Activators of oncogenic pathways to reduce non-cancer cells with cancer mutations in order to prevent development of cancer

Abstract

This patent application relates to methods of reducing a risk of developing cancer in an individual, comprising providing the individual with an inhibitor of Protein Phosphatase 2A (PP2A), either alone or in combination with a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor and / or a GSK3β inhibitor.

Description

[0001] ACTIVATORS OF ONCOGENIC PATHWAYS TO REDUCE NON-CANCER CELLS WITH CANCER MUTATIONS IN ORDER TO PREVENT DEVELOPMENT OF CANCER

[0002] FIELD OF THE INVENTION

[0003] This disclosure relates to the use of a compound or a pharmaceutically acceptable salt of the compound for prophylactic treatment of an individual.

[0004] BACKGROUND

[0005] Cancer cells differ from normal cells in that the former have mutations that activate mitogenic signaling and this creates a therapeutic window for cancer cells to selectively respond to agents that further activate mitogenic signaling. However, many cells in an aged human body carry cancer-causing mutations in apparently normal epithelia (Martincorena et al., 2018. Science 362: 911-917), suggesting that cancer initiating mutations may not be the rate limiting step in human carcinogenesis, but rather the promotion of initiated cells by other agents. This is supported by recent studies on the promotion of lung cancer in mice carrying lung cancer-predisposing EGFR mutations by air pollutants (Hill et al., 2023. Nature 616: 159-167).

[0006] Hyper- activation of oncogenic signaling could be more detrimental to cancer cells than normal cells. Normal cells are more resistant to perturbations in signaling, due to the many feedback and cross talk loops that operate in normal cells to maintain homeostasis. Supporting this notion, transgenic mouse models overexpressing growth factors (Zhang et al., 2012. Elife 1: e00065; Santoni-Rugiu et al., 1996. Proc Natl Acad Sci U S A 93: 9577-9582; Coffin et al., 1995. Mol Biol Cell 6: 1861-1873; Chan and Wong, R, 2000. J Biol Chem 275: 38693-38698, or displaying tonic ERK1 / 2 activation due to DUSP6 knockout (Maillet et al., 2008. J Biol Chem 283: 31246-31255) do not show increased tumorigenesis, when compared to normal mice.

[0007] Reinforcement of mitogenic signaling may exploit a fundamental defect of cancer cells and in doing so creates a window for therapeutic intervention. For example, there is epidemiological evidence that long-term treatment with the GSK3β inhibitor lithium for bipolar disorder decreases cancer risk rather than increasing it, arguing that chronic pharmacological WNT activation may not leadto cancer in humans (Ge and Jakobsson, 2019. Frontiers Oncol 9: 296; Cohen et al., 1998. Med Oncol 15, 32-36).

[0008] It may be concluded that cells that carry cancer-initiating mutations could potentially be more sensitive to agents that activate mitogenic signaling than their normal counterparts, as they resemble cancer cells in some aspects of their genotype. There is thus a need to study how drugs that hyperactivate oncogenic signaling affect normal cells, when compared to cells carrying such cancer-initiating mutations.

[0009] SUMMARY OF THE INVENTION

[0010] Protein Phosphatase 2A (PP2A) has been recognized as a potentially important target for cancer therapy because of its regulatory role in cell division, DNA damage response, homologous recombination repair, and mitotic exit.

[0011] However, inhibition of PP2A has long been considered likely too toxic for clinical use. Surprisingly, recent clinical experience with an inhibitor of PP2A, LB- 100, was found to be favorable in terms of toxicity (Chung et al., 2017. Clin Cancer Res 23: 3277-3284). In addition, cells that carry mutations that are normally found only in cancer cells, hereinafter termed “initiated cells”, may be eliminated by stimulation of an oncogenic pathway, thereby hyperactivating said initiated cells.

[0012] In embodiments, the individual is provided with an inhibitor of Protein Phosphatase 2A (PP2A), a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor, a GSK3β inhibitor, an activator of PIK3CA, and / or activator of RAS in order to hyperactivate, and thereby eliminate, initiated cells.

[0013] In embodiments, the individual has cells with one or more mutations in one of three RAS genes, (HRAS, KRAS, NRAS), HER2, EGFR, PDGFR, VEGFR, BCR / ABL1, MYC, MYCN, EML4-ALK, BRAF, PTEN, CTNNB, PIK3CA, MUC16, APC, TP53, BRCA1, BRCA2, SRC family kinase, ABL, SYK-ZAP-70 family kinase, and the BTK family of tyrosine kinases. Said individual is not known to have cancer. The stimulation of an oncogenic pathway to hyperactivate and eliminate initiated cells is to reduce a risk of developing cancer in said individual, and / or to prevent the development of a cancer in said individual.The invention provides a method for reducing a risk of developing cancer in an individual, comprising providing the individual with an inhibitor of Protein Phosphatase 2A (PP2A). Said the inhibitor of PP2A may help in eliminating noncancer cells that carry mutations found in cancer cells, hereinafter termed “initiated cells”. In embodiments, the PP2A inhibitor is selected from cantharidin, norcantharidin, fostriecin, a compound with formula I:

[0014]

[0015] o

[0016] Formula I

[0017] or a pharmaceutically acceptable salt thereof.

[0018] In embodiments, the individual is provided with an inhibitor of PP2A, in combination with one or more other active ingredients. Said one or more other active ingredients may be selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor and / or a GSK3β inhibitor, an activator of PIK3CA or activator of RAS.

[0019] In embodiments, the PP2A inhibitor is LB- 100. In embodiments, LB- 100 is provided to the individual at a dose of 0.1 mg / m2to 3.5 mg / m2. In embodiments, LB- 100 is provided to the individual at a daily dose. In embodiments, LB- 100 is provided intravenously to the individual.

[0020] In embodiments, the individual is provided with LB- 100, in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor and / or a GSK3β inhibitor.

[0021] In embodiments, the WEE1 kinase inhibitor is selected from adavosertib (AZD1775, MK1755), PD407824, ZN-c3 (azenosertib), Debio-0123, SY-4835, ACR-2316, SDGR2, NUV-569 or IMP7086. In embodiments, the WEE1 kinase inhibitor is provided to the individual at a dose of 20 mg to 400 mg.In embodiments, a checkpoint kinase 1 inhibitor is selected from GDC-0575, prexasertib, rabusertib, SCH-900776, CCT-245737, AZD-7762, PF-477736, GDC-0425, or SRA737. In embodiments, a checkpoint kinase 1 inhibitor is provided to the individual at a dose of 1 mg to about 600 mg.

[0022] In embodiments, a protein kinase C activator such as prostratin is provided at a dose of 0.01-10 mg / kg.

