Technical Field

[0001] The present invention relates to treatment of cancer, in particular to methods for sensitising cancer cells to a NEDD8 activating enzyme (NAE) inhibitor by administering an effective amount of an ABCG2 inhibitor and a NAE inhibitor, such as pevonedistat.

Background

[0002] Cancer is a great burden to our society and there were an estimated 18 million cancer cases around the world in 2018, of these 9.5 million cases were in men and 8.5 million in women. Despite the introduction of many new treatment modalities/options, de novo or acquired resistance to the applied treatments still represents the major cause of death from cancer. Thus, the resistance of cancer cells to anti-cancer drugs is a major reason for the failure of traditional cancer treatment.

[0003] A variety of chemo-sensitizing agents has been developed and several have been shown to be effective in experimental models in vitro and in vivo. These sensitizing agents include, among many others, tyrosine kinase inhibitors, anti-ABC transporters, angiogenesis inhibitors and modulators and/or inhibitors of volume regulated anion channels (VRACs).

[0004] Pevonedistat is a potent, selective, first-in-class NEDD8 activating enzyme (NAE) inhibitor undergoing clinical trials to investigate its anticancer effect against certain cancer types.

[0005] ATP-binding cassette (ABC) transporters are membrane proteins that are involved in mediating multidrug resistance (MDR).

[0006] Pevonedistat is a substrate of ABCG2 which decreases the therapeutic effect of pevonedistat in cancer cells expressing ABCG2. Hence, the cytotoxicity of pevonedistat is significantly weakened in ABCG2-overexpressing cells.

[0007] W017198700 disclose a method of sensitising cancer cells to an anti-cancer treatment by administering to a patient in need thereof an effective amount of a VRAC modulator such as SCO-101. [0008] Thus, characterisation and clinically validation of new drug combinations and treatment regimens for treatment of cancer and in particular drug resistant cancers constitute a highly unmet medical need.

Summary

[0009] Over this background art the present invention provides a combination drug for improving the anti-cancer effects of NEDD8 activating enzyme (NAE) inhibitors, such as pevonedistat by administering an effective amount of a potent ABCG2 inhibitor, such as SCO-101 or SCO-201 to a patient.

[0010] As described in working example 1 herein, the inventors surprinsingly and unexpectedly found that co-administering an effective amount of an ABCG2 inhibitor, such as SCO-101 or SCO-201, significantly increased the anti-cancer effect of the NEDD8 inhibitor pevonedistat toward both parental and resistant cancer cells. The treatment was shown to be much more effective than just treatment with the the NEDD8 inhibitor or the ABCG2 inhibitor alone. In some embodiments of the present disclosure, the NEDD8 inhibitor is an inhibitor of the NEDD8 activating enzyme (NAE).

[0011] Accordingly, in a first aspect the present invention provides a combination drug, comprising separately or together, (a) an ABCG2 inhibitor, and (b) a NEDD8 inhibitor; for use in the treatment of a cancer. In particular, the cancer treated is in a patient.

[0012] In a further aspect, the present invention provides a pharmaceutical composition comprising, separately or together, (a) an ABCG2 inhibitor, (b) a NEDD8 inhibitor, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.

Description of drawings and figures

[0013] The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out. When using reference numerals in drawings, throughout the specification, claims and drawings the same reference numerals are used for identical or corresponding parts. The figures and drawing include:

[0014] Figure 1 is a schematic representation of the anti-cancer effect of pevonedistat alone, SCO- 101 alone, and the combination of pevonedistat and SCO-101 on resistant cancer cells, respectively.

[0015] Figure 2 is a schematic representation of the anti-cancer effect of pevonedistat alone, SCO- 201 alone, and the combination of pevonedistat and SCO-201 on resistant cancer cells, respectively. [0016] Figure 3 is a schematic representation of the synergy plot from combination treatment of SCO- 101 and pevonedistat with a ZIP synergy score of 29.8.

[0017] Figure 4 is a schematic representation of the synergy plot from combination treatment of SCO- 201 and pevonedistat with a ZIP synergy score of 38.5.

[0018] Figure 5 shows survival curves for HT29 and HT29 SN38Res cells with Pevonedistat. IC50 calculation using linear regression, based on 2 replicates. Data represents mean plus/minus SEM.

[0019] Figure 6 shows survival curves for HT29 and HT29 SN38Res cells with Azacytidine. IC50 calculation using linear regression, based on 2 replicates. Data represents mean plus/minus SEM.

[0020] Figure 7 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of the ABCG2 inhibitor SCO-101 in combination with different doses of Pevonedistat. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in two biological replicates.

[0021] Figure 8 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of the ABCG2 inhibitor SCO-201, in combination with different doses of Pevonedistat. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in three biological replicates.

[0022] Figure 9 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of the ABCG2 inhibitor Ko 143, in combination with different doses of Pevonedistat. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in three biological replicates.

[0023] Figure 10 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of Azacytidine, in combination with different doses of Pevonedistat. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in two biological replicates.

[0024] Figure 11 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of Azacytidine in combination with different doses of Pevonedistat. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in two biological replicates.

[0025] Figure 12 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of Azacytidine in combination with different doses of Pevonedistat or SCO-201 or triple combination of all three compounds. Viability was measured at 72h using the CCK8 assay. Error bars are shown as SEM. Experiment was performed in a single biological replicate.

[0026] Figure 13 shows the relative viability of HT29 SN38Res cells (compared to untreated control) of 72h treatment with different doses of Azacytidine in combination with fixed doses of Pevonedistat (1 pM) or SCO-201 (2.5 pM) or triple combination of all three compounds. Viability was measured at 72h using the CCK8 assay. Experiment was performed in a single biological replicate.

[0027] Figure 14 shows a synergy plot for combination of SCO-101 and pevonedistat with a ZIP synergy score of 27.308.

[0028] Figure 15 shows a synergy plot for combination of SCO-201 and pevonedistat with a ZIP synergy score of 38.456.

[0029] Figure 16 shows a synergy plot for combination of Kol43 and pevonedistat with a ZIP synergy score of 44.769.

[0030] Figure 17 shows a synergy plot for combination of Azacytidine and pevonedistat with a ZIP synergy score of 11.473.

[0031] Figure 18 shows a synergy plot for combination of Azacytidine and SCO-201 with a ZIP synergy score of 0.202.

[0032] Figure 19 shows an analysis of ABCG2 (BCRP) protein expression of the cells used in Example 4. The HL60-ABCG2 cells and the HL60 parental cells were stained by either the ABCG2 recognizing antibody 5D3 or by an isotype control that does not recognize ABCG2 (negative control). The readout is phycoerythrin (PE) signal. Top part: A clear increase in the signal is observed in the ABCG2 expressing cells when the 5D3 staining (solid line, b) is compared to the IgG control (a). Lower part (black lines): Almost no difference in the signal in the parental cells comparing c and d. The difference in ABCG2 levels between the two cell lines is calculated to app. 33-fold. Real time quantitative PCR analyses demonstrates that the level of ABCG2 mRNA is app. 65,000 fold up-regulated in the HL60-ABCG2 cell line compared to the parental HL60 cell line. Analyses of the protein expression demonstrated a 33- fold up-regulation of ABCG2 protein (BCRP) in the HL60-ABCG2 cell line compared to the parental HL60 cell line.

