phospholipid components of cell membranes, and their activation through PI3K-mediated phosphorylation allows the recruitment of AKT to the plasma membrane, where it is in turn activated.
  • AKT/protein kinase B is a serine/threonine-specific protein kinase that enhances the survival of cells by preventing apoptosis through blocking pro-apoptotic proteins and processes, such as through negatively regulating Bcl-2 family members and p53.
  • AKT also promotes cell cycle advancement through its phosphorylation and inhibition of G1 state associated factors P21/Waf1/Cip1 and P27/Kip2. Additionally, AKT promotes cell growth through inhibition of TSC2 and indirect activation of the mTOR complex 1.
  • a review of the PI3K pathway can be found in Hemmings, B. A., & Restuccia, D. F., Cold Spring Harbor Perspectives in Biology, 4(9), a011189.
  • Inhibitors of PI3K signalling may interact with and inhibit the activity or function, or downregulate the expression, of a PI3K and one or more target proteins involved in the PI3K pathway selected from RTK, PIP2, PIP3, and/or AKT.
  • the inhibitor may interact with and inhibit the activity or function, or downregulate the expression, of one or more target proteins involved in the PI3K pathway selected from RTK, PIP2, PIP3, and/or AKT, and may show no interaction or effect on PI3K itself.
  • An inhibitor of PI3K signalling may be a PI3K inhibitor.
  • a PI3K inhibitor may in particular directly interact with and inhibit the activity or function of, one or more PI3K isoform.
  • PI3Ks are members of a family of kinases capable of phosphorylating the hydroxyl group of the inositol ring of phosphatidylinositol. All PI3Ks consist of two domains: a catalytic domain P1 10 and a regulatory domain P85. They are divided into classes I to III.
  • PI3K inhibitor preferably interacts with and inhibits activity or function of one or more Class I PI3K isoform.
  • Class I PI3K isoforms function as heterodimers consisting of one of four catalytic p110 subunits (PIK3CA/p110a, PIK3CB/p110b, PIK3CD/p110d, or PIK3CG/p110y) and a regulatory subunit selected from PIK3R1/p85a (or its splice variants p55a and p50a), PIK3R2/p85p, PIK3R3/p55y, PIK3R5/p101 or PIK3R6/p84.
  • Some PI3K inhibitors interact with and inhibit activity or function of PIK3CA/p110a.
  • Some PI3K inhibitors interact with and inhibit activity or function of PIK3CB/p110p. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CD/p110d. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CG/p1 10y. Some PI3K inhibitors interact with and inhibit activity or function of one or more, two or more, three or more, or all PI3K isoforms selected from PIK3CA/p110a, PIK3CB/p110b, PIK3CG/p110y and/or PIK3CD/p110d. Preferably, the PI3K inhibitor interacts with and inhibits activity or function of PIK3CA/p110a, RIK3 ⁇ B/r110b and PIK3CD/p1106.
  • a PI3K inhibitor may not interact with and may have no effect on the activity or function of one or more Class I PI3K isoform, e.g. one, two, three, or four selected from PIK3CA/p110a, PIK3CB/p110b, PIK3CG/p110y and/or PIK3CD/p110d.
  • the PI3K inhibitor optionally and additionally inhibits activity or function of Class III PI3K isoform PIK3C3/Vps34.
  • the PI3K inhibitor inhibits activity or function of PIK3CA/p110a, PIK3CB/p110b and PIK3CD/p110d, but does not substantially inhibit activity or function of PIK3CG/p110y.
  • An inhibitor of PI3K signalling may, in addition or as an alternative to inhibition of Class I PI3K isoform as described above, interact with and inhibit activity or function of one or more Class II PI3K isoform.
  • the class II family has three members in humans - PIK3C2A/PI3KC2a, RIK3 ⁇ 2B/RI3K ⁇ 2b and
  • the inhibitor of PI3K signalling interacts with and inhibits one, preferably two, or all three, selected from
  • the inhibitor of PI3K signalling may interact with and inhibit activity or function of Class III PI3K isoform PIK3C3/Vps34. This is the only class III PI3K in humans, which exists as a dimer of catalytic PIK3C3/Vps34 and its regulatory subunit PIK3R4/Vps15.
  • Some PI3K inhibitors exhibit different inhibitory activity or function for different classes of PI3K isoforms, and may inhibit a group of PI3K isoforms selected from a one or more Class I PI3K isoform, one or more Class II PI3K isoform, one or more Class I PI3K isoform and one or more Class II PI3K isoform, one or more Class I PI3K isoform and the Class III PI3K isoform, one or more Class II PI3K isoform and the Class III PI3K isoform, or one or more PI3K isoform from Classes I, II and III.
  • Inhibitor of PI3K signalling may additionally or alternatively interact with and inhibit activity or function of a phosphatidylinositol 4-kinase alpha (PI4K) selected from phosphatidylinositol 4-kinase alpha (PI4KA), latidylinositol 4-kinase beta (PI4KB), phosphatidylinositol 4-kinase 2-alpha (PI4K2A), and/or phosphatidylinositol 4-kinase 2-beta (PI4K2B).
  • PI4K phosphatidylinositol 4-kinase alpha
  • PI4KA phosphatidylinositol 4-kinase alpha
  • PI4KB latidylinositol 4-kinase beta
  • PI4K2A phosphatidylinositol 4-kinase 2-alpha
  • An inhibitor of PI3K signalling may be a non-selective pan-PI3K inhibitor.
  • a“non-selective pan-PI3K inhibitor” refers to a substance or compound which inhibits, either by inhibiting activity or function of or by reducing expression of, multiple targets within the PI3K signalling pathway. In this way, the inhibitor effectively downregulates signalling through the pathway.
  • a non-selective pan- PI3K inhibitor may target one, two, three, four, or more targets selected from a RTK, a PI3K, PIP2, PIP3, and/or AKT.
  • a non-selective pan-PI3K inhibitor may inhibit one or more additional targets outside of the PI3K signalling pathway, either by interacting with them and inhibiting their activity or function, or by reducing their expression.
  • the additional targets are selected from casein kinase 2 (CK2), Myosin Light-chain Kinase (MLCK, and/or mammalian target of rapamycin (mTOR).
  • CK2 casein kinase 2
  • MLCK Myosin Light-chain Kinase
  • mTOR mammalian target of rapamycin
  • a non-selective pan-PI3K inhibitor may target, for example may interact with and inhibit the activity or function of at least a PI3K class I, a PI3K class II and a PI4K.
  • Some PI3K inhibitors do not interact with and inhibit the activity or function of PI3K class I, II, III, and/or PI4K. In particular, they may not interact with and inhibit the activity or function of PI3K class III.
  • Exemplary non-selective pan-PI3K inhibitors are wortmannin, BEZ235 (Dactolisib), PI-103, Buparlisib (BKM120, NVP-BKM120), GDC-0941 (Pictilisib), PI828, and 2-Morpholin-4-yl-8-phenylchromen-4-one (LY-294002) (Gharbi et al., 2007).
  • LY-294002 is a relatively weak, pan-PI3K inhibitor showing inhibition in cell-free assays of the first 3 PI3K isoforms (Liu et al., 2017).
  • LY294002 It inhibits RI3Ka/d/b with IC50 of 0.5 pM/0.57 pM/0.97 pM, respectively. Similar to LY294002, BEZ235 and GDC-0941 also show pan-PI3K activity and are currently in clinical trials for glioblastoma. The structure of LY294002 is shown below:
  • the inhibitor of PI3K signalling is BEZ235 (Dactolisib) or a salt, conjugate, prodrug, or derivative thereof.
  • the inhibitor of PI3K signalling is GDC-0941 (Pictilisib), or a salt, conjugate, prodrug, or derivative thereof.
  • the inhibitor of PI3K signalling is wortmannin, or a salt, conjugate, prodrug, or derivative thereof.
  • the inhibitor of PI3K signalling is LY-294002, or a salt, conjugate, prodrug, or derivative thereof.
  • the inhibitor of PI3K signalling has similar chemical and biological properties to LY-294002.
  • the inhibitor of PI3K signalling may show pan-PI3K selectivity similar to
  • a PI3K inhibitor is a PI3K inhibitor other than fucoxanthin.
  • an“inhibitor of Janus kinase (JAK) activity or function” is a compound that reduces or inhibits the activity of one or more member of the JAK family of non-receptor protein-tyrosine kinases.
  • JAK1 JAK1
  • JAK2, JAK3, and TYK2 tyrosine kinase-2
  • JAK/STAT pathway is a key signalling mechanism for a wide array of cytokines and growth factors, and JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis.
  • An inhibitor of JAK activity or function may inhibit activity or function of one, two, three or all four of JAK1 , JAK2, JAK3 and/or TYK3.
  • the inhibitor of JAK activity or function inhibits JAK1 , JAK2, and/or TYK3 activity or function.
  • the inhibitor of JAK activity or function may be an inhibitor of JAK2 activity or function. More preferably, the inhibitor of JAK activity or function preferentially inhibits JAK2 activity or function relative to JAK1 activity or function. Still more preferably, the inhibitor of JAK activity or function may inhibit JAK2 activity or function but may not inhibit JAK1 activity or function.
  • Inhibitors of JAK activity or function may prevent or reduce activating phosphorylation of Janus kinases, preferably of JAK2. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling will be diminished.
  • Inhibitors of JAK activity or function include a protein kinase inhibitor.
  • Protein kinases are ATP-dependent enzymes, which phosphorylate suites of target sites.
  • the inhibitor of JAK activity or function may be a Type I protein kinase inhibitor which inhibits the activity or function of one or more of JAK1 , JAK2, JAK3 and/or TYK2, more preferably JAK2.
  • An inhibitor of JAK activity or function may be an ATP-competitive inhibitor of JAK1 , JAK2, JAK3 and/or TYK2, and preferably the inhibitor of JAK activity or function may be an ATP-competitive inhibitor of JAK2.
  • the inhibitor of JAK activity or function may be Ruxolitinib, or a salt, conjugate, prodrug, or derivative thereof.
  • Ruxolitinib is an ATP-competitive inhibitor of JAK1 and JAK2 (ICso-s of 3.3 ⁇ 1 .2 nM and 2.8 ⁇ 1 .2 nM, respectively) and inhibition occurs regardless of the JAK2 V617F mutational status.
  • JAK1 and JAK2 downregulates the JAK-signal transducer and activator of transcription (STAT) pathway, inhibiting proliferation, inducing apoptosis, and reducing numerous cytokine plasma levels.
  • STAT JAK-signal transducer and activator of transcription
  • the inhibitor of JAK activity or function has similar chemical and biological properties to ruxolitinib.
  • the inhibitor of JAK activity or function may have JAK2-selective inhibitory properties similar to ruxolitnib, a comparable selectivity and/or affinities for JAK2 as ruxolitinib, the same or similar binding mode in x-ray crystallography and/or may compete with ruxolitinib for binding sites on JAK2.
  • the inhibitor of JAK activity or function may be 5-Chloro-N2-[(1 S)-1 -(5-fluoro-2- pyrimidinyl)ethyl]-N4-(5-methyl-1 H-pyrazol-3-yl)-2,4-pyrimidine-2, 4-diamine (AZD1480), or a salt, conjugate, prodrug, or derivative thereof.
  • AZD1480 4-diamine
  • the structure of AZD1480 is shown below:
  • the inhibitor of JAK activity or function has similar chemical and biological properties to AZD1480.
  • the inhibitor of JAK activity or function may have a comparable selectivity and/or affinities for JAK2 as AZD1480, may have the same or similar binding mode in x-ray crystallography and/or may compete with AZD1480 for binding sites on JAK2.
  • such an inhibitor has an improved dose-limiting toxicity relative to AZD1480.
  • JAK inhibitors without either JAK1 or JAK3 inhibitory activities may be preferable, since chronic dosing required for many treatments carries the potential risk of immunosuppressive side effects related to inhibition of JAK1 , JAK3, or TYK2, suggesting that identification of a JAK2 selective inhibitor may offer increased safety.
  • the inhibitor of JAK activity or function is not Ruxolitinib.
  • the combination of an inhibitor of PI3K signalling and an inhibitor of JAK activity or function finds use in the treatment or prevention of cancer.
  • compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • Treatment includes preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease and/or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.
  • “prevention of cancer” refers to preventing the progression of a tumour. Tumours may be classified by grade (“The 2016 World Health Organisation Classification of Tumours of the Central Nervous System: a summary". Acta Neuropathologica. 131 (6): 803-820). A cancer may be prevented from progression from low-grade (WHO grade I or II) to high-grade (WHO grade III or IV).“Prevention of cancer” may include preventing a benign or pre-cancerous tumour from progressing to a malignant or cancerous state. “Prevention of cancer” may include preventing a cancer or tumour from arising from healthy tissue.
  • prevention of cancer may also include preventing a cancer from spreading, for example from metastasising.
  • the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part, such as from the lung to the brain.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumour and has one or more secondary tumours.
  • non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumour but not one or more secondary tumours.
  • metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumour and with one or more secondary tumours at a second location or multiple locations, e.g., in the brain.
  • ition of cancer may also include preventing the recurrence of a cancer which is in partial or complete remission.
  • cancer in remission refers to a decrease in or disappearance of signs and symptoms of cancer.
  • Partial remission refers to a cancer which has a decrease in some, but not all signs and symptoms have decreased or disappeared.
  • a cancer in partial remission may exhibit decreases in tumour sise, cancer cell count, metastasis rate, etc.
  • the active compounds may be administered to a subject or patient simultaneously or sequentially, by any suitable route or administration.
  • Patient “subject” or “subject in need thereof refers to a living organism suffering from or prone to a disease or condition that can be treated by using the methods provided herein.
  • the term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.
  • a subject or patient is human.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • compositions described herein are administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy.
  • additional therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy.
  • the compounds of the invention can be administered alone or can be coadministered to the patient.
  • Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
  • compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • “Simultaneous” administration refers to administration of the agents together, for example as a pharmaceutical composition containing the agents (i.e. a combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
  • the inhibitor of PI3K signalling and the inhibitor of JAK activity or function may be administered simultaneously in a combined preparation.
  • the two or more of the agents may be administered via different routes of administration.
  • Simultaneous administration may refer to administration at the same time, or within e.g.
  • ntial administration refers to administration of one or more of the agents followed after a given time interval by separate administration of another of the agents. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval, including hours, days, weeks, months, or years.
  • Sequential administration may refer to administrations separated by a time interval of one of at least 10 min, 30 min, 1 hr, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months or 6 months.
  • the inhibitor of PI3K signalling and/or the inhibitor of Janus kinase (JAK) activity or function may be administered simultaneously or sequentially with one or more further compounds.
  • the further compound is an inhibitor of Insulin Like Growth Factor 1 Receptor (IGF1 R) activity or function and/or an inhibitor of Serine/threonine-protein kinase 1 (SGK1) activity or function.
  • IGF1 R Insulin Like Growth Factor 1 Receptor
  • SGK1 Serine/threonine-protein kinase 1
  • Suitable compounds may include; 2-Amino-3H-phenoxazin-3-one, 2-Hydroxyoleic acid, 3-(3,4- dichlorophenyl)-1-(3,4-dimethylphenyl)-1-(5-methyl-4,5-dihydro-1 ,3-thiazol-2-yl)urea, 3-Deazaneplanocin, 4egi-1 , 5-Nonyloxytryptamine, 6-Hydroxyquinoline-4-carboxylic acid, 7-Ethyl-10-hydroxycamptothecin, 7- Hydroxystaurosporine, 8-Bromo-cyclic AMP, 8-Hydroxy-2-methyl-1 H-quinazolin-4-one, 9-ING-41 , A- 966492, Abemaciclib, Abt-737, AC1 MMYR2, Acalabrutinib, Acetazolamide,
  • the inhibitor of PI3K signalling and/or the inhibitor of JAK activity or function may be delivered across the blood-brain barrier into the brain, for example through intracranial administration.
  • blood-brain barrier refers to a highly selective semipermeable membrane barrier that separates the circulating blood from the brain and extracellular fluid in the central nervous system. The barrier provides tight regulation of the movement of ions, molecules and cells between the blood and the brain, see e.g. Daneman and Prat, Cold Spring Harb Perspect Biol. 2015;7(1):a020412. Many therapeutic molecules are generally excluded from transport from blood to brain due to their negligible permeability over the brain capillary endothelial wall.
  • Inhibitors of PI3K signalling and/or of JAK activity or function may be capable of crossing the blood- brain barrier into the brain, for example by virtue of a targeting domain and/or encapsulation in a liposomal or similar carrier, and are delivered for example by intravenous injection or through the gastrointestinal or oral route.
  • an effective prophylactic or therapeutic treatment regimen can be j that does not cause substantial toxicity and yet is effective to treat the clinical symptoms demonstrated by the particular patient.
  • This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent.
  • an “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, reduce one or more symptoms of a disease or condition, reduce viral replication in a cell).
  • An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount”.
  • a “reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • a “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
  • An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme or protein relative to the absence of the antagonist.
  • a “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist.
  • an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and
  • the compounds described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoural, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration which may include injection or infusion.
  • Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the
  • the invention provides a pharmaceutical composition comprising an inhibitor of PI3K signalling and an inhibitor of JAK activity or function.
  • the inhibitor of PI3K signalling and an inhibitor of JAK activity or function may be as described herein.
  • the pharmaceutical composition may comprise one or more pharmaceutically acceptable binders, diluents, or excipients.
  • the pharmaceutical compositions may be formulated for delivery across the blood-brain barrier, for example via intracranial injection.
  • the pharmaceutical composition may be for the treatment or prevention of a cancer, preferably glioblastoma multiforme.
  • the invention provides a kit comprising (i) an inhibitor of PI3K signalling and (ii) a inhibitor of JAK activity or function.
  • the inhibitor of PI3K signalling and an inhibitor of JAK activity or function may be as described herein.
  • the inhibitor of PI3K signalling and/or inhibitor of JAK activity or function may be formulated as pharmaceutical compositions which may comprise one or more pharmaceutically acceptable binders, diluents, or excipients.
  • the pharmaceutical compositions may be formulated for delivery across the blood-brain barrier, for example via intracranial injection.
  • a kit may contain multiple doses of the inhibitors, each of which may be packaged together or separately.
  • Inhibitors may be supplied as aqueous formulations, liquids, powders, pastes, gels, vials, tablets, pills, capsules, or any other formulation described herein.
  • the kit may be for the treatment or prevention of a cancer, preferably glioblastoma multiforme.
  • the kit may comprise instructions for sequential or simultaneous administration of the inhibitors in order to treat or prevent cancer according to the methods described herein.
  • the kit may further comprise means for administration, such as syringes, salt, buffers, or pharmaceutically acceptable carriers or excipients.
  • the kit may comprise additional compounds as listed above.
  • a “combination therapy”, or a“therapeutic combination” relates to two or more compounds useful in treating or preventing a disease when delivered simultaneously or sequentially to a subject.
  • the combination therefore comprises at least a first and a second compound, which may be for sequential or simultaneous administration.
  • “Compounds”, in this context, comprise at least one therapeutic agent, for example a cytotoxic or anti-cancer agent.
  • Test cells refers to any suitable cells in vivo, ex vivo or in vitro.
  • Test cells may be pathological cells, i.e. cells having or displaying symptoms of a dysfunction or disease.
  • test cells may be cancer cells, such as glioblastoma cells, although cells of any of the cancers described herein will also be suitable.
  • Suitable cancer cell lines or glioblastoma cell lines may include primary human cancer cells, such as low-passage patient-derived glioma and glioblastoma cell lines, or established cell lines such as the well characterised cell lines A172, LN18, LN229, LNZ308, T98G, U118, U138, U251 , U343, U373 and U87, or selected glioma and glioblastoma cell lines from more extensive cell culture collections such as the well-known ATCC and ECACC cell ons, as well as cell collections dedicated to GBM such as the HGCC.
  • a particularly preferred GBM cell line is U87MG.
  • test cells may be cancer cells, preferably glioblastoma cells, which are obtained from a patient for whom the cancer combination therapy is intended. In these embodiments, the methods provide a personalised cancer combination therapy which is tailored to the patient’s cancer.
  • Test cells may be contacted with a first compound in any concentration.
  • the concentration may be equal to or below the anti-proliferative EC50 of the first compound for the test cells.
  • Half maximal effective concentration refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time.
  • the antiproliferative EC50 is the concentration of the first compound which induces an anti-proliferative response halfway between the baseline and maximum after a specified exposure time.
  • EC50 is usually described in relation to a time point, e.g. 24h-EC50 is the EC50 calculated after a 24h exposure time. The EC50 may be calculated based on an exposure time which is longer than the length of the contacting step.
  • test cells are contacted with the first compound at the 72h-EC50 concentration.
  • test cells are contacted with the first compound at the 72h-EC50 concentration, and measurement occurs no later than 48hrs after exposure to the first compound.
  • test cells are contacted with the first compound at the 72h-EC50 concentration, and measurement occurs no sooner than 24h and no later than 48hrs after exposure to the first compound.
  • Measurement may entail obtaining a sample of mRNA from the test cells and quantifying the levels of transcripts, for example through qPCR or microarray analysis. Measurement may involve comparing the quantified levels of transcripts to a reference. Suitable references include untreated cells, i.e. cells of the same cell line which are not exposed to the first compound. The method may involve measuring and quantifying the levels of transcripts in cells identical to the test cells in all aspects except their exposure to the first compound, so as to create a reference.
  • the method may employ a known or pre-generated standard reference.
  • Transcript levels may be“normalised” to the expression levels of a“housekeeping gene”.
  • Housekeeping genes are those that are always expressed because they are constantly required by the cell, hence, they are always present under any conditions. Examples include as ubiquitin, actin, GAPDH, or other housekeeping genes which will be known to the skilled person.
  • the transcriptomic profile is constructed for all transcripts, for example by using a universal probe or primer mix, and is a“whole” transcriptome profile.
  • the transcriptomic profile is constructed only for a subset of transcripts.
  • a transcriptomic profile may include one or more genes which are implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • a transcriptomic profile may be limited by pathway, for example to genes in the PI3K or retinoblastoma pathways.
  • a transcriptomic profile may be limited to genes for which inhibitory and/or approved drugs are known.
  • a transcriptomic profile may additionally include one or more housekeeping genes.
  • transcriptomic profile reports the changes in the transcriptome induced by a treatment.
  • the method measures the effects of the compound on the transcriptome of the test cells at a first and a second time-point.
  • the transcriptomic profile that is constructed includes time as a variable alongside expression, capturing not just whether but when expression is affected.
  • the first time point may be before the second time point, and may be separated by an incubation in the presence of the test compound.
  • Such time points may represent an“early” and a“late” drug response.
  • the time points may be separated by a period of 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 48, 60, or 72 hours or more.
  • the first time point may be 24 hr after contacting the test cells with the compound and the second time point may be 48 hr after contacting the test cells with the compound, for a separation of 24 hr.
  • “Early” drug responses may include shock responses and immediate anti-toxin effects, whilst“late” drug responses will tend to include “escape” response, such as regulation of alternative pathways to compensate for the action of the first compound.
  • a gene whose expression is perturbed in both the“early” and“late” time-points is a candidate for selection as a druggable target, as this may reveal a sustained reliance on the gene when challenged by the first compound, and its disruption may enhance the effect of the first compound.
  • a gene whose expression is perturbed only in the early time-point may be a druggable target, as it may represent an immediate or“front-line” response to the first compound, and its disruption may enhance the effect of the first compound.
  • a gene whose expression is perturbed only in the late time- point may be a druggable target, as it may represent a downstream response to the first compound, the disruption of which may restore sensitivity to and enhance the effect of the first compound.
  • a gene is “perturbed” if its expression changes. Perturbation may be an increase or a decrease in expression.
  • a perturbed gene may be expressed at 150%, 160%, 170%, 180%, 190% or 200% or more of its wild-type or reference expression.
  • a perturbed gene may be expressed at 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, or 20 times or more of the level of its wild- type or reference expression.
  • a perturbed gene may be expressed at 60%, 50%, 40%, 30%, 20%, 15%,
  • a perturbed gene may be expressed at 1/2 times, 1/3 times, 1/4 times, 1/5 times, 1/6 times, 1/7 times, 1/8 times, 1/9 times, 1/10 times, 1/15 times, or 1/20 times or less of the level of its wild-type or reference expression.
  • a druggable target gene is, by virtue of its implication in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, expected to enhance cell sensitivity to the first compound if administered in combination with the first compound.
  • a cell When a cell is challenged with a compound, it attempts to“escape” the effects by (i) upregulating expression of genes which positively upregulate compensatory pathways, and (ii) downregulating expression of genes which negatively regulate compensatory pathways.
  • Compensatory pathways are those which substitute for the function conveyed by the gene targeted by the first compound.
  • the cell will respond by upregulating genes which promote alternative proliferative genes and pathways, whilst downregulating anti-proliferative regulators.
  • the first compound promotes a pro-apoptotic pathway
  • the cell will respond by upregulating anti-apoptotic genes and pathways, and downregulating pro-apoptotic genes and pathways.
  • a gene implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation may be a gene which is a known regulator of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • the step of selecting a gene whose expression is perturbed as a druggable target may comprise comparing the expression in the test cells to expression in normal cells - either to control cells or to a set of known standards - in order to determine relative changes in expression.
  • the gene regulates angiogenesis, mTOR signalling, or the NFKB signalling.
  • Druggable targets may be genes implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, which are up-regulated (i.e. for which relative expression increases) in the cell.
  • a positive regulator whose expression is upregulated following treatment may be a candidate druggable target.
  • a druggable target that is upregulated is expressed at 150%, 160%, 170%, 180%, 190% or 200% or more of its wild-type or reference expression. In some embodiments, a druggable target that is upregulated is expressed at 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, or 20 times or more of the level of its wild-type or reference expression.
  • druggable targets may be genes implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, which are down-regulated (i.e. for which relative expression decreases) in the cell.
  • a negative regulator whose expression is downregulated following treatment may be a candidate druggable target.
  • a druggable target that is downregulated is expressed at 60%, 50%, 40%, 30%, 20%,
  • a druggable target’s expression is perturbed in both a first and second time point.