[0023] In embodiments, a DUSP1 / 6 inhibitor is BCI, also termed (E / Z)-BCI.

[0024] In embodiments, a GSK3β inhibitor is selected from lithium, AZD1080, LY2090314, tideglusib, BRD3731, or an isoorientin analog.

[0025] In embodiments, the PP2A inhibitor reduces a risk of the individual for developing colorectal cancer, pancreatic cancer, breast cancer or ovarian cancer.

[0026] LEGENDS TO THE FIGURES

[0027] Figure 1. In vivo effect of paradoxical activation of oncogenic signaling on adenoma formation and progression to adenocarcinoma. (A) Treatment of Lgr5-CreERT2; Apcfl / fl mice with lithium chloride to activate WNT signaling 7 days prior to Ape deletion leads to less adenoma formation. (B) Macroscopic images of adenomas in the small intestine with and without lithium treatment. (C) Quantification of data shown in B. (D) Schedule of drug treatment in relation to start of adenoma induction (TAM Day 0). All animals will be sacrificed at day 60 (D). Drug treatments started at day -7 or +7 will be continued until sacrifice at day 60. Data obtained from van Neerven et al., 2021 (van Neerven et al., 2021. Nature 594: 436-441).

[0028] Figure 2. Reduction of poly number in LB-100-treated mice, when compared to controls. (A) Treatment of Lgr5-CreERT2; Apcfl / fl mice with LB- 100, lithium chloride, or vehicle. (B) Reduction in polyp number was observed in the LB- 100-treated group and the lithium chloride -treated group, compared to controls.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Definitions

[0031] The term “proto-oncogene”, as is used herein refers to a gene that, when activated in a cell, may help that cell to become a cancer cell. Activation of an proto-oncogene may result from mutation, chromosomal rearrangement, geneamplification or from epigenetic mechanisms, as is known to a person skilled in the art. Proto-oncogenes may be classified in different categories, such as growth factor receptors, for example epidermal growth factor receptor (EGFR), ERBB2 receptor tyrosine kinase 2 and platelet- derived growth factor receptor (PDGFR); regulatory GTPases, such as RAS GTPase; cytoplasmic tyrosine kinases, for example SRC-family, SYK, ZAP- 70, Bruton's tyrosine kinase, and ABL; cytoplasmic serine / threonine kinases, for example RAS kinase; transcription factors, for example MYC; and from inactivation of tumor suppressors such as APC, retinoblastoma 1 (RB1), BRCA1 or BRCA2 andTP53.

[0032] The term “oncogene”, as is used herein, refers to a proto-oncogene that has been activated in a cell, as a result of a mutation, chromosomal rearrangement, gene amplification, or by an epigenetic mechanism. A non-cancerous cell that an oncogene as a result of a genomic alteration of a proto-oncogene is hereinafter termed “initiated cell”.

[0033] The term “activation”, as is used herein, refers to a genomic alteration of a gene such as a proto-oncogene, or to an epigenetic mechanism of that gene, that may result in the uncontrolled, continuous activity of the product of the gene such as the protein that is encoded by the gene. For the avoidance of doubts, inactivation of a tumor suppressor gene as a result of a mutation, chromosomal rearrangement, gene amplification, or by an epigenetic mechanism is included herein under the term “activation”, as will be clear to a person skilled in the art.

[0034] The term “epigenetic mechanism”, as is used herein, refers to non-genomic alterations that may activate a proto-oncogene to become an oncogene. Examples of such epigenetic mechanisms include DNA methylation, histone modification, chromatin remodeling, alterations in microRNA expression levels, and / or alteration of other chromatin components.

[0035] The term “pharmaceutically acceptable salt”, as is used herein, can be obtained by treating a compound useful in the methods of the invention and having a basic, e.g., an amino group, with an inorganic or organic acid to form a salt, for example, a salt of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p -toluenesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc.Pharmaceutically acceptable salts can also be obtained by treating a compound useful in methods of the invention and having an acidic, e.g., a carboxyl group, with an inorganic or organic base to form a salt, for example, a salt of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, ammonia, isopropylamine, trimethylamine, etc. In some embodiments, the pharmaceutically acceptable salt is a sodium salt, potassium salt, a calcium salt, an ammonium salt, or magnesium salt. In some embodiments, the pharmaceutically acceptable salt is a zinc salt. Pharmaceutically acceptable salts can also be obtained by treating a compound useful in the methods of the invention and having an acidic, e.g., a carboxyl, group with a basic amino acid, including but not limited to a D, L-amino acid, an L-amino acid, and a D-amino acids. Basic amino acids useful for preparing pharmaceutically acceptable salts can be natural amino acids, or synthetic amino acids. In some embodiments, the basic amino acids include, but are not limited to, histidine (H), arginine (R), lysine (K), glutamine (Q), 2,3-diaminopropionic acid (Dpr), ornithine (Orn), homoarginine (hArg), 2,4-diaminobutyric acid (Dbu), 2,3-diaminobutyric acid (Dab), or p-aminophenylalanine (Phe(p-NH2)). In some embodiments, the pharmaceutically acceptable salt is a meglumine (N-methyl-D-glucamine) salt, an eglumine (N-ethyl-D-glucamine) salt, D-glucamine salt, glucosamine salt, a choline salt, a lysine salt, an arginine salt, a histidine salt, or a glutamine salt. In some embodiments, the pharmaceutically acceptable salt is an L-lysine salt, an L- arginine salt, an L-histidine salt, or an L-glutamine salt. Those skilled in the art will further recognize that pharmaceutically acceptable salts can be prepared by reaction of the compounds of the invention with an appropriate inorganic or organic acid or base via any of a number of known methods.

[0036] The term “effective amount”, as is used herein, refers to an amount of a compound or pharmaceutically acceptable salt of a compound disclosed herein that is effective. An “effective amount” when used in connection with a PP2A inhibitor such as LB- 100, or a pharmaceutically acceptable salt thereof, means an amount of the compound that, when administered to a subject is effective to reduce or prevent the development of a cancer, alone or in combination with another agent.

[0037] Activation of oncogenic pathwayHyper activation of one or more oncogenic pathways in cells of an individual that carry an oncogenic mutation, hereinafter termed “initiated cells”, may lead to a reduction or even elimination of said cells.

[0038] Said hyperactivation of one or more oncogenic pathways may result from providing the individual with an inhibitor of Protein Phosphatase 2A (PP2A), a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor, a GSK3β inhibitor, an activator of PIK3CA, and / or activator of RAS.