[0033] Figure 20 shows the response to Pevonedistat +/- 20 pM SCO-101 in AML HL60 parental cells (without ABCG2) and HL60-ABCG2 cells (with ABCG2 expression). A) HL60-ABCG2 cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 7.9 pM). B) HL60-ABCG2 cells were exposed to the indicated concentrations of Pevonedistat and 20 pM SCO-101 (IC50 = 0.6 pM), C) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 0.1 pM), D) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat + 20 pM SCO-101 (IC50 = 0.1 pM). [0034] Figure 21 shows the response to Pevonedistat, Kol43 or the combination in (A) HL60-ABCG2 cells (with ABCG2 expression) and (B) AML HL60 parental cells (without ABCG2). Kol43 is a positive control for ABCG2 inhibition.

[0035] Figure 22 shows the response to Pevonedistat, SCO-101 or the combination in (A) HL60-ABCG2 cells (with ABCG2 expression) or (B) AML HL60 parental cells (without ABCG2).

[0036] Figure 23 shows synergy plots of the SCO-101/pevonedistat combination based on the data from the Example 4 that combined 4 concentrations of Pevonedistat and SCO-101. The synergy plots were constructed by SynergyFinder to reveal synergistic interactions between these two compounds. In the HL60-ABCG2 cells a very strong synergy was observed - Figure 23A (ZIP synergy score of 76). In the HL60 parental cells a small synergistic effect was observed - Figure 23B (ZIP synergy score of 16). The scores can be interpreted as following: less than -10 is antagonistic; -10 to 10 is additive; larger than 10 is synergistic.

[0037] Figure 24 shows the response to Pevonedistat +/- 5 pM SCO-201 in AML HL60 parental cells (without ABCG2) and HL60-ABCG2 cells (with ABCG2 expression). In Figure 24A) HL60-ABCG2 cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 7.6 pM). In Figure 24B) HL60- ABCG2 cells were exposed to the indicated concentrations of Pevonedistat and 5 pM SCO-201 (IC50 = 0.2 pM), In Figure 24C) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 0.2 pM), In Figure 24D) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat + 5 pM SCO-201 (IC50 = 0.2 pM).

Incorporation by reference

[0038] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.

Detailed description

[0039] The features and advantages of the present invention is readily apparent to a person skilled in the art by the below detailed description of embodiments and examples of the invention with reference to the figures and drawings included herein.

[0040] The present disclosure provides a method for treatment of cancer in a patient using a) an ABCG2 inhibitor, and b) a NEDD8 activating enzyme (NAE) inhibitor.

[0041] In some embodiments, the method further involves administering to the patient a hypomethylating agent, such as azacytidine or decitabine, for example azacytidine.

ABCG2 inhibitor

[0042] ABCG2 is also known as breast cancer resistance protein (bcrp) and is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transporters. ABCG2 is further known to confer resistance to a wide range of chemotherapeutic agents including mitoxantrone, topotecan and irinotecan. ABCG2 is highly expressed in normal and cancer stem cells and has also been detected in a wide variety of untreated human solid tumors. Further, ABCG2 has been shown to decrease the therapeutic effect of pevonedistat leading to development of resistance toward pevonedistat, in particular for cancer cells overexpressing ABCG2.

[0043] The term "ABCG2 inhibitor" as used herein refers to a molecule which is capable of binding to the ABCG2 efflux pump in human or animal cells and thereby reduce its pumping capacity. Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. They are active transporters, meaning that they require a source of chemical energy to perform their function. Hence, the present disclosure provides a combination drug comprising, separately or together, an ABCG2 inhibitor and a NEDD8 inhibitor, such as pevonedistat for use in the treatment of cancer.

[0044] In an still further embodiment, the ABCG2 inhibitor is selected from the group consisting of:

SCO-101 having CAS number 265646-85-3, SCO-201, Febuxostrat having CAS number 14406-53-7, Benzbromarone having CAS number 3562-84-3, Atorvastatin having CAS number 134523-0-5, Rosuvastatin having CAS number 287714-41-4, Losartan having CAS number 114798-26-4, Tacrolimus having CAS number 10498-11-3, Topiroxostat having CAS number 577778-58-6, Fenofibrate having CAS number 49562-8-9, Cyclosporine having CAS number 59865-13-3, Furosemide having CAS number 54-31-9 and Chlorothiazide having CAS number 58-94-6.

[0045] In one embodiment, the ABCG2 inhibitor is selected from the group consisting of: SCO-101, SCO-201, Quercetin, Naringenin, Novobiocin, Estrone, Saquinavir, Omeprazole, Hesperetin, Genistein, Nelfinavir, Diethylstilbestrol, Ritonavir, Pantoprazole, Cyclosporine, Taurocholic acid, Imatinib, Estradiol, Daidzin, Sorafenib, Sunitinib, Dronabinol, Buprenorphine, Sulfasalazine, Telmisartan, Dexamethasone, Dasatinib, Rabeprazole, Elacridar, Nilotinib, Erlotinib, Vandetanib, Cabazitaxel, Rilpivirine, Vismodegib, Regorafenib, Ponatinib, Dabrafenib, Afatinib, Cobicistat, Rolapitant, Daclatasvir, Alectinib, Dasabuvir, Velpatasvir, Simeprevir, Topiroxostat, Voxilaprevir, Enasidenib, Pibrentasvir, Glecaprevir, Abemaciclib, Letermovir, Brigatinib, Rucaparib, Baricitinib, Cannabidiol, Estradiol acetate, Estradiol benzoate, Estradiol cypionate, Estradiol dienanthate, Estradiol valerate, Isavuconazole, Nabiximols, Avatrombopag, Gefitinib, Safinamide, Paritaprevir, Fostamatinib, Lansoprazole, Pravastatin, Dovitinib, Eltrombopag, Teriflunomide, Vemurafenib, Progesterone, Fusidic acid, Netupitant, Ledipasvir, Osimertinib, Grazoprevir, Elbasvir, Sapropterin, Tedizolid, Dacomitinib, Venetoclax, Gilteritinib, Glasdegib, Olaparib, Itraconazole, Palbociclib, Alpelisib, Beclomethasone dipropionate, Fedratinib, Istradefylline, Caffeine, Lasmiditan, Darolutamide, Upadacitinib, Tafamidis, Capmatinib, Selpercatinib, Ripretinib, Clofazimine, Dexamethasone acetate, Fostemsavir, Pralsetinib, Lonafarnib, Relugolix, Tepotinib, Febuxostat, Avanafil, Tivozanib, Infigratinib, Sotorasib, Tecovirimat, Avapritinib, Brincidofovir, Belumosudil, Lorlatinib, Mobocertinib, Asciminib, Maribavir, Sotagliflozin, Roxadustat, Cannabinol, Pacritinib, Oteseconazole, Stiripentol, Baloxavir marboxil, and Deucravacitinib.