  • the perturbation is in the same direction in both time points, i.e. is upregulated at both time points, or is downregulated in both time points.
  • ip of selecting a gene as a druggable target may comprise selecting a gene (i) that is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, (ii) that has perturbed expression in following exposure to the first compound and (iii) to which an inhibitor compound is known.
  • the inhibitor compound has achieved regulatory approval for use as a medicine in the treatment of the cancer.
  • a gene which is a“positive regulator” is one which functions to promote, accelerate, increase or enhance a process.
  • a positive regulator of angiogenesis works to increase angiogenesis.
  • a gene which is a“negative regulator” is one which functions to inhibit, supress or prevent a process. Therefore, a negative regulator of angiogenesis would work to reduce angiogenesis.
  • the compounds may alter cell viability, for example the first or second compound may comprise compounds that are cytotoxic to mammalian cells. Suitable compounds may include for example active anti-cancer agents. In other embodiments, the compounds may alter phenotypic properties displayed by mammalian cells, causing cell cycle perturbation, apoptosis enhancement, or changes in antigen display.
  • the compounds may alter a phenotypic property of mammalian cells.
  • the compounds may reduce or inhibit the proliferation, viability, migration, invasion and/or angiogenesis of mammalian cells, such as cancer cells; increase or promote apoptosis and/or radio-sensitisation; and/or may alter a cell surface phenotype, such as EGFRvlll or other neoantigen, or a molecular characteristic associated with differentiation and apoptotic processes, antigen display and cell renewal.
  • the compounds may regulate, for example reduce or inhibit, angiogenesis, mTOR signalling, or the NFKB signalling.
  • chemotherapeutic agents for example alkylating agents such as platinum complexes including cisplatin, mono(platinum),
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime
  • nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard
  • nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine
  • antibiotics such as aclacino
  • aldophosphamide glycoside aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;
  • defofamine demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate;
  • phenamet pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine;
  • TAXOL paclitaxel
  • TXOTERE docetaxel
  • chlorambucil gemcitabine
  • 6-thioguanine mercaptopurine
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • etoposide VP- 16
  • ifosfamide mitomycin C; mitoxantrone; vincristine; vinorelbine; binblastine; vindesine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA);
  • Topoisomerase inhibitors such as doxorubicin HCI, daunorubicin citrate, mitoxantrone HCI, actinomycin D, etoposide, topotecan HCI, teniposide (VM-26), and irinotecan and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • a first or second compound may include one or more compounds shown to be active in preclinical models of GBM. Suitable compounds may include; 2-Amino-3H-phenoxazin-3-one, 2- Hydroxyoleic acid, 3-(3,4-dichlorophenyl)-1-(3,4-dimethylphenyl)-1 -(5-methyl-4,5-dihydro-1 ,3-thiazol-2- yl)urea, 3-Deazaneplanocin, 4egi-1 , 5-Nonyloxytryptamine, 6-Hydroxyquinoline-4-carboxylic acid, 7-Ethyl- 10-hydroxycamptothecin, 7-Hydroxystaurosporine, 8-Bromo-cyclic AMP, 8-Hydroxy-2-methyl-1 H- quinazolin-4-one, 9-ING-41 , A-966492, Abemaciclib, Abt-737, AC1 MMYR2, Acalabrutini
  • Arctigenin Ardipusilloside I, Ascochlorin, Astemizole, AT13387, Atorvastatin, Aurintricarboxylic acid, Axitinib, AXL1717, AZD2014, AZD-7451 , AZD8055, BAY 1 1-7082, BDBM86691 , Bendamustine, BI2536, Bimiralisib, Binimetinib, Birinapant, BIX-01294, BMS-536924, BMS-777607, Bortezomib, Bosutinib,
  • Bufalin Buparlisib, Cabazitaxel, Caffeic acid phenethyl ester, Caffeine, Camptothecin, Capecitabine, Capmatinib, Captopril, Carboplatin, Cardamonin, Carmustine, carnosol, Carvacrol, Casticin, Cathepsin S Inhibitor, Caudatin, CBL-0137, CC-115, CC-223, Cediranib, Celastrol, Celecoxib, Ceritinib, Cerivastatin, Chaetocin, Chloroquine, Chlorpromazine, Cilengitide, Cisplatin, Cladribine, Clioquinol, Clofazimine, Clorgiline, Cordycepin, Crenolanib, Crizotinib, Cryptotanshinone, Cucurbitacin I, CUDC-101 , Curcumin, Cycloheximide, Cyclophosphamide, D609, Da
  • GSK461364 Guggulsterone, Hispolon, Honokiol, Hsp-990, Hydroxyurea, Hypericin, Ibrutinib, Ibuprofen, Idarubicin, Imatinib, Imiquimod, Indatraline, Indirubin Derivative E804, Indomethacin, Infigratinib, Iniparib, Irinotecan, Isobutylmethylxanthine, Isoliquiritigenin, Isotretinoin, Itraconazole, Ixazomib, JQ1 , JS-K, Juglone , Karenitecin, KPT251 , KPT276, KU55933, KU-55933, KU60019, Lactacystin, Lanatoside C, Lapatinib, Larotrectinib, LB-100 , Lenalidomide, Lenalidomide, Lenvatinib, LEQ506, Letrozole,
  • the methods of the tenth and eleventh aspects may be extended to generate additional transcriptomic profiles in order to provide more extensive combination therapies.
  • the method is extended to identify further druggable targets by: d. selecting an inhibitor of the druggable target for combination therapy as the second compound e. contacting test cells with the second compound, and optionally the first compound, f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
  • said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • test cells with the further compound, optionally sequentially or simultaneously with any and all of the other compounds used in the previous stages,
  • said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • the method may provide a series of druggable targets by:
  • the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation,
  • the method provides an anti-cancer therapeutic combination comprising a first, a second and a third compound, and comprises the additional steps of: e. contacting test cells with the second compound, and optionally the first compound f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
  • this embodiment of the eleventh aspect may be extended to provide a fourth compound by repeating the contacting and measuring steps with the third compound, optionally in combination with the first and/or second compound, to create a third transcriptomic profile; selecting a further druggable target that is perturbed in the third transcriptomic profile; and selecting an inhibitor of this target as a fourth compound in the combination therapy. This may be repeated to provide a fifth, sixth, etc. compound for the combination.
  • the method may be extended to provide an anti-cancer therapeutic combination comprising a first compound and any number of further compounds, comprising the steps of: a. contacting test cells with a first compound,
  • steps a to d optionally repeating steps a to d one or more times, each time supplementing or substituting the compound used in step a with the further compound identified in the previous step d, until the desired number of further compounds has been identified.
  • the method may be used to sequentially identify compounds for use in a treatment which is tailored to the individual and their cancer, which is dynamic, heterogeneous and adapts in response to treatment.
  • the second compound may additionally be selected on the basis of synergy with the first compound.
  • step d may comprise the sub-steps of:
  • a combination with a“synergistic” effect is one for which the effect is greater than the sum of the components provided in isolation.
  • a second compound that exhibits a synergistic effect one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radiosensitisation when provided in combination with the first compound is one where the effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation is greater than the sum of the effect seen for the first and second compounds administered in isolation.
  • the test cells may be the same cells that are contacted with the first compound in step a), in order to model ongoing therapy.
  • the test cells may be of the same type as those in step a) but may have not previously been contacted with the first compound.
  • the invention also provides a therapeutic combination provided by the method of the eleventh aspect.
  • the invention also provides a therapeutic combination provided by the method of the eleventh aspect, for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the first compound simultaneously or sequentially with the second compound.
  • the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
  • the invention also provides the use therapeutic combination provided by the method of the eleventh aspect in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament administers an effective amount of the first compound simultaneously or sequentially with an effective amount of the second compound to a subject in need thereof.
  • the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
  • the invention also provides a method of treating or preventing cancer with the therapeutic combination according to the eleventh aspect, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of the first compound and an effective amount of second compound.
  • the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
  • the method may comprise sequentially administering the compounds in combination, for example by (i) administering the ripound, followed by (ii) the second compound simultaneously with the first compound, followed by (iii) the third compound simultaneously with the second and/or first compound, followed by (iv) a further compound simultaneously with any combination of the previous compounds. Steps (i) to (iv) may be separated by any length of time.
  • the first and second compounds may be any contemplated herein, the first compound is preferably an inhibitor of PI3K signalling, and the second compound is preferably an inhibitor of JAK activity or function, or the first compound may be an inhibitor of JAK activity or function and the second compound may be an inhibitor of PI3K signalling.
  • the cancer is sequentially challenged with new drugs during ongoing therapy.
  • a compound may be administered at an initial effective dose, before reverting to a lower dose for subsequent stages - i.e.
  • the first compound may be administered at a higher dose in step (i) than (ii)-iv), the second at a higher dose in step (ii) than steps (iii)-(iv), the third compound in a higher dose in step (iii) than in step (iv), etc.
  • the cancer is challenged with an initial effective dose of a compound, and ongoing therapy at a lower dose to avoid dose limiting toxicity or side effects.
  • a therapeutic combination provided by the method according to the eleventh aspect for use in the treatment or prevention of cancer, preferably for the treatment of GBM.
  • the combination may be for sequential or simultaneous administration.
  • the invention also provides a method, preferably an in vitro method, of identifying druggable targets for combination therapy, comprising the steps of:
  • the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • the invention also provides a method, preferably an in vitro method, of providing a therapeutic combination, the method comprising
  • the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
  • a“treatment” may comprise or consist of a therapeutic compound as defined above.
  • a treatment may comprise a suite of therapeutic compounds.
  • a treatment may comprise or consist of additional therapies, such as radiotherapy.
  • A“first treatment” may comprise or consist of any therapeutic compounds and/or therapies.
  • A“second treatment” comprises or consists of an inhibitor of the druggable target, and may comprise additional compounds or therapies, so long as it includes an inhibitor of the druggable target.
  • the second treatment may include one or more therapeutic compound and/or therapy which constituted the first treatment.
  • a diagnostic process using similar transcriptomic techniques to identify further dysregulated genes and pathways as diagnostic targets which may comprise biomarkers of relevance to the treatments is provided herein.
  • a method of identifying diagnostic targets in a cancer comprising:
  • test cells are cancer cells
  • test cells are cells from a patient with a cancer, more preferably the cells are in vivo.
  • the second compound may be selected on the basis of selectivity screening.
  • a compound may be provided, or developed specifically, on the basis of the transcriptomic profile induced by it on test cells, either alone or in combination with the first compound, or on the basis of its inhibitory profile.
  • the second compound may have a similar or the same transcriptomic or inhibitory profile as a candidate inhibitor for which a synergistic effect with the first compound has been shown.
  • step d may comprise the sub-steps of:
  • ii) contacting test cells with the candidate inhibitor in combination with the first compound, and iii) measuring the effects of the combination on the transcriptome of the test cells, so as to create a transcriptomic profile for the combination, if the combination exhibits a synergistic effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation,
  • a cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumour or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumour.
  • the cancer may be benign or malignant and may be primary or secondary (metastatic).
  • a neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue.
  • tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • lymph node including abdominal lymph node, axillary lymph node, cervical lymph node, inguinal lymph node, mediastinal lymph node, pelvic lymph node, periaortic lymph node
  • lymphoblast maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentume, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells
  • Cancers may be of a particular type.
  • types of cancer include astrocytoma, carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g. angiosarcoma, chrondrosarcoma, osteosarcoma).
  • carcinoma e.g. adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma
  • glioma e.g. adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma
  • glioma e.g. adenocarcinoma, hepat
  • the cancer to be treated or prevented is a cancer of the brain or central nervous system (CNS).
  • Brain cancers include primary brain tumours (tumours which start in the brain) and secondary brain tumours (i.e. cancerous tumours which have metastasised to the brain from another location in the body, also known as brain metastasis tumours).
  • the brain cancer is a primary brain tumour.
  • Primary brain tumours may originate in astrocytes,
  • oligodendrocytes oligodendrocytes, ependyma, neurons or meninges.
  • Brain cancers may be subdivided into glioma (tumours of the glial cells), meningioma (tumours of the meninges), pituitary adenoma (tumours of the pituitary gland) and nerve sheath tumours.
  • the cancer to be treated or prevented is a glioma.
  • a glioma may be selected from an ependymoma, an astrocytoma, an oligodendroglioma, or a brain stem glioma.
  • Gliomas may be categorised according to their grade, as determined according to WHO Classification of Tumours of the Central Nervous System (“The 2016 World Health Organisation Classification of Tumours of the Central Nervous System: a summary". Acta Neuropathologica. 131 (6): 803-820).
  • the cancer to be treated or prevented may be a low-grade (WHO grade II or lower) glioma or, more preferably, a high-grade (WHO grade lll-IV) glioma.
  • the cancer to be treated or prevented is glioblastoma multiforme (GBM).
  • GBM is a primary cancer of the astrocytes, and is the most common primary malignant brain tumour in adults (Ostrom et al., 2017).
  • the GBM has one or more mutations in a signalling pathway selected from: RB, TP53, and receptor tyrosine kinase (RTK)/mitogen activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K).
  • RTK receptor tyrosine kinase
  • MAPK mitogen activated protein kinase
  • PI3K phosphoinositide 3-kinase
  • GBM can be stratified into 4 molecular subtypes based on gene expression according to Verhaak et al., 2010.
  • the cancer to be treated or prevented may be a GBM of the Classical subtype.
  • Classical GBM is characterised by extra copies of the EGFR, higher than normal expression of EGFR, retention of wild- type p53, loss of heterozygosity in chromosome 10, and chromosome 7 amplification.
  • the cancer to be treated or prevented may be a GBM of the Mesenchymal subtype.
  • Mesenchymal GBM can be characterised by high rates of mutations or other alterations in NF1 , the gene encoding Neurofibromin 1 and fewer alterations in the EGFR gene and less expression of EGFR than classical GBM.
  • the cancer to be treated or prevented may be a GBM of the proneural subtype.
  • Proneural GBM can be characterised by high rates of alterations in TP53 (p53), and in PDGFRA, the gene encoding a-type platelet-derived growth factor receptor, and in IDH1 , the gene encoding isocitrate dehydrogenase-1.
  • the cancer to be treated or prevented may be a GBM of the Neural subtype.
  • Neural GBM can be characterised by the expression of neuron markers. The characterisation of GBM subtypes by biomarker is a matter of routine.
  • the cancer to be treated or prevented is a metastatic GBM i.e. one which has
  • the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In some embodiments, about means the specified value.
  • Fucoxanthin and LY-294002 were selected as reagents for investigation.
  • Fucoxanthin is a member of the xanthophyll class of carotenoids, and is present at high concentrations in the brown alga Saccharina latissimi (Galasso et al., 2017) where it plays an accessory role in light harvesting and radiation protection. Fucoxanthin has been suggested to act in cancer by suppressing invasion and inducing apoptosis through PI3K/Akt pathway inhibition (Liu et al., 2016; Satomi, 2017) and JAK/STAT pathway inhibition (Kanno et al., 2013; Szymanska et al., 2015).
  • Fucoxanthin has a particularly interesting and unique molecular structure ( Figure 1 C), exhibiting antioxidant properties due to a long conjugated backbone characteristic of all carotenoids, and possessing unusual terminal allenic bond and conjugated carbonyl groups (Dembitsky and Maoka, 2007; Sangeetha et al., 2009).
  • Figure 1 C a particularly interesting and unique molecular structure
  • Fucoxanthin has been shown to inhibit migration and invasion of metastatic melanoma and osteosarcoma cells in vitro and in vivo (Liu et al., 2016).
  • Fucoxanthin is metabolically unstable, upon metabolism producing
  • Fucoxanthinol (Zhang et al., 2015), a factor to take into account when evaluating its transcriptomic effects.
  • LY-294002 is one of the PI3K/Akt inhibitors annotated as active in GBM annotated in the GBM Drug Bank, a public domain resource developed by IOTA Pharmaceuticals based on compounds known to influence the growth and development of glioblastoma (Svensson et al., 2018) http://www.gbmdrugbank.com/db.php).
  • man glioblastoma cell line U87MG expressing the wild-type p53 gene (U87MG, p53wt, female, obtained from European Collection of Authenticated Cell Cultures) was maintained in DMEM/F12 media (Gibco, ThermoFisher, UK) supplemented with 10% foetal bovine serum (FBS) (Sigma, UK) and 5% antibiotic/antimycotic solution (Sigma, UK).
  • FBS foetal bovine serum
  • FBS foetal bovine serum
  • antibiotic/antimycotic solution (Sigma, UK).
  • Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 LY-294002 and Fucoxanthin were purchased from Sigma, UK and dissolved in dimethyl sulfoxide (DMSO) to obtain 0.1 M and 0.05 M stock solutions, respectively.
  • DMSO dimethyl sulfoxide
  • cells were seeded into T25 flasks at a density of 5x10 5 cells/flask and allowed to adhere and grow for 24h.
  • the culture medium was removed, and fresh medium containing compound for test was added to each flask at the EC50 concentration for inhibiting proliferation at 72h, previously determined as 199.5 pM for LY-294002 and 19.9 pM for Fucoxanthin.
  • Control cells were treated with medium containing 1 % DMSO alone. All experiments were performed in triplicate. Cells were visualised during culture using the EVOS Cell Imaging System (Thermo Fisher Scientific, UK).
  • the cytotoxicity of Fucoxanthin and LY-294002 in U87MG cell line was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma).
  • U87MG cells were seeded at a density of 8,000 cells/well in 96- well plates and allowed to adhere overnight at 37°C in a humidified atmosphere of 95% air and 5% CO2.
  • Both Fucoxanthin and LY-294002 were tested at concentrations of 1- 1000 pM in triplicate in at least two separate experiments. All initial dilutions of Fucoxanthin and LY-294002 were made in DMSO and then further diluted in the medium at 1 : 100 ratio.
  • the culture medium was removed from the plates, and the fresh medium containing tested compounds at different dilutions was added to the plates.
  • Control cells were treated with vehicle solution containing 1 % DMSO. Blank controls without cells were also prepared.
  • 72h after treatment 5 pL of CCK-8 was added to every well containing 100 pL of tested compounds, controls or blank.
  • the plates were read using a Mithras LB940 multimode microplate reader (Berthold Technologies), and the absorbance values were determined at 490 nm. The percentage of surviving cells was calculated for each well using the formula:
  • At absorbance of the medium with tested compound
  • a c absorbance of control medium
  • Ab absorbance of blank medium
  • Control cells were tested at concentrations of 19.9 pM and 199.5 pM, respectively.
  • Control cells were treated with vehicle solution containing 1 % DMSO. After 48h of treatment, the cells were trypsinised, centrifuged, resuspended in 500 pL of binding buffer followed by the addition of 5 pL Annexin V-FITC and 5 pL propidium iodide (PI) according to the manufacturer's instruction. The samples were incubated at room temperature for 5 min in the dark and then analysed using a BD AccuriTM C6 Flow Cytometer. A total of 10,000 events were counted for each sample. Fluorescence was measured at an excitation wavelength of 480 nm with detection for PI at 530 nm and Annexin V at 585 nm.
  • RNA samples were further prepared by ATLAS Biolabs (Berlin, Germany) using their Affymetrix WT Expression Profiling Standard Service. The analysis was conducted on a Clariom S Human array with a fixed number of probes per transcript as probe sets consisting of a subset of 10 probes per gene (yielding >20,000 annotated genes, as documented by the NetAffx Analysis Center).
  • RNA sample was determined using the Agilent 2100 Bioanalyzer and Nanodrop.
  • Agilent 2100 Bioanalyzer and Nanodrop For library preparation (cDNA synthesis, amplification and labeling) GeneChip ® WT PLUS reagents were used.
  • 96-array plates were processed on an Applied Biosystems GeneChip 3000 instrument system followed by hybridisation, dyeing, washing, and scanning using the GeneChipTM Hybridisation, Wash, and Stain Kit from Affymetrix. Hybridisation controls, quality control parameters and primary data analysis was performed using Expression Console v1 .4 (also from Affymetrix).
  • the average signal intensities for each probe set were analysed and an expression matrix was created by applying the RMA (Robust Multi-array Average) algorithm as a multi-chip model (Irizarry et al., 2003).
  • RMA Robot Multi-array Average
  • a threshold of 0.8 area under the receiver operating curve was applied to compare the exon and intron probe sets of housekeeping genes, to ensure a good separation within the data.
  • Microarray gene expression profiling and visualisation results were performed using the Affymetrix Transcriptome Analysis Console (TAC) software (ThermoFisher) which uses the limma package (Ritchie et al., 2015).
  • TAC Affymetrix Transcriptome Analysis Console
  • DEGs differentially expressed genes
  • a gene log 2 Fold Change (FC) level has to reach the threshold of ⁇ -1 or > 1 based on Tukey's bi-weight average between treatment and time matching controls (Kohl and Deigner, 2010). Tukey’s bi-weight averaging makes the average less sensitive to outliers (Tukey, 1973).
  • DEGs differentially expressed genes
  • WikiPathways map representations were used in which the signal intensities of DEGs involved in one particular enriched pathway can be shown as down-regulated or up- regulated in response to specific treatments (Kutmon et al., 2016). For pathway analysis, Fisher exact tests were used.
  • the Connectivity Map approach is based on the most significant differentially expressed genes characteristic of compound molecular response. Significantly differentially expressed genes were analysed using Connectivity Map 2. Differentially expressed genes IDs were mapped to Affymetrix HGU133-plus2 probe set IDs, which were then input into Connectivity Map. The down- and up-regulated genes responding to Fucoxanthin and LY-294002 treatment were used as the query signatures for comparing transcriptomic profiles to other compounds in CMap. The connectivity score was calculated based on Kolmogorov-Smirnov statistics (Smalley et al., 2010). This approach is described in detail in Alexander-Dann et al., 2018; Iwata and Yamanishi, 2019; and Oerton and Bender, 2017. Significant findings were cross-referenced to the Glioblastoma Drug Bank (Svensson et al., 2018).
  • the target cell population U87MG glioblastoma cells
  • each compound was treated with each compound at its 72h antiproliferative EC50, determined in an initial drug response study (Figure 2) in order to see“early” gene expression changes in response to drug challenge within 24h, a time point where no growth inhibition was evident ( Figure 2A), with“late” gene expression changes accompanying treatments at subsequent time points.
  • The“late” time point was 48h, prior to any overt toxicities emerging as the cells reached the EC50s of the compounds at 72h.
  • Fucoxanthin or LY-294002 at these concentrations clearly differed from those of DMSO-treated controls (Figure 3).
  • the cells grew as monolayers in the cell culture flasks before treatment and formed some spheres.
  • After 24h of treatment with Fucoxanthin most adherent cells were round, with some showing signs of nuclear fragmentation, although some cells with pseudopod-like protrusions were also observed (Figure 3C).
  • Figure 3D After 48h of treatment, a mixture of effects was seen: some cells remained adherent, while others floated off their plastic substrates, often as aggregates (Figure 3D).
  • PCA Principal Component Analysis
  • Affymetrix Clariom S microarrays contain probe sets for both non-coding and coding genes.
  • FDR false discovery rate
  • top 25 expressed genes in response to LY-294002 at 24h and 48h were compared with those seen in Fucoxanthin treatments at the same time points ( Figure 7).
  • the top 25 expressed genes in response to Fucoxanthin at 24h and 48h were compared with those seen in LY-294002 treatments at the same time points ( Figure 8). Clear differences were seen between the gene expression profiles amongst the top 25 expressed genes in each treatment.
  • Fucoxanthin has a transcriptomic effect on PI3K/Akt pathway, increasing the expression of 10 of its component genes after 24h, 5 of which are remain up-regulated after 48h treatment, while decreasing the expression of 24 genes after 24h, with 19 of these genes remaining downregulated after 48h treatment (p ⁇ 0.05 Benjamin Hochberg corrected Fisher exact test).
  • LY-294002 had no significant transcriptomic effect at the pathway level on the PI3K/Akt pathway (p>0.05 Benjamin Hochberg corrected Fisher exact test), even though it acts directly on this pathway by inhibition of PI3K. Only 4 genes at 24h and 8 genes at 48h were up-regulated from this large pathway while 17 and 19 genes, respectively, were down-regulated after 24h and 48h treatment (see Figure 10 for the wiring diagrams of the genes involved in PI3K/Akt pathway). In contrast, Fucoxanthin has a clear effect at 24h on the expression of components of the PI3K/Akt pathway, although this is not id by their continuing expression at 48h.
  • JAK2 modulates the PI3K/mTOR pathway (Rane and Reddy, 2000). Up-regulation of JAK2 could reflect a pro-proliferative role within the glioblastoma cells as they react to the growth inhibition caused by down-regulation of the PI3K pathway.
  • pro-proliferative genes Bcl-2, SGK1 , IGF1 R show similar induction patterns.
  • Fucoxanthin has a marked transcriptomic effect on the Retinoblastoma pathway, downregulating all the genes in the pathway at 24h with a considerable number remaining down-regulated after 48h treatment.
  • LY-294002 similarly down-regulates this pathway, with 35 genes showing down-regulation at 24h, 19 of which remain down-regulated at the 48h treatment time ( Figure 11).
  • TP53 is the only gene up-regulated in both the PI3K/Akt and Retinoblastoma pathways, and this only in response to
  • PI3K No proteins except isoforms of PI3K were identified using PIDGIN that also occurred within the set of up- or down-regulated genes seen using transcriptomics, and PI3K was only identified as a putative target for LY-294002, not Fucoxanthin or Fucoxanthinol. Additional proteins showing putative LY-294002 binding included Topoisomerase 1 and the GABA receptor P subunit. A further protein, the kinase PIM1 , was identified as a weak hit (seen in the transcriptomics data at 48h of LY-294002 treatment).
  • Connectivity Map is a method used routinely to compare the effects of drugs on gene expression space (Lamb et al., 2006). Besides providing clues to enable the determination of drug mode of action at a molecular level, the method also enables an objective comparison of drug properties (Alexander-Dann et al., 2018). Here, we used CMap to compare the gene expression“signatures” of LY-294002 and Fucoxanthin seen in our studies with U87MG cells, to drug-like compounds in CMap with similar gene expression signatures from other cells and tissues.