[0039] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by a WEE1 kinase inhibitor, such as adavosertib (AZD1775, MK1755; 1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-l-yl)anilino]-2-(prop-2-en- 1-yl)- l,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one), PD407824 (9-hydroxy-4-phenyl-6H-pyrrolo[3,4-c]carbazole- 1,3-dione), ZN-c3 (azenosertib; 1-[(7R)-7-ethyl-7-hydroxy-5,6-dihydrocyclopenta[b]pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one), Debio-0123 (6-(2,6-dichlorophenyl)-8-methyl-2-[3-methyl-4-(l-methylpiperidin-4-yl)anilino]-7H-pyrimido[4,5-d]pyrimidin-5-one), SY-4835 (Shouyao Holdings (Beijing)), ACR-2316 (Acrivon Therapeutics), SDGR2, NUV-569 (Nuvation Bio Inc), or IMP7086 (Impact Therapeutics, Inc.).

[0040] In embodiments, a WEE1 kinases is provided at a dose of 20 mg to 400 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, and including all values and subranges therebetween.

[0041] In embodiments, adavosertib may be provided at a dose of 30 mg to about 400 mg, such as 50-200 mg, such as about 40 mg, about 50 mg, about 60 mg. and about 70 mg. In embodiments, azenosertib may be provided at a dose of 50-400 mg, such as 60-200 mg, such as about 70 mg, about 80 mg, about 90 mg, about 100 mg, and about 120 mg.

[0042] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by a checkpoint kinase inhibitor such as GDC-0575 (N-[4-[(3R)-3-aminopiperidin-l-yl]-5-bromo-lH-pyrrolo[2,3-b]pyridin-3-yl]cyclopropanecarboxamide;dihydrochloride), prexasertib (5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-lH-pyrazol-3-yl] amino]pyrazine-2-carbonitrile), rabusertib (l-[5-bromo-4-methyl-2-[[(2S)-morpholin-2-yl]methoxy]phenyl]-3-(5-methylpyrazin-2-yl)urea), SCH-900776 (6-bromo-3-(l-methylpyrazol-4-yl)-5-[(3R)-piperidin-3-yl]pyrazolo[l,5-a]pyrimidin-7-amine), CCT-245737 (5-[[4-[[(2R)-morphohn-2-yl]methylamino]-5-(trifluoromethyl)pyridin-2-yl]amino]pyrazine-2-carbonitrile), AZD-7762 (3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide), PF-477736 ((2R)-2-amino-2-cyclohexyl-N-[2-(l-methylpyrazol-4-yl)-9-oxo-3, 10, ll-triazatricyclo[6.4.1.04, 13]trideca- 1,4,6,8(13), 11-pentaen-6-yl] acetamide), GDC-0425 (3-(l-ethylpiperidin-4-yl)oxy-5,8, 10-triazatricyclo[7.4.0.02,7]trideca-l(9),2,4,6, 10, 12-hexaene-4-carbonitrile), or SRA737 (5-[[4-[[(2R)-morpholin-2-yl]methylamino]-5-(trifluoromethyl)pyridin-2-yl] amino]pyr azine-2-carbonitrile).

[0043] In embodiments, a checkpoint kinase inhibitor is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0044] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by a protein kinase C activator such as prostratin

[0045] ([(1R,2S,6R, 10S, HR, 13S, 15R)-l,6-dihydroxy-8-(hydroxymethyl)-4, 12, 12, 15-tetramethyl- 5-oxo- 13-tetracyclo[8.5.0.02, 6.011,13]pentadeca-3, 8-dienyl] acetate; 12-deoxyphorbol- 13- acetate).

[0046] In embodiments, a protein kinase C activator such as prostratin is provided at a dose of 0.01-10 mg / kg, including 0.05-5 mg / kg or 0.05-2 mg / kg, e.g., about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg. 1.7 mg / kg, 1.8 mg / kg, and 1.9 mg / kg, and including all values and subranges there-between.

[0047] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by a DUSP1 / 6 inhibitor such as BCI (E / Z-BCI; (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-lH-inden- 1-one) ((E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-lH-inden- 1-one).

[0048] In embodiments, a DUSP1 / 6 inhibitor such as BCI is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0049] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by a GSK3P inhibitor such as lithium, AZD1080 (2-hydroxy-3-[5-(morpholin-4-ylmethyl)pyridin-2-yl]-1H-indole-5-carbonitrile), LY2090314 (3-[6-fluoro-10-(piperidine-1-carbonyl)-1,10-diazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-3-yl]-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione), tideglusib (4-benzyl-2-naphthalen- 1-yl- 1, 2, 4-thiadiazolidine-3, 5-dione), BRD3731 ((4S)-3-(2,2-dimethylpropyl)-4,7,7-trimethyl-4-phenyl-2,6,8,9-tetrahydropyrazolo[3,4-b] quinolin- 5 -one), or an isoorientin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]chromen-4-one), or analog thereof (Liang and Li, 2018. ACS Chem Neurosci 9: 1166–1183).

[0050] In embodiments, a GSK3β inhibitor such as lithium may be orally provided at a dose of 100-1800 mg / day, including 200-1800 mg / day, such as 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, or 900 mg / day.

[0051] In embodiments, a GSK3β inhibitor such as AZD1080 may be provided parenterally at a dose of 1-20 mg / kg, including 2-10 mg / kg, such as 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 9 mg / kg.

[0052] In embodiments, a GSK3β inhibitor such as LY2090314 may be provided intravenously at a dose of 1-100 mg, including 2-50 mg, such as 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg.

[0053] In embodiments, a GSK3β inhibitor such as tideglusib may be provided orally at a dose of 50-500 mg, including 100-400 mg, such as 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 450 mg.In embodiments, a GSK3β inhibitor such as BRD3731 may be provided parenterally at a dose of 1-20 mg / kg, including 2-10 mg / kg, such as 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 9 mg / kg.

[0054] In embodiments, a GSK3β inhibitor such as isoorientin, or analog thereof, may be provided parenterally or orally at a dose of 1-100 mg, including 2-50 mg, such as 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg.

[0055] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by an activator of PI3KCA such as calycosin (7-hydroxy-3-(3-hydroxy-4-methoxyphenyl)chromen-4-one), as described in Wang et al., 2022 (Wang et al., 2022. Front Pharmacol 13: 828061), UCL-TRO-1938 1-[7-[[2-[4-(4-ethylpiperazin-1-yl)anilino]pyridin-4-yl]amino]-2,3-dihydroindol-1-yl]ethenone), as described in Gong et al., 2023 (Gong et al., 2023. Nature 618: 159–168), and / or 740 Y-P, a cell-permeable phosphopeptide activator of PI3KCA, comprising the amino acid sequence RQIKIWFQNRRMKWKKSDGGYMDMS (Derossi et al., 1998. Biochem Biophys Res Comm 251: 148-152).