[0046] In one embodiment, the ABCG2 inhibitor is selected from the group consisting of: SCO-101, SCO-201, Febuxostrat, Benzbromarone, Atorvastatin, Rosuvastatin, Losartan, Tacrolimus, Topiroxostat, Fenofibrate, Cyclosporine, Furosemide, and Chlorothiazide.

Determining ABCG2 inhibition

[0047] It is well known in the art how to identify modulators of ABCG2 activity for example described in Henrich et al. A High-Throughput Cell-Based Assay for Inhibitors of ABCG2 Activity" Journal of Biomolecular Screening 11(2); 2006. In a further embodiment the ABCG2 inhibitor inhibits, i.e is capable of inhibiting the efflux of the NEDD8 inhibitor from the cancer cell, or the expression of the ABCG2 transporters compared to similar non-cancer cells.

Specific ABCG2 inhibitors

[0048] In another embodiment the ABCG2 inhibitor is a compound of general formula I: or a pharmaceutically acceptable salt thereof, wherein R2 represents tetrazolyl; and

R3, R4, R5, R6, R12, R13, R14, R15, and R16 independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NRaRb, -Nra-CO-Rb, phenyl or heteroaryl; which phenyl is optionally substituted with halo, trifluoromethyl, nitro, -CO-NHRc, CO-O-Rc or -CO- NR'R"; wherein Rc is hydrogen, alkyl, or phenyl;

R' and R" independently of each other are hydrogen or alkyl; or

R' and R" together with the nitrogen to which they are attached form a 5- to 7-membered heterocyclic ring, which ring may optionally comprise as a ring member, one oxygen atom, and/or one additional nitrogen atom, and/or one carbon-carbon double bond, and/or one carbon-nitrogen double bond; and which heterocyclic ring may optionally be substituted with alkyl;

Ra and Rb independently of each other are hydrogen or alkyl; or

R15 and R16, or R14 and R15 together with the phenyl ring to which they are attached form a naphthyl ring or an indanyl ring; and R3, R4, R5, R6, R12 and R13 and the remaining one of R14, R15 and R16 are as defined above.

[0049] In a further embodiment R3, R5, and R6 represent hydrogen; and R4 represents halo. [0050] In a still further embodiment R3, R5, and R6 represent hydrogen; and R4 represents phenyl substituted with trifluoromethyl, nitro or -CO-NHRc; wherein Rc is phenyl.

[0051] In a still further embodiment, the ABCG2 inhibitor is selected from the group consisting ofSCO- 101, SCO-201, NS3623, and NS3749. [0052] In some embodiments, the ABCG2 inhibitor is SCO-101:

(SCO-101); or a pharmaceutically acceptable salt, and/or hydrate thereof.

[0053] In one embodiment, the ABCG2 inhibitor is SCO-201 of formula (III): or a pharmaceutically acceptable salt, and/or hydrate thereof.

[0054] In other embodiments the ABCG2 inhibitor is also an inhibitor of UGT1A1, optionally with an IC50 of less than 0.5 pM.

UGT1A1 inhibitior [0055] The term "UGT1A1 inhibitor" as used herein refers to a molecule which is capable of binding to human uridine diphosphate (UDP)-glycuronosyl transferase (UGT1A1), particularly in humans or animals and decrease the enzymatic activity of the UGT1A1. UGT1A1, for example found in the liver of humans, gluronidation a range of different molecules hereinunder several anti-cancer agents, bilirubin, and other molecules usually converting them to less active and more water-soluble forms. Whether or not a particular molecule is an UGT1A1 inhibitor can be determined by examining the molecule's IC50 in vitro in a UGT1A1 inhibition assays known in the art. In one embodiment, the ABCG2 inhibitor is also a UGT1A1 inhibitor.

[0056] Inhibitors described herein typically have an IC50 of 20 pM or less, such as 15 pM or less, such as 10 pM or less, such as 5 pM or less against the inhibitor target. In one embodiment, the IC50 is 5 pM or less, such as 4 pM or less, such as 3 pM or less, such as 2 pM or less, for example between 0.1 pM and 2.0 pM. In some embodiments, the inhibitor is non-competitive, while in other embodiments the inhibitor is competitive.

Pharmaceutically Acceptable Salts

[0057] The ABCG2 inhibitor as described herein may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts, and pre- or prodrug forms. Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate af derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2- sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, and the like. Such salts may be formed by procedures well known and described in the art. Other acids such as oxalic acid, which may not be considered pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining a chemical compound for use according to the invention and its pharmaceutically acceptable acid addition salt. Examples of pharmaceutically acceptable cationic salts of a chemical compound of the invention include, without limitation, the sodium, the potassium, the calcium, the magnesium, the zinc, the aluminium, the lithium, the choline, the lysine, and the ammonium salt, and the like, of a chemical compound of the invention containing an anionic group.

[0058] Such cationic salts may be formed by procedures well known and described in the art. In the context of this invention the "onium salts" of N-containing compounds are also contemplated as pharmaceutically acceptable salts (aza-onium salts). Preferred azaonium salts include the alkylonium salts, in particular the methyl- and the ethylonium salts; the cycloalkylonium salts, in particular the cyclopropylonium salts; and the cycloalkylalkylonium salts, in particular the cyclopropyl-methylonium salts.

NEDD8 activating enzyme inhibitor

[0059] Cancer cells depend on signals that promote cell cycle progression and prevent programmed cell death that would otherwise result from cumulative, aberrant stress. These activities require the temporally controlled destruction of specific intracellular proteins by the ubiquitin-proteasome system (UPS). To a large extent, the control points in this process include a family of E3 ubiquitin ligases called cullin-RING ligases (CRLs). The ligase activity of these multicomponent complexes requires modification of the cullin protein situated at their core with a ubiquitin-like protein called NEDD8. Neddylation results in conformational rearrangements within the CRL, which are necessary for ubiquitin transfer to a substrate. The NEDD8 pathway thus has a critical role in mediating the ubiquitination of numerous CRL substrate proteins involved in cell cycle progression and survival including the DNA replication licensing factor Cdt-1, the NF-KB transcription factor inhibitor pIxBa, and the cell cycle regulators cyclin E and p27. The initial step required for attachment of NEDD8 to a cullin is catalyzed by the El, NEDD8-activating enzyme (NAE). Inhibition of of NAE has been shown to prevent the subsequent neddylation of cullins, being of high relevance in cancer therapy. Hence, in some embodiments, the present disclosure provides a NEDD8 inhibitor for use in the treatment of cancer. In some embodiments, the NEDD8 inhibitor of the present disclosure is an inhibitor of the NEDD8 activating enzyme, i.e. a NAE inhibitor. Hence, in all aspects and embodiments disclosed herein the NEDD8 inhibitor is preferably a NAE inhibitor.