  • the gene expression signatures from U87MG cells treated with LY-294002 show very similar gene expression profiles to other PI3K drugs already deposited in CMap, including the PI3K pharmacological tools Quinostatin (which inhibits the lipid-kinase activity of the catalytic subunits of class la PI3Ks), and Wortmannin (a potent, selective and irreversible inhibitor of PI3K).
  • Quinostatin which inhibits the lipid-kinase activity of the catalytic subunits of class la PI3Ks
  • Wortmannin a potent, selective and irreversible inhibitor of PI3K
  • Fucoxanthin exhibits higher similarity to more compounds in the CMap archive with similar gene expression signatures (384 compounds, Figure 12), than does LY-294002 (182 compounds, Figure 12), possibly representing more interactions with cellular components.
  • 294002 with JAK2 inhibitors Ruxolitinib or AZD1480 was investigated.
  • Cells were exposed to LY294002 in concentrations ranging from 10 _6 M to 10 _3 M, alone or in combination with JAK inhibitors at 10, 25 or 50 pM.
  • Reference treatments were performed, comprising JAK inhibitors alone at 10, 25 and 50 pM, and a negative control treatment of 1 .1 % DMSO. Cell survival as a percentage of DMSO control was measured at 72 hrs following treatment.
  • microarray analysis When combined with the deployment of the method using patient-derived tumour tissue and the cell lineages therein, microarray analysis will allow identification of newly emergent targets, allowing more precise treatments to be deployed on a patient-by-patient basis (personalised therapies).
  • LY-294002 For LY-294002, within 24h of treatment at the 72h EC50, a clear gene expression pattern is seen in response to the drug. The pattern observed for U87MG cells parallels that seen when other established cancer cell lines are treated with LY-294002. Moreover, many of these“early” changes are recapitulated at the“late” 48h time point, indicative of a sustained drug response to LY-294002: over 44% of highly induced genes are shared at 24h and 48h of treatment. Even more distinctively, 61 % of the down- regulated genes at 24h remain down-regulated at 48h.
  • Fucoxanthin treatments at the 72h EC50 for 24h and 48h show less correspondence, with only 28% of the highly induced genes seen at 24h remaining at 48h.
  • the disparity of gene expression responses between the 24- and 48-hour time points for Fucoxanthin may indicate that this compound exhibits more complex drug-induced gene expression effects over time than LY-294002, a hypothesis that requires further investigation but which is consistent with both Fucoxanthin’s metabolic lability and its production of a new and powerfully antiproliferative active metabolite, Fucoxanthinol (Terasaki et al., 2018).
  • the retinoblastoma pathway is at the top most significantly down-regulated pathway. After closer scrutiny the pathways effector endpoints are down-regulated suggesting the cells are going into a dormant state.
  • IGF1 R As well as up-regulation of JAK2, up-regulation of IGF1 R, BCL2L11 and SGK1 can be seen, all of which can be involved in growth promotion and cell proliferation (Basnet et al., 2018; Peng et al., 2016).
  • LY294002 enhances cytotoxicity of temozolomide in glioma by down-regulation of the PI3K/Akt pathway. Mol. Med. Report. 5, 575-579.
  • Apatinib exerts antitumour effects on ovarian cancer cells. Gynecol. Oncol. 153, 165-174.
  • the IGFR1 inhibitor NVP-AEW541 disrupts a prosurvival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells. Biochem. Pharmacol. fin 4 ⁇ -462.
  • PI3K-AKT pathway negatively controls EGFR-dependent DNA-binding activity of Stat3 in glioblastoma multiforme cells. Oncogene 24, 7290-7300.
  • Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002. J. Biol. Chem. 280, 13728-13734.
  • Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling. Brain 135, 1055-1069.
  • VHL tumour suppressor protein regulates tumourigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signalling pathway. Int. J. Oncol. 42, 881-886.
  • the Connectivity Map using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929-1935.
  • Fucoxanthin Activates Apoptosis via Inhibition of PI3K/Akt/mTOR Pathway and Suppresses Invasion and Migration by Restriction of p38-MMP-2/9 Pathway in Human Glioblastoma Cells. Neurochem. Res. 41, 2728-2751.
  • CHAF1A Over-expression of CHAF1A promotes cell proliferation and apoptosis resistance in glioblastoma cells via AKT/F0X03a/Bim pathway. Biochem. Biophys. Res. Commun. 469, 11 11-1116.
  • a next generation connectivity map L1000 platform and the first 1 ,000,000 profiles. Cell 171, 1437-1452.e17.
  • Glycine and succinic acid are effective indicators of the suppression of epithelial- mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells. Oncol. Rep. 40, 414-424.
  • Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterised by abnormalities in PDGFRA, IDH1 , EGFR, and NF1. Cancer Cell 17, 98- 1 10.

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Abstract

A method of identifying druggable targets for cancer combination therapy is provided, whereby test cells are contacted with a compound, and a druggable target selected from amongst the genes whose transcription is affected. A method of providing an anti-cancer therapeutic combination is further provided, as well as therapeutic combination as identified by this method, and methods of using the same in the treatment or prevention of cancer. An exemplary therapeutic combination which is provided is that of an inhibitor of phosphoinositide 3-kinase (RISK) signalling and an inhibitor of Janus kinase (JAK) activity or function.

Description

TRANSCRIPTOMICS FOR SELECTING DRUGGABLE TARGETS FOR COMBINATION
THERAPY IN CANCER
Field of the Invention
The present invention relates to combination therapy for cancer and particularly to methods for identifying new anti-cancer combinations. The invention also relates to the combination of an inhibitor of phosphoinositide 3-kinase (PI3K) signalling with an inhibitor of Janus kinase (JAK) activity or function.
Background
Glioblastoma multiforme (GBM) is the most common and aggressive malignant glioma, with patients having a median survival of just over one year (Batash et al., 2017). First line therapy remains empiric and consists of surgical resection followed by radiation with concurrent and adjuvant temozolomide, a DNA damaging agent (Stupp et al., 2005). Clinical trials of inhibitors targeting the pathways frequently mutated in GBM have had disappointing results for a variety of reasons, including drug resistance and inclusion of molecularly heterogeneous patients (Cloughesy et al., 2014; Mendelsohn, 2013). Current chemotherapies, together with surgery and radiotherapy, provide only minor patient benefit, and there is a considerable need for development of effective new therapies.
Many key cellular signalling pathways are dysregulated in GBM and provide attractive targets for drug therapy (Pearson and Regad, 2017). In addition, the genetic alterations found repeatedly in patients with glioblastoma multiforme point to specific pathways involved in GBM proliferation, survival and migration such as angiogenesis, mTOR signalling or the NFKB signalling (Tuncbag et al., 2016). One of the most important pathways mutated in GBM is the PI3K/Akt pathway, which appears particularly important in glioblastoma proliferation but also plays a central role in the regulation of tumour cell survival, motility, angiogenesis and metabolism (Zhao et al., 2017). This has led to many attempts to target the PI3K/Akt pathway as a potential treatment option for glioblastoma (Cancer Genome Atlas Research Network,
2008; Pridham et al., 2017). So far, this has met with limited success.
An early PI3K inhibitor to be discovered was LY-294002 (Figure 1A). LY-294002 is a non-selective pan- PI3K inhibitor, interacting with PI3K class I, III and PI4K proteins, among other unrelated proteins (Gharbi et al., 2007). LY-294002 has previously been shown to enhance the cytotoxicity of temozolomide in U87MG glioma cells by down-regulating genes involved in the PI3K/Akt pathway (Chen et al., 2012). In drug-resistant leukemia cells, LY-294002 also blocks the cyclin-dependent kinases (CDKs), as well as PKC and other PI3K pathway components, in a manner similar to other drugs such as Flavopiridol, Roscovitine, Wortmannin (Figure 1 B) and UCN-01 , leading to augmentation of apoptosis (Cory et al., 2005).
In parallel, natural products targeting PI3K emerged as early cancer therapeutics: Wortmannin was isolated as a fungal metabolite, while LY-294002, the first synthetic PI3K inhibitor, was derived from the natural product Quercetin (Kong and Yamori, 2008). Another natural product with potential in a variety of cancers is Fucoxanthin (Mbaveng et al., 2019; Panossian et al., 2018).
Chemical inhibitors of the PI3K pathway have been instrumental in understanding the role of PI3K enzymes in signal transduction and validating them as therapeutic targets, with the early pan-specific PI3K inhibitors Wortmannin and LY294002 being used extensively to probe the activity of the PI3K/Akt pathway in cancer.
However, neither LY-294002 nor Wortmannin are PI3K-selective inhibitors, showing additional Casein Kinase 2 (CK2) and Myosin Light-chain Kinase (MLCK) activities, respectively (Kong and Yamori, 2008). A detailed analysis of LY-294002 binding proteins using a chemical proteomics approach based on the close chemical analog PI828 (Gharbi et al., 2007)), together with earlier biochemical studies (Davies et al., 2000; Jacobs et al., 2005), suggests several non-lipid kinases as additional biochemical targets of LY- 294002, including CK2, PIM-1 , mTOR and GSK3p.
The feasibility of using gene expression for digital transcriptome profiling of normal and glioblastoma- derived neural stem cells has already been reported (Engstrom et al., 2012). Microarray gene expression analysis, coupled with Pathway and Connectivity Map (CMap) analyses has been previously used for functional drug response analysis and comparison (Babcock et al., 2013; Subramanian et al., 2017a).
The CMap method considers the most highly differentially expressed genes as query signatures and compares them to the reference signatures of drugs tested in a wide variety of biological systems, to date most particularly human cancer cell lines. Using this method, both pre-existing and novel signatures indicative of a compound’s functional effects, molecular modes of action, or toxicity can be defined (Alexander-Dann et al., 2018; lorio et al., 2010).
Summary of the Invention
The present invention provides inhibitors of the phosphoinositide 3-kinase (PI3K) signalling pathway for use in combination with inhibitors of Janus kinase (JAK) activity or function. These find use, for example, in methods of treating or preventing a cancer.
In a first aspect, the invention provides a method of treating or preventing cancer, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of an inhibitor of phosphoinositide 3-kinase (PI3K) signalling simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
In a second aspect, the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling for a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the inhibitor of PI3K signalling simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function. In a third aspect, the invention provides an inhibitor of Janus kinase (JAK) activity or function for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the inhibitor of PI3K signalling simultaneously or sequentially with an inhibitor of phosphoinositide 3-kinase (PI3K) signalling.
In a fourth aspect, the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling for use in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament is administered simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
In a fifth aspect, the invention provides an inhibitor of Janus kinase (JAK) activity or function for use in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament is administered simultaneously or sequentially with an inhibitor of phosphoinositide 3-kinase (PI3K) signalling
In a sixth aspect, the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and an inhibitor of Janus kinase (JAK) activity or function for use in a method of treating or preventing cancer in a subject.
In a seventh aspect, the invention provides an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and an inhibitor of Janus kinase (JAK) activity or function for use in the manufacture of a medicament for treating or preventing cancer in a subject.
In a eighth aspect, the invention provides a composition comprising an inhibitor of phosphoinositide 3- kinase (PI3K) signalling, an inhibitor of Janus kinase (JAK) activity or function, and a pharmaceutically acceptable excipient.
In a ninth aspect, the invention provides a kit comprising (i) an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and (ii) an inhibitor of Janus kinase (JAK) activity or function.
In some embodiments of any of the first to ninth aspects above, the inhibitor of PI3K signalling interacts with and inhibits the activity or function of PI3Kpreferably a PI3K isoform selected from PIK3CA/p110a, PIK3CB/p1 10b and/or PIK3CD/p1 10d. Optionally, the inhibitor of PI3K signalling may additionally interact with and inhibit the activity or function of one or more of Casein Kinase 2 (CK2) and/or Myosin Light-chain Kinase (MLCK).
In a preferred embodiment of any of the aspects above, the inhibitor of PI3K signalling is 2-Morpholin-4- enylchromen-4-one (LY-294002).
In some embodiments of any of the aspects above, the inhibitor of JAK activity or function is an inhibitor of JAK2 activity or function, preferably AZD1480. In some embodiments of any of the aspects listed above, the cancer is a cancer of the brain, for example a primary brain tumour, preferably a glioma, most preferably glioblastoma multiforme.
In a preferred embodiment of the invention according to the first to ninth aspect, the inhibitor of PI3K signalling is LY-294002, or a salt, conjugate, prodrug, or derivative thereof, and the inhibitor of JAK activity or function is AZD1480, or a salt, conjugate, prodrug, or derivative thereof, and the cancer is glioblastoma multiforme.
In another set of related aspects, the invention relates to methods useful in the development of combination therapies for cancer and, in particular, for glioblastoma multiforme.
In a tenth aspect, the invention provides a method, preferably an in vitro method, of identifying druggable targets for combination therapy, comprising the steps of:
a. contacting test cells with a first compound,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is perturbed in the transcriptomic profile,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation. Preferably, the combination therapy is combination cancer therapy, more preferably a glioblastoma multiforme combination therapy.
In an eleventh aspect, the invention provides a method, preferably an in vitro method, of providing a combination therapy, said combination comprising a first and a second compound, the method comprising
a. contacting test cells with the first compound,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is perturbed in the transcriptomic profile,
d. selecting as the second compound an inhibitor of the druggable target for combination therapy,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation. The combination therapy is preferably a cancer combination therapy, preferably where the cancer is glioblastoma multiforme.
'ention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. Summary of the Figures
Figure 1. Chemical structures of A. LY-294002; B. Wortmannin; C. Fucoxanthin; and D. Fucoxanthinol. Structures taken from PubChem and redrawn by ChemDraw
Figure 2. Dose-response measurements for A. Fucoxanthin and B. LY-294002 using cell survival compared to no-drug controls as a measure of proliferation in U87MG glioblastoma cells over 24, 48 and 72 hours of treatment.
Figure 3. Morphology of U87MG cells treated with Fucoxanthin or LY-294002. Panels A, B: U87MG cells without compound treatment (controls), visualised at 24h and 48h, respectively; Panels C, D: U87MG cells treated with Fucoxanthin, visualised at 24h and 48h respectively; Panels E, F: U87MG cells treated with LY-294002, visualised at 24h and 48h, respectively. Treatments with LY-294002 and Fucoxanthin were performed at concentrations of 19.9 pM and 199.5 pM, respectively. FACS analysis. G is control, H is Fucoxanthin and I is LY-294002 48h after treatment. Apoptotic cells were detected by Annexin V- FITC/PI staining and death cells were detected by PI. Viable cells are shown in the lower left quarter (Q3- LL), early apoptotic cells are shown in the lower right quarter (Q4-LR), late apoptotic cells are shown in the upper right quarter (Q2-UR) and necrotic or mechanically damaged cells are shown in the upper left quarter (Q1 -UL).
Figure 4. PCA plots of the gene expression data. The first 3 Principal Components (PCs) plotted contain 73.5% of the variance. Each of the 3 PCs are indicated with their representative variances on the axes of the graphs, together with what they represent in the analysis. Note that the samples cluster tightly with respect to treatment and time conditions emphasising concordance within the analysis.
Figure 5. Euclidean distance based heatmap and clustering of the samples. The samples were clustered based on treatment first and then by time. LY-294002 24h (L24), LY-294002 48h (L48) treatments, Fucoxanthin 24h (F24) and Fucoxanthin 48h (F48) treatments, Control 24h (C24), Control 48h (C48). Up- regulated genes are shown in red; down-regulated genes are shown in green. Only significantly differentially expressed genes are shown. The colors used are the same as those used in the PCA analysis.
Figure 6. Venn Diagram showing the in-common and unique responses of the two treatments in gene expression space. (A) Gene up-expression at 24 h and 48 h for LY294002 and Fucoxanthin treated U87MG cells. (B) Gene down-expression at 24 h and 48 h for LY294002 and Fucoxanthin treated U87MG cells. Note that few up-regulated genes are shared by the two treatments, in contrast to much higher in down-regulated genes. Figure 7. Comparison of gene expression signature in response to LY-294002 and Fucoxanthin at 24h and 48h. (A) Top 25 up-regulated genes in LY-294002 treatment at 24h (L24, left side) and at 48h (L48, right side) relative to no-treatment controls, and comparison to the expression signature seen in fucoxanthin at the same timepoints. (B) Top 25 down-regulated genes in LY-294002 treatment at 24h (L24, left side) and at 48h (L48, right side) relative to no-treatment controls, and comparison to the expression signature seen in fucoxanthin at the same timepoints.
Figure 8. Comparison of gene expression signature in response to LY-294002 and Fucoxanthin at 24h and 48h. (A) Top 25 up-regulated genes in Fucoxanthin treatment at 24h (F24, left side) and at 48h (F48, right side) relative to no-treatment controls, and comparison to the expression signature seen in LY- 294002 at the same timepoints. (B) Top 25 down-regulated genes in fucoxanthin treatment at 24h (F24, left side) and at 48h (F48, right side) relative to no-treatment controls, and comparison to the expression signature seen in LY-294002 at the same timepoints.
Figure 9. Volcano plot of the 4 treatments showing the top 25 down-regulated genes (left side, in green) and the top 25 up-regulated genes (right side, in red) accompanied by their level of expression, expressed as logarithm-based 2 fold changes (Log2FC, < -1 or > 1) and corrected p-value as logarithm- based 10 false discovery rate (Logl OFDR P-value, <0.05). A. top 25 differentially expressed genes in U87MG responding to LY-294002 at 24h treatment; B. top 25 differentially expressed genes in U87MG responding to LY-294002 at 48h treatment; C. top 25 differentially expressed genes in U87MG responding to Fucoxanthin at 24h; D. top 25 differentially expressed genes in U87MG responding to Fucoxanthin at 48h.
Figure 10. PI3K/Akt signalling Pathway WikiPathway map representations. (A) Down-regulated (in green) and up-regulated genes (in red) in response to individual treatments; (B) L24, PI3K/Akt signalling pathway affected by LY-294002 at 24h; (C) L48, PI3K/Akt signalling Pathway affected by LY-294002 at 48h; (D) F24, PI3K/Akt signalling Pathway affected by Fucoxanthin at 24h; (E) F48, PI3K/Akt signalling Pathway affected by Fucoxanthin 48h.
Figure 11. Retinoblastoma gene in cancer pathway, WikiPathway map representations. (A) Down- regulated (in green) and up-regulated genes (in red) in response to individual treatments; (B) L24.
Retinoblastoma gene in cancer pathway affected by LY-294002 24h (C) L48. Retinoblastoma gene in cancer pathway affected by LY-294002 48h (D) F24. Retinoblastoma gene in cancer pathway affected by Fucoxanthin 24h (E) F48. Retinoblastoma gene in cancer pathway affected by Fucoxanthin 48h.
12. Network representation of the CMap analysis. Yellow lines are perturbagens with significantly correlating gene signatures and blue lines perturbagens with anti-correlating signatures. Blue squares are compounds from IOTA'S GBM Drug Bank. LY-294002 and Fucoxanthin have drug counterparts with similar gene signatures, including groups of antibiotics and anti-protozoal / antifungal agents as well as antipsychotics and antidepressants and compounds with described effects in GBM cell lines. They also share similar gene expression signatures with other PI3K inhibitors such as Quinostatin and Wortmannin, as indicated.
Figure 13. Summary of differential drug induced gene expression. U87MG cells treated for 24h (L24,
F24) and 48h (L48, F48) with LY-294002 and Fucoxanthin were analysed for differential gene expression. Shown are the top 25 up-regulated genes expressed at both time points and with both compounds (left and right) and the specific genes involved in the PI3K pathway (middle). Note the major differences between the genes induced by each compound and the sustained changes observed in the PI3K pathway genes for LY-294002 but not Fucoxanthin.
Figure 14. Differential drug induced gene expression in the PI3K pathway. Shown are members of the PI3K pathway which are among the top 25 upregulated genes in LY-294002 (left) and Fucoxanthin (right) treated cells, at 24h and 48h. Note that JAK2 is up-regulated 24 h and 48 h following LY-294002 treatment. JAK2 is also up-regulated 24 h but not 48 h after Fucoxanthin treatment.
Figure 15. Combination of LY-294002 with JAK2 inhibitors (A) ruxolitinib and (B) AZD1480. Cells were exposed to LY294002 in concentrations ranging from 10_6M to 10_3M, alone or in combination with JAK inhibitor at 10, 25 or 50 pM. Cell survival after 72h is given as a percentage of 1 .1 % DMSO control. Also shown is an analysis of the synergy of the two combinations, from which it can be seen that the combination of AZD1480 and LY-294002 is synergistic (indicated in blue).
Detailed Description of the Invention
Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures.
The claims relate to inhibitors and uses thereof. As used herein,“inhibitor” relates to a compound or substance which reduces or suppresses the activity or function of a target. A target may be one or more proteins or nucleic acids (such as mRNAs). An inhibitor may have multiple targets, which may share structural homology (e.g. in the case of related proteins) or a shared function (e.g. in the case of a pathway inhibitor). Inhibitors include a compound or substance which interacts with its target, for example a competitive or non-competitive/allosteric inhibitor. A competitive inhibitor competes with a substrate for the active site, whist an allosteric inhibitor binds to a site other than the active site and prevents substrate binding for example by stabilising a conformation which abolishes or disrupts the active form of the target. interactions are typically reversible, however an inhibitor may be an irreversible inhibitor, for example an inhibitor which covalently links to a target, blocking or disrupting the active form. Alternatively, an inhibitor may be a transcriptional inhibitor, which reduces or abolishes the expression of a target. As used herein, a“phosphatidylinositol 3-kinase (PI3K) pathway inhibitor”, an“inhibitor of phosphatidylinositol 3-kinase (PI3K) signalling” or an“inhibitor of the phosphatidylinositol 3-kinase (PI3K) signalling pathway” are used interchangeably and refer to a compound which inhibits, downregulates or abolishes signalling through the PI3K signalling pathway. It is therefore an inhibitor which targets one or more components in the PI3K pathway and, as a consequence, downregulates or abolishes PI3K signalling. The inhibitor’s action may comprise or consist of direct interaction with and inhibition of PI3K activity or function.
The PI3K pathway is an intracellular signal transduction pathway that functions to stimulate cell to proliferation and growth, and simultaneously inhibit cell apoptosis. The pathway involves many members, however key proteins involved include receptor tyrosine kinases (RTKs), phosphatidylinositol 3-kinases (PI3Ks), phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-bisphosphate (PIP3) and AKT/protein kinase B. RTKs are cell surface receptors for multiple growth factors, cytokines and hormones. Ligands binding to RTKs promote activation of PI3Ks. PI3K family kinases are capable of phosphorylating the 3’-hydroxyl group of the inositol ring of phosphatidylinositol. Phosphatidylinositol containing PIP2 and PIP3 are minor phospholipid components of cell membranes, and their activation through PI3K-mediated phosphorylation allows the recruitment of AKT to the plasma membrane, where it is in turn activated. AKT/protein kinase B is a serine/threonine-specific protein kinase that enhances the survival of cells by preventing apoptosis through blocking pro-apoptotic proteins and processes, such as through negatively regulating Bcl-2 family members and p53. AKT also promotes cell cycle advancement through its phosphorylation and inhibition of G1 state associated factors P21/Waf1/Cip1 and P27/Kip2. Additionally, AKT promotes cell growth through inhibition of TSC2 and indirect activation of the mTOR complex 1. A review of the PI3K pathway can be found in Hemmings, B. A., & Restuccia, D. F., Cold Spring Harbor Perspectives in Biology, 4(9), a011189.
Inhibitors of PI3K signalling may interact with and inhibit the activity or function, or downregulate the expression, of a PI3K and one or more target proteins involved in the PI3K pathway selected from RTK, PIP2, PIP3, and/or AKT. Alternatively, the inhibitor may interact with and inhibit the activity or function, or downregulate the expression, of one or more target proteins involved in the PI3K pathway selected from RTK, PIP2, PIP3, and/or AKT, and may show no interaction or effect on PI3K itself.
An inhibitor of PI3K signalling may be a PI3K inhibitor. A PI3K inhibitor may in particular directly interact with and inhibit the activity or function of, one or more PI3K isoform. PI3Ks are members of a family of kinases capable of phosphorylating the hydroxyl group of the inositol ring of phosphatidylinositol. All PI3Ks consist of two domains: a catalytic domain P1 10 and a regulatory domain P85. They are divided into classes I to III. A review of the PI3K family can be found in Jean & Kiger, (2014), Classes of phosphoinositide 3-kinases at a glance, Journal of Cell Science, 127( Pt 5), 923-928. In vitro, all classes nerate phosphatidylinositol 3-phosphate (Ptdlns(3)P), class I and II can synthesise
phosphatidylinositol (3,4)-bisphosphate (Ptdlns(3,4)P2), and only class I can produce phosphatidylinositol (3 ,4 , 5)-trisphosph ate (Ptd I ns(3 ,4 , 5) P3) . An PI3K inhibitor preferably interacts with and inhibits activity or function of one or more Class I PI3K isoform. Class I PI3K isoforms function as heterodimers consisting of one of four catalytic p110 subunits (PIK3CA/p110a, PIK3CB/p110b, PIK3CD/p110d, or PIK3CG/p110y) and a regulatory subunit selected from PIK3R1/p85a (or its splice variants p55a and p50a), PIK3R2/p85p, PIK3R3/p55y, PIK3R5/p101 or PIK3R6/p84. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CA/p110a. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CB/p110p. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CD/p110d. Some PI3K inhibitors interact with and inhibit activity or function of PIK3CG/p1 10y. Some PI3K inhibitors interact with and inhibit activity or function of one or more, two or more, three or more, or all PI3K isoforms selected from PIK3CA/p110a, PIK3CB/p110b, PIK3CG/p110y and/or PIK3CD/p110d. Preferably, the PI3K inhibitor interacts with and inhibits activity or function of PIK3CA/p110a, RIK3ΰB/r110b and PIK3CD/p1106.