[0056] In embodiments, an activator of PI3KCA is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0057] In embodiments, said hyperactivation of one or more oncogenic pathways is provided by an activator of a RAS such as KRA-533 (4-[4-[(2-bromoacetyl)amino]butyl]benzoic acid), as described in Xu et al., 2019 (Xu et al., 2019. Mol Cancer 18: 85), and compound 11, as described in Liu et al., 2023 (Liu et al., 2023. Bioorganic Med Chem 93: 117457).

[0058] In embodiments, an activator of RAS is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg,about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0059] PP2A inhibitor

[0060] The number of cells in an individual that carry an oncogenic mutation that is normally present only in cancer cells, hereinafter termed “initiated cells”, can be reduced or even eliminated by treatment with a PP2A inhibitor. Without being bound by theory, pharmacological hyperactivation of Wnt / KRAS signaling using LB- 100 may paradoxically prevent APC- / — driven tumor initiation by pushing early transformed cells beyond a tolerable signaling threshold, triggering differentiation, stress responses, or elimination before polyps can form.

[0061] In embodiments, the PP2A inhibitor has the structure of Formula I:

[0062]

[0063] o

[0064] Formula I

[0065] or a pharmaceutically acceptable salt thereof,

[0066] wherein:

[0067] R1is H, alkyl, hydroxyalkyl, alkenyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, C(O)O-t-Bu or -CH2CN;

[0068] Y is OR2, wherein R2is H, C1 -C10 alkyl, C2 -C10 alkenyl or phenyl.

[0069] In embodiments, Ri is H, methyl, ethyl, CH₂CH₂OH, CH₂(phenyl); and R₂ is H or C1 -C10 alkyl.

[0070] In embodiments, Ri is methyl; and R2 is H or C1-C4 alkyl.

[0071] In embodiments, Ri is methyl; and R2 is H (LB- 100).

[0072] In embodiments, the PP2A inhibitor is selected from cantharidin, norcantharidin, fostriecin, or LB- 1.2, as described in Lu et al., 2009. Proc Natl Acad Sci USA 106: 11697-11702), which is incorporated herein by reference.A preferred PP2A inhibitor is LB- 100. LB- 100 may be provided to an individual at an effective amount.

[0073] In embodiments, a PP2A inhibitor such as LB- 100 may be provided to an individual at a dose of 0.1 mg / m2to 3.5 mg / m2.

[0074] It is understood that where a numerical range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, "0.1 mg / m2to 3.5 mg / m2is a disclosure of 0.1 mg / m2, 0.2 mg / m2, 0.3 mg / m2, 0.4 mg / m2, 0.5 mg / m2, 0.6 mg / m2, etc., up to 3.5 mg / m2.

[0075] In embodiments, the PP2A inhibitor such as LB- 100 is administered to an individual at regular intervals, for example twice daily, daily, two days / week, three days per week, four days per week, five days per week or six days per week. In embodiments, the PP2A inhibitor such as LB- 100 is provided one day every other week, two days every other week, three days every other week, four days every other week, five days every other week, or six days every other week. In embodiments, the PP2A inhibitor such as LB- 100 is provided one day every three weeks, two days every three weeks, three days three weeks, four days every three weeks, five days every three weeks, or six days every three weeks. In embodiments, the PP2A inhibitor such as LB- 100 is provided one day every four weeks, two days every four weeks, three days four weeks, four days every four weeks, five days every four weeks, or six days every four weeks.

[0076] In embodiments, the PP2A inhibitor such as LB- 100 is administered to an individual for a number of is provided to an individual at a daily dose. In embodiments, the PP2A inhibitor such as LB- 100 is administered to an individual in an amount ranging from 0.02-5 mg / kg body weight per day. 0.05-3 mg / kg body weight per day, 0.1- 1 mg / kg body weight per day, 0.2-0.9 mg / kg body weight per day, 0.3-0.8 mg / kg body weight per day, or 0.4-0.7 mg / kg body weight per day, such as at 0.5 or 0.6 mg / kg body weight per day.

[0077] A PP2A inhibitor such as LB- 100 can be administered, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma orintraparenchymally. In embodiments, a PP2A inhibitor such as LB- 100 is orally administered to an individual. In embodiments, the PP2A inhibitor such as LB- 100 is intravenously administered to the individual. In embodiments, the PP2A inhibitor such as LB- 100 is administered by infusion to the individual.

[0078] In embodiments, a pharmaceutical composition comprising a PP2A inhibitor such as LB- 100 may comprise monosodium glutamate, as described in granted patent US 10,532,050 B2, which is hereby incorporated by reference.

[0079] In embodiments, a pharmaceutical composition comprising a PP2A inhibitor such as LB- 100 may have a pH of 9-11, such as about 9.5, about 10 and about 10.5, as described in granted patent US 10,532,050 B2, which is hereby incorporated by reference.

[0080] The invention further provides an inhibitor of Protein Phosphatase 2A (PP2A), for use in prophylactic treatment of an individual. Said inhibitor of PP2A is preferably for use in reducing a risk of developing cancer in said individual, more preferably for preventing the development of cancer in said individual. Said inhibitor of PP2A may help in eliminating non-cancer cells that carry mutations found in cancer cells, hereinafter termed “initiated cells”. Said inhibitor of PP2A may be combined with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor.

[0081] The invention further provides a use of an inhibitor of Protein Phosphatase 2A (PP2A), in the preparation of medicament for prophylactic treatment of an individual. Said inhibitor of PP2A is preferably for use in reducing a risk of developing cancer in said individual, more preferably for preventing the development of cancer in said individual. Said inhibitor of PP2A may help in eliminating non-cancer cells that carry mutations found in cancer cells, hereinafter termed “initiated cells”. Said inhibitor of PP2A may be combined with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, prostratin, a DUSP1 / 6 inhibitor, a GSK3P inhibitor, an activator of PI3KCA and / or an activator of a RAS protein.

[0082] Combinatorial useIn embodiments, a PP2A inhibitor such as LB- 100 is provided to an individual in combination with one or more other active ingredients. The PP2A inhibitor such as LB- 100 and the one or more other active ingredients are each periodically administered to the subject. A PP2A inhibitor such as LB- 100 and the one or more other active ingredients may be provided as a single pharmaceutical composition, or as separate pharmaceutical compositions. The PP2A inhibitor such as LB- 100 and the one or more other active ingredients may be administered to the individual simultaneously, separately or sequentially.

[0083] In embodiments, a PP2A inhibitor such as LB- 100 is provided to an individual in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor.