[0060] In some embodiment, the NEDD8 inhibitor is a selective NEDD8 inhibitor. A NEDD8 inhibitor has an IC50 of I 20 pM or less against NEDD8, such as 15 pM or less, such as 10 pM or less, such as 5 pM or less. A selective NEDD8 inhibitor has an IC50 of I 20 pM or less against NEDD8, such as 15 pM or less, such as 10 pM or less, such as 5 pM or less. In some embodiments, the IC50 value is based on inhibition of NEDD8 activating enzyme (NAE).

[0061] In some embodiment, the NEDD8 inhibitor is pevonedistat of formula (I); or a pharmaceutically acceptable salt, and/or hydrate thereof.

Hypomethylating agent

[0062] In some embodiments, the method for treating cancer or combination drug for use in cancer treatment also involves using a hypomethylating agent for the treatment of the cancer. As shown in Example 2, combining a hypomethylating agent, such as azacytidine or decitabine, for example azacytidine with a NAE inhibitor and/or a a ABCG2 inhibitor provides a synergistic treatment effect. In particular, the treatment is effective in treatment of resistant cancers, such as resistant colorectal cancer.

[0063] In some embodiments, the combination drug for use in the treatment of cancer as disclosed herein is combined with azacytidine. Azacytidine can be administered prior to, simultaneously with, or subsequent to any of the ingredients of the combination drug.

[0064] In some embodiments, a pharmaceutical composition is provided according to the present disclosure further comprising a hypomethylating agent, such as azacytidine Cancers

[0065] The cancers encompassed herein include those cancers where the ABCG2 inhibitor improves the effect of the NEDD8 inhibitor on the cancer. In some embodiments the cancer is resistant to treatment with the NEDD8 inhibitor, such as pevonedistone. If the cancer is resistant, co-treatment with the ABCG2 inhibitor is capable of re-sensitising the cancer to the NEDD8 inhibitor in question. Resistance of cancers may be either de novo resistance or acquired resistance. In general, a cancer is regarded as resistant to a particular NEDD8 inhibitor if a patient treated with the clinically accepted dosage of the NEDD8 inhibitor does not respond as expected to the NEDD8 inhibitor, i.e. in case of worsening, growth, or spread of the cancer (progressive disease). Whether a cancer is drug-sensitive or resistant can be determined by the skilled person using methodology known in the art.

[0066] In one embodiment, wherein the ABCG2 inhibitor re-sensitises the cancer to an anti-cancer agent and/or the NEDD8 inhibitor, i.e. the ABCG2 inhibitor is capable of re-sensitizing the cancer to an anti-cancer agent and/or the NEDD8 inhibitor.

[0067] In certain embodiments, the cancer is selected from the group consisting of adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T- lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0068] In another embodiment, the cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer (non small cell lung cancer and small cell lung cancer), one or more glioblastomas, one or more Head and neck cancers, one or more malignant melanomas, basal cell skin cancer, squamous cell skin cancer, liver cancer, pancreatic cancer, prostate cancer, anal cancer, cervix uteri cancer, bladder cancer, corpus uteri cancer, ovarian cancer, gall bladder cancer, one or more sarcomas, one or more leukemias (myeloid and lymphatic), one or more lymphomas, myelomatosis, cholangiocarcinoma, gastric cancer, testicular cancer, uveal cancer, mesothelioma, merkel cell carcinoma, and one or more myelodysplastic syndromes (MDS).

[0069] In one embodiment, the cancer is a solid tumor, such as a solid tumour selected from sarcoma, carcinoma and lymphoma, while in other embodiments the cancer is not a solid tumour, such as hematological malignancy including but not limited to leukemia. In some embodiments, the cancer is one or more advanced solid tumors. An advanced solid tumor is a type of solid tumor that has spread from where it first started to nearby tissue, lymph nodes, or distant parts of the body of a patient. The present disclosure demonstrates that the treatments disclosed herein work effectively on both solid tumors and hematological malignancies, see Examples 1-4. Examples 1-2 demonstrates the effective treatment of solid tumors, in particular treatment of resistant solid tumors, whereas Example 4 demonstrates effective treatment of resistant AML.

[0070] In some embodiments, the cancer is selected from the group consisting of: myeloma, lymphoma, melanoma, one or more leukemias, one or more myelodysplastic syndromes, and one or more solid tumors.

[0071] In some embodiments, the cancer is selected from the group consisting of: mantle cell lymphoma (MCL), acute myeloid leukemia (AML), and myelomonocytic leukaemia, such as chronic myelomonocytic leukaemia, and one or more myelodysplastic syndromes. In some embodiments, the one or more myelodysplastic syndromes are MDS with single lineage dysplasia, MDS with multilineage dysplasia, or MDS with excess blasts.

[0072] In some embodiments, the present disclosure provides a treatment of a cancer resistant to a NAE inhibitor. In some embodiments, the resistant cancer is AML.

[0073] In particular embodiments, the present disclosure provides a combination treatment comprising the ABCG2 inhibitor SCO-101 and the NAE inhibitor pevonedistat. In particular embodiments, this combination treatment is highly effective against resistant AML, such as AML resistant to NAE inhibitors if administered without an ABCG2 inhibitor. The synergistic effects of this particular combination in AML are demonstrated at least by Example 4 of the present disclosure.

[0074] In particular embodiments, the present disclosure provides a combination treatment comprising the ABCG2 inhibitor SCO-201 and the NAE inhibitor pevonedistat. In particular embodiments, this combination treatment is highly effective against resistant AML, such as AML resistant to NAE inhibitors if administered without an ABCG2 inhibitor. The highly beneficial effects of this particular combination in AML are demonstrated at least by Example 4 of the present disclosure. [0075] In some embodiments, the cancer is relapsed refractory multiple myeloma or relapsed refractory lymphoma.

[0076] In some embodiments, the cancer is metastatic melanoma.

[0077] In some embodiments the cancer is metastatic, such as metastatic colorectal cancer, metastatic prostate cancer, and/or metastatic breast cancer. In other embodiments the cancer is glioblastoma.

[0078] In a further embodiment the cancer is a steroid hormone receptor positive cancer, e.g. an estrogen receptor positive cancer, a progesterone receptor positive cancer or an androgen receptor positive cancer.

[0079] In a still further embodiment, the cancer is undifferentiated colon carcinoma cancer.

[0080] In some embodiments the cancer comprises cancer cells having an increased expression of ABCG2 compared to a corresponding non-cancer cell. In particular the cancer can comprise ABCG2+ cancer cells. ABCG2+ cancer cells for which the treatment described herein is effective are ABCG2+ cancer cells proficient in genetic mismatch repair a well as ABCG2+ cancer cell that are deficient in genetic mismatch repair. ABCG2+ are well known in the art.

[0081] Further types of cancers are described in EP1907000 and US20200323862 incoporated herein by refercence. Specific combinations

[0082] In one embodiment, the ABCG2 inhibitor is SCO-101 or a pharmaceutically acceptable salt, and/or hydrate thereof, and the NEDD8 inhibitor is pevonedistat.