Not all PI3K inhibitors target all PI3K isoforms. A PI3K inhibitor may not interact with and may have no effect on the activity or function of one or more Class I PI3K isoform, e.g. one, two, three, or four selected from PIK3CA/p110a, PIK3CB/p110b, PIK3CG/p110y and/or PIK3CD/p110d. The PI3K inhibitor optionally and additionally inhibits activity or function of Class III PI3K isoform PIK3C3/Vps34. In preferred embodiments, the PI3K inhibitor inhibits activity or function of PIK3CA/p110a, PIK3CB/p110b and PIK3CD/p110d, but does not substantially inhibit activity or function of PIK3CG/p110y.
An inhibitor of PI3K signalling may, in addition or as an alternative to inhibition of Class I PI3K isoform as described above, interact with and inhibit activity or function of one or more Class II PI3K isoform. The class II family has three members in humans - PIK3C2A/PI3KC2a, RIK3ΰ2B/RI3Kΰ2b and
PIK3C2G/PI3KC2y. There is no known obligatory regulatory subunit. In some embodiments, the inhibitor of PI3K signalling interacts with and inhibits one, preferably two, or all three, selected from
P I K3C2A/P 13KC2a , RIK3ΰ2B/RI3Kΰ2b and PIK3C2G/PI3KC2y. Additionally or alternatively, the inhibitor of PI3K signalling may interact with and inhibit activity or function of Class III PI3K isoform PIK3C3/Vps34. This is the only class III PI3K in humans, which exists as a dimer of catalytic PIK3C3/Vps34 and its regulatory subunit PIK3R4/Vps15.
Some PI3K inhibitors exhibit different inhibitory activity or function for different classes of PI3K isoforms, and may inhibit a group of PI3K isoforms selected from a one or more Class I PI3K isoform, one or more Class II PI3K isoform, one or more Class I PI3K isoform and one or more Class II PI3K isoform, one or more Class I PI3K isoform and the Class III PI3K isoform, one or more Class II PI3K isoform and the Class III PI3K isoform, or one or more PI3K isoform from Classes I, II and III.
Inhibitor of PI3K signalling may additionally or alternatively interact with and inhibit activity or function of a phosphatidylinositol 4-kinase alpha (PI4K) selected from phosphatidylinositol 4-kinase alpha (PI4KA), latidylinositol 4-kinase beta (PI4KB), phosphatidylinositol 4-kinase 2-alpha (PI4K2A), and/or phosphatidylinositol 4-kinase 2-beta (PI4K2B). The PI4K family catalyses phosphorylation of
phosphatidylinositol at the D-4 position, and are part of the PI3K signalling pathway. An inhibitor of PI3K signalling may be a non-selective pan-PI3K inhibitor. As used herein, a“non-selective pan-PI3K inhibitor” refers to a substance or compound which inhibits, either by inhibiting activity or function of or by reducing expression of, multiple targets within the PI3K signalling pathway. In this way, the inhibitor effectively downregulates signalling through the pathway. For example, a non-selective pan- PI3K inhibitor may target one, two, three, four, or more targets selected from a RTK, a PI3K, PIP2, PIP3, and/or AKT. Optionally, a non-selective pan-PI3K inhibitor may inhibit one or more additional targets outside of the PI3K signalling pathway, either by interacting with them and inhibiting their activity or function, or by reducing their expression. Preferably, the additional targets are selected from casein kinase 2 (CK2), Myosin Light-chain Kinase (MLCK, and/or mammalian target of rapamycin (mTOR). Even more preferably a non-selective pan-PI3K inhibitor may target, for example may interact with and inhibit the activity or function of at least a PI3K class I, a PI3K class II and a PI4K.
Some PI3K inhibitors do not interact with and inhibit the activity or function of PI3K class I, II, III, and/or PI4K. In particular, they may not interact with and inhibit the activity or function of PI3K class III.
Exemplary non-selective pan-PI3K inhibitors are wortmannin, BEZ235 (Dactolisib), PI-103, Buparlisib (BKM120, NVP-BKM120), GDC-0941 (Pictilisib), PI828, and 2-Morpholin-4-yl-8-phenylchromen-4-one (LY-294002) (Gharbi et al., 2007). LY-294002 is a relatively weak, pan-PI3K inhibitor showing inhibition in cell-free assays of the first 3 PI3K isoforms (Liu et al., 2017). It inhibits RI3Ka/d/b with IC50 of 0.5 pM/0.57 pM/0.97 pM, respectively. Similar to LY294002, BEZ235 and GDC-0941 also show pan-PI3K activity and are currently in clinical trials for glioblastoma. The structure of LY294002 is shown below:
Figure imgf000011_0001
In preferred embodiments, the inhibitor of PI3K signalling is BEZ235 (Dactolisib) or a salt, conjugate, prodrug, or derivative thereof. In some embodiments, the inhibitor of PI3K signalling is GDC-0941 (Pictilisib), or a salt, conjugate, prodrug, or derivative thereof. In some embodiments, the inhibitor of PI3K signalling is wortmannin, or a salt, conjugate, prodrug, or derivative thereof. In some embodiments, the inhibitor of PI3K signalling is LY-294002, or a salt, conjugate, prodrug, or derivative thereof.
In some embodiments, the inhibitor of PI3K signalling has similar chemical and biological properties to LY-294002. For example, the inhibitor of PI3K signalling may show pan-PI3K selectivity similar to
)02, and may have a comparable selectivity and/or affinity to LY-294002, may have the same or similar binding mode in x-ray crystallography, or may compete with LY-294002 for substrate binding sites. Preferably, such an inhibitor has a lower dose-limiting toxicity than LY-294002. Preferably, a PI3K inhibitor is a PI3K inhibitor other than fucoxanthin.
As used herein, an“inhibitor of Janus kinase (JAK) activity or function” is a compound that reduces or inhibits the activity of one or more member of the JAK family of non-receptor protein-tyrosine kinases.
The family consists of JAK1 , JAK2, JAK3, and TYK2 (tyrosine kinase-2), all of which are involved in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The JAK/STAT pathway is a key signalling mechanism for a wide array of cytokines and growth factors, and JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis. The pathway is discussed in detail in Aaronson and Horvath, 2002; Heinrich et al., 2003; Kisseleva et al., 2002; O'Shea et al., 2002; Rawlings et al 2004 Journal of Cell Science 1 17:1281 -1283; and Roskoski, Pharmacol Res. 2016 Sep;1 1 1 :784-803.
An inhibitor of JAK activity or function may inhibit activity or function of one, two, three or all four of JAK1 , JAK2, JAK3 and/or TYK3. For example, the inhibitor of JAK activity or function inhibits JAK1 , JAK2, and/or TYK3 activity or function. Preferably, the inhibitor of JAK activity or function may be an inhibitor of JAK2 activity or function. More preferably, the inhibitor of JAK activity or function preferentially inhibits JAK2 activity or function relative to JAK1 activity or function. Still more preferably, the inhibitor of JAK activity or function may inhibit JAK2 activity or function but may not inhibit JAK1 activity or function.
Inhibitors of JAK activity or function may prevent or reduce activating phosphorylation of Janus kinases, preferably of JAK2. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling will be diminished.
Inhibitors of JAK activity or function include a protein kinase inhibitor. Protein kinases are ATP-dependent enzymes, which phosphorylate suites of target sites.
Preferably, the inhibitor of JAK activity or function may be a Type I protein kinase inhibitor which inhibits the activity or function of one or more of JAK1 , JAK2, JAK3 and/or TYK2, more preferably JAK2.
An inhibitor of JAK activity or function may be an ATP-competitive inhibitor of JAK1 , JAK2, JAK3 and/or TYK2, and preferably the inhibitor of JAK activity or function may be an ATP-competitive inhibitor of JAK2.
In some embodiments, the inhibitor of JAK activity or function may be Ruxolitinib, or a salt, conjugate, prodrug, or derivative thereof. Ruxolitinib is an ATP-competitive inhibitor of JAK1 and JAK2 (ICso-s of 3.3 ± 1 .2 nM and 2.8 ± 1 .2 nM, respectively) and inhibition occurs regardless of the JAK2V617F mutational status. Ruxolitinib is a moderately potent inhibitor of the related JAK, TYK2 (ICso = 19 ± 3.2 nM) but is /e versus JAK3 (ICso = 428 ± 243 nM). It is also selective versus a panel of 26 other kinases at concentrations approximately 100-fold the ICso of JAK1 and JAK2. Inhibition of JAK1 and JAK2 downregulates the JAK-signal transducer and activator of transcription (STAT) pathway, inhibiting proliferation, inducing apoptosis, and reducing numerous cytokine plasma levels. In some embodiments, the inhibitor of JAK activity or function has similar chemical and biological properties to ruxolitinib. For example, the inhibitor of JAK activity or function may have JAK2-selective inhibitory properties similar to ruxolitnib, a comparable selectivity and/or affinities for JAK2 as ruxolitinib, the same or similar binding mode in x-ray crystallography and/or may compete with ruxolitinib for binding sites on JAK2.
In preferred embodiments, the inhibitor of JAK activity or function may be 5-Chloro-N2-[(1 S)-1 -(5-fluoro-2- pyrimidinyl)ethyl]-N4-(5-methyl-1 H-pyrazol-3-yl)-2,4-pyrimidine-2, 4-diamine (AZD1480), or a salt, conjugate, prodrug, or derivative thereof. The structure of AZD1480 is shown below:
Figure imgf000013_0001
In some embodiments, the inhibitor of JAK activity or function has similar chemical and biological properties to AZD1480. For example, the inhibitor of JAK activity or function may have a comparable selectivity and/or affinities for JAK2 as AZD1480, may have the same or similar binding mode in x-ray crystallography and/or may compete with AZD1480 for binding sites on JAK2. Preferably, such an inhibitor has an improved dose-limiting toxicity relative to AZD1480.
Other JAK inhibitors without either JAK1 or JAK3 inhibitory activities may be preferable, since chronic dosing required for many treatments carries the potential risk of immunosuppressive side effects related to inhibition of JAK1 , JAK3, or TYK2, suggesting that identification of a JAK2 selective inhibitor may offer increased safety.
In some embodiments, the inhibitor of JAK activity or function is not Ruxolitinib.
The combination of an inhibitor of PI3K signalling and an inhibitor of JAK activity or function finds use in the treatment or prevention of cancer.
Cancer cells exposed to PI3K inhibitors need to escape from the inhibitory effect and activate alternative growth through JAK. Challenge with PI3K signalling inhibitors results in up-regulation of JAK2, reflecting a pro-proliferative role within the glioblastoma cells as they react to the growth inhibition caused by down- ion of the PI3K pathway. By further challenging cells with an inhibitor of JAK2 activity or function, it is possible to potently and effective block cell proliferation. As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. Treatment includes preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease and/or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.
As used herein,“prevention of cancer” refers to preventing the progression of a tumour. Tumours may be classified by grade (“The 2016 World Health Organisation Classification of Tumours of the Central Nervous System: a summary". Acta Neuropathologica. 131 (6): 803-820). A cancer may be prevented from progression from low-grade (WHO grade I or II) to high-grade (WHO grade III or IV).“Prevention of cancer” may include preventing a benign or pre-cancerous tumour from progressing to a malignant or cancerous state. “Prevention of cancer” may include preventing a cancer or tumour from arising from healthy tissue.
“Prevention of cancer” may also include preventing a cancer from spreading, for example from metastasising. As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part, such as from the lung to the brain. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumour and has one or more secondary tumours. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumour but not one or more secondary tumours. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumour and with one or more secondary tumours at a second location or multiple locations, e.g., in the brain. ition of cancer” may also include preventing the recurrence of a cancer which is in partial or complete remission. As used herein, the term“cancer in remission” refers to a decrease in or disappearance of signs and symptoms of cancer.“Partial remission” refers to a cancer which has a decrease in some, but not all signs and symptoms have decreased or disappeared. For example, a cancer in partial remission may exhibit decreases in tumour sise, cancer cell count, metastasis rate, etc.
In“complete remission”, all signs and symptoms of cancer have disappeared, although cancer still may be in the body. As described herein, the active compounds may be administered to a subject or patient simultaneously or sequentially, by any suitable route or administration.
"Patient",“subject” or "subject in need thereof refers to a living organism suffering from or prone to a disease or condition that can be treated by using the methods provided herein. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. In preferred embodiments, a subject or patient is human.
As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
“Simultaneous” administration refers to administration of the agents together, for example as a pharmaceutical composition containing the agents (i.e. a combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. In particular embodiments, the inhibitor of PI3K signalling and the inhibitor of JAK activity or function may be administered simultaneously in a combined preparation. In certain embodiments upon simultaneous administration the two or more of the agents may be administered via different routes of administration. Simultaneous administration may refer to administration at the same time, or within e.g. 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs or 48 hrs. ntial” administration refers to administration of one or more of the agents followed after a given time interval by separate administration of another of the agents. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval, including hours, days, weeks, months, or years. Sequential administration may refer to administrations separated by a time interval of one of at least 10 min, 30 min, 1 hr, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months or 6 months.
The inhibitor of PI3K signalling and/or the inhibitor of Janus kinase (JAK) activity or function may be administered simultaneously or sequentially with one or more further compounds. Preferably, the further compound is an inhibitor of Insulin Like Growth Factor 1 Receptor (IGF1 R) activity or function and/or an inhibitor of Serine/threonine-protein kinase 1 (SGK1) activity or function.
In particular, the further compounds may have been shown to be active in preclinical models of GBM. Suitable compounds may include; 2-Amino-3H-phenoxazin-3-one, 2-Hydroxyoleic acid, 3-(3,4- dichlorophenyl)-1-(3,4-dimethylphenyl)-1-(5-methyl-4,5-dihydro-1 ,3-thiazol-2-yl)urea, 3-Deazaneplanocin, 4egi-1 , 5-Nonyloxytryptamine, 6-Hydroxyquinoline-4-carboxylic acid, 7-Ethyl-10-hydroxycamptothecin, 7- Hydroxystaurosporine, 8-Bromo-cyclic AMP, 8-Hydroxy-2-methyl-1 H-quinazolin-4-one, 9-ING-41 , A- 966492, Abemaciclib, Abt-737, AC1 MMYR2, Acalabrutinib, Acetazolamide, Adavosertib, AEE788, Afatinib, AG-120, Aldoxorubicin, Alexidine, Alisertib, Altiratinib, Alvespimycin, AMG 232, AMG-925, Amlexanox, Anisomycin, Antroquinonol, Apatinib, Apigenin, Arctigenin, Ardipusilloside I, Ascochlorin, Astemizole, AT13387, Atorvastatin, Aurintricarboxylic acid, Axitinib, AXL1717, AZD2014, AZD-7451 , AZD8055, BAY 11-7082, BDBM86691 , Bendamustine, BI2536, Bimiralisib, Binimetinib, Birinapant, BIX- 01294, BMS-536924, BMS-777607, Bortezomib, Bosutinib, Bufalin , Buparlisib, Cabazitaxel, Caffeic acid phenethyl ester, Caffeine, Camptothecin, Capecitabine, Capmatinib, Captopril, Carboplatin, Cardamonin, Carmustine, carnosol, Carvacrol, Casticin, Cathepsin S Inhibitor, Caudatin, CBL-0137, CC-115, CC-223, Cediranib, Celastrol, Celecoxib, Ceritinib, Cerivastatin, Chaetocin, Chloroquine, Chlorpromazine, Cilengitide, Cisplatin, Cladribine, Clioquinol, Clofazimine, Clorgiline, Cordycepin, Crenolanib, Crizotinib, Cryptotanshinone, Cucurbitacin I, CUDC-101 , Curcumin, Cycloheximide, Cyclophosphamide, D609, Dabrafenib, Dacomitinib, Dactinomycin, Dactolisib, Dasatinib, DB-074971 , Demethoxycurcumin, Deoxyglucose, Dexanabinol, Dibutyryl cAMP, Diclofenac, Digitoxigenin, Digitoxin, Digoxigenin, Digoxin, Dihydroartemisinin, Dimethylaminomicheliolide, Dinaciclib, Disufenton sodium, Disufenton sodium, Docetaxel, Dorsomorphin, Dovitinib, Doxazosin, Doxorubicin, Eflorn ithine, Embelin, Enasidenib, Entrectinib, Enzastaurin, Epigallocathecin, Epigallocathecin Gallate, Epirubicin, Erdafitinib, Erlotinib, Etoposide, Everolimus, Evodiamine, Evofosfamide, Farnesylthiosalicylic acid, Fasudil, Fenofibrate, Fingolimod, Fingolimod, Flavopiridol, Flubendazole, Fluorouracil, Fluvoxamine, Foretinib, Forskolin, Fotemustine, Fucoxanthin, Galunisertib, Gamma-Secretase Inhibitor IX, GANT61 , Gartanin, GDC-0084, GDC-0941 , Gedatolisib, Gefitinib, Genistein, Germacrone, GK921 , Glasdegib, Glaucocalyxin A, Golvatinib, Gossypol, GSK J4, GSK1838705A, GSK1904529A, GSK3326595, GSK461364,
Guggulsterone, Hispolon, Honokiol, Hsp-990, Hydroxyurea, Hypericin, Ibrutinib, Ibuprofen, Idarubicin, Imatinib, Imiquimod, Indatraline, Indirubin Derivative E804, Indomethacin, Infigratinib, Iniparib, Irinotecan, Imethylxanthine, Isoliquiritigenin, Isotretinoin, Itraconazole, Ixazomib, JQ1 , JS-K, Juglone , Karenitecin, KPT251 , KPT276, KU55933, KU-55933, KU60019, Lactacystin, Lanatoside C, Lapatinib, Larotrectinib, LB-100 , Lenalidomide, Lenalidomide, Lenvatinib, LEQ506, Letrozole, LGK974, Lomustine, Lonafarnib, Lorlatinib, Lovastatin, LOXO-195, Luteolin, LY294002, LY341495, Marizomib, MAZ51 , Mebendazole, Melatonin, Mepacrine, Metformin, Methotrexate, Methyl gallate, MG-132, Mibefradil, Midazolam, Mitoxantrone, MK-2206, ML00253764 , MLN4924, Monensin, MRK003, Naringin, Navitoclax, NBQX, Nelfinavir, Neratinib, Niclosamide, Nilotinib, Nintedanib, Nisoldipine, NKTR-102, NMS-P715, Nobiletin, NSC141562, NSC23766, NVP-AEW541 , NVX-207, Olanzapine, Olaparib, Oleuropein, Oligomycin, Omacetaxine, ON123300, Oroxindin, Oroxylin A, Otx-008, OTX-015, Ouabain, Oxaliplatin, Oxamate, Paclitaxel, Palbociclib, Palomid 529, Pamiparib, Panobinostat, Pazopanib, PCI-24781 , PD0325901 , PD173074, Pegdinetanib, Perifosine, Perilla alcohol, Pexidartinib, PF-04691502, PF- 06840003, PF-429242, PF8380, Phenformin, PI-103, Pimozide, Piplartine, Pitavastatin, PJ34, Plumbagin, PLX-4720, Pomolic acid, Ponatinib, PP242, PQ401 , Pregnenolone, Procarbazine, Propofol, Proscillaridin A, Punicalagin, PX-866, Pyrvinium, Quercetin, R428, Ralimetinib, RapaLink-1 , Regorafenib, Repaglinide, Resveratrol, Reversan, RG2833, RG7112, Ribociclib, Riluzole, Ritonavir, RO4929097, Rolipram, Romidepsin, Roscovitine, Rottlerin, Rucaparib, Ruxolitinib, Salinomycin, Salvianolic acid B, Sanguinarine, Satraplatin, Savolitinib, Schizandrin B, Scriptaid, Selinexor, Selumetinib, Semustine, Sepantronium bromide, Sertraline, Sevoflurane, SF1126, SGX-523, SH-4-54, SH5-07, Shikonin, Silibinin, Silmitasertib, Sinomenine, Sirolimus, SKI II, Sonidegib, Sorafenib, SSR128129E, STX-0119, Sulfasalazine, Sunitinib, T56-LIMKi, Talampanel, Tamoxifen, Tandutinib, Tanespimycin, Telomestatin, Temozolomide,
Temsirolimus, Terfenadine, Tesevatinib, Tetraethylammonium, Tetrandrine, TG02, TG101209, TGX-221 , Thalidomide, Thapsigargin, Theobromine, Thymoquinone, THZ1 , Tipifarnib, Tivozanib, Tnp-470, Tocladesine, Topotecan, Tozasertib, Tpi-287, Trametinib, Transcrocetinate sodium, Tricetin, Trichostatin, Trifluoperazine, Triptolide, UMI-77, UNC2025, UNII-2AW48LAZ4I, USL311 , VAL-083, Valproic Acid, Vandetanib, Vatalanib, Veliparib, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,
Vinpocetine, Vismodegib, Visudyne, Vorinostat, Wortmannin, WP1066, WP1130, XL-184, XL765, Xyloketal B, YU238259, ZSTK474.
The inhibitor of PI3K signalling and/or the inhibitor of JAK activity or function may be delivered across the blood-brain barrier into the brain, for example through intracranial administration. The term“blood-brain barrier” refers to a highly selective semipermeable membrane barrier that separates the circulating blood from the brain and extracellular fluid in the central nervous system. The barrier provides tight regulation of the movement of ions, molecules and cells between the blood and the brain, see e.g. Daneman and Prat, Cold Spring Harb Perspect Biol. 2015;7(1):a020412. Many therapeutic molecules are generally excluded from transport from blood to brain due to their negligible permeability over the brain capillary endothelial wall. Inhibitors of PI3K signalling and/or of JAK activity or function may be capable of crossing the blood- brain barrier into the brain, for example by virtue of a targeting domain and/or encapsulation in a liposomal or similar carrier, and are delivered for example by intravenous injection or through the gastrointestinal or oral route.
Utilising the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be j that does not cause substantial toxicity and yet is effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent.
An "effective amount" is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, reduce one or more symptoms of a disease or condition, reduce viral replication in a cell). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a "therapeutically effective amount". A "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An "activity decreasing amount," as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme or protein relative to the absence of the antagonist. A "function disrupting amount," as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist.
Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and
Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,
Williams & Wilkins).
The compounds described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoural, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration which may include injection or infusion. Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the
or animal body.
In some aspects, the invention provides a pharmaceutical composition comprising an inhibitor of PI3K signalling and an inhibitor of JAK activity or function. The inhibitor of PI3K signalling and an inhibitor of JAK activity or function may be as described herein. The pharmaceutical composition may comprise one or more pharmaceutically acceptable binders, diluents, or excipients. The pharmaceutical compositions may be formulated for delivery across the blood-brain barrier, for example via intracranial injection. The pharmaceutical composition may be for the treatment or prevention of a cancer, preferably glioblastoma multiforme.
In some aspects, the invention provides a kit comprising (i) an inhibitor of PI3K signalling and (ii) a inhibitor of JAK activity or function. The inhibitor of PI3K signalling and an inhibitor of JAK activity or function may be as described herein. The inhibitor of PI3K signalling and/or inhibitor of JAK activity or function may be formulated as pharmaceutical compositions which may comprise one or more pharmaceutically acceptable binders, diluents, or excipients. The pharmaceutical compositions may be formulated for delivery across the blood-brain barrier, for example via intracranial injection. A kit may contain multiple doses of the inhibitors, each of which may be packaged together or separately. Inhibitors may be supplied as aqueous formulations, liquids, powders, pastes, gels, vials, tablets, pills, capsules, or any other formulation described herein. The kit may be for the treatment or prevention of a cancer, preferably glioblastoma multiforme. The kit may comprise instructions for sequential or simultaneous administration of the inhibitors in order to treat or prevent cancer according to the methods described herein. The kit may further comprise means for administration, such as syringes, salt, buffers, or pharmaceutically acceptable carriers or excipients. The kit may comprise additional compounds as listed above.
The tenth and eleventh aspects of the invention relate to identifying druggable targets, and providing therapeutic combinations against said targets, for cancer combination therapy. As used herein, a “combination therapy”, or a“therapeutic combination” relates to two or more compounds useful in treating or preventing a disease when delivered simultaneously or sequentially to a subject. The combination therefore comprises at least a first and a second compound, which may be for sequential or simultaneous administration.“Compounds”, in this context, comprise at least one therapeutic agent, for example a cytotoxic or anti-cancer agent.
The methods comprise contacting test cells with a first compound.“Test cells” refers to any suitable cells in vivo, ex vivo or in vitro. Test cells may be pathological cells, i.e. cells having or displaying symptoms of a dysfunction or disease. In particular, test cells may be cancer cells, such as glioblastoma cells, although cells of any of the cancers described herein will also be suitable. Suitable cancer cell lines or glioblastoma cell lines may include primary human cancer cells, such as low-passage patient-derived glioma and glioblastoma cell lines, or established cell lines such as the well characterised cell lines A172, LN18, LN229, LNZ308, T98G, U118, U138, U251 , U343, U373 and U87, or selected glioma and glioblastoma cell lines from more extensive cell culture collections such as the well-known ATCC and ECACC cell ons, as well as cell collections dedicated to GBM such as the HGCC. A particularly preferred GBM cell line is U87MG. Alternatively, test cells may be cancer cells, preferably glioblastoma cells, which are obtained from a patient for whom the cancer combination therapy is intended. In these embodiments, the methods provide a personalised cancer combination therapy which is tailored to the patient’s cancer.
Test cells may be contacted with a first compound in any concentration. The concentration may be equal to or below the anti-proliferative EC50 of the first compound for the test cells. Half maximal effective concentration (EC50) refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. In this context, the antiproliferative EC50 is the concentration of the first compound which induces an anti-proliferative response halfway between the baseline and maximum after a specified exposure time. EC50 is usually described in relation to a time point, e.g. 24h-EC50 is the EC50 calculated after a 24h exposure time. The EC50 may be calculated based on an exposure time which is longer than the length of the contacting step. For example, if the effects of the first compound are to be measured at 24h and/or 48h, the 36h or 72h EC50 is suitable. This allows the transcriptomic effects of the drug to be measured without resulting in excessive cell death, preventing measurement. In some embodiments of the tenth and eleventh aspects, test cells are contacted with the first compound at the 72h-EC50 concentration. In some particular embodiments, test cells are contacted with the first compound at the 72h-EC50 concentration, and measurement occurs no later than 48hrs after exposure to the first compound. In some specific embodiments, test cells are contacted with the first compound at the 72h-EC50 concentration, and measurement occurs no sooner than 24h and no later than 48hrs after exposure to the first compound.