[0084] In embodiments, the further active ingredient is a WEE1 kinase inhibitor, such as adavosertib (AZD1775, MK1755; l-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-l-yl)anilino]-2-(prop-2-en-l-yl)-l,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one), PD407824 (9-hydroxy-4-phenyl-6H-pyrrolo[3,4-c]carbazole- 1,3-dione), ZN-c3 (azenosertib; l-[(7R)-7-ethyl-7-hydroxy-5,6-dihydrocyclopenta[b]pyridin-2-yl]-6-[4-(4-methylpiperazin-l-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one), Debio-0123 (6-(2,6-dichlorophenyl)-8-methyl-2-[3-methyl-4-(l-methylpiperidin-4-yl)anilino]-7H-pyrimido[4,5-d]pyrimidin-5-one), SY-4835 (Shouyao Holdings (Beijing)), ACR-2316 (Acrivon Therapeutics), SDGR2, NUV-569 (Nuvation Bio Inc), or IMP7086 (Impact Therapeutics, Inc.).

[0085] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a WEE1 kinase inhibitor to an individual, whereby the WEE1 kinases is provided at a dose of 20 mg to 400 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, and including all values and subranges therebetween.

[0086] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with adavosertib to an individual, whereby adavosertib is provided at a dose of 30 mg to about 400 mg, such as 50-200 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with adavosertib to an individual, whereby the WEE1 kinase is provided at a dose about 40 mg. In embodiments, a PP2A inhibitorsuch as LB- 100 is provided in combination adavosertib to an individual, whereby adavosertib is provided at a dose of 50 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with adavosertib to an individual, whereby adavosertib is provided at a dose of 60 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with adavosertib to an individual, whereby the adavosertib is provided at a dose of 70 mg.

[0087] In embodiments, the WEE1 kinase inhibitor is azenosertib. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 50-400 mg, such as 60-200 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 70 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 80 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 90 mg. In em-bodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 100 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 100 mg. In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with azenosertib to an individual, whereby azenosertib is provided at a dose of 120 mg.

[0088] In embodiments, the further active ingredient is a checkpoint kinase inhibitor such as GDC-0575 (N-[4-[(3R)-3-aminopiperidin-l-yl]-5-bromo-lH-pyrrolo[2,3-b]pyridin-3-yl]cyclopropanecarboxamide;dihydrochloride), prexasertib (5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-lH-pyrazol-3-yl] amino]pyrazine-2-carbonitrile), rabusertib (l-[5-bromo-4-methyl-2-[[(2S)-morpholin-2-yl]methoxy]phenyl]-3-(5-methylpyrazin-2-yl)urea), SCH-900776 (6-bromo-3-(l-methylpyrazol-4-yl)-5-[(3R)-piperidin-3-yl]pyrazolo[l,5-a]pyrimidin-7-amine), CCT-245737 (5-[[4-[[(2R)-morpholin-2-yl]methylamino]-5-(trifluoromethyl)pyridin-2-yl]amino]pyrazine-2-carbonitrile), AZD-7762 (3-(carbamoylamino)-5-(3-fluorophenyl)-N-[(3S)-piperidin-3-yl]thiophene-2-carboxamide), PF-477736 ((2R)-2-amino-2-cyclohexyl-N-[2-(l-methylpyrazol-4-yl)-9-oxo-3, 10, ll-triazatricyclo[6.4.1.04, 13]trideca- 1,4,6,8(13), 11-pentaen-6-yl] acetamide), GDC-0425 (3-(l-ethylpiperidin-4-yl)oxy-5,8, 10-triazatricyclo[7.4.0.02,7]trideca-l(9),2,4,6, 10, 12-hexaene-4-carbonitrile), or SRA737 (5-[[4-[[(2R)-morpholin-2-yl]methylamino]-5-(trifluoromethyl)pyridin-2-yl] amino]pyr azine-2-carbonitrile).

[0089] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a checkpoint kinase inhibitor to an individual, whereby the checkpoint kinase inhibitor is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0090] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a protein kinase C activator such as prostratin ([(1R,2S,6R, 10S, HR, 13S,15R)-l,6-dihydroxy-8-(hydroxymethyl)-4, 12, 12, 15-tetramethyl-5-oxo- 13-tetracyclo[8.5.0.02, 6.01 l,13]pentadeca-3, 8-dienyl] acetate; 12-deoxyphorbol-13-acetate) to an individual.

[0091] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a protein kinase C activator such as prostratin to an individual, whereby the protein kinase C activator such as prostratin is provided at a dose of 0.01-10 mg / kg, including 0.05-5 mg / kg or 0.05-2 mg / kg, e.g., about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg. 1.7 mg / kg, 1.8 mg / kg, and 1.9 mg / kg, and including all values and subranges therebetween.

[0092] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a DUSP1 / 6 inhibitor such as BCI (E / Z-BCI; (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-lH-inden- 1-one) ((E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-lH-inden- 1-one).

[0093] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a DUSP1 / 6 inhibitor such as BCI to an individual, whereby the DUSP1 / 6 inhibitor is provided at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0094] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as lithium, AZD1080 (2-hydroxy-3-[5-(morpholin-4-ylmethyl)pyridin-2-yl] - lH-indole-5-carbonitrile), LY2090314 (3- [6-fhioro- 10-(piperidine-l-carbonyl)-l,10-diazatricyclo[6.4.1.04, 13]trideca-2,4,6,8(13)-tetraen-3-yl]-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione), tideglusib (4-benzyl-2-naphthalen-l-yl-1, 2, 4-thiadiazolidine-3, 5-dione), BRD3731 ((4S)-3-(2,2-dimethylpropyl)-4,7,7-trimethyl-4-phenyl-2,6,8,9-tetrahydropyrazolo[3,4-b] quinolin- 5 -one), or an isoorientin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]chromen-4-one), or analog thereof (Liang and Li, 2018. ACS Chem Neurosci 9: 1166–1183).

[0095] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as lithium, whereby lithium is orally provided at a dose of 100-1800 mg / day, including 200-100 mg / day, such as 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, or 900 mg / day.

[0096] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3p inhibitor such as AZD1080, whereby AZD1080 is provided parenterally at a dose of 1-20 mg / kg, including 2-10 mg / kg, such as 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 9 mg / kg.

[0097] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as LY2090314, whereby LY2090314 is provided intravenously at a dose of 1-100 mg, including 2-50 mg, such as 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg.

[0098] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as tideglusib, whereby tideglusib is provided orally at a dose of 50-500 mg, including 100-400 mg, such as 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 450 mg.

[0099] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as BRD3731, whereby BRD3731 is providedparenterally at a dose of 1-20 mg / kg, including 2-10 mg / kg, such as 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 9 mg / kg.