[0083] In one embodiment, the ABCG2 inhibitor is SCO-201 or a pharmaceutically acceptable salt, and/or hydrate thereof, and the NEDD8 inhibitor is pevonedistat.

[0084] In one embodiment, the ABCG2 inhibitor is SCO-101, or a pharmaceutically acceptable salt, and/or hydrate thereof, and the NEDD8 inhibitor is pevonedistat, and the cancer is mantle cell lymphoma (MCL) or acute myeloid leukemia (AML), or one or more myelodysplastic syndromes.

Compositions and formulations

[0085] In the combination drug, pharmaceutical composition and methods disclosed herein, the NEDD8 inhibitor is preferably pevonedistat; and the ABCG2 inhibitor is preferably SCO-101 or SCO-201 as described herein.

[0086] The present disclosure also provides for a method for the preparation of a medicament for treating a disease, suitably a cancer, more suitably one or more of the cancers described herein, wherein the medicament, separately or together, comprises

(a) an ABCG2 inhibitor, and

(b) a NEDD8 inhibitor; for use in the treatment of a cancer. In some embodiments, the cancer is treated in a patient.

[0087] The pharmaceutical composition or combination drug disclosed herein may comprise, separately or together, the ABCG2 inhibitor and the NEDD8 inhibitor together with one or more pharmaceutically acceptable carriers therefore, and, optionally, other therapeutic and/or prophylactic ingredients known and used in the art. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. In a further embodiment, the pharmaceutical composition comprises more than one compound or prodrug for use according to the invention, such as two different compounds or prodrugs for use according to the invention.

[0088] Pharmaceutical compositions of the invention may be those suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.

[0089] The ABCG2 inhibitor, and the NEDD8 inhibitor together with a conventional adjuvant, carrier, or diluent, may thus be placed separately or together into the form of pharmaceutical compositions and unit dosages thereof. Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The ABCG2 inhibitor, and the NEDD8 inhibitor as described herein can be administered separately or together in a wide variety of oral and parenteral dosage forms.

[0090] For preparing pharmaceutical compositions from a chemical compound of the present invention, pharmaceutically acceptable carriers can be either solid or liquid.

[0091] Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets suitably contain from 5 or 10 to about 70 percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration. For preparing suppositories, a low melting wax, such as a mixture of fatty acid glyceride or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify.

[0092] Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. The ABCG2 inhibitor, the NEDD8 inhibitor may thus be formulated separately or together for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the ABCG2 inhibitor may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0093] Also included are solid form preparations, intended for conversion shortly before use to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. In addition to the active component such preparations may comprise colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0094] For topical administration to the epidermis the ABCG2 inhibitor, and the NEDD8 inhibitor may be formulated separately or together as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0095] Solutions or suspensions may be applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in single or multidose form.

[0096] Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve. Alternatively, the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatine, or blister packs from which the powder may be administered by means of an inhaler.

[0097] In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.

[0098] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these inpackaged form. [0099] Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.

[0100] Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA).

[0101] A therapeutically effective dose refers to that amount of active ingredient, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity, e.g. ED50 and LD50, may be determined by standard pharmacological procedures in cell cultures or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index and may be expressed by the ratio LD50/ED50. Pharmaceutical compositions exhibiting large therapeutic indexes are preferred.

[0102] The dose administered must of course be carefully adjusted to the age, weight and condition of the individual being treated, as well as the route of administration, dosage form and regimen, and the result desired, and the exact dosage should of course be determined by the practitioner.

[0103] The actual dosage of the ABCG2 inhibitor, and the NEDD8 inhibitor depends on the chosen active ingredients as well as the nature and severity of the disease being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances to produce the desired therapeutic effect.

[0104] For example the ABCG2 inhibitor, and the NEDD8 inhibitor may be administered separately or together in one or several doses per day. Exemplary ranges are from 10-200 mg/day p.o. (per oral) administered in one or two doses, such as from 25-50 mg p.o. twice a day for the ABCG2 inhibitor.

[0105] The patient to be treated according to the present disclosure may be any human or animal patient, preferably a human suffering from cancer.

[0106] In special embodiments the ABCG2 inhibitor, and the NEDD8 inhibitor are suitably administered together in any order of sequence providing optimal therapeutic efficacy.

[0107] In some embodiments, the combination drug is provided, wherein (i) the ABCG2 inhibitor is formulated as a tablet or a capsule for enteral oral administration or as a liquid for parenteral administration, optionally intravenous administration or continuous infusion, (ii) the NEDD8 inhibitor is formulated as a tablet or a capsule for enteral oral administration or as a liquid for parenteral administration, optionally intravenous administration or continuous infusion.

[0108] In some embodiments, a pharmaceutical composition is provided comprising, separately or together, (a) an ABCG2 inhibitor,

(b) a NEDD8 inhibitor, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.

[0109] In some embodiments, the ABCG2 inhibitor is SCO-101 or a pharmaceutically acceptable salt and/or hydrate thereof; and the NEDD8 inhibitor is pevonedistat.

[0110] In some embodiments, the ABCG2 inhibitor is SCO-201 or a pharmaceutically acceptable salt and/or hydrate thereof; and the NEDD8 inhibitor is pevonedistat.

Administration

[0111] In one embodiment, the ABCG2 inhibitor is administered prior to and/or simultaneously with and/or after the NEDD8 inhibitor.

[0112] In one embodiment, administration of the SCO-101 enhances and/or potentiates the therapeutical effect of the NEDD8 inhibitor, optionally wherein the therapeutic effect is an anti-cancer effect.

[0113] In one embodiment, administration of the SCO-201 enhances and/or potentiates the therapeutical effect of the NEDD8 inhibitor, optionally wherein the therapeutic effect is an anti-cancer effect.

Kit of parts

[0114] In a separate aspect of the present invention, a kit of parts is provided, wherein the kit of parts comprises an (a) ABCG2 inhibitor, and (b) a NEDD8 inhibitor; for use in the treatment of a cancer.

[0115] In one embodiment of the present invention, the ABCG2 inhibitor is SCO-101 or SCO-201, and the NEDD8 inhibitor is pevonedistat.

[0116] In some embodiments, the kit of parts further comprises a hypomethylating agent, such as azacytidine.

Examples

Example 1 - Combination treatment of pevonedistat and ABCG2 inhibitors

Materials and methods Compounds

[0117] Pevonedistat was purchased from MedChem Express (HY-70062), SCO-101 was sourced from API (Cambrex, Karslkoga, Sweden 219003), SCO-201 was sourced from stock OBR-5-340, while Ko-143 was purchased from Sigma (K2144). All compounds were dissolved in dimethyl sulfoxide (DMSO) and kept at -20°C.