Following contact, the effects of the first compound on the transcriptome of the test cells are measured. This measurement may occur after an incubation period, for example a 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 48, 60, or 72 hour incubation period. Measurement may entail obtaining a sample of mRNA from the test cells and quantifying the levels of transcripts, for example through qPCR or microarray analysis. Measurement may involve comparing the quantified levels of transcripts to a reference. Suitable references include untreated cells, i.e. cells of the same cell line which are not exposed to the first compound. The method may involve measuring and quantifying the levels of transcripts in cells identical to the test cells in all aspects except their exposure to the first compound, so as to create a reference. Alternatively, the method may employ a known or pre-generated standard reference. Transcript levels may be“normalised” to the expression levels of a“housekeeping gene”. Housekeeping genes are those that are always expressed because they are constantly required by the cell, hence, they are always present under any conditions. Examples include as ubiquitin, actin, GAPDH, or other housekeeping genes which will be known to the skilled person.
In some embodiments of the tenth and eleventh aspects, the transcriptomic profile is constructed for all transcripts, for example by using a universal probe or primer mix, and is a“whole” transcriptome profile.
9 embodiments of the tenth and eleventh aspects, the transcriptomic profile is constructed only for a subset of transcripts. For example, a transcriptomic profile may include one or more genes which are implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation. A transcriptomic profile may be limited by pathway, for example to genes in the PI3K or retinoblastoma pathways. A transcriptomic profile may be limited to genes for which inhibitory and/or approved drugs are known. A transcriptomic profile may additionally include one or more housekeeping genes.
The effects on the transcriptome are used to construct a transcriptomic profile. A“transcriptomic profile” reports the changes in the transcriptome induced by a treatment.
In some embodiments of the tenth and eleventh aspects, the method measures the effects of the compound on the transcriptome of the test cells at a first and a second time-point. In these methods, the transcriptomic profile that is constructed includes time as a variable alongside expression, capturing not just whether but when expression is affected. The first time point may be before the second time point, and may be separated by an incubation in the presence of the test compound. Such time points may represent an“early” and a“late” drug response. The time points may be separated by a period of 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 48, 60, or 72 hours or more. For example, the first time point may be 24 hr after contacting the test cells with the compound and the second time point may be 48 hr after contacting the test cells with the compound, for a separation of 24 hr. “Early” drug responses may include shock responses and immediate anti-toxin effects, whilst“late” drug responses will tend to include “escape” response, such as regulation of alternative pathways to compensate for the action of the first compound. A gene whose expression is perturbed in both the“early” and“late” time-points is a candidate for selection as a druggable target, as this may reveal a sustained reliance on the gene when challenged by the first compound, and its disruption may enhance the effect of the first compound. Alternatively, a gene whose expression is perturbed only in the early time-point may be a druggable target, as it may represent an immediate or“front-line” response to the first compound, and its disruption may enhance the effect of the first compound. Alternatively, a gene whose expression is perturbed only in the late time- point may be a druggable target, as it may represent a downstream response to the first compound, the disruption of which may restore sensitivity to and enhance the effect of the first compound.
Based on the transcriptomic profile, druggable target genes are identified amongst those whose expression is perturbed when the cells are exposed to the first compound. As used herein, a gene is “perturbed” if its expression changes. Perturbation may be an increase or a decrease in expression. For example, a perturbed gene may be expressed at 150%, 160%, 170%, 180%, 190% or 200% or more of its wild-type or reference expression. A perturbed gene may be expressed at 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, or 20 times or more of the level of its wild- type or reference expression. A perturbed gene may be expressed at 60%, 50%, 40%, 30%, 20%, 15%,
10%, 5%, 4%, 3%, 2%, or 1 % or lower of its wild-type or reference expression. A perturbed gene may be expressed at 1/2 times, 1/3 times, 1/4 times, 1/5 times, 1/6 times, 1/7 times, 1/8 times, 1/9 times, 1/10 times, 1/15 times, or 1/20 times or less of the level of its wild-type or reference expression.
A druggable target gene is, by virtue of its implication in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, expected to enhance cell sensitivity to the first compound if administered in combination with the first compound. When a cell is challenged with a compound, it attempts to“escape” the effects by (i) upregulating expression of genes which positively upregulate compensatory pathways, and (ii) downregulating expression of genes which negatively regulate compensatory pathways. Compensatory pathways are those which substitute for the function conveyed by the gene targeted by the first compound. For example, if the first compound inhibits a pro-proliferative pathway, the cell will respond by upregulating genes which promote alternative proliferative genes and pathways, whilst downregulating anti-proliferative regulators. Similarly, if the first compound promotes a pro-apoptotic pathway, the cell will respond by upregulating anti-apoptotic genes and pathways, and downregulating pro-apoptotic genes and pathways.
A gene implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation may be a gene which is a known regulator of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
The step of selecting a gene whose expression is perturbed as a druggable target may comprise comparing the expression in the test cells to expression in normal cells - either to control cells or to a set of known standards - in order to determine relative changes in expression. In some embodiments, the gene regulates angiogenesis, mTOR signalling, or the NFKB signalling. Druggable targets may be genes implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, which are up-regulated (i.e. for which relative expression increases) in the cell. In particular, a positive regulator whose expression is upregulated following treatment may be a candidate druggable target. In some embodiments, a druggable target that is upregulated is expressed at 150%, 160%, 170%, 180%, 190% or 200% or more of its wild-type or reference expression. In some embodiments, a druggable target that is upregulated is expressed at 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, or 20 times or more of the level of its wild-type or reference expression.
Alternatively, druggable targets may be genes implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, which are down-regulated (i.e. for which relative expression decreases) in the cell. In particular, a negative regulator whose expression is downregulated following treatment may be a candidate druggable target. In some embodiments, a druggable target that is downregulated is expressed at 60%, 50%, 40%, 30%, 20%,
10%, 5%, 1 % or less of its wild-type or reference expression.
In some embodiments of the tenth and eleventh aspects, a druggable target’s expression is perturbed in both a first and second time point. Preferably the perturbation is in the same direction in both time points, i.e. is upregulated at both time points, or is downregulated in both time points. ip of selecting a gene as a druggable target may comprise selecting a gene (i) that is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation, (ii) that has perturbed expression in following exposure to the first compound and (iii) to which an inhibitor compound is known. Preferably, the inhibitor compound has achieved regulatory approval for use as a medicine in the treatment of the cancer.
As used herein, a gene which is a“positive regulator” is one which functions to promote, accelerate, increase or enhance a process. For example, a positive regulator of angiogenesis works to increase angiogenesis. Similarly, a gene which is a“negative regulator” is one which functions to inhibit, supress or prevent a process. Therefore, a negative regulator of angiogenesis would work to reduce angiogenesis.
In some embodiments of the tenth and eleventh aspects, the compounds may alter cell viability, for example the first or second compound may comprise compounds that are cytotoxic to mammalian cells. Suitable compounds may include for example active anti-cancer agents. In other embodiments, the compounds may alter phenotypic properties displayed by mammalian cells, causing cell cycle perturbation, apoptosis enhancement, or changes in antigen display.
The compounds may alter a phenotypic property of mammalian cells. For example, the compounds may reduce or inhibit the proliferation, viability, migration, invasion and/or angiogenesis of mammalian cells, such as cancer cells; increase or promote apoptosis and/or radio-sensitisation; and/or may alter a cell surface phenotype, such as EGFRvlll or other neoantigen, or a molecular characteristic associated with differentiation and apoptotic processes, antigen display and cell renewal. In some embodiments, the compounds may regulate, for example reduce or inhibit, angiogenesis, mTOR signalling, or the NFKB signalling.
Exemplary compounds for use as a first or second compound herein are chemotherapeutic agents, for example alkylating agents such as platinum complexes including cisplatin, mono(platinum),
bis(platinum), tri-nuclear platinum complexes and carboplatin, thiotepa and cyclosphosphamide
(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- racil (5-FU) gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, pitavastatin, fludarabine phosphate, and cladribine; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; binblastine; vindesine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA);
Topoisomerase inhibitors such as doxorubicin HCI, daunorubicin citrate, mitoxantrone HCI, actinomycin D, etoposide, topotecan HCI, teniposide (VM-26), and irinotecan and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In particular, a first or second compound may include one or more compounds shown to be active in preclinical models of GBM. Suitable compounds may include; 2-Amino-3H-phenoxazin-3-one, 2- Hydroxyoleic acid, 3-(3,4-dichlorophenyl)-1-(3,4-dimethylphenyl)-1 -(5-methyl-4,5-dihydro-1 ,3-thiazol-2- yl)urea, 3-Deazaneplanocin, 4egi-1 , 5-Nonyloxytryptamine, 6-Hydroxyquinoline-4-carboxylic acid, 7-Ethyl- 10-hydroxycamptothecin, 7-Hydroxystaurosporine, 8-Bromo-cyclic AMP, 8-Hydroxy-2-methyl-1 H- quinazolin-4-one, 9-ING-41 , A-966492, Abemaciclib, Abt-737, AC1 MMYR2, Acalabrutinib,
Acetazolamide, Adavosertib, AEE788, Afatinib, AG-120, Aldoxorubicin, Alexidine, Alisertib, Altiratinib, Alvespimycin, AMG 232, AMG-925, Amlexanox, Anisomycin, Antroquinonol, Apatinib, Apigenin,
Arctigenin, Ardipusilloside I, Ascochlorin, Astemizole, AT13387, Atorvastatin, Aurintricarboxylic acid, Axitinib, AXL1717, AZD2014, AZD-7451 , AZD8055, BAY 1 1-7082, BDBM86691 , Bendamustine, BI2536, Bimiralisib, Binimetinib, Birinapant, BIX-01294, BMS-536924, BMS-777607, Bortezomib, Bosutinib,
Bufalin , Buparlisib, Cabazitaxel, Caffeic acid phenethyl ester, Caffeine, Camptothecin, Capecitabine, Capmatinib, Captopril, Carboplatin, Cardamonin, Carmustine, carnosol, Carvacrol, Casticin, Cathepsin S Inhibitor, Caudatin, CBL-0137, CC-115, CC-223, Cediranib, Celastrol, Celecoxib, Ceritinib, Cerivastatin, Chaetocin, Chloroquine, Chlorpromazine, Cilengitide, Cisplatin, Cladribine, Clioquinol, Clofazimine, Clorgiline, Cordycepin, Crenolanib, Crizotinib, Cryptotanshinone, Cucurbitacin I, CUDC-101 , Curcumin, Cycloheximide, Cyclophosphamide, D609, Dabrafenib, Dacomitinib, Dactinomycin, Dactolisib, Dasatinib, DB-074971 , Demethoxycurcumin, Deoxyglucose, Dexanabinol, Dibutyryl cAMP, Diclofenac,
igenin, Digitoxin, Digoxigenin, Digoxin, Dihydroartemisinin, Dimethylaminomicheliolide, Dinaciclib, Disufenton sodium, Disufenton sodium, Docetaxel, Dorsomorphin, Dovitinib, Doxazosin, Doxorubicin, Eflornithine, Embelin, Enasidenib, Entrectinib, Enzastaurin, Epigallocathecin, Epigallocathecin Gallate, Epirubicin, Erdafitinib, Erlotinib, Etoposide, Everolimus, Evodiamine, Evofosfamide, Farnesylthiosalicylic acid, Fasudil, Fenofibrate, Fingolimod, Fingolimod, Flavopiridol, Flubendazole, Fluorouracil, Fluvoxamine, Foretinib, Forskolin, Fotemustine, Fucoxanthin, Galunisertib, Gamma-Secretase Inhibitor IX, GANT61 , Gartanin, GDC-0084, GDC-0941 , Gedatolisib, Gefitinib, Genistein, Germacrone, GK921 , Glasdegib, Glaucocalyxin A, Golvatinib, Gossypol, GSK J4, GSK1838705A, GSK1904529A, GSK3326595,
GSK461364, Guggulsterone, Hispolon, Honokiol, Hsp-990, Hydroxyurea, Hypericin, Ibrutinib, Ibuprofen, Idarubicin, Imatinib, Imiquimod, Indatraline, Indirubin Derivative E804, Indomethacin, Infigratinib, Iniparib, Irinotecan, Isobutylmethylxanthine, Isoliquiritigenin, Isotretinoin, Itraconazole, Ixazomib, JQ1 , JS-K, Juglone , Karenitecin, KPT251 , KPT276, KU55933, KU-55933, KU60019, Lactacystin, Lanatoside C, Lapatinib, Larotrectinib, LB-100 , Lenalidomide, Lenalidomide, Lenvatinib, LEQ506, Letrozole, LGK974, Lomustine, Lonafarnib, Lorlatinib, Lovastatin, LOXO-195, Luteolin, LY294002, LY341495, Marizomib, MAZ51 , Mebendazole, Melatonin, Mepacrine, Metformin, Methotrexate, Methyl gallate, MG-132, Mibefradil, Midazolam, Mitoxantrone, MK-2206, ML00253764 , MLN4924, Monensin, MRK003, Naringin, Navitoclax, NBQX, Nelfinavir, Neratinib, Niclosamide, Nilotinib, Nintedanib, Nisoldipine, NKTR-102, NMS- P715, Nobiletin, NSC141562, NSC23766, NVP-AEW541 , NVX-207, Olanzapine, Olaparib, Oleuropein, Oligomycin, Omacetaxine, ON123300, Oroxindin, Oroxylin A, Otx-008, OTX-015, Ouabain, Oxaliplatin, Oxamate, Paclitaxel, Palbociclib, Palomid 529, Pamiparib, Panobinostat, Pazopanib, PCI-24781 , PD0325901 , PD173074, Pegdinetanib, Perifosine, Perilla alcohol, Pexidartinib, PF-04691502, PF- 06840003, PF-429242, PF8380, Phenformin, PI-103, Pimozide, Piplartine, Pitavastatin, PJ34, Plumbagin, PLX-4720, Pomolic acid, Ponatinib, PP242, PQ401 , Pregnenolone, Procarbazine, Propofol, Proscillaridin A, Punicalagin, PX-866, Pyrvinium, Quercetin, R428, Ralimetinib, RapaLink-1 , Regorafenib, Repaglinide, Resveratrol, Reversan, RG2833, RG7112, Ribociclib, Riluzole, Ritonavir, RO4929097, Rolipram, Romidepsin, Roscovitine, Rottlerin, Rucaparib, Ruxolitinib, Salinomycin, Salvianolic acid B, Sanguinarine, Satraplatin, Savolitinib, Schizandrin B, Scriptaid, Selinexor, Selumetinib, Semustine, Sepantronium bromide, Sertraline, Sevoflurane, SF1126, SGX-523, SH-4-54, SH5-07, Shikonin, Silibinin, Silmitasertib, Sinomenine, Sirolimus, SKI II, Sonidegib, Sorafenib, SSR128129E, STX-01 19, Sulfasalazine, Sunitinib, T56-LIMKi, Talampanel, Tamoxifen, Tandutinib, Tanespimycin, Telomestatin, Temozolomide,
Temsirolimus, Terfenadine, Tesevatinib, Tetraethylammonium, Tetrandrine, TG02, TG101209, TGX-221 , Thalidomide, Thapsigargin, Theobromine, Thymoquinone, THZ1 , Tipifarnib, Tivozanib, Tnp-470, Tocladesine, Topotecan, Tozasertib, Tpi-287, Trametinib, Transcrocetinate sodium, Tricetin, Trichostatin, Trifluoperazine, Triptolide, UMI-77, UNC2025, UNII-2AW48LAZ4I, USL311 , VAL-083, Valproic Acid, Vandetanib, Vatalanib, Veliparib, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,
Vinpocetine, Vismodegib, Visudyne, Vorinostat, Wortmannin, WP1066, WP1130, XL-184, XL765, Xyloketal B, YU238259, ZSTK474.
The methods of the tenth and eleventh aspects may be extended to generate additional transcriptomic profiles in order to provide more extensive combination therapies.
In some embodiments of the tenth aspect, the method is extended to identify further druggable targets by: d. selecting an inhibitor of the druggable target for combination therapy as the second compound e. contacting test cells with the second compound, and optionally the first compound, f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
g. selecting as a further druggable target for combination therapy a gene whose expression is perturbed in the second transcriptomic profile,
wherein said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
The skilled person will appreciate that this may be repeated to identify further druggable targets, for example by:
g. selecting an inhibitor of the further druggable target for combination therapy
h. contacting test cells with the further compound, optionally sequentially or simultaneously with any and all of the other compounds used in the previous stages,
i. measuring the effects of the further compound, or the further and additional compounds, on the transcriptome of the test cells, so as to create a further transcriptomic profile,
j. selecting as a yet further druggable target for combination therapy a gene whose expression is perturbed in the further transcriptomic profile,
wherein said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
The skilled person will appreciate that this process may be repeated any number of times to identify any number of further druggable targets. For example, the method may provide a series of druggable targets by:
a. contacting test cells with a compound,
b. measuring the effects of the compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is
perturbed in the transcriptomic profile, wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation,
d. repeating steps a to c one or more times, each time supplementing or substituting the
compound in the previous step a with an inhibitor of the druggable target identified in previous claim c, until the desired number of druggable targets has been provided.
In some embodiments of the eleventh aspect, the method provides an anti-cancer therapeutic combination comprising a first, a second and a third compound, and comprises the additional steps of: e. contacting test cells with the second compound, and optionally the first compound f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
g. selecting as a second druggable target for combination therapy a gene whose expression is perturbed in the second transcriptomic profile, wherein said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
h. selecting an inhibitor of the second druggable target as a third compound for the combination therapy.
The skilled person will appreciate how this embodiment of the eleventh aspect may be extended to provide a fourth compound by repeating the contacting and measuring steps with the third compound, optionally in combination with the first and/or second compound, to create a third transcriptomic profile; selecting a further druggable target that is perturbed in the third transcriptomic profile; and selecting an inhibitor of this target as a fourth compound in the combination therapy. This may be repeated to provide a fifth, sixth, etc. compound for the combination.
The method may be extended to provide an anti-cancer therapeutic combination comprising a first compound and any number of further compounds, comprising the steps of: a. contacting test cells with a first compound,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is
perturbed in the transcriptomic profile, wherein said gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
d. selecting an inhibitor of the druggable target for combination therapy as a further compound, and
e. optionally repeating steps a to d one or more times, each time supplementing or substituting the compound used in step a with the further compound identified in the previous step d, until the desired number of further compounds has been identified.
In this way, the method may be used to sequentially identify compounds for use in a treatment which is tailored to the individual and their cancer, which is dynamic, heterogeneous and adapts in response to treatment.
In the tenth and eleventh aspects, the second compound may additionally be selected on the basis of synergy with the first compound. For example, step d may comprise the sub-steps of:
i) identifying a candidate inhibitor of the druggable target
ii) contacting test cells with the candidate inhibitor in combination with the first compound, and iii) selecting the candidate inhibitor as the second compound if it exhibits a synergistic effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation when provided in combination with the first compound. As used herein a combination with a“synergistic” effect is one for which the effect is greater than the sum of the components provided in isolation. Therefore, a second compound that exhibits a synergistic effect one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radiosensitisation when provided in combination with the first compound is one where the effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation is greater than the sum of the effect seen for the first and second compounds administered in isolation.
In some embodiments of the tenth and eleventh aspects, the test cells may be the same cells that are contacted with the first compound in step a), in order to model ongoing therapy. Alternatively, the test cells may be of the same type as those in step a) but may have not previously been contacted with the first compound.
The invention also provides a therapeutic combination provided by the method of the eleventh aspect.
The invention also provides a therapeutic combination provided by the method of the eleventh aspect, for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the first compound simultaneously or sequentially with the second compound. Where the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
The invention also provides the use therapeutic combination provided by the method of the eleventh aspect in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament administers an effective amount of the first compound simultaneously or sequentially with an effective amount of the second compound to a subject in need thereof. Where the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
The invention also provides a method of treating or preventing cancer with the therapeutic combination according to the eleventh aspect, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of the first compound and an effective amount of second compound. Where the therapeutic combination comprises further compounds, for example a fourth, fifth, etc. compound, these may independently be administered simultaneously or sequentially with the first and/or second compound.
Where any of the therapeutic combinations described herein are used to treat a patient, the method may comprise sequentially administering the compounds in combination, for example by (i) administering the ripound, followed by (ii) the second compound simultaneously with the first compound, followed by (iii) the third compound simultaneously with the second and/or first compound, followed by (iv) a further compound simultaneously with any combination of the previous compounds. Steps (i) to (iv) may be separated by any length of time. Whilst the first and second compounds may be any contemplated herein, the first compound is preferably an inhibitor of PI3K signalling, and the second compound is preferably an inhibitor of JAK activity or function, or the first compound may be an inhibitor of JAK activity or function and the second compound may be an inhibitor of PI3K signalling. In these embodiments, the cancer is sequentially challenged with new drugs during ongoing therapy. A compound may be administered at an initial effective dose, before reverting to a lower dose for subsequent stages - i.e. the first compound may be administered at a higher dose in step (i) than (ii)-iv), the second at a higher dose in step (ii) than steps (iii)-(iv), the third compound in a higher dose in step (iii) than in step (iv), etc. In this way, the cancer is challenged with an initial effective dose of a compound, and ongoing therapy at a lower dose to avoid dose limiting toxicity or side effects.
Also provided herein is a therapeutic combination provided by the method according to the eleventh aspect for use in the treatment or prevention of cancer, preferably for the treatment of GBM. The combination may be for sequential or simultaneous administration.
The invention also provides a method, preferably an in vitro method, of identifying druggable targets for combination therapy, comprising the steps of:
a. contacting test cells with a first treatment,
b. measuring the effects of the first treatment on the transcriptome of the test cells, so as to create a transcriptomic profile for the first treatment,
c. selecting as a druggable target for combination therapy a gene whose expression is perturbed in the transcriptomic profile,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
The invention also provides a method, preferably an in vitro method, of providing a therapeutic combination, the method comprising
a. contacting test cells with a first treatment,
b. measuring the effects of the first treatment on the transcriptome of the test cells, so as to create a transcriptomic profile for the first treatment,
c. selecting as a druggable target for combination therapy a gene whose expression is perturbed in the transcriptomic profile,
d. selecting as the second treatment at least an inhibitor of the druggable target for combination therapy,
so as to provide a combination therapy comprising the first and second treatment,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
9 aspects a“treatment” may comprise or consist of a therapeutic compound as defined above. Alternatively, a treatment may comprise a suite of therapeutic compounds. A treatment may comprise or consist of additional therapies, such as radiotherapy. A“first treatment” may comprise or consist of any therapeutic compounds and/or therapies. A“second treatment” comprises or consists of an inhibitor of the druggable target, and may comprise additional compounds or therapies, so long as it includes an inhibitor of the druggable target. The second treatment may include one or more therapeutic compound and/or therapy which constituted the first treatment.
Also, a diagnostic process using similar transcriptomic techniques to identify further dysregulated genes and pathways as diagnostic targets which may comprise biomarkers of relevance to the treatments is provided herein. For example, a method of identifying diagnostic targets in a cancer in provided, comprising:
a. contacting test cells with a first compound, wherein the test cells are cancer cells,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound, and
c. selecting as a diagnostic target a gene whose expression is perturbed in the transcriptomic
profile,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation. Preferably, the test cells are cells from a patient with a cancer, more preferably the cells are in vivo.
Also provided herein is a drug discovery process using specific combinations of dysregulated genes and pathways revealed by similar transcriptomic techniques as those discussed herein in order to identify specific drug discovery targets and drug target combinations, for example specific protein kinase targets and other biochemical activities, and develop them as biochemical screens of direct relevance to the development of further medicinal treatments. Therefore, in the tenth or eleventh aspects, the second compound may be selected on the basis of selectivity screening. For example, a compound may be provided, or developed specifically, on the basis of the transcriptomic profile induced by it on test cells, either alone or in combination with the first compound, or on the basis of its inhibitory profile. The second compound may have a similar or the same transcriptomic or inhibitory profile as a candidate inhibitor for which a synergistic effect with the first compound has been shown. For example, step d may comprise the sub-steps of:
i) identifying a candidate inhibitor of the druggable target
ii) contacting test cells with the candidate inhibitor in combination with the first compound, and iii) measuring the effects of the combination on the transcriptome of the test cells, so as to create a transcriptomic profile for the combination, if the combination exhibits a synergistic effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation,
iv) providing or developing an inhibitor with the same or similar transcriptomic or inhibitory profile as the candidate inhibitor, and,
selecting the inhibitor of sub-step iv) as the second compound.
The present combination of an inhibitor of PI3K signalling with an inhibitor of Janus kinase (JAK) activity or function is effective in the treatment of cancer. As used herein, a cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumour or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumour. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumour may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node (including abdominal lymph node, axillary lymph node, cervical lymph node, inguinal lymph node, mediastinal lymph node, pelvic lymph node, periaortic lymph node), lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentume, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells
Cancers may be of a particular type. Examples of types of cancer include astrocytoma, carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g. angiosarcoma, chrondrosarcoma, osteosarcoma).
In preferred embodiments of the invention, the cancer to be treated or prevented is a cancer of the brain or central nervous system (CNS). Brain cancers include primary brain tumours (tumours which start in the brain) and secondary brain tumours (i.e. cancerous tumours which have metastasised to the brain from another location in the body, also known as brain metastasis tumours). In preferred embodiments, the brain cancer is a primary brain tumour. Primary brain tumours may originate in astrocytes,
oligodendrocytes, ependyma, neurons or meninges.
Brain cancers may be subdivided into glioma (tumours of the glial cells), meningioma (tumours of the meninges), pituitary adenoma (tumours of the pituitary gland) and nerve sheath tumours. Preferably, the cancer to be treated or prevented is a glioma. A glioma may be selected from an ependymoma, an astrocytoma, an oligodendroglioma, or a brain stem glioma. Gliomas may be categorised according to their grade, as determined according to WHO Classification of Tumours of the Central Nervous System (“The 2016 World Health Organisation Classification of Tumours of the Central Nervous System: a summary". Acta Neuropathologica. 131 (6): 803-820). The cancer to be treated or prevented may be a low-grade (WHO grade II or lower) glioma or, more preferably, a high-grade (WHO grade lll-IV) glioma.