[0100] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with a GSK3P inhibitor such as isoorientin, or analog thereof, whereby isoorientin, or analog thereof, is provided parenterally or orally at a dose of 1-100 mg, including 2-50 mg, such as 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg.

[0101] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with an activator of PI3KCA such as calycosin, UCL-TRO-1938, and / or 740 Y-P.

[0102] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with an activator of PI3KCA at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0103] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with an activator of a RAS such as KRA-533 and compound 11.

[0104] In embodiments, a PP2A inhibitor such as LB- 100 is provided in combination with an activator of RAS at a dose of 1 mg to 600 mg, including 10 mg-500 mg, e.g., about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, and including all values and subranges therebetween.

[0105] Proto-oncogenes

[0106] Alteration of growth factor receptors, such as HER2, EGFR, ALK1 and PDGFR, may result in constitutive stimulation of the proliferative properties of cells. Said alteration may be the result of a mutation, for example deletion of exon 19 and mutation L858R in exon 21 of EGFR, a translocation, for example of ALK1 in non-small cell lung cancer, or an amplification, for example of HER2 in breast cancer.As is used herein, HER2 is characterized by HUGO Gene Nomenclature Committee (HGNC) access number 3430, NCBI gene number 2064, and Ensembl number ENSG00000141736. EGFR is characterized by HGNC access number 3236, NCBI gene number 1956, and Ensembl number ENSG00000146648. ALK1 is characterized by HGNC access number 427, NCBI gene number 238, and Ensembl number ENSG00000171094. PDGFRA is characterized by HGNC access number 8803, NCBI gene number 5156, and Ensembl number ENSG00000134853.

[0107] RAS, including Harvey Ras (HRAS), neuroblastoma Ras (NRAS) and Kirsten Ras (KRAS) is a GTPase. Mutations in the RAS family of proteins have frequently been observed in several cancer types, including colorectal cancer and follicular thyroid cancer. The amino acid positions G12, G13 and Q61 account for the overwhelming majority of these mutations. HRAS is characterized by HGNC access number 5173, NCBI gene number 3265, and Ensembl number ENSG00000174775. NRAS is characterized by HGNC access number 7989, NCBI Gene number 4893, and Ensembl number ENSG00000213281. KRAS is characterized by HGNC access number 6407, NCBI gene number 3845, and Ensembl number ENSG00000133703.

[0108] Non receptor tyrosine kinases, including members of the SRC-family, such as SRC, YES, FYN, FGR, LYN, BLK, HCK, AND LCK, SYK, ZAP-70, Bruton tyrosine kinase (BTK) and ABL1, may be activated by mutation or translocation and acquire transforming functions that may lead to malignancy. SRC is characterized by HGNC access number 11283, NCBI gene number 6714, and Ensembl number ENSG00000197122. YES is characterized by HGNC access number 12841, NCBI Gene number 7525, and Ensembl number ENSG00000176105. FYN is characterized by HGNC access number 4037, NCBI gene number 2534, and Ensembl number ENSG00000010810. FGR is characterized by HGNC access number 3697, NCBI gene number 2268, and Ensembl number ENSG00000000938. LYN is characterized by HGNC access number 6735, NCBI Gene number 4067, and Ensembl number ENSG00000254087. BLK is characterized by HGNC access number 1057, NCBI gene number 640, and Ensembl number ENSG00000136573. HCK is characterized by HGNC access number 4840, NCBI gene number 3055, and Ensembl number ENSG00000101336. LCK is characterized by HGNC access number 6524, NCBI Gene number 3932, and Ensembl number ENSG00000182866. SYK is characterized by HGNC access number 11491, NCBI gene number 6850,and Ensembl number ENSG00000165025. ZAP-70 is characterized by HGNC access number 12858, NCBI gene number 7535, and Ensembl number ENSG00000115085. BTK is characterized by HGNC access number 1133, NCBI Gene number 695, and Ensembl number ENSG00000010671. ABL1 is characterized by HGNC access number 76, NCBI gene number 25, and Ensembl number ENSG00000097007.

[0109] MYC encodes a transcriptional regulator. Genomic alterations, including amplification and translocation are associated with Burkitt lymphoma and multiple myeloma in human patients. MYC is characterized by HGNC access number 7553, NCBI gene number 4609, and Ensembl number ENSG00000136997.

[0110] BRAF encodes a serine / threonine kinase that plays a role in the MAP kinase / ERK signaling pathway, which affects cell division. Several mutations and gene fusions are found in epithelial tumors such as colorectal cancer and non-small cell lung cancer. BRAF is characterized by HGNC access number 1097, NCBI gene number 673, and Ensembl number ENSG00000157764.

[0111] PIK3CA encodes the catalytic subunit of phosphatidylinositol 3-kinase, which plays a role in an intracellular signaling pathway important in regulating the cell cycle. This gene has been found to be oncogenic and has been implicated in cervical cancers. BRAF is characterized by HGNC access number 8975, NCBI gene number 5290, and Ensembl number ENSG00000121879.

[0112] APC encodes a tumor suppressor that participates in Wnt signaling.

[0113] Mutations in APC cause familial adenomatous polyposis, an autosomal dominant pre-malignant disease that usually progresses to malignancy. TP53 encodes a tumor suppressor protein that is capable of inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome. BRCA1 and BRCA2 encode proteins that play a role in maintaining genomic stability. Mutations in these genes have become a hallmark for hereditary breast and ovarian cancers. APC is characterized by HGNC access number 583, NCBI gene number 324, and Ensembl number ENSG00000134982. TP53 is characterized by HGNC access number 11998, NCBI gene number 7157, and Ensembl number ENSG00000141510. BRCA1 is characterized by HGNC access number 1100, NCBI gene number 672, and Ensembl numberENSG00000012048. BRCA2 is characterized by HGNC access number 1101, NCBI gene number 675, and Ensembl number ENSG00000139618.

[0114] In embodiments, a PP2A inhibitor such as LB- 100 is provided to an individual either alone or in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor as a prophylactic treatment to prevent to occurrence of cancer in the individual. Said individual may comprise non-cancer cells that carry mutations found in cancer cells, termed “initiated cells”. Said individual may or may not have been typed as having or carrying such “initiated cells”. In embodiments, the individual is an otherwise healthy individual who does not suffer from a cancer. In embodiments, the individual does not have, or is not known to have, a hereditary cancer syndrome, such as hereditary breast and ovarian cancer syndrome, Lynch syndrome, familial adenomatous polyposis syndrome and von Hippel-Lindau syndrome.