Cell culture

[0118] The colorectal adenocarcinoma cell line HT-29 was obtained from the NCI/Development Therapeutics program. The generation of the SN38 acquired resistance cell line, HT29-SN38R, was previously described in Jensen et al, 2015 (1). Both HT-29 and HT29-SN38R were cultured in RPMI1640 with GlutaMAX (Gibco, 61870 044) supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher, 15140122). Cell lines were kept in a humidified incubator at 37°C, with 5% CO2. All cell lines were checked for Mycoplasma through Eurofins genomics, using the standardized qPCR test for mycoplasma performed under ISO17025 accreditation. Cell line identification by STR analyses was performed by Eurofins genomics according to ANSI/ATCC standard ASN-0002 and confirmed the cell line identity. To avoid artefacts of long-term culture of immortalized cell lines, cell culture passage numbers were tracked and annotated for each experiment and culturing was interrupted after 8-12 weeks post thawing.

Cell survival assay and IC50 determination

[0119] For the HT-29 cell line, 4000 cells were plated in a clear bottom 96-well plate and treated with increasing drug doses. For the HT29-SN38R cell line, the same procedure was done using 6000 cells. Cells were allowed to attach for 24 hours before treatment was initiated. Cell viability was measured after 72 hours of drug exposure, using the Cell counting Kit 8 (MedChemExpress, HY-K0301), according to manufacturer's instructions. Absorbance was measured at 450 nM using a VERSAMax plate reader (Molecular Devices). IC50 values were determined in GraphPad Prism v9.2, using nonlinear regression analysis (log inhibitor vs. response, four parameters).

Combinatorial treatment and evaluation of drug synergy

[0120] For the combinatorial treatment, HT29-SN38R cells were plated at 6000 cells per well in a 96- well plate. In all cases, cells were treated with one drug combination per plate (Pevonedistat+SCO- 101, Pevonedistat+SCO-201 or Pevonedistat+Kol43). Each drug was administered in four different concentrations, determined by their respective IC50 values, and given as single treatment and as combinatorial treatment in all combinations (48 combinations in total). The synergy of inhibitor combinations was calculated utilizing the SynergyFinder web application 2.0 (2), according to publishers' instructions.

1. Jensen, Niels F., Jan Stenvang, Mette K. Beck, Barbora Hanakova, Kirstine C. Belling, Khoa N. Do, Birgitte Viuff, et al. 2015. "Establishment and Characterization of Models of Chemotherapy Resistance in Colorectal Cancer: Towards a Predictive Signature of Chemoresistance." Molecular Oncology 9 (6): 1169-85.

2. lanevski, Aleksandr, Anil K. Giri, and Tero Aittokallio. 2020. "SynergyFinder 2.0: Visual Analytics of Multi-Drug Combination Synergies." Nucleic Acids Research 48 (Wl): W488-93.

Results

[0121] SCO-101 and SCO-201 are both strong inhibitors of the efflux pump ABCG2. Both molecules are able to potentiate the anti-cancer effect of pevonedistat in cancer cells that overexpress ABCG2 (pevonedistat resistant cancer cells).

Conclusion

[0122] The present example demonstrates that ABCG2 inhibitors, such as SCO-101 or SCO-201 are surprisingly effective in potentiating the effect of pevonedistat in cancer therapy, in particular towards cells resistant to common chemotherapy, such as pevonedistat and/or SN-38 resistant cells.

Example 2 - Standalone and combinatorial treatment with pevonedistat and SCO-201 in HT29 SN38 resistant colorectal cancer model system

[0123] The aim of the present example was to determine the IC50 values of Pevonedistat and Azacytidine in HT29P and HT29 SN38-resitant cell lines and to determine the potential of ABCG2 inhibitors in enhancing the efficiency of NAE inhibitors, such as pevonedisate and/or the anti-cancer agent Azacytidine. Further, the aim was to Investigate the potential of SCO-201 with respect to improving the response of the cancer cells to a previously investigated Azacytidine and Pevonedistat combination.

Material and Reagents

Table 1: Compounds used in Example 2.

Cell lines: Colorectal cancer cell lines, HT29 Parental and HT29 SN38-resistant.

Reagents: Media (RPMI1640, Thermo Fisher 61870 044), DMSO (Dimethyl Sulfoxide, Sigma Aldrich), CCK8 (Cell counting kit 8, Med Chem Express HY-K0301).

Instruments: LAF bench, incubator, plate reader (Molecular Devices VERSAMax).

Software: SoftmaxPro, Office Excel, SynergyFinder, GraphPad Prism.

Methods

[0124] To determine the IC50 values of Pevonedistat and Azacytidine in the HT29 and HT29 SN38-res cells, as well as to determine the ability of SCO-201 to enhance efficiency of Pevonedistat (and Azacytidine) in combination with various ABCG2 inhibitors, a series of viability assays was performed in a 96 well format. CCK8 was used to determine viability. HT29 parental cells were plated at the density of 4000 cells, while HT29 SN38-Res cells were plated at the density of 6000 cells per well into a 96-well plate. The following day, the cells were treated using increasing doses of Pevonedistat for the IC50 determination or using a 4x4 setup for combined treatment with SCO-101, SCO-201 or Kol43. For the triple combination of Pevonedistat, Azacytidine and SCO-201, two approaches were attempted. In the first one, two different concentrations of each of the compounds were used and combined against all compounds. In the other approach a dose response curve was made for azacytidine, either alone or with the addition of a fixed dose of either Pevonedistat or SCO-201, or both. Control conditions consisted of either growth medium (referred to as 'untreated') or DMSO treated cells, at the dose corresponding to the highest amount of DMSO among compound treated cells. All combination therapy experiments included the equivalent doses of single drugs. All experiments were performed twice on two separate days (biological duplicates), unless otherwise stated. For each experiment, treatments were performed in technical triplicates. Cells were kept in a humidified incubator at 37°C, with 5% CO2. lOOpI 1:10 CCK-8 diluted in media is added to each well. After addition of CCK-8, cells were incubited for l-2h at 37°C. Optical densities (Ods) are measured at 450 nM with VersaMax microplate spectrophotometer (Molecular Devices). The Ods measured for media alone were subtracted from the OD values of the samples, prior to further analysis. IC50 values were determined using GraphPad Prism and the in-built non-linear regression (log (inhibitor) vs response -variable slope (four parameters)) calculation. For determination of synergy, the online tool Synergy finder was used.

Results

Monotherapy: Pevonedistat and Azacytidine IC50 determination

[0125] HT29 and HT29 SN38Res cells were treated with increasing doses of Pevonedistat or Azacytidine and we determined the sensitivity of HT29 parental and SN38 Resistant cells to Pevonedistat or Azacytidine upon monotherapy treatment, see figures 5 and 6. The resulting IC50 values for HT29 parental and SN38Res cells can be found in Table 2.

Table 2: Overview of determined IC50 values in HT29 Parental and SN38 resistant cells, as determined by non-linear regression in Graph Pad Prism. Values are shown in pM. last column shows number of biological replicates made for each drug.

[0126] From the determined IC50 values, it is evident that HT29 SN38 Resistant cells have a higher resistance to Pevonedistat than HT29 Parental cells.