In most preferred embodiments, the cancer to be treated or prevented is glioblastoma multiforme (GBM). GBM is a primary cancer of the astrocytes, and is the most common primary malignant brain tumour in adults (Ostrom et al., 2017). In some embodiments, the GBM has one or more mutations in a signalling pathway selected from: RB, TP53, and receptor tyrosine kinase (RTK)/mitogen activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K). The nature of these mutations will be known to the skilled person, and can be found in Cancer Genome Atlas Research Network, 2008.
GBM can be stratified into 4 molecular subtypes based on gene expression according to Verhaak et al., 2010. The cancer to be treated or prevented may be a GBM of the Classical subtype. Classical GBM is characterised by extra copies of the EGFR, higher than normal expression of EGFR, retention of wild- type p53, loss of heterozygosity in chromosome 10, and chromosome 7 amplification. The cancer to be treated or prevented may be a GBM of the Mesenchymal subtype. Mesenchymal GBM can be characterised by high rates of mutations or other alterations in NF1 , the gene encoding Neurofibromin 1 and fewer alterations in the EGFR gene and less expression of EGFR than classical GBM. The cancer to be treated or prevented may be a GBM of the proneural subtype. Proneural GBM can be characterised by high rates of alterations in TP53 (p53), and in PDGFRA, the gene encoding a-type platelet-derived growth factor receptor, and in IDH1 , the gene encoding isocitrate dehydrogenase-1. The cancer to be treated or prevented may be a GBM of the Neural subtype. Neural GBM can be characterised by the expression of neuron markers. The characterisation of GBM subtypes by biomarker is a matter of routine. In some embodiments, the cancer to be treated or prevented is a metastatic GBM i.e. one which has
metastasised.
The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.
For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.
ction headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the claims which follow, unless the context requires otherwise, the word“comprise” and“include”, and variations such as“comprises”,“comprising”, and“including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent“about,” it will be understood that the particular value forms another embodiment. The term“about” in relation to a numerical value is optional and means for example +/- 10%.
While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.
The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & 20 Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present rlicrlncure.
As used herein, the term "about" means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In some embodiments, about means the specified value.
Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Examples
MATERIALS AND METHODS
Fucoxanthin and LY-294002
Fucoxanthin and LY-294002 were selected as reagents for investigation.
Fucoxanthin is a member of the xanthophyll class of carotenoids, and is present at high concentrations in the brown alga Saccharina latissimi (Galasso et al., 2017) where it plays an accessory role in light harvesting and radiation protection. Fucoxanthin has been suggested to act in cancer by suppressing invasion and inducing apoptosis through PI3K/Akt pathway inhibition (Liu et al., 2016; Satomi, 2017) and JAK/STAT pathway inhibition (Kanno et al., 2013; Szymanska et al., 2015). In addition, its metabolites, including Fucoxanthinol, show modulatory actions on the NF-KB pathway, caspase activity, Bcl-2 proteins, MAPK, PI3K/Akt, JAK/STAT, AP-1 , GADD45 (Martin, 2015).
Fucoxanthin has a particularly interesting and unique molecular structure (Figure 1 C), exhibiting antioxidant properties due to a long conjugated backbone characteristic of all carotenoids, and possessing unusual terminal allenic bond and conjugated carbonyl groups (Dembitsky and Maoka, 2007; Sangeetha et al., 2009). In addition to its effects on the PI3K/Akt pathway, Fucoxanthin has been shown to inhibit migration and invasion of metastatic melanoma and osteosarcoma cells in vitro and in vivo (Liu et al., 2016). Importantly, Fucoxanthin is metabolically unstable, upon metabolism producing
Fucoxanthinol (Zhang et al., 2015), a factor to take into account when evaluating its transcriptomic effects.
Since Fucoxanthin’s Mechanism of Action (MoA) is incompletely understood, we have used genome-wide expression analysis to compare it with the well-characterised PI3K/Akt pathway inhibitor LY-294002 (Chen et al., 2012). LY-294002 is one of the PI3K/Akt inhibitors annotated as active in GBM annotated in the GBM Drug Bank, a public domain resource developed by IOTA Pharmaceuticals based on compounds known to influence the growth and development of glioblastoma (Svensson et al., 2018) http://www.gbmdrugbank.com/db.php).
Cell culture and treatment
man glioblastoma cell line U87MG expressing the wild-type p53 gene (U87MG, p53wt, female, obtained from European Collection of Authenticated Cell Cultures) was maintained in DMEM/F12 media (Gibco, ThermoFisher, UK) supplemented with 10% foetal bovine serum (FBS) (Sigma, UK) and 5% antibiotic/antimycotic solution (Sigma, UK). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 LY-294002 and Fucoxanthin were purchased from Sigma, UK and dissolved in dimethyl sulfoxide (DMSO) to obtain 0.1 M and 0.05 M stock solutions, respectively.
For treatments, cells were seeded into T25 flasks at a density of 5x105 cells/flask and allowed to adhere and grow for 24h. The culture medium was removed, and fresh medium containing compound for test was added to each flask at the EC50 concentration for inhibiting proliferation at 72h, previously determined as 199.5 pM for LY-294002 and 19.9 pM for Fucoxanthin. Control cells were treated with medium containing 1 % DMSO alone. All experiments were performed in triplicate. Cells were visualised during culture using the EVOS Cell Imaging System (Thermo Fisher Scientific, UK).
Proliferation assay
The cytotoxicity of Fucoxanthin and LY-294002 in U87MG cell line was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma). U87MG cells were seeded at a density of 8,000 cells/well in 96- well plates and allowed to adhere overnight at 37°C in a humidified atmosphere of 95% air and 5% CO2. Both Fucoxanthin and LY-294002 were tested at concentrations of 1- 1000 pM in triplicate in at least two separate experiments. All initial dilutions of Fucoxanthin and LY-294002 were made in DMSO and then further diluted in the medium at 1 : 100 ratio. Then, the culture medium was removed from the plates, and the fresh medium containing tested compounds at different dilutions was added to the plates. Control cells were treated with vehicle solution containing 1 % DMSO. Blank controls without cells were also prepared. 72h after treatment, 5 pL of CCK-8 was added to every well containing 100 pL of tested compounds, controls or blank. After 3 h of incubation at 37°C in the dark, the plates were read using a Mithras LB940 multimode microplate reader (Berthold Technologies), and the absorbance values were determined at 490 nm. The percentage of surviving cells was calculated for each well using the formula:
Figure imgf000035_0001
where At is absorbance of the medium with tested compound, Ac is absorbance of control medium, and Ab is absorbance of blank medium.
Concentration-effect relationships for both compounds were analysed using Prism software version 8.0.1 (GraphPad, Inc., San Diego, CA). Data were fitted using a four-parameter logistic equation.
Apoptosis assay
The extent of cell apoptosis was measured using the Annexin V-FITC apoptosis detection kit (Abeam, Cambridge, UK). U87MG cells were seeded at a density of 80,000 cells/well in 6-well plates and allowed to adhere overnight at 37°C in a humidified atmosphere of 95% air and 5% C02. Fucoxanthin and LY-
_ _ ! were tested at concentrations of 19.9 pM and 199.5 pM, respectively. Control cells were treated with vehicle solution containing 1 % DMSO. After 48h of treatment, the cells were trypsinised, centrifuged, resuspended in 500 pL of binding buffer followed by the addition of 5 pL Annexin V-FITC and 5 pL propidium iodide (PI) according to the manufacturer's instruction. The samples were incubated at room temperature for 5 min in the dark and then analysed using a BD Accuri™ C6 Flow Cytometer. A total of 10,000 events were counted for each sample. Fluorescence was measured at an excitation wavelength of 480 nm with detection for PI at 530 nm and Annexin V at 585 nm.
Microarrays
After 24h and 48h of treatment, the cells were trypsinised, and total RNA was isolated using the RNeasy Mini kit (Qiagen, UK) according to the manufacturer's recommendation. A Thermo Scientific™ NanoDrop instrument was used to quantify the RNA and test the purity of each sample. Aliquots of RNA were frozen at -80°C prior to analysis at ATLAS Biolabs (Berlin, Germany) for microarray experiments.
The total RNA samples were further prepared by ATLAS Biolabs (Berlin, Germany) using their Affymetrix WT Expression Profiling Standard Service. The analysis was conducted on a Clariom S Human array with a fixed number of probes per transcript as probe sets consisting of a subset of 10 probes per gene (yielding >20,000 annotated genes, as documented by the NetAffx Analysis Center).
The quality and quantity of each RNA sample was determined using the Agilent 2100 Bioanalyzer and Nanodrop. For library preparation (cDNA synthesis, amplification and labeling) GeneChip® WT PLUS reagents were used. 96-array plates were processed on an Applied Biosystems GeneChip 3000 instrument system followed by hybridisation, dyeing, washing, and scanning using the GeneChip™ Hybridisation, Wash, and Stain Kit from Affymetrix. Hybridisation controls, quality control parameters and primary data analysis was performed using Expression Console v1 .4 (also from Affymetrix).
Microarray data analysis
The average signal intensities for each probe set were analysed and an expression matrix was created by applying the RMA (Robust Multi-array Average) algorithm as a multi-chip model (Irizarry et al., 2003). A threshold of 0.8 area under the receiver operating curve was applied to compare the exon and intron probe sets of housekeeping genes, to ensure a good separation within the data.
Gene expression analysis
Microarray gene expression profiling and visualisation results were performed using the Affymetrix Transcriptome Analysis Console (TAC) software (ThermoFisher) which uses the limma package (Ritchie et al., 2015). In defining differentially expressed genes (DEGs), we used two criteria. First, a gene log 2 Fold Change (FC) level has to reach the threshold of < -1 or > 1 based on Tukey's bi-weight average between treatment and time matching controls (Kohl and Deigner, 2010). Tukey’s bi-weight averaging makes the average less sensitive to outliers (Tukey, 1973). Secondly, the Benjamini Hochberg corrected p-value of the ANOVA (Analysis of Variance) test was applied to compare conditions (either time or treatment) with a threshold lower than 0.05 (Hochberg, 1995). The ANOVA tests were calculated with an ebayes (Empirical Bayes Statistics for Differential Expression) variance calculation (Phipson et al., 2016). ariom S chip’s probe sets were mapped to UniProt SwissProt identifiers (UniProt Consortium,
2018), keeping only the protein coding genes. If a gene had more than one probe set, the most differentially expressed probe sets were used in all cases. Gene pathway analysis
After defining the differentially expressed genes (DEGs), pathway analysis was performed to identify compound response. For each treatment, untreated U87MG cells were used as control samples for each corresponding time point.
To show the most significant pathways, WikiPathways map representations were used in which the signal intensities of DEGs involved in one particular enriched pathway can be shown as down-regulated or up- regulated in response to specific treatments (Kutmon et al., 2016). For pathway analysis, Fisher exact tests were used.
Compound and gene expression signatures
To compare gene expression profiles by signature-matching between LY-294002 and Fucoxanthin treatments, we used Connectivity mapping applying the Connectivity Map 02 tool provided by The Broad Institute (Subramanian et al., 2017b).
The Connectivity Map approach is based on the most significant differentially expressed genes characteristic of compound molecular response. Significantly differentially expressed genes were analysed using Connectivity Map 2. Differentially expressed genes IDs were mapped to Affymetrix HGU133-plus2 probe set IDs, which were then input into Connectivity Map. The down- and up-regulated genes responding to Fucoxanthin and LY-294002 treatment were used as the query signatures for comparing transcriptomic profiles to other compounds in CMap. The connectivity score was calculated based on Kolmogorov-Smirnov statistics (Smalley et al., 2010). This approach is described in detail in Alexander-Dann et al., 2018; Iwata and Yamanishi, 2019; and Oerton and Bender, 2017. Significant findings were cross-referenced to the Glioblastoma Drug Bank (Svensson et al., 2018).
EXAMPLE 1 - Both Fucoxanthin and LY-294002 reduce proliferation of U87MG cells
We compared the effects of Fucoxanthin (Figure 1 C) and LY-294002 (Figure 1A) on the cellular proliferation of U87MG cells. When tested from 10_6M to 10_3M concentrations, cell survival remained at approximately 100% for 24h upon treatment with both compounds. However, at concentrations above 10_ 5M at between 24h and 48h, cell survival decreased, most markedly for Fucoxanthin and to a lesser extent for LY-294002. Cell survival was severely reduced after 72h at compound concentrations higher than 10_5M (Figure 2).
The EC50 value for growth inhibition for Fucoxanthin (Figure 2A) was approximately 19.9 pM after 72h of treatment. The EC50 value for growth inhibition for LY-294002 was 199.5 pM for LY-294002 (Figure 2B) after 72h of treatment. EXAMPLE 2 - Morphological effects of Fucoxanthin and LY-294002 on U87MG cells
The target cell population, U87MG glioblastoma cells, was treated with each compound at its 72h antiproliferative EC50, determined in an initial drug response study (Figure 2) in order to see“early” gene expression changes in response to drug challenge within 24h, a time point where no growth inhibition was evident (Figure 2A), with“late” gene expression changes accompanying treatments at subsequent time points. The“late” time point was 48h, prior to any overt toxicities emerging as the cells reached the EC50s of the compounds at 72h.
Morphological studies were performed for Fucoxanthin and LY-294002 at 24h and 48h time points, using the 72h EC50 concentrations of 19.9 pM and 199.5 pM respectively, to observe both early and late drug responses.
The cellular morphology of the glioblastoma U87MG cell line after 24h and 48h of treatment with
Fucoxanthin or LY-294002 at these concentrations clearly differed from those of DMSO-treated controls (Figure 3). The cells grew as monolayers in the cell culture flasks before treatment and formed some spheres. After 24h of treatment with Fucoxanthin, most adherent cells were round, with some showing signs of nuclear fragmentation, although some cells with pseudopod-like protrusions were also observed (Figure 3C). After 48h of treatment, a mixture of effects was seen: some cells remained adherent, while others floated off their plastic substrates, often as aggregates (Figure 3D).
The morphology of cells treated with LY-294002 was slightly different, with some cell membrane blebbing observed at both 24h and 48h (Figure 3, E & F).
Fluorescence-activated cell sorting (Figure 3, panels G-J) showed that while LY-294002 increases apoptosis compared to control, Fucoxanthin does not induce apoptosis (Figure 3, G, H & I) at the concentrations used in our study, although Fucoxanthin marginally increased cell death through necrosis compared to control.
EXAMPLE 3 - Gene Expression analysis
Gene expression studies were performed for Fucoxanthin and LY-294002 at 24h and 48h time points, using the 72h EC50 concentrations of 19.9 pM and 199.5 pM respectively, to observe both early and late drug responses. Principal Component Analysis (PCA) was performed after normalising the gene expression results (Figure 4). The first three PCs covered 73.5% of the variance in the gene expression response (Figure 4). The first and third PCs were correlated with the time variable of the gene expression measurement, while the second PC was correlated with the drug treatments themselves. PCA clearly differentiates the effects of Fucoxanthin from those of LY-294002 within gene expression space, at the same time demonstrating a very high degree of experimental reproducibility.
The reproducibility of these results was further confirmed by clustering the samples and deriving a heatmap based on Euclidean distance-based analysis. Again, this demonstrated very high reproducibility of the gene expression profiles. Importantly, both PCA and hierarchical clustering indicate very high reproducibility within the data sets (Figure 4). Also, according to both PCA and cluster analysis, a clear difference can be observed between the transcriptomic profiles exhibited by Fucoxanthin and LY-294002. LY-294002 produced more defined differences when compared to Fucoxanthin, indicating a more specific mechanism of action for LY- 294002.
EXAMPLE 4 - Differentially expressed gene overview of LY-294002 and Fucoxanthin treatments
Affymetrix Clariom S microarrays contain probe sets for both non-coding and coding genes. Here, we consider modulation of expression of the coding genes and calculate the false discovery rate (FDR) using their adjusted p-values to reduce false positive results.
Down-regulation of U87MG gene expression was observed more frequently than up-regulation: in all, 887 genes were up-regulated by the two compounds, while 1429 genes were down-regulated.
For LY-294002 treatment at 24h, there are in total 687 down-regulated genes and 338 up-regulated genes, and for LY-294002 at 48h, 836 down-regulated and 319 up-regulated genes (. For Fucoxanthin, there are a total of 470 down-regulated genes and 380 up-regulated genes at 24h and 325 down- regulated and 169 up-regulated genes at 48h. These results are summarised in Table 1 and in the Venn diagram in Figure 6.
Amongst the 381 genes that were up-regulated at 24h by Fucoxanthin, 106 (28%) remained up-regulated at 48h, with 63 new overexpressed genes appearing at this time point. In contrast, amongst the 339 genes that were up-regulated at 24h by LY-294002, 148 (44%) remained up-regulated at 48h with 172 new over-expressed genes appearing.
Only 45 genes were up-regulated at 24h in both treatments, and only 6 genes were up-regulated at both 24h and 48h in both treatments, emphasising the disparity of the transcriptional responses to the two compound treatments.
Amongst the 460 genes that were down-regulated at 24h by Fucoxanthin, 152 (30%) remained down- regulated at 48h, while 236 new genes were down-regulated. In contrast, amongst the 750 genes that were down-regulated at 24h by LY-294002, 458 (61 %) remained down-regulated at 48h, with an additional 368 genes not seen at 24h being down-regulated by LY-294002 at 48h. In all, 265 genes were down-regulated at 24h in both treatments, with 68 genes showing down-regulation at both 24h and 48h in both treatments, again illustrating more commonalities amongst the down-regulated genes within the transcriptional response to the two compounds. EXAMPLE 5 - Analysis of the Top 25 genes from LY-294002 and Fucoxanthin treatments
The top 25 expressed genes in response to LY-294002 at 24h and 48h were compared with those seen in Fucoxanthin treatments at the same time points (Figure 7). Similarly, the top 25 expressed genes in response to Fucoxanthin at 24h and 48h were compared with those seen in LY-294002 treatments at the same time points (Figure 8). Clear differences were seen between the gene expression profiles amongst the top 25 expressed genes in each treatment.
The top 25 up- and down-regulated genes were compared upon LY-294002 and Fucoxanthin treatments at 24h and 48h (Figure 7 and Figure 8). A suite of co-ordinately regulated genes can be seen, with only a few genes differing in their levels of expression at 48h. This concordance is higher in the case of LY- 294002 where common down-regulated genes are seen (CCNE2, POLE2, CDK1).
EXAMPLE 6 - Pathways differentially modulated by Fucoxanthin and LY-294002 treatments
Following analysis of individual gene expression changes, we used WikiPathway analysis to obtain an overview of the main changes in signalling pathways characterising the response to the two compounds (Table 2).
Many well-defined cellular signalling pathways are significantly modulated in common by the two compounds. For example, both compounds decreased the expression of members of the PI3K/Akt, retinoblastoma gene in cancer and multiple cell cycle related pathways, suggesting a cytostatic effect on U87MG cells.
Detailed changes in gene expression within the PI3K/Akt and retinoblastoma signalling pathways are shown in Figures 10 and 11 , mapping the genes involved directly on to the schematic pathways.
Fucoxanthin has a transcriptomic effect on PI3K/Akt pathway, increasing the expression of 10 of its component genes after 24h, 5 of which are remain up-regulated after 48h treatment, while decreasing the expression of 24 genes after 24h, with 19 of these genes remaining downregulated after 48h treatment (p<0.05 Benjamin Hochberg corrected Fisher exact test).
Surprisingly, LY-294002 had no significant transcriptomic effect at the pathway level on the PI3K/Akt pathway (p>0.05 Benjamin Hochberg corrected Fisher exact test), even though it acts directly on this pathway by inhibition of PI3K. Only 4 genes at 24h and 8 genes at 48h were up-regulated from this large pathway while 17 and 19 genes, respectively, were down-regulated after 24h and 48h treatment (see Figure 10 for the wiring diagrams of the genes involved in PI3K/Akt pathway). In contrast, Fucoxanthin has a clear effect at 24h on the expression of components of the PI3K/Akt pathway, although this is not id by their continuing expression at 48h.
Both compounds increased JAK2 expression: LY-294002 at 24h and 48h, Fucoxanthin only at the 24h time point. JAK2 modulates the PI3K/mTOR pathway (Rane and Reddy, 2000). Up-regulation of JAK2 could reflect a pro-proliferative role within the glioblastoma cells as they react to the growth inhibition caused by down-regulation of the PI3K pathway. Several other pro-proliferative genes (Bcl-2, SGK1 , IGF1 R) show similar induction patterns. The observation of SGK1 up-regulation is of particular interest since this kinase has recently been shown to be a key survival kinase for glioblastoma stem cells (Kulkarni et al., 2018). This can be seen in detail in Figure 14.
This reveals that combination of inhibitors of the PI3K pathway with inhibitors of JAK, and optionally SGK1 and/or IGF1 R would be expected to have enhanced anti-proliferative effects.
Additionally, Fucoxanthin has a marked transcriptomic effect on the Retinoblastoma pathway, downregulating all the genes in the pathway at 24h with a considerable number remaining down-regulated after 48h treatment. LY-294002 similarly down-regulates this pathway, with 35 genes showing down-regulation at 24h, 19 of which remain down-regulated at the 48h treatment time (Figure 11). TP53 is the only gene up-regulated in both the PI3K/Akt and Retinoblastoma pathways, and this only in response to
Fucoxanthin at 48h treatment.
Many other signalling pathways were affected by both treatments, listed in Table 2. Amongst those of particular interest were the apoptosis and necrosis pathways, cell cycle and the EGFR pathways, elements of which characterise the U87MG growth response (Ghosh et al., 2005)
EXAMPLE 7- In silico target identification for LY-294002 and Fucoxanthin
Some consideration needs to be given to the way in which compounds exert their effects in a cellular context. Where a drug has been discovered using target-based drug discovery techniques (e.g. fragment- based and structure-based drug discovery approaches), there is some logic to suggest that it exerts its functional effects via that target. However, for natural products such as Fucoxanthin, where molecular mode of action is speculative, it may be necessary to use other screening techniques, such as in silico target prediction to uncover mode of action.
Using the structures of Fucoxanthin, Fucoxanthinol and LY-294002 as shown in Figure 1 , a target prediction algorithm, PIDGIN v2 (Mervin et al., 2015), was run to identify potential drug targets with which the ligands might interact.
No proteins except isoforms of PI3K were identified using PIDGIN that also occurred within the set of up- or down-regulated genes seen using transcriptomics, and PI3K was only identified as a putative target for LY-294002, not Fucoxanthin or Fucoxanthinol. Additional proteins showing putative LY-294002 binding included Topoisomerase 1 and the GABA receptor P subunit. A further protein, the kinase PIM1 , was identified as a weak hit (seen in the transcriptomics data at 48h of LY-294002 treatment).
In the case of Fucoxanthin and Fucoxanthinol, the Retinoic Acid Receptor (RAR) Protein Phosphatase 1 B (PTP1 B) and Mitogen-activated protein kinase kinase kinase 13 (MAP3K13) were indicated as possible targets. Interestingly PTP1 B only in the case of Fucoxanthin meanwhile the upstream MAP kinase in the case of Fucoxanthinol. The limitations of fingerprint based target prediction include biases introduced by large molecular structure such as steroids or a retinoic acid chains (Sydow et al., 2019).
EXAMPLE 8 - Connectivity Map analysis of Fucoxanthin and LY-294002 gene expression patterns
Connectivity Map (CMap) is a method used routinely to compare the effects of drugs on gene expression space (Lamb et al., 2006). Besides providing clues to enable the determination of drug mode of action at a molecular level, the method also enables an objective comparison of drug properties (Alexander-Dann et al., 2018). Here, we used CMap to compare the gene expression“signatures” of LY-294002 and Fucoxanthin seen in our studies with U87MG cells, to drug-like compounds in CMap with similar gene expression signatures from other cells and tissues.
Initial CMap studies shown in Figure 12 show that LY-294002 elicits similar gene expression signatures in U87MG to those seen with LY-294002 in other cancer cells such as the prostate cancer cell line PC3 and the breast cancer cell line MCF7. This not only validates the CMap approach, but also validates our data set, demonstrating both internal consistency with independent gene expression studies and suggests that LY-294002 most likely has a similar mode of action in U87MG cells as it has in the cells within the other CMap cell types. Moreover, the gene expression signatures from U87MG cells treated with LY-294002 show very similar gene expression profiles to other PI3K drugs already deposited in CMap, including the PI3K pharmacological tools Quinostatin (which inhibits the lipid-kinase activity of the catalytic subunits of class la PI3Ks), and Wortmannin (a potent, selective and irreversible inhibitor of PI3K).
More broadly, we observe that many entries in CMap show similar perturbation to both LY-294002 and Fucoxanthin. There are 116 compounds in CMap related to LY-294002 and Fucoxanthin by their specific gene expression effects, including certain tricyclic antidepressants, canonical antipsychotics, anthelmintic, antibiotics, calcium channel blockers and vasodilators (Figure 12). The widespread gene expression changes in the PI3K pathway demonstrated by such a wide variety of compounds indicates the central role that this pathway plays in cell growth and differentiation. We noted that a number of compounds revealed by CMap analysis were also overrepresented significantly in the GBM Drug Bank (Chi square test p<0.000001), indicating previous studies of these drugs in glioblastoma.
Fucoxanthin exhibits higher similarity to more compounds in the CMap archive with similar gene expression signatures (384 compounds, Figure 12), than does LY-294002 (182 compounds, Figure 12), possibly representing more interactions with cellular components.
EXAMPLE 9 - Combinations of LY-294002 with two JAK2 inhibitors
As LY-294002 increased JAK2 expression at 24h and 48h, it was hypothesised that combination therapy
. . ,K2 inhibitors would further reduce proliferation in U87MG cells. The effect of a combination of LY-
294002 with JAK2 inhibitors Ruxolitinib or AZD1480 was investigated. Cells were exposed to LY294002 in concentrations ranging from 10_6M to 10_3M, alone or in combination with JAK inhibitors at 10, 25 or 50 pM. Reference treatments were performed, comprising JAK inhibitors alone at 10, 25 and 50 pM, and a negative control treatment of 1 .1 % DMSO. Cell survival as a percentage of DMSO control was measured at 72 hrs following treatment.
