Field of the Invention:

The invention relates to a pharmacy preparation for malignant melanoma treatment by combining three known active ingredients. The application generally solves the issue of targeted treatment for tumor cells. Specifically, the issue of annihilation of cells of one type of tumor (melanoma) is addressed, employing a synergic combination of active ingredients, described below in the application.

Background of the Invention:

Targeted treatment for neoplastic diseases means treatment by an active chemical (and/or chemicals), certified for use in human medicine by a state authority, for instance by the American agency FDA (Food and Drug Administration) or a transnational agency such as EMEA. In addition, consent granted by Statni listav pro kontrolu leciv (SUKL - State Institute for Drug Control) must be sought to medical use of active ingredients in the Czech Republic. So far, in the vast majority of cases only one compound has been used both for targeted treatment and clinical studies of the stage I-IV (immediately preceding the treatment), for example, an inhibitor of some cell signaling pathway, an inhibitor of function of mutated oncogene, an inhibitor of epigenetic regulation, an inhibitor of anti-apoptotic proteins etc. Currently, more than 100,000 various chemical inhibitors exist with at least marginal (though statistically significant) negative effect on growth of tumor cells (and/or directly eradicating cells predominantly by apoptosis) in the experimental testing. However, many fewer ingredients (ca. 200-300) were used and tested (either experimentally in cell cultures, preclinical studies in immunodeficient so-called nude mice as well as clinical studies carried out directly on patients treated with any tumor).

Nevertheless, resistance is the problem of clinical use of single ingredient, acquired ordinarily after several months of“successful” treatment, in essence each time (exceptionally, a tumor is inherently resistant to medication from the very beginning, which cannot be identified without experimental verification). For instance, resistance to vemurafenib or dabrafenib, used to inhibit activity of BRAF oncogene mutation, is typical. Mutations of this oncogene (“driver mutations”) are recognized in malignant melanoma approximately in 60 % of cases (1). NRA.S represents another typically mutated oncogene in melanoma, approximately in 15 % of cases (2). After completed treatment with the relevant inhibitor, tumor acquires (owing to many mutations occurring during tumor progression) resistance to the original treatment and becomes even“addicted” to administered medication (this condition is labelled as“drug addiction”) (3-5) and produces a pro-oncogenic“secretome”, supporting tumorous growth (6). It is therefore more complicated to respond to this situation compared to administration of the first medication. Generally, administration of the first medication is naturally discontinued, and second medication is put on a course to continue therapy. Gradual resistance to this second medication develops very often. Melanoma shows strong phenotypic heterogeneity already since the early stages (7, 8) and heterogeneity gradually increases. Presence of many various subpopulations of cells builds up its resistance as only a small part of subpopulations is sensitive to administered medication. Microheterogeneity and plasticity of phenotype are general treatment problems indicated also in other types of tumors (9,10).

Recently, tendency is reinforced to administer two and/or more medications simultaneously already as the initial treatment (11-13). These medications should be carefully selected on the basis of results from previous experimental research on cell cultures of the same type of tumor and