Combination therapy [0127] HT29 SN38R cells were treated with Pevonedistat in combination with three different ABCG2 inhibitors - SCO-101, SCO-201 and Kol43, of which Kol43 was used as a positive control. As HT29 SN38-Resistant cells overexpress ABCG2, the combined treatments should increase their sensitivity to Pevonedistat. Indeed, all three ABCG2 inhibitors in combination with Pevonedistat enhanced its cytotoxicity. Additionally, Azacytidine was tested in combination with Pevonedistat to evaluate the clinically tested combination in our model. Expectedly, Pevonedistat enhanced the cell killing capabilities of Azacytidine, even though colorectal cancer is not the intended indication for these compounds. For control, Azacytidine was tested in combination with SCO201 and did not show any enhancement of cell killing compared to Azacytidine alone.

[0128] Furthermore, the triple combination of Azacytidine with Pevonedistat and SCO-201 was tested at a reduced number of concentrations. In the first approach (Figure 12) we used two concentrations for each compound and combined them in all possible combinations. Additionally, we used Kol43 as a positive control (only one dose). In the second approach (Figure 13) we used a fixed dose of either Pevonedistat (at lpM) or SCO-201 (at 2.5 pM) (or both) and added it to a dose response curve of Azacytidine ranging from 10 pM to 0.16 pM. Importantly, both triple combination experiments were done only once, and the second approach was performed with only technical triplicates due to space constrain. Based on the present example, it seems plausible that a triple combination of Azacytidine, Pevonedistat and SCO-201 provides even better results than the combination of Azacytidine and Pevonedistat alone.

[0129] Finally, for all the double treatment combinations that did seem promising, a synergy score analysis was performed. Pevonedistat was highly synergistic with all ABCG2 inhibitors, SCO-101, SCO- 201 and Kol43, supporting that ABCG2 inhibition may increase the efficacy of Pevonedistat. Pevonedistat was also synergistic with Azacytidine, however the combination of Azacytidine and SCO- 201 was not synergistic (and it was barely additive) in the resistant cells.

Discussion

[0130] Pevonedistat is an NAE inhibitor that has recently failed a Phase 3 clinical trial in older adults with high risk MDS, CMML or AML, due to lack of improvement over azacytidine alone. Presumably, this could be due to ABCG2 overexpression and Pevonedistat being pumped out of cancer cells through ABCG2, as it has been shown that indeed Pevonedistat interacts with ABCG2 and could potentially be a substrate. The HT29 colorectal cancer cell line (parental) as well as the HT29 SN38- Res (ABCG2 overexpressing) cells was exposed to increasing doses of Pevonedistat for 72h. The SN38 Resistant, ABCG2 overexpressing cells were observed to have an almost 20 times higher Pevonedistat IC50 compared to their parental counterparts, not overexpressing ABCG2. Hence, two ABCG2 inhibitors, namely SCO-101 and SCO-201 were investigated in combination with Pevonedistat and whether this could increase the potential of Pevonedistat to kill cancer cells. The results of the ABCG2 inhibitors were compared to a known ABCG2 inhibitor (Kol43). Furthermore, when testing Pevonedistat in combination with any of the used ABCG2 inhibitors, resensitization was observed, compared to either molecule alone. When analyzing the synergistic effects, all ABCG2 inhibitors were synergistic with Pevonedistat, however SCO-201 and Kol43 caused a more profound effect compared to SCO-101. Additionally, the original treatment combination of Azacytidine and Pevonedistat in the tested model system was investigated as well as the interaction between Azacytidine and SCO-201 in the HT29 SN38-Res cells. Expectedly, a synergistic effect was observed for the combination of Azacytidine and Pevonedistat, even though the model system was not an AML or MDS model. However, there was no synergism (or additivity) observed between Azacytidine and SCO-201. This was expected as Azacytidine has not been reported to be an ABCG2 substrate. Finally, two initial investigations were performed of triple combinations of Azacytidine, Pevonedistat, and SCO-201.

Conclusion

[0131] The present example demonstrates that NAE inhibitors, such as Pevonedistat, and ABCG2 inhibitors, such as SCO-101, SCO-201 or Kol43 exhibit much stronger synergistic anti-cancer effects than combination treatment of Pevonedistat with Azacytidine.

Example 3 - Combination treatment of azacytidine and SCO-201 in non-resistant cancer

[0132] Azacytidine and SCO-201 obtained from the same source as in Example 2 is subjected to parental cancer cells, such as the HT29 colorectal cancer cell line and otherwise identical methodology of Example 2. The combination of azacytidine and SCO-201 shows an anti-cancer effect in the non- resistant cancer cells. Further, the anti-cancer effect demonstrated is synergistic. Example 4 - Combination treatment of pevonedistat and ABCG2 inhibitors in AML with or without ABCG2

Materials and methods

Reagents: RPMI medium (ThermoFisher cat no A1049101), Fetal calf serum (ThermoFisher cat no 10270-106), Pen/Strep (ThermoFisher cat no 15140-122), Pevonedistat (MedChemExpress cat no HY- 70062), Kol43 (Sigma cat no K2144), SCO-101 (batch 68251), SCO-201 (batch 23.06.11), CCK8 (MedChemExpress cat no HY-K0301)

Cell lines: The HL60 parental and the HL60-ABCG2 cell lines were obtained from Associate Professor Martina Ceckova, Charles University, Faculty of Pharmacy, Hradec Kralove, Czech Republic.

References:

Sorf et. al. Cancers, 2020 Jun 16;12(6):1596. Targeting Pharmacokinetic Drug Resistance in Acute Myeloid Leukemia Cells with CDK4/6 Inhibitors (PMID 32560251).

Hollo Z., Homolya L., Hegedus T., Sarkadi B. Transport properties of the multidrug resistance- associated protein (MRP) in human tumour cells. FEBS Lett. 1996;383:99-104. doi: 10.1016/0014- 5793(96)00237-2

Ozvegy-Laczka C., Hegedus T., Varady G., Ujhelly O., Schuetz J.D., Varadi A., Keri G., Orfi L., Nemet K., Sarkadi B. High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter. Mol. Pharmacol. 2004;65:1485-1495.

Ujhelly O., Ozvegy C., Varady G., Cervenak J., Homolya L., Grez M., Scheffer G., Roos D., Bates S.E., Varadi A., et al. Application of a human multidrug transporter (ABCG2) variant as selectable marker in gene transfer to progenitor cells. Hum. Gene Ther. 2003;14:403-412.

Ozvegy et al 2002: Characterization of Drug Transport, ATP Hydrolysis, and Nucleotide Trapping by the Human ABCG2 Multidrug Transporter. MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS | VOLUME Til , ISSUE 50, P47980-47990, DECEMBER 2002

Sucha, et al. 2022 ABCB1 as a potential beneficial target of midostaurin in acute myeloid leukemia. Biomed Pharmacother . 2022 Jun;150:112962. PMID: 35462331 Culture conditions: Cells were cultured in RPMI medium, supplemented with 10% fetal calf serum, 50 units/ml penicillin and streptomycin at 37 °C in humidified 5% CO2.