Cell survival was severely reduced after 72h at compound concentrations higher than 10_5M (Figure 15).
In all treatments, higher concentration of LY294002 reduced cell survival (Figure 15 A & B).
Enhanced effects on cell survival were observed for both LY294002 with Ruxolitinib and LY294002 with AZD1480 relative to LY294002 alone. This effect was dose-responsive, with higher concentrations of JAK inhibitors resulting in further reductions in cell survival.
For LY294002 + 10 pM Ruxolitinib, the effect on cell survival is relatively muted compared to LY294002 alone (Figure 15A) However, an enhanced response can be seen for 25 and 50 pM Ruxolitinib.
The combination of LY294002 + AZD1480 had a more marked effect. Even with 10 pM AZD1480, the effect on cell survival is pronounced, and shows an even greater difference as JAK inhibitor concentration increases. Interestingly, and importantly, the dose-response curve and further statistical analyses for LY294002 + AZD1480 reveals that this combination has a pronounced synergistic effect on the reduction in cell survival (Figure 15B).
EXAMPLE 10 - Identifying further combination therapies
Our work has characterised some of the genes and pathways deployed by glioblastoma cells in their response to drugs. An initial focus on the pro-proliferative PI3K/Akt/mTOR pathway, known to be central to glioblastoma growth and invasion, using the pan-PI3K-targeted inhibitor LY-294002, highlighted the key gene expression responses to this well-characterised PI3K inhibitor, summarised in Figure 13.
While highlighting the co-ordinated responses that U87MG glioblastoma cells display upon drug challenge, the approach outlined in examples 1 to 10 allows the prediction of further drugs that might be used in combination to block glioblastoma cell proliferation. As demonstrated and experimentally verified herein, one such combination is the co-administration of inhibitors of both PI3K and JAK2.
Many cancers show pronounced heterogeneity in the cells which evolve from them, suggesting that several alternative drug combinations may be required to inhibit the growth of the majority of malignant cells emerging from the tumour. Our method allows the sequential determination of emerging drug targets, allowing the deployment of existing drugs targeting such emerging targets, and laying the c - >"tion for the discovery of new lineage-specific drugs by those skilled in the art of drug discovery. An additional aspect of the method is the possibility of immediately determining the preferred subtype selectivity of the drugs required to treat drug-resistant cancers, in the case of GBM JAK2 selectivity over JAK1 or JAK3.
When combined with the deployment of the method using patient-derived tumour tissue and the cell lineages therein, microarray analysis will allow identification of newly emergent targets, allowing more precise treatments to be deployed on a patient-by-patient basis (personalised therapies).
Moreover, subsequent beneficial drug combinations can be identified using a wide variety of initial drug treatments. In the examples presented in this application, a relatively non-selective PI3K inhibitor (LY- 294002) was used to identify newly emerging molecular targets specifically induced by that compound. When used in the method herein, other selective or non-selective drugs will yield their own unique spectra of drug targets. Thus, our method allows the tailoring of combination therapies to particular initiating drugs and drug combinations, not only in GBM but a wide variety of other cancers and therapeutic situations.
EXAMPLE 11 - Comparison of LY-294002 with Fucoxanthin
For LY-294002, within 24h of treatment at the 72h EC50, a clear gene expression pattern is seen in response to the drug. The pattern observed for U87MG cells parallels that seen when other established cancer cell lines are treated with LY-294002. Moreover, many of these“early” changes are recapitulated at the“late” 48h time point, indicative of a sustained drug response to LY-294002: over 44% of highly induced genes are shared at 24h and 48h of treatment. Even more distinctively, 61 % of the down- regulated genes at 24h remain down-regulated at 48h.
For Fucoxanthin, treatments at the 72h EC50 for 24h and 48h show less correspondence, with only 28% of the highly induced genes seen at 24h remaining at 48h. The disparity of gene expression responses between the 24- and 48-hour time points for Fucoxanthin may indicate that this compound exhibits more complex drug-induced gene expression effects over time than LY-294002, a hypothesis that requires further investigation but which is consistent with both Fucoxanthin’s metabolic lability and its production of a new and powerfully antiproliferative active metabolite, Fucoxanthinol (Terasaki et al., 2018).
Both compounds arrest cell cycle by down-regulating the cyclin dependent kinase CDK2 and CDK1 and cyclin D4 and D6 and the main cell cycle initiator transcription factor E2F. In all time point-compound pairs the retinoblastoma pathway is at the top most significantly down-regulated pathway. After closer scrutiny the pathways effector endpoints are down-regulated suggesting the cells are going into a dormant state.
1 into the growth factors and receptors from the top genes revealed JAK2 and insulin receptor substrate 2 in the up-regulated genes after 24h or 48h of LY-294002 treatment. However, these could not be found amongst the up-regulated genes after 48h of Fucoxanthin treatment and only JAK2 after 24h of Fucoxanthin treatment. The glioblastoma cells probably want to escape from the PI3K inhibitors effect and activate alternative growth through JAK2. This reveals novel combinatorial therapeutic approaches of LY-294002 and JAK2 inhibitors, as borne out by the experiments with AZD1480.
Direct comparison of the changes in gene expression induced in U87MG cells by Fucoxanthin with those induced by LY-294002 allowed the identification of specific components of the PI3K pathway as down- regulated by both Fucoxanthin and LY-294002. However, these compounds also show pronounced parallel effects on other processes such as the cell cycle and the retinoblastoma gene pathways.
Importantly, co-ordinate up-regulation of other pro-proliferative genes can be seen after both LY-294002 and Fucoxanthin treatments. Specifically, JAK2 within the PI3K/Akt pathway is up-regulated in the case of LY-294002 at both 24h and 48h treatment times, and at the 24h treatment time in the case of
Fucoxanthin.
As well as up-regulation of JAK2, up-regulation of IGF1 R, BCL2L11 and SGK1 can be seen, all of which can be involved in growth promotion and cell proliferation (Basnet et al., 2018; Peng et al., 2016). In fact, the features of cell proliferation and growth promotion are shared by the majority of genes up-regulated by LY-294002, including IGF-1 R (Gariboldi et al., 2010; Zhou et al., 2016), IL-6 (Jin et al., 2012) (Qiu et al., 2013; West et al., 2018) , BAG-1 (Roth et al., 2000), IRS2 (Knobbe and Reifenberger, 2003) (Figure 13).
Table 1. Quantitative differential expressed genes description. The total number of down- and up- regulated coding genes (based upon unique Swiss-Prot IDs)
Figure imgf000046_0001
Table 2. Top 25 significantly enriched pathways in U87MG cells upon LY-294002 and Fucoxanthin treatment for 24h (A) and 48h (B), using WikiPathway analysis.
Table 2A. Top 25 pathways significantly enriched as response to 24h compounds treatment
Figure imgf000047_0001
Table 2B. Top 25 pathways significantly enriched as response to 48h compounds treatment
Figure imgf000048_0001
References
For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Alexander-Dann, B., Pruteanu, L.L., Oerton, E., Sharma, N., Berindan-Neagoe, I., Modos, D., and Bender, A. (2018). Developments in toxicogenomics: understanding and predicting compound-induced toxicity from gene expression data. Mol. Omics 14, 218-236.
Babcock, J.J., Du, F., Xu, K., Wheelan, S.J., and Li, M. (2013). Integrated analysis of drug-induced gene expression profiles predicts novel hERG inhibitors. PLoS ONE 8, e69513.
Basnet, R., Gong, G.Q., Li, C., and Wang, M.-W. (2018). Serum and glucocorticoid inducible protein kinases (SGKs): a potential target for cancer intervention. Acta Pharm. Sin. B 8, 767-771.
Batash, R., Asna, N., Schaffer, P., Francis, N., and Schaffer, M. (2017). Glioblastoma multiforme, diagnosis and treatment; recent literature review. Curr. Med. Chem. 24, 3002-3009.
Cancer Genome Atlas Research Network (2008). Comprehensive genomic characterisation defines human glioblastoma genes and core pathways. Nature 455, 1061-1068.
Chen, L, Han, L, Shi, Z., Zhang, K., Liu, Y„ Zheng, Y„ Jiang, T„ Pu, P„ Jiang, C., and Kang, C. (2012). LY294002 enhances cytotoxicity of temozolomide in glioma by down-regulation of the PI3K/Akt pathway. Mol. Med. Report. 5, 575-579.
Cloughesy, T.F., Cavenee, W.K., and Mischel, P.S. (2014). Glioblastoma: from molecular pathology to targeted treatment. Annu. Rev. Pathol. 9, 1-25.
Cory, A.H., Somerville, L., and Cory, J.G. (2005). Blockage of cy cl in cdk’s, PKC and phosphoinositol 3- kinase pathways leads to augmentation of apoptosis in drug-resistant leukemia cells: evidence for interactive effects of flavopiridol, LY 294002, roscovitine.wortmannin and UCN-01. Anticancer Res. 25, 101-106.
Davies, S.P., Reddy, H., Caivano, M., and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95-105.
Dembitsky, V.M., and Maoka, T. (2007). Allenic and cumulenic lipids. Prog. Lipid Res. 46, 328-375.
Ding, J., Cheng, X.-Y., Liu, S., Ji, H.-Y., Lin, M., Ma, R., and Meng, F.-L. (2019). Apatinib exerts antitumour effects on ovarian cancer cells. Gynecol. Oncol. 153, 165-174.
Engstrom, P.G., Tommei, D., Strieker, S.H., Ender, C., Pollard, S.M., and Bertone, P. (2012). Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival. Genome Med. 4, 76.
Galasso, C., Corinaldesi, C., and Sansone, C. (2017). Carotenoids from Marine Organisms: Biological Functions and Industrial Applications. Antioxidants (Basel) 6.
Gariboldi, M.B., Ravizza, R., and Monti, E. (2010). The IGFR1 inhibitor NVP-AEW541 disrupts a prosurvival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells. Biochem. Pharmacol. fin 4^ -462.
Gharbi, S.I., Zvelebil, M.J., Shuttleworth, S.J., Hancox, T., Saghir, N., Timms, J.F., and Waterfield, M.D. (2007). Exploring the specificity of the PI3K family inhibitor LY294002. Biochem. J. 404, 15-21 .
Ghosh, M.K., Sharma, P., Harbor, P.C., Rahaman, S.O., and Hague, S.J. (2005). PI3K-AKT pathway negatively controls EGFR-dependent DNA-binding activity of Stat3 in glioblastoma multiforme cells. Oncogene 24, 7290-7300.
Hochberg, B. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289-300.
lorio, F., Bosotti, R., Scacheri, E., Belcastro, V., Mithbaokar, P., Ferriero, R., Murino, L, Tagliaferri, R., Brunetti-Pierri, N., Isacchi, A., et al. (2010). Discovery of drug mode of action and drug repositioning from transcriptional responses. Proc Natl Acad Sci USA 107, 14621-14626.
Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. (2003). Exploration, normalisation, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264.
Iwata, M., and Yamanishi, Y. (2019). The Use of Large-Scale Chemically-Induced Transcriptome Data Acquired from LINCS to Study Small Molecules. Methods Mol. Biol. 1888, 189-203.
Jacobs, M.D., Black, J., Futer, O., Swenson, L., Hare, B., Fleming, M., and Saxena, K. (2005). Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002. J. Biol. Chem. 280, 13728-13734.
Jin, X., Kim, S.-H., Jeon, H.-M., Beck, S„ Sohn, Y.-W., Yin, J., Kim, J.-K., Lim, Y.C., Lee, J.-H., Kim, S.- H., et al. (2012). Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling. Brain 135, 1055-1069.
Kanno, H., Sato, H., Yokoyama, T.-A., Yoshizumi, T., and Yamada, S. (2013). The VHL tumour suppressor protein regulates tumourigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signalling pathway. Int. J. Oncol. 42, 881-886.
Knobbe, C.B., and Reifenberger, G. (2003). Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3’-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol. 13, 507-518.
Kohl, M., and Deigner, H.-P. (2010). Preprocessing of gene expression data by optimally robust estimators. BMC Bioinformatics 11, 583.
Kong, D., and Yamori, T. (2008). Phosphatidylinositol 3-kinase inhibitors: promising drug candidates for cancer therapy. Cancer Sci. 99, 1734-1740.
Kulkarni, S., Goel-Bhattacharya, S., Sengupta, S., and Cochran, B.H. (2018). A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells. Mol. Cancer Res. 16, 103-114.
Kutmon, M., Riutta, A., Nunes, N., Hanspers, K., Willighagen, E.L., Bohler, A., Melius, J., Waagmeester, A., Sinha, S.R., Miller, R., et al. (2016). WikiPathways: capturing the full diversity of pathway knowledge. Nucleic Acids Res. 44, D488-94.
Lamb, J., Crawford, E.D., Peck, D., Modell, J.W., Blat, I.C., Wrobel, M.J., Lerner, J., Brunet, J.-P., Subramanian, A., Ross, K.N., et al. (2006). The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929-1935.
Liu, Y., Zheng, J., Zhang, Y., Wang, Z., Yang, Y., Bai, M., and Dai, Y. (2016). Fucoxanthin Activates Apoptosis via Inhibition of PI3K/Akt/mTOR Pathway and Suppresses Invasion and Migration by Restriction of p38-MMP-2/9 Pathway in Human Glioblastoma Cells. Neurochem. Res. 41, 2728-2751.
Liu, Y., Wan, W.-Z., Li, Y., Zhou, G.-L., and Liu, X.-G. (2017). Recent development of ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents. Oncotarget 8, 7181-7200.
Martin L.J. (2015). Fucoxanthin and its metabolite fucoxanthinol in cancer prevention and treatment. Mar.
13, 4784-4798.
Mbaveng, A.T., Damen, F., elik, L, Tane, P., Kuete, V., and Efferth, T. (2019). Cytotoxicity of the crude extract and constituents of the bark of Fagara tessmannii towards multi-factorial drug resistant cancer cells. J. Ethnopharmacol. 235, 28-37. Mendelsohn, J. (2013). Personalising oncology: perspectives and prospects. J. Clin. Oncol. 31, 1904- 1911.
Mervin, L.H., Afzal, A.M., Drakakis, G., Lewis, R., Engkvist, O., and Bender, A. (2015). Target prediction utilising negative bioactivity data covering large chemical space. J. Cheminform. 7, 51.
Oerton, E., and Bender, A. (2017). Concordance analysis of microarray studies identifies representative gene expression changes in Parkinson’s disease: a comparison of 33 human and animal studies. BMC Neurol. 17, 58.
Ostrom, Q.T., Gittleman, H., Liao, P., Vecchione-Koval, T., Wolinsky, Y., Kruchko, C., and Barnholtz- Sloan, J.S. (2017). CBTRUS Statistical Report: Primary brain and other central nervous system tumours diagnosed in the United States in 2010-2014. Neuro Oncol. 19, v1-v88.
Panossian, A., Seo, E.-J., and Efferth, T. (2018). Novel molecular mechanisms for the adaptogenic effects of herbal extracts on isolated brain cells using systems biology. Phytomedicine 50, 257-284.
Pearson, J.R.D., and Regad, T. (2017). Targeting cellular pathways in glioblastoma multiforme. Signal Transduct. Target. Ther. 2, 17040.
Peng, H., Du, B., Jiang, H., and Gao, J. (2016). Over-expression of CHAF1A promotes cell proliferation and apoptosis resistance in glioblastoma cells via AKT/F0X03a/Bim pathway. Biochem. Biophys. Res. Commun. 469, 11 11-1116.
Phipson, B., Lee, S., Majewski, I.J., Alexander, W.S., and Smyth, G.K. (2016). Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann. Appl. Stat. 10, 946-963.
Pridham, K.J., Varghese, R.T., and Sheng, Z. (2017). The Role of Class IA Phosphatidylinositol-4,5- Bisphosphate 3-Kinase Catalytic Subunits in Glioblastoma. Front. Oncol. 7, 312.
Qiu, B., Zhang, D., Wang, Y., Ou, S., Wang, J., Tao, J., and Wang, Y. (2013). Interleukin-6 is overexpressed and augments invasiveness of human glioma stem cells in vitro. Clin. Exp. Metastasis 30, 1009-1018.
Rane, S.G., and Reddy, E.P. (2000). Janus kinases: components of multiple signalling pathways. Oncogene 19, 5662-5679.
Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47.
Roth, W., Grimmel, C., Rieger, L., Strik, H., Takayama, S., Krajewski, S., Meyermann, R., Dichgans, J., Reed, J.C., and Weller, M. (2000). Bag-1 and Bcl-2 gene transfer in malignant glioma: modulation of cell cycle regulation and apoptosis. Brain Pathol. 10, 223-234.
Sangeetha, R.K., Bhaskar, N., and Baskaran, V. (2009). Comparative effects of beta-carotene and fucoxanthin on retinol deficiency induced oxidative stress in rats. Mol. Cell. Biochem. 331, 59-67.
Satomi, Y. (2017). Antitumour and Cancer-preventative Function of Fucoxanthin: A Marine Carotenoid. Anticancer Res. 37, 1557-1562.
Smalley, J.L., Gant, T.W., and Zhang, S.-D. (2010). Application of connectivity mapping in predictive toxicology based on gene-expression similarity. Toxicology 268, 143-146.
Stupp, R., Mason, W.P., van den Bent, M.J., Weller, M., Fisher, B., Taphoorn, M.J.B., Belanger, K., Brandes, A.A., Marosi, C., Bogdahn, U., et al. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987-996.
lanian, A., Narayan, R., Corsello, S.M., Peck, D.D., Natoli, T.E., Lu, X., Gould, J., Davis, J.F., l uDeiii, A.A., Asiedu, J.K., et al. (2017a). A next generation connectivity map: L1000 platform and the first 1 ,000,000 profiles. BioRxiv.
Subramanian, A., Narayan, R., Corsello, S.M., Peck, D.D., Natoli, T.E., Lu, X., Gould, J., Davis, J.F., Tubelli, A.A., Asiedu, J.K., et al. (2017b). A next generation connectivity map: L1000 platform and the first 1 ,000,000 profiles. Cell 171, 1437-1452.e17.
Svensson, F., Westerman, B., Wiirdinger, T., and Bailey, D. (2018). GBM Drug Bank-a new resource for glioblastoma drug discovery and informatics research. Neuro Oncol. 20, 1680-1681.
Sydow, D., Burggraaff, L, Szengel, A., van Vlijmen, H.W.T., IJzerman, A.P., van Westen, G.J.P., and Volkamer, A. (2019). Advances and challenges in computational target prediction. J. Chem. Inf. Model. 59, 1728-1742.
Szymanska, J., Smolewski, P., Majchrzak, A., Cebula-Obrzut, B., Chojnowski, K., and Trelinski, J. (2015). Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines. Adv. Clin. Exp. Med. 24, 195-202.
Tasian, S.K., Teachey, D.T., Li, Y., Shen, F., Harvey, R.C., Chen, l.-M., Ryan, T., Vincent, T.L., Willman, C.L., Perl, A.E., et al. (2017). Potent efficacy of combined PI3K/mTOR and JAK or ABL inhibition in murine xenograft models of Ph-like acute lymphoblastic leukemia. Blood 129, 177-187.
Terasaki, M., Mima, M., Kudoh, S., Endo, T., Maeda, H., Hamada, J., Osada, K., Miyashita, K., and Mutoh, M. (2018). Glycine and succinic acid are effective indicators of the suppression of epithelial- mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells. Oncol. Rep. 40, 414-424.
Tukey, J.W. (1973). The Estimators of the Princeton Robustness Study (Department of Statistics, Univ.).
Tuncbag, N., Milani, P., Pokorny, J.L., Johnson, H., Sio, T.T., Dalin, S., lyekegbe, D.O., White, F.M., Sarkaria, J.N., and Fraenkel, E. (2016). Network Modeling Identifies Patient-specific Pathways in Glioblastoma. Sci. Rep. 6, 28668.
UniProt Consortium, T. (2018). UniProt: the universal protein knowledgebase. Nucleic Acids Res. 46, 2699.
Verhaak, R.G.W., Hoadley, K.A., Purdom, E„ Wang, V., Qi, Y., Wilkerson, M.D., Miller, C.R., Ding, L., Golub, T., Mesirov, J.P., et al. (2010). Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterised by abnormalities in PDGFRA, IDH1 , EGFR, and NF1. Cancer Cell 17, 98- 1 10.
Wang, M., and Yuang-Chi Chang, A. (2018). Molecular mechanism of action and potential biomarkers of growth inhibition of synergistic combination of afatinib and dasatinib against gefitinib-resistant non-small cell lung cancer cells. Oncotarget 9, 16533-16546.
West, A.J., Tsui, V., Stylli, S.S., Nguyen, H.P.T., Morokoff, A.P., Kaye, A.H., and Luwor, R.B. (2018). The role of interleukin-6-STAT3 signalling in glioblastoma. Oncol. Lett. 16, 4095-4104.
9.
Zhang, H., Tang, Y., Zhang, Y., Zhang, S., Qu, J., Wang, X., Kong, R., Han, C., and Liu, Z. (2015). Fucoxanthin: A promising medicinal and nutritional ingredient. Evid. Based Complement. Alternat. Med. 2015, 723515.
Zhao, H.-F., Wang, J., Shao, W„ Wu, C.-P., Chen, Z.-P., To, S.-S.T., and Li, W.-P. (2017). Recent advances in the use of PI3K inhibitors for glioblastoma multiforme: current preclinical and clinical development. Mol. Cancer 16, 100.
Zhou, X., Zhao, X., Li, X., Ping, G„ Pei, S., Chen, M., Wang, Z., Zhou, W„ and Jin, B. (2016). PQ401 , an IGF-1 R inhibitor, induces apoptosis and inhibits growth, proliferation and migration of glioma cells. J. Chemother. 28, 44-49.

Claims

Claims:
1 . A method of identifying druggable targets for cancer combination therapy, comprising the steps of: a. contacting test cells with a first compound,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is
perturbed in the transcriptomic profile,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
2. The method of claim 1 , further comprising the steps of:
d. selecting an inhibitor of the druggable target for combination therapy as a second compound e. contacting test cells with the second compound, optionally the first and second compound, f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
g. selecting as a further druggable target for combination therapy a gene whose expression is perturbed in the second transcriptomic profile and is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radiosensitisation.
3. A method of providing an anti-cancer therapeutic combination comprising at least a first and a second compound, the method comprising:
a. contacting test cells with the first compound,
b. measuring the effects of the first compound on the transcriptome of the test cells, so as to create a transcriptomic profile for the first compound,
c. selecting as a druggable target for combination therapy a gene whose expression is
perturbed in the transcriptomic profile,
d. selecting as the second compound an inhibitor of the druggable target for combination
therapy,
wherein the gene is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
4. The method according to claim 3, wherein the combination further comprises a third compound, comprising the further steps of:
e. contacting test cells with the second compound, and optionally the first compound f. measuring the effects of the second, or first and second, compound on the transcriptome of the test cells, so as to create a second transcriptomic profile,
g. selecting as a second druggable target for combination therapy a gene whose expression is perturbed in the second transcriptomic profile and is implicated in the regulation of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radiosensitisation, and
h. selecting an inhibitor of the second druggable target as a third compound for the combination therapy.
5. The method according to any of claims 1 to 4, wherein the test cells are contacted with a first
compound at a concentration equal to or below its anti-proliferative EC50.
6. The method according to any of claims 1 to 5, wherein step b) comprises measuring the effects of the first compound on the transcriptome of the test cells at a first and a second time-point, so as to create a transcriptomic profile for the first compound.
7. The method according to claim 6, wherein step c) comprises selecting as a druggable target for combination therapy a gene whose expression is perturbed in the transcriptomic profile in both the first and second time-points.
8. The method according to any of claims 1 to 7, wherein the gene whose expression is perturbed is upregulated in the transcriptomic profile.
9. The method according to claim 8, wherein the gene is a positive regulator of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
10. The method according to any of claims 1 to 7, wherein the gene whose expression is perturbed is down-regulated in the transcriptomic profile.
1 1. The method according to claim 10, wherein the gene is a negative regulator of one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation.
12. The method according to any of claims 1 to 11 , wherein the first compound inhibits one or more of cell proliferation, viability, migration, invasion and angiogenesis, or promotes apoptosis or radiosensitisation.
13. The method according to any of claims 1 to 12, wherein the first compound is cytotoxic.
14. The method according to any of claims 1 to 13, wherein the first compound is an anticancer agent.
15. The method according to claim 1 to 14, wherein the first compound is an inhibitor of PI3K signalling, ferably 2-Morpholin-4-yl-8-phenylchromen-4-one (LY-294002) or an inhibitor of PI3K which has similar chemical and biological properties to LY-294002.
16. The method according to any of claims 1 to 15, wherein the test cells are cancer cells.
17. The method according to any of claims 1 to 16, wherein the test cells are glioblastoma multiforme cells, and the cancer is glioblastoma.
18. The method according to claim 17, wherein the test cells are glioblastoma cells derived from a patient for whom the cancer combination therapy is intended.
19. A therapeutic combination provided by a method according to claim 3, or according to claims 4 to 18 as dependent on claim 3.
20. The therapeutic combination according to claim 19, for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of administering the first compound simultaneously or sequentially with the second compound.
21 . Use of the therapeutic combination according to claim 19 in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament administers an effective amount of the first compound simultaneously or sequentially with an effective amount of the second compound to a subject in need thereof.
22. A method of treating or preventing cancer with the therapeutic combination according to claim 19, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of the first compound and an effective amount of second compound.
23. An inhibitor of phosphoinositide 3-kinase (PI3K) signalling for use in a method of treating or
preventing cancer in a subject, wherein the method comprises the step of administering the inhibitor of PI3K signalling simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
24. Use of an inhibitor of phosphoinositide 3-kinase (PI3K) signalling in the manufacture of a medicament for treating or preventing cancer in a subject, wherein the medicament is administered simultaneously or sequentially with an inhibitor of Janus kinase (JAK) activity or function.