[0115] In embodiments, the individual comprises an alteration in a proto-oncogene including a growth factor receptor, such as HER2, EGFR, ALK1 and PDGFR, For example, said alteration may be the result of a mutation, for example deletion of exon 19 and mutation L858R in exon 21 of EGFR, a translocation, for example of ALK1 in non-small cell lung cancer, or an amplification, for example of HER2 in breast cancer. For example, the individual has a point mutation causing an amino acid alteration at codon 12, 13, or 61 of a Ras gene such as KRAS. Databases of genomic alterations in a proto-oncogene that may be present in “initiated cells” are known to a skilled person, including cosmic (available at cancer.sanger.ac.uk / cosmic); the Cancer Genome Atlas (available at portal.gdc.cancer.gov); and Network of Cancer Genes (available at ncg-colcc.cs.ucl.ac.uk / ).

[0116] In embodiments, a PP2A inhibitor such as LB- 100 is provided to an individual either alone or in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor may be useful for prophylactic treatment to prevent the occurrence of a cancer, wherein the cancer is hepatocellular carcinoma (hepatoma), cholangiocarcinoma, colorectal carcinoma, small cell lung cancer, non-small cell lung cancer, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial carcinoma, Fallopian tube cancer, prostate cancer, gastrointestinal stromal tumor (GIST), esophageal cancer, gallbladder cancer, gastrointestinal carcinoid tumor, duodenal cancer, gastroesophageal junction cancer, islet cell cancer, gastric cancer, anal cancer, cancer of the small intestine, pseudomyxoma peritonei, head and neck squamous cell carcinoma, Merkel cell carcinoma, tumor mutational burden-high cancer (TMB-H), microsatellite stable (MSS), mismatch repair proficient colon cancer, thyroid cancer, renal cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, urothelial carcinoma, testicular cancer, bladder carcinoma, glioma, glioblastoma, multiforme, astrocytoma, medulloblastoma, craniopharyngioma, oligodendroglioma, malignant meningioma, diffuse intrinsic pontine glioma, melanoma, neuroblastoma, retinoblastoma, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, or polycythemia vera.

[0117] In some embodiments, the cancer is colorectal cancer, cholangiocarcinoma, pancreatic cancer, breast cancer or ovarian cancer. In some embodiments, the nonsmall cell lung cancer is lung adenocarcinoma, lung squamous carcinoma or lung large cell carcinoma.

[0118] EXAMPLES

[0119] Example 1Hyper- activation of oncogenic signaling should be more detrimental to cancer cells than normal cells, given that the targeted pathways are already activated by cancer-causing mutations in the cancer cells. Normal cells are more resistant to perturbations in signaling, due to the many feedback and cross talk loops that operate in normal cells to maintain homeostasis. However, the situation may be different in pre-malignant lesions in which there is a partial perturbation of normal homeostasis. It is therefore conceivable that agents that hyperactivate oncogenic signaling have a tumor-promoting effect on such pre-malignant lesions

[0120] Multiple animal models of cancer predisposition exist. We will start by testing drugs that activate WNT and MAPK signaling, pathways that contribute to colon cancer development. We will, therefore, study the effects of these drugs on intestinal adenomas, which are a precursor lesions to full blown intestinal cancers. Intestinal adenomas develop following loss of the APC tumor suppressor gene and this process can be readily modeled in mice (Li et al., 2022. Biomedicines 10: 1710). To model intestinal adenoma development, mice will be used that are heterozygous for an Lgr5-EGFP-IRES-CreERT2 "knock-in" allele that directs the synthesis of both EGFP and CreERT2 fusion protein. When these mice are bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Lgr5 -expressing cells of the offspring. Thus, crossing these mice with mice homozygous for an allele carrying the Ape (adenomatosis polyposis coli) gene exon 14 flanked by loxP sites will result in homozygous deletion of the floxed region, resulting in an adenoma-prone mouse like ApcMin animals. Both strains are commercially available.

[0121] Moreover, the number of adenomas can be titrated by using different concentrations of tamoxifen to activate the Cre recombinase (van Neerven et al., 2021. Nature 594: 436-441; see Figure 1). This finding is important as it suggests that further activation on WNT signaling in adenomas is not promoting the number of initiating events.

[0122] Briefly, Lgr5-CreERT2; Apcfl / fl mice will be treated with low dose tamoxifen and day 0 to initiate Ape deletion. Animals will be sacrificed at day 60 and the number of adenomas will be counted. Histopathology will be used to ask if drug treatment stimulates progression of adenomas to adenocarcinoma or whether signs ofoncogene-induced senescence are present. The effects of drug addition one week prior to tamoxifen addition to initiate Ape deletion and thus adenoma formation and drug addition one week after tamoxifen addition will be tested (see schematic in Fig. ID).

[0123] To study effects on normal cells, immunocompetent wild type mice will be treated with agents to activate mitogenic signaling with or without their synthetic lethal stress response inhibitors at concentrations that show anti-cancer effects. Mice will be treated long-term (2-4 months) and tissues from multiple organs will be collected for analyses. A focus will be on tissues that are frequently affected by cancer therapies, such as bone marrow and colonic epithelia, using approaches as described (Fernandes Neto et al., 2020. Nature Commun 11: 3157). In addition, we will collect RNA from control and treated mouse tissues and subject these to RNAseq to study transcriptomic effects of drug treatment on activation of mitogenic signaling and activation of stress responses. Immunohistochemistry will be performed for proliferation markers (KI67, p-histone H3), apoptosis and mitogenic signaling (e.g. p-ERK, pS6-kinase, p4E-BP1). These data will inform us on the effects of the drugs on normal cells. It will be important to ask if increases in mitogenic signaling result in increases in proliferation. In earlier mouse studies, activation of p-ERK through deletion of DUSP6 did not result in dysregulation of normal tissue architecture, pointing towards a remarkable buffering capacity of normal cells (Maillet et al., 2008. J Biol Chem 283: 31246-31255).

[0124] It is possible that drug treatment will mediate progression to adenocarcinoma, but that more time is required to observe this than 60 days. The Lgr5-CreERT2; Apcfl / flmice develop so many adenomas that animals cannot be maintained for more than 60 days. This can be solved by use of heterozygous Lgr5-CreERT2; Apcfl / wtmice that develop fewer adenomas and will live longer after treatment with low dose tamoxifen. Such heterozygous mice can then be treated with schemes indicated in Figure ID for more than 60 days to allow potential progression to adenocarcinoma to proceed.

[0125] Together, these experiments will give us a comprehensive view of how paradoxical activation of oncogenic signaling affects normal cells and pre-malignant adenomas.

[0126] Example 2Materials and methods

[0127] An Ape- / -; Lgr5-Cre transgenic mouse model (L5R; VCM; dT; APF) was employed to evaluate whether paradoxical activation of the oncogenic signaling pathway by LB- 100 enhances elimination of Apc-mutant cells and suppresses tumor initiation. Ape loss was induced using low-dose tamoxifen (0.05 mg per 30 g mouse, intraperitoneally), which reliably results in polyp formation within approximately two months. LiCl treatment (300 mg / L in drinking water), previously validated to activate the Wnt pathway and reduce polyp formation, was included as a positive control (Van Neerven et al., 2021. Nature 594: 436-441).

[0128] A total of 5 mice were pretreated with LB- 100 (1 mg / kg, intraperitoneally, three times per week), or with saline vehicle (4 mice), beginning one week prior to tamoxifen administration and continuing for a total duration of 67 days. LiCl-treated animals (4 mice) received LiCl-containing water, refreshed every 2-3 days, over the same time period.

[0129] Results

[0130] At 60 days post-tamoxifen induction, mice were euthanized, and intestinal tissues were harvested for analysis (see Fig. 2A). Blinded pathological assessment of representative tissue sections revealed no visible intestinal polyps in three mice in the LB- 100 group, although one sample was designated as an outlier. A reduction in polyp number was observed in the LB-100-treated group compared to controls (Fig. 2B), indicating that LB- 100 administration prior to and during polyp induction is capable of suppressing premalignant lesion development in Apc-mutant models. No obvious decrease in body weight was observed in any of the mice, indicating good tolerance to the treatment.

[0131] Normal tissues, including blood, liver, and bone marrow, were collected for subsequent analysis to evaluate potential toxicity or off-target effects associated with treatment.

Claims

Claims1. A method for reducing a risk of developing cancer in an individual, comprising providing the individual with an activator of an oncogenic pathway in order to reduce or eliminate initiated cells, which are non-cancer cells that carry mutations found in cancer cells.

2. The method of claim 1, whereby the activator of an oncogenic pathway is selected from an inhibitor of Protein Phosphatase 2A (PP2A), a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor, a GSK3β inhibitor, an activator of PIK3CA, and / or activator of RAS.

3. The method of claim 1 or claim 2, wherein the individual has cells with one or more mutations in RAS, including HRAS, NRAS and KRAS, HER2, EGFR, PDGFR, VEGFR, BCR / ABL1, cMYC gene, nMYC gene, EML4AK, BRAF, PIK2CA, MUC16, APC, TP53, BRCA1, BRCA2, SRC family kinase, ABL, SYK-ZAP-70 family kinase, and BTK family of tyrosine kinases.

4. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is a WEE1 kinase inhibitor selected from adavosertib (AZD1775, MK1755), PD407824, ZN-c3 (azenosertib), Debio-0123, SY-4835, ACR-2316, SDGR2, NUV-569 or IMP7086.

5. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is a checkpoint kinase 1 inhibitor selected from GDC-0575, prexasertib, rabusertib, SCH-900776, CCT-245737, AZD-7762, PF-477736, GDC-0425, or SRA737.

6. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is a protein kinase C activator such as prostratin.

7. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is a DUSP1 / 6 inhibitor such as BCE8. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is a GSK3P inhibitor selected from lithium, AZD1080, LY2090314, tideglusib, BRD3731, or an isoorientin analog.

9. The method of any one of claims 1-3, wherein the activator of an oncogenic pathway is an inhibitor of Protein Phosphatase 2A (PP2A).

10. The method of any one of claims 1, 2,3 or 8, wherein the PP2A inhibitor isoor a pharmaceutically acceptable salt thereof,wherein:R1is H, alkyl, hydroxyalkyl, alkenyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, C(O)O-t-Bu or -CH2CN;Y is OR2, wherein R2is H, C1 -C10 alkyl, C2 -C10 alkenyl or phenyl.

11. The method of any one of claims 1, 2, 3, 8, or 9, wherein the individual is provided with an inhibitor of PP2A, in combination with one or more other active ingredients.

12. The method of claim 10, wherein the individual is provided with an inhibitor of PP2A, in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor.

13. The method of claim 10 or 11, wherein the PP2A inhibitor is LB- 100.

14. The method of claim 12, wherein LB- 100 is provided to the individual at a dose of 0.1 mg / m2to 3.5 mg / m2.

15. The method of claim 12 or 13, wherein LB- 100 is provided to the individual at a daily dose.

16. The method of any one of claims 12-14, wherein LB- 100 is provided intravenously to the individual.

17. The method of any one of claims 1-3 or 12-15, wherein the individual is provided with LB- 100, in combination with a further active ingredient selected from a WEE1 kinase inhibitor, a checkpoint kinase 1 inhibitor, a protein kinase C activator such as prostratin, a DUSP1 / 6 inhibitor and / or a GSK3P inhibitor.

18. The method of any one of claims 1-3 or 12-16, wherein the WEE1 kinase inhibitor is adavosertib (AZD1775, MK1755), PD407824, ZN-c3 (azenosertib), Debio-0123, SY-4835, ACR-2316, SDGR2, NUV-569 or IMP7086.

19. The method of claim 17, wherein the WEE1 kinase inhibitor is provided to the individual at a dose of 20 mg to 400 mg.

20. The method of any one of claims 1-3 or 12-16, wherein the checkpoint kinase 1 inhibitor is GDC-0575, prexasertib, rabusertib, SCH-900776, CCT-245737, AZD-7762, PF-477736, GDC-0425, or SRA737, which is preferably provided to the individual at a dose of 1 mg to about 600 mg.

21. The method of any one of claims 1-3 or 12-16, wherein the protein kinase C activator such as prostratin is provided at a dose of 0.01-10 mg / kg.

22. The method of any one of claims 1-3 or 12-16, wherein the DUSP1 / 6 inhibitor is BCI.

23. The method of any one of claims 1-3 or 12-16, wherein the GSK3P inhibitor is lithium, AZD1080, LY2090314, tideglusib, BRD3731, or an isoorientin analog.

24. The method of any one of claim 1-22, wherein a risk of developing colorectal cancer, pancreatic cancer, breast cancer or ovarian cancer is reduced in the individual.1 / 2P138742PC00FiguresFigure 1TAMDI + LOfllwl0gQ TAM1 T LB-1OO ■ i *-7 0 60 TAM BI + WEEli i_A____™^ ®3n,ro* -7 0 60TAM, 1 + LB-100 + WEEli ■ * 1-7 0 60 TAM I TC _L_J _L0 +7 60 TAM, 1 t WEEli i ■ _ U 0 +7 60 TAM| t LB-100+WEEli t i _ L.0 +7 602 / 2Figure 2LB-100 Tamoxifen sac -7d 0d 60dLiCl Tamoxifen sac -7d 0d 60dVehicle Tamoxifen sac -7d 0d 60dB• LB-100 • LiCl • Saline

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