Drug treatment and viability read out: Cells were plated with a density of 50,000 cells/well in 96-well format. After addition of drug(s), the cells were kept in the incubator at 37 °C in humidifeied 5% CO2. After 72 hours drug exposure a CCK8 viability assay was performed according to the providers instructions. The optical densities (OD) was expressed in percentage of untreated cells, which was defined as 100%. The standard deviation (N=3) was calculated in percentage and visualized as error bars.

[0133] Synergy between drugs was calculated by "SynergyFinder Plus" as recommended by the provider SynergyFinder.

References: Zheng, S.; Wang, W.; Aldahdooh, J.; Malyutina, A.; Shadbahr, T.; Tanoli, Z.; Passia, A.; Tang, J. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics, Proteomics & Bioinformatics 2022, 20 (3), 587-596. doi:

10.1016/j.gpb.2022.01.004.

Analysis of ABCG2 (BCRP) protein expression by flow cytometry:

1. The HL60-ABCG2 cells and the HL60 parental cells were plated at 1x105 cells/well.

2. Cells were washed with PBS (Ca ² /Mg ² ) then stained with eF780 viability dye (Invitrogen, 65-0865- 14) for 30 min at 4°C following supplier's recommendations. Thereafter, cells were washed in FACS buffer (1% BSA, 0.1% sodium azide in PBS (Ca ² /Mg ² then resuspended in Fc receptor block (Miltenyi 130-059-901) for 10 minutes then without washing the FC block, cells were incubated with 0.5pg of PE-labelled anti-ABCG2 clone 5D3 (Biolegend) or 0.5pg PE Mouse lgG2b, k Isotype control (Biolegend) at 4°C for 30 minutes. Cells were then washed 2x in FACS buffer and fixed at 1% paraformaldehyde (PFA), kept at 4°C and analysed the following day.

3. A Fortessa X20 (BD) was used for the analysis, PE was excited with a 561nm laser and emission was collected using a 586/15 band pass filter. Effect of Pevonedistat +/- SCO-101 on HL60AML with or without ABCG2

[0134] The AML HL60 parental cells (without ABCG2) and HL60-ABCG2 cells (with ABCG2 expression) were exposed to the indicated concentrations (Figure 20A-D) of Pevonedistat and SCO-101 for 72h. The cell viability was meassured by the CCK8 viability assay. Data are expressed as percentage of untreated cells (Y-axis). Error bars indicate the standard deviation in percentage (N=3).

[0135] The effect of 20 pM SCO-101 without Pevonedistat was 82% of untreated controls for the HL60-ABCG2 cells and 75% for the HL60 parental cells.

[0136] The ABCG2 expressing HL60 cells are approximately 13-fold more resistant to Pevonedistat than the HL60 parental cells. By adding 20 pM SCO101 to Pevonedistat, the sensitivity to Pevonedistat was restored (IC50 = 0.6 pM) to approximately the same level as the HL60 parental cells (IC-50 = 0.1 pM). There was no effect of adding SCO-101 to Pevonedistat in the HL60 parental cells. The results can be seen in Figures 20A-D and in Table 3, and the synergistic effect demonstrated in Figure23A-B.

Table 3: Effect of Pevoneditat +/- SCO-101 on HL60 AML with or without ABCG2

Effect of Pevoneditat and Ko 143 on HL60AML with or without ABCG2

[0137] The effect of pevonedistat was further studied in combination with the model compound Kol43 (a known ABCG2 inhibitor) on AML cells with or without ABCG2 to elucidate whether the reversal in resistance results from ABCG2 inhibition. The cells were exposed to the indicated concentrations of Pevonedistat, Kol43 or the combination for 72h (Figure 21).

[0138] The cell viability was meassured by the CCK8 viability assay. Data are expressed as percentage of untreated cells. Error bars indicate the standard deviation in percentage (N=3).

[0139] The ABCG2 expressing HL60 cells were clearly sensitive to Kol43 mediated ABCG2 inhibition - which re-sensitize the cells to Pevonedistat. In the HL60 parental cells no effect of Pevonedistat + Kol43 was observed compared to either drug alone. Effect of Pevoneditat and SCO-101 on HL60-ABCG2 or HL60 parental cells

[0140] The effect of Pevonedistat response was further studied with/without SCO-101 in HL60- ABCG2 cells (with ABCG2 expression) and in AML HL60 parental cells (without ABCG2) at selected concentrations, see Figure 22A-B. The cells were exposed to the indicated concentrations of Pevonedistat, SCO-101 or the combination for 72h.

[0141] The cell viability was meassured by the CCK8 viability assay. Data are expressed as percentage of untreated cells. Error bars indicate the standard deviation in percentage (N=3).

The ABCG2 expressing HL60 cells are clearly sensitive to SCO-101 mediated ABCG2 inhibition - which re-sensitize the cells to Pevonedistat. In the HL60 parental cells no effect of Pevonedistat + SCO-101 is observed compared to either drug alone.

Effect of Pevoneditat with/without SCO-201 on HL60AML with/without ABCG2 expression

[0142] The effect of Pevonedistat response was further studied with/without SCO-201 in HL60 AML cells (with ABCG2 expression) and in AML HL60 parental cells (without ABCG2) at selected concentrations, see Figure 24A-D. HL60-ABCG2 cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 7.6 pM). B) HL60-ABCG2 cells were exposed to the indicated concentrations of Pevonedistat and 5 pM SCO-201 (IC50 = 0.2 pM), C) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat (IC50 = 0.2 pM), D) HL60-parental cells were exposed to the indicated concentrations of Pevonedistat + 5 pM SCO-201 (IC50 = 0.2 pM). The effect of 5 pM SCO-201 without Pevonedistat was 81% of untreated controls for the HL60-ABCG2 cells and 107% for the HL60 parental cells. The cells were exposed to the on Figures 24A-D indicated concentrations of Pevonedistat and SCO-201 for 72h. The cell viability was meassured by the CCK8 viability assay. Data are expressed as percentage of untreated cells (Y-axis, Figures 24A-D). Error bars indicate the standard deviation in percentage (N=3).

[0143] The ABCG2 expressing HL60 cells are approximately 38-fold more resistant to Pevonedistat than the HL60 parental cells.

Adding 5 pM SCO-201 to Pevonedistat restores the sensitivity to Pevonedistat (IC50 = 0.2) to the same level as the HL60 parental cells (IC-50 = 0.2 pM). There is no effect of adding SCO-201 to Pevonedistat in the HL60 parental cells. The results can be seen in Figures 24A-D and in Table 4.

Table 4: Effect of Pevoneditat +/- SCO-201 on HL60 AML with or without ABCG2

Conclusion [0144] The present examples demonstrates that ABCG2 inhibitors, such as SCO-101 or SCO-201 significantly revert cancer resistance toward NAE inhibitors, such as pevonedistat in AML cells that express ABCG2. Further, the combination of SCO-101 and pevonedisat is highly synergistic in AML cells expressing ABCG2 as shown in Figure 23A. These findings render ABCG2 inhibitors, such as SCO-101 or SCO-201 promising clinical candidates for treatment of resistant AML in combination with NAE inhibitors, such as pevonedistat.