25. A method of treating or preventing cancer, the method comprising administering simultaneously or sequentially to a subject in need thereof an effective amount of an inhibitor of phosphoinositide 3- kinase (PI3K) signalling and an inhibitor of Janus kinase (JAK) activity or function.
26. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 25, wherein the ibitor of PI3K signalling is a non-selective pan-PI3K inhibitor.
27. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 26, wherein the Inhibitor of PI3K signalling interacts with PI3K and inhibits PI3K activity or function.
28. The inhibitor of PI3K signalling, use, or method according to claim 27, wherein the inhibitor of PI3K signalling interacts with and inhibits the activity or function of PI3K class I, PI3K class II, and/or PI4K.
29. The inhibitor of PI3K signalling, use, or method according to claim 27, wherein the inhibitor of PI3K signalling interacts with and inhibits the activity or function of one or more PI3K selected from PIK3CA/p110a, PIK3CB/p110b and/or PIK3CD/p110d.
30. The inhibitor of PI3K signalling, use, or method according to any of claims 27 to 29, wherein the inhibitor of PI3K signalling additionally interacts with and inhibits the activity or function of one or more of Casein Kinase 2 (CK2) and/or Myosin Light-chain Kinase (MLCK).
31. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 30, wherein the inhibitor of PI3K signalling is 2-Morpholin-4-yl-8-phenylchromen-4-one (LY-294002), or an inhibitor of PI3K which has similar chemical and biological properties to LY-294002.
32. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 31 , wherein the inhibitor of JAK activity or function is an inhibitor of JAK2 activity or function.
33. The inhibitor of PI3K signalling, use, or method according to claim 32, wherein the inhibitor of JAK activity or function is an ATP-competitive inhibitor of JAK2 activity or function.
34. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 33, wherein the inhibitor of JAK activity or function is a Type I protein kinase inhibitor.
35. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 34, wherein the inhibitor of JAK activity or function is 5-Chloro-N2-[(1 S)-1-(5-fluoro-2-pyrimidinyl)ethyl]-N4-(5-methyl- 1 H-pyrazol-3-yl)-2,4-pyrimidine-2, 4-diamine (AZD1480), or an inhibitor of JAK activity or function which has similar chemical and biological properties to AZD1480.
36. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 35, wherein the cancer is a cancer of the brain.
37. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 36, wherein the cancer is a primary brain tumour.
38. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 37, wherein the icer is a glioma.
39. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 38, wherein the cancer is glioblastoma multiforme.
40. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 39, wherein the method comprises the additional step of administering an inhibitor of Insulin Like Growth Factor 1 Receptor (IGF1 R) activity or function and/or an inhibitor of Serine/threonine-protein kinase 1 (SGK1) activity or function.
41. A pharmaceutical composition comprising an inhibitor of phosphoinositide 3-kinase (PI3K) signalling, an inhibitor of Janus kinase (JAK) activity or function, and a pharmaceutically acceptable excipient.
42. A kit comprising (i) an inhibitor of phosphoinositide 3-kinase (PI3K) signalling and (ii) an inhibitor of Janus kinase (JAK) activity or function.
43. The therapeutic combination according to claim 20, the use according to claim 21 , or the method according to claim 22, wherein the method comprises the steps of (i) administering an effective amount of the first compound, and (ii) administering an effective amount of the second compound simultaneously with an amount of the first compound, wherein the amount of the first compound administered in step (i) is larger than that administered in step (ii).
44. The inhibitor of PI3K signalling, use, or method according to any of claims 23 to 40, wherein the
method comprises the steps of (i) administering an effective amount of the inhibitor of PI3K signalling, and (ii) administering an effective amount of the inhibitor of JAK activity or function simultaneously with an amount of the inhibitor of PI3K signalling, wherein the amount of the inhibitor of PI3K signalling administered in step (i) is larger than that administered in step (ii).
45. The method according to claim 2 or claim 3, wherein step d comprises the sub-steps of:
i) identifying a candidate inhibitor of the druggable target,
ii) contacting test cells with the candidate inhibitor in combination with the first compound, and iii) selecting the candidate inhibitor as the second compound if it exhibits a synergistic effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation when provided in combination with the first compound.
46. The method according to claim 2 or claim 3, wherein step d comprises the sub-steps of:
i) identifying a candidate inhibitor of the druggable target,
ii) contacting test cells with the candidate inhibitor in combination with the first compound, iii) measuring the effects of the combination on the transcriptome of the test cells, so as to create a transcriptomic profile for the combination, if the combination exhibits a synergistic effect on one or more of cell proliferation, viability, migration, invasion and angiogenesis, apoptosis or radio-sensitisation,
iv) providing or developing an inhibitor with the same or similar transcriptomic or inhibitory profile as the candidate inhibitor, and
v) selecting the inhibitor provided or developed by sub-step iv) as the second compound.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015109286A1 (en) * 2014-01-20 2015-07-23 Gilead Sciences, Inc. Therapies for treating cancers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015109286A1 (en) * 2014-01-20 2015-07-23 Gilead Sciences, Inc. Therapies for treating cancers

Non-Patent Citations (79)

* Cited by examiner, † Cited by third party
Title
"Cancer Genome Atlas Research Network", vol. 455, 2008, article "Comprehensive genomic characterisation defines human glioblastoma genes and core pathways. Nature", pages: 1061 - 1068
"The 2016 World Health Organisation Classification of Tumours of the Central Nervous System: a summary", ACTA NEUROPATHOLOGICA., vol. 131, no. 6, pages 803 - 820
ALEXANDER-DANN, B.PRUTEANU, L.L.OERTON, E.SHARMA, N.BERINDAN-NEAGOE, I.MODES, D.BENDER, A.: "Developments in toxicogenomics: understanding and predicting compound-induced toxicity from gene expression data", MOL. OMICS, vol. 14, 2018, pages 218 - 236
BABCOCK, J.J.DU, F.XU, K.WHEELAN, S.J.LI, M.: "Integrated analysis of drug-induced gene expression profiles predicts novel hERG inhibitors", PLOS ONE, vol. 8, 2013, pages e69513
BASNET, R.GONG, G.Q.LI, C.WANG, M.-W.: "Serum and glucocorticoid inducible protein kinases (SGKs): a potential target for cancer intervention", ACTA PHARM. SIN. B, vol. 8, 2018, pages 767 - 771
BATASH, R.ASNA, N.SCHAFFER, P.FRANCIS, N.SCHAFFER, M.: "Glioblastoma multiforme, diagnosis and treatment; recent literature review", CURR. MED. CHEM., vol. 24, 2017, pages 3002 - 3009
CHEN, L.HAN, L.SHI, Z.ZHANG, K.LIU, Y.ZHENG, Y.JIANG, T.PU, P.JIANG, C.KANG, C.: "LY294002 enhances cytotoxicity of temozolomide in glioma by down-regulation of the PI3K/Akt pathway", MOL. MED. REPORT., vol. 5, 2012, pages 575 - 579
CLOUGHESY, T.F.CAVENEE, W.K.MISCHEL, P.S.: "Glioblastoma: from molecular pathology to targeted treatment", ANNU. REV. PATHOL., vol. 9, 2014, pages 1 - 25
CORY, A.H.SOMERVILLE, L.CORY, J.G.: "Blockage of cyclin cdk's, PKC and phosphoinositol 3-kinase pathways leads to augmentation of apoptosis in drug-resistant leukemia cells: evidence for interactive effects of flavopiridol, LY 294002, roscovitine,wortmannin and UCN-01", ANTICANCER RES., vol. 25, 2005, pages 101 - 106
DAVIES, S.P.REDDY, H.CAIVANO, M.COHEN, P.: "Specificity and mechanism of action of some commonly used protein kinase inhibitors", BIOCHEM. J., vol. 351, 2000, pages 95 - 105, XP000979108, DOI: 10.1042/0264-6021:3510095
DEMBITSKY, V.M.MAOKA, T.: "Allenic and cumulenic lipids", PROG. LIPID RES., vol. 46, 2007, pages 328 - 375, XP022277214, DOI: 10.1016/j.plipres.2007.07.001
DING, J.CHENG, X.-Y.LIU, S.JI, H.-Y.LIN, M.MA, R.MENG, F.-L.: "Apatinib exerts antitumour effects on ovarian cancer cells", GYNECOL. ONCOL., vol. 153, 2019, pages 165 - 174, XP085639586, DOI: 10.1016/j.ygyno.2019.01.010
ENGSTROM, P.G.TOMMEI, D.STRICKER, S.H.ENDER, C.POLLARD, S.M.BERTONE, P.: "Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival", GENOME MED., vol. 4, 2012, pages 76, XP021129498, DOI: 10.1186/gm377
GALASSO, C.CORINALDESI, C.SANSONE, C.: "Carotenoids from Marine Organisms: Biological Functions and Industrial Applications", ANTIOXIDANTS (BASEL, 2017, pages 6
GARIBOLDI, M.B.RAVIZZA, R.MONTI, E.: "The IGFR1 inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells", BIOCHEM. PHARMACOL., vol. 80, 2010, pages 455 - 462, XP027092938
GHARBI, S.I.ZVELEBIL, M.J.SHUTTLEWORTH, S.J.HANCOX, T.SAGHIR, N.TIMMS, J.F.WATERFIELD, M.D.: "Exploring the specificity of the PI3K family inhibitor LY294002", BIOCHEM. J., vol. 404, 2007, pages 15 - 21, XP055076776, DOI: 10.1042/BJ20061489
GHOSH, M.K.SHARMA, P.HARBOR, P.C.RAHAMAN, S.O.HAQUE, S.J.: "PI3K-AKT pathway negatively controls EGFR-dependent DNA-binding activity of Stat3 in glioblastoma multiforme cells", ONCOGENE, vol. 24, 2005, pages 7290 - 7300, XP055586108, DOI: 10.1038/sj.onc.1208894
HEMMINGS, B. A.RESTUCCIA, D. F., COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY, vol. 4, no. 9, pages a011189
HOCHBERG, B.: "Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing", JOURNAL OF THE ROYAL STATISTICAL SOCIETY. SERIES B (METHODOLOGICAL, vol. 57, 1995, pages 289 - 300
IRIZARRY, R.A.HOBBS, B.COLLIN, F.BEAZER-BARCLAY, Y.D.ANTONELLIS, K.J.SCHERF, U.SPEED, T.P.: "Exploration, normalisation, and summaries of high density oligonucleotide array probe level data", BIOSTATISTICS, vol. 4, 2003, pages 249 - 264, XP002466228, DOI: 10.1093/biostatistics/4.2.249
IWATA, M.YAMANISHI, Y.: "The Use of Large-Scale Chemically-Induced Transcriptome Data Acquired from LINCS to Study Small Molecules", METHODS MOL. BIOL., vol. 1888, 2019, pages 189 - 203
JACOBS, M.D.BLACK, J.FUTER, O.SWENSON, L.HARE, B.FLEMING, M.SAXENA, K.: "Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002", J. BIOL. CHEM., vol. 280, 2005, pages 13728 - 13734
JEANKIGER: "Classes of phosphoinositide 3-kinases at a glance", JOURNAL OF CELL SCIENCE, vol. 127, no. 5, 2014, pages 923 - 928
JIN, X.KIM, S.-H.JEON, H.-M.BECK, S.SOHN, Y.-W.YIN, J.KIM, J.-K.LIM, Y.C.LEE, J.-H.KIM, S.-H. ET AL.: "Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling", BRAIN, vol. 135, 2012, pages 1055 - 1069
KANNO, H.SATO, H.YOKOYAMA, T.-A.YOSHIZUMI, T.YAMADA, S.: "The VHL tumour suppressor protein regulates tumourigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signalling pathway", INT. J. ONCOL., vol. 42, 2013, pages 881 - 886
KNOBBE, C.B.REIFENBERGER, G.: "Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas", BRAIN PATHOL., vol. 13, 2003, pages 507 - 518, XP002690127
KOHL, M.DEIGNER, H.-P.: "Preprocessing of gene expression data by optimally robust estimators", BMC BIOINFORMATICS, vol. 11, 2010, pages 583, XP021085857, DOI: 10.1186/1471-2105-11-583
KONG, D.YAMORI, T.: "Phosphatidylinositol 3-kinase inhibitors: promising drug candidates for cancer therapy", CANCER SCI., vol. 99, 2008, pages 1734 - 1740, XP055323420, DOI: 10.1111/j.1349-7006.2008.00891.x
KULKARNI, S.GOEL-BHATTACHARYA, S.SENGUPTA, S.COCHRAN, B.H.: "A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells", MOL. CANCER RES., vol. 16, 2018, pages 103 - 114
KUTMON, M.RIUTTA, A.NUNES, N.HANSPERS, K.WILLIGHAGEN, E.L.BOHLER, A.MELIUS, J.WAAGMEESTER, A.SINHA, S.R.MILLER, R. ET AL.: "WikiPathways: capturing the full diversity of pathway knowledge", NUCLEIC ACIDS RES., vol. 44, 2016, pages D488 - 94
LAMB, J.CRAWFORD, E.D.PECK, D.MODELL, J.W.BLAT, I.C.WROBEL, M.J.LERNER, J.BRUNET, J.-P.SUBRAMANIAN, A.ROSS, K.N. ET AL.: "The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease", SCIENCE, vol. 313, 2006, pages 1929 - 1935, XP055537824, DOI: 10.1126/science.1132939
LAVINIA-LORENA PRUTEANU ET AL: "Transcriptomics predicts compound synergy in drug and natural product treated glioblastoma cells", PLOS ONE, vol. 15, no. 9, 18 September 2020 (2020-09-18), pages e0239551, XP055737871, DOI: 10.1371/journal.pone.0239551 *
LIEBERMAN, PHARMACEUTICAL DOSAGE FORMS, vol. 1-3, 1992
LIU, Y.WAN, W.-Z.LI, Y.ZHOU, G.-L.LIU, X.-G.: "Recent development of ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents", ONCOTARGET, vol. 8, 2017, pages 7181 - 7200
LIU, Y.ZHENG, J.ZHANG, Y.WANG, Z.YANG, Y.BAI, M.DAI, Y.: "Fucoxanthin Activates Apoptosis via Inhibition of PI3K/Akt/mTOR Pathway and Suppresses Invasion and Migration by Restriction of p38-MMP-2/9 Pathway in Human Glioblastoma Cells", NEUROCHEM. RES., vol. 41, 2016, pages 2728 - 2751, XP036076804, DOI: 10.1007/s11064-016-1989-7
LLOYD, THE ART, SCIENCE AND TECHNOLOGY OF PHARMACEUTICAL COMPOUNDING, 1999
LORIO, F.BOSOTTI, R.SCACHERI, E.BELCASTRO, V.MITHBAOKAR, P.FERRIERO, R.MURINO, L.TAGLIAFERRI, R.BRUNETTI-PIERRI, N.ISACCHI, A. ET : "Discovery of drug mode of action and drug repositioning from transcriptional responses", PROC NATL ACAD SCI USA, vol. 107, 2010, pages 14621 - 14626, XP055057022, DOI: 10.1073/pnas.1000138107
MARTIN, L.J.: "Fucoxanthin and its metabolite fucoxanthinol in cancer prevention and treatment", MAR. DRUGS, vol. 13, 2015, pages 4784 - 4798
MBAVENG, A.T.DAMEN, F.QELIK, I.TANE, P.KUETE, V.EFFERTH, T.: "Cytotoxicity of the crude extract and constituents of the bark of Fagara tessmannii towards multi-factorial drug resistant cancer cells", J. ETHNOPHARMACOL., vol. 235, 2019, pages 28 - 37
MENDELSOHN, J.: "Personalising oncology: perspectives and prospects", J. CLIN. ONCOL., vol. 31, 2013, pages 1904 - 1911
MERVIN, L.H.AFZAL, A.M.DRAKAKIS, G.LEWIS, R.ENGKVIST, O.BENDER, A.: "Target prediction utilising negative bioactivity data covering large chemical space", J. CHEMINFORM., vol. 7, 2015, pages 51, XP021230997, DOI: 10.1186/s13321-015-0098-y
OERTON, E.BENDER, A.: "Concordance analysis of microarray studies identifies representative gene expression changes in Parkinson's disease: a comparison of 33 human and animal studies", BMC NEUROL., vol. 17, 2017, pages 58
OSTROM, Q.T.GITTLEMAN, H.LIAO, P.VECCHIONE-KOVAL, T.WOLINSKY, Y.KRUCHKO, C.BARNHOLTZ-SLOAN, J.S.: "CBTRUS Statistical Report: Primary brain and other central nervous system tumours diagnosed in the United States in 2010-2014", NEURO ONCOL., vol. 19, 2017, pages v1 - v88
PANOSSIAN, A.SEO, E.-J.EFFERTH, T.: "Novel molecular mechanisms for the adaptogenic effects of herbal extracts on isolated brain cells using systems biology", PHYTOMEDICINE, vol. 50, 2018, pages 257 - 284, XP085539089, DOI: 10.1016/j.phymed.2018.09.204
PEARSON, J.R.D.REGAD, T.: "Targeting cellular pathways in glioblastoma multiforme", SIGNAL TRANSDUCT. TARGET. THER., vol. 2, 2017, pages 17040
PENG, H.DU, B.JIANG, H.GAO, J.: "Over-expression of CHAF1A promotes cell proliferation and apoptosis resistance in glioblastoma cells via AKT/FOX03a/Bim pathway", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 469, 2016, pages 1111 - 1116, XP029390086, DOI: 10.1016/j.bbrc.2015.12.111
PHIPSON, B.LEE, S.MAJEWSKI, I.J.ALEXANDER, W.S.SMYTH, G.K.: "Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression", ANN. APPL. STAT., vol. 10, 2016, pages 946 - 963
PICKAR, DOSAGE CALCULATIONS, 1999
PRIDHAM, K.J.VARGHESE, R.T.SHENG, Z.: "The Role of Class IA Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunits in Glioblastoma", FRONT. ONCOL., vol. 7, 2017, pages 312
QIU, B.ZHANG, D.WANG, Y.OU, S.WANG, J.TAO, J.WANG, Y.: "Interleukin-6 is overexpressed and augments invasiveness of human glioma stem cells in vitro", CLIN. EXP. METASTASIS, vol. 30, 2013, pages 1009 - 1018
RANE, S.G.REDDY, E.P.: "Janus kinases: components of multiple signalling pathways", ONCOGENE, vol. 19, 2000, pages 5662 - 5679
RAWLINGS ET AL., JOURNAL OF CELL SCIENCE, vol. 117, 2004, pages 1281 - 1283
RITCHIE, M.E.PHIPSON, B.WU, D.HU, Y.LAW, C.W.SHI, W.SMYTH, G.K.: "limma powers differential expression analyses for RNA-sequencing and microarray studies", NUCLEIC ACIDS RES., vol. 43, 2015, pages e47
ROSKOSKI, PHARMACOL RES., vol. 111, September 2016 (2016-09-01), pages 784 - 803
ROTH, W.GRIMMEL, C.RIEGER, L.STRIK, H.TAKAYAMA, S.KRAJEWSKI, S.MEYERMANN, R.DICHGANS, J.REED, J.C.WELLER, M.: "Bag-1 and Bcl-2 gene transfer in malignant glioma: modulation of cell cycle regulation and apoptosis", BRAIN PATHOL., vol. 10, 2000, pages 223 - 234
SAMBROOK ET AL.: "MOLECULAR CLONING, A LABORATORY MANUAL", 1989, COLD SPRINGS HARBOR PRESS
SAMBROOK, J.RUSSEL, D.W.: "Molecular Cloning, A Laboratory Manual.", 2001, COLD SPRING HARBOR
SANGEETHA, R.K.BHASKAR, N.BASKARAN, V.: "Comparative effects of beta-carotene and fucoxanthin on retinol deficiency induced oxidative stress in rats", MOL. CELL. BIOCHEM., vol. 331, 2009, pages 59 - 67, XP019751914, DOI: 10.1007/s11010-009-0145-y
SATOMI, Y.: "Antitumour and Cancer-preventative Function of Fucoxanthin: A Marine Carotenoid", ANTICANCER RES., vol. 37, 2017, pages 1557 - 1562
SINGLETON ET AL.: "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY", 1994, J. WILEY & 20 SONS
SMALLEY, J.L.GANT, T.W.ZHANG, S.-D.: "Application of connectivity mapping in predictive toxicology based on gene-expression similarity", TOXICOLOGY, vol. 268, 2010, pages 143 - 146, XP026913566, DOI: 10.1016/j.tox.2009.09.014
STUPP, R.MASON, W.P.VAN DEN BENT, M.J.WELLER, M.FISHER, B.TAPHOORN, M.J.B.BELANGER, K.BRANDES, A.A.MAROSI, C.BOGDAHN, U. ET AL.: "Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma", N. ENGL. J. MED., vol. 352, 2005, pages 987 - 996
SUBRAMANIAN, A.NARAYAN, R.CORSELLO, S.M.PECK, D.D.NATOLI, T.E.LU, X.GOULD, J.DAVIS, J.F.TUBELLI, A.A.ASIEDU, J.K. ET AL.: "A next generation connectivity map: L1000 platform and the first 1,000,000 profiles", BIORXIV, 2017
SUBRAMANIAN, A.NARAYAN, R.CORSELLO, S.M.PECK, D.D.NATOLI, T.E.LU, X.GOULD, J.DAVIS, J.F.TUBELLI, A.A.ASIEDU, J.K. ET AL.: "A next generation connectivity map: L1000 platform and the first 1,000,000 profiles", CELL, vol. 171, 2017, pages 1437 - 1452.e17
SUMI NATALIA J ET AL: "Divergent Polypharmacology-Driven Cellular Activity of Structurally Similar Multi-Kinase Inhibitors through Cumulative Effects on Individual Targets", CELL CHEMICAL BIOLOGY , ELSEVIER, AMSTERDAM, NL, vol. 26, no. 9, 27 June 2019 (2019-06-27), pages 1240, XP085836310, ISSN: 2451-9456, [retrieved on 20190627], DOI: 10.1016/J.CHEMBIOL.2019.06.003 *
SVENSSON, F.WESTERMAN, B.WURDINGER, T.BAILEY, D.: "GBM Drug Bank-a new resource for glioblastoma drug discovery and informatics research", NEURO ONCOL., vol. 20, 2018, pages 1680 - 1681
SYDOW, D.BURGGRAAFF, L.SZENGEL, A.VAN VLIJMEN, H.W.T.IJZERMAN, A.P.VAN WESTEN, G.J.P.VOLKAMER, A.: "Advances and challenges in computational target prediction", J. CHEM. INF. MODEL., vol. 59, 2019, pages 1728 - 1742
SZYMARISKA, J.SMOLEWSKI, P.MAJCHRZAK, A.CEBULA-OBRZUT, B.CHOJNOWSKI, K.TRELIRISKI, J.: "Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines", ADV. CLIN. EXP. MED., vol. 24, 2015, pages 195 - 202
TASIAN, S.K.TEACHEY, D.T.LI, Y.SHEN, F.HARVEY, R.C.CHEN, I.-M.RYAN, T.VINCENT, T.L.WILLMAN, C.L.PERL, A.E. ET AL.: "Potent efficacy of combined PI3K/mTOR and JAK or ABL inhibition in murine xenograft models of Ph-like acute lymphoblastic leukemia", BLOOD, vol. 129, 2017, pages 177 - 187
TERASAKI, M.MIMA, M.KUDOH, S.ENDO, T.MAEDA, H.HAMADA, J.OSADA, K.MIYASHITA, K.MUTOH, M.: "Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells", ONCOL. REP., vol. 40, 2018, pages 414 - 424
TUKEY, J.W.: "The Estimators of the Princeton Robustness Study", 1973, DEPARTMENT OF STATISTICS, UNIV.
TUNCBAG, N.MILANI, P.POKORNY, J.L.JOHNSON, H.SIO, T.T.DALIN, S.LYEKEGBE, D.O.WHITE, F.M.SARKARIA, J.N.FRAENKEL, E.: "Network Modeling Identifies Patient-specific Pathways in Glioblastoma", SCI. REP., vol. 6, 2016, pages 28668
UNIPROT CONSORTIUM, T.: "UniProt: the universal protein knowledgebase", NUCLEIC ACIDS RES., vol. 46, 2018, pages 2699
VERHAAK, R.G.W.HOADLEY, K.A.PURDOM, E.WANG, V.QI, Y.WILKERSON, M.D.MILLER, C.R.DING, L.GOLUB, T.MESIROV, J.P. ET AL.: "Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterised by abnormalities in PDGFRA, IDH1, EGFR, and NF1", CANCER CELL, vol. 17, 2010, pages 98 - 110, XP055219370, DOI: 10.1016/j.ccr.2009.12.020
WANG, M.YUANG-CHI CHANG, A.: "Molecular mechanism of action and potential biomarkers of growth inhibition of synergistic combination of afatinib and dasatinib against gefitinib-resistant non-small cell lung cancer cells", ONCOTARGET, vol. 9, 2018, pages 16533 - 16546
WEST, A.J.TSUI, V.STYLLI, S.S.NGUYEN, H.P.T.MOROKOFF, A.P.KAYE, A.H.LUWOR, R.B.: "The role of interleukin-6-STAT3 signalling in glioblastoma", ONCOL. LETT., vol. 16, 2018, pages 4095 - 4104
ZHANG, H.TANG, Y.ZHANG, Y.ZHANG, S.QU, J.WANG, X.KONG, R.HAN, C.LIU, Z.: "Fucoxanthin: A promising medicinal and nutritional ingredient", EVID. BASED COMPLEMENT. ALTERNAT. MED., 2015, pages 723515
ZHAO, H.-F.WANG, J.SHAO, W.WU, C.-P.CHEN, Z.-P.TO, S.-S.T.LI, W.-P.: "Recent advances in the use of PI3K inhibitors for glioblastoma multiforme: current preclinical and clinical development", MOL. CANCER, vol. 16, 2017, pages 100
ZHOU, X.ZHAO, X.LI, X.PING, G.PEI, S.CHEN, M.WANG, Z.ZHOU, W.JIN, B.: "PQ401, an IGF-1 R inhibitor, induces apoptosis and inhibits growth, proliferation and migration of glioma cells", J. CHEMOTHER., vol. 28, 2016, pages 44 - 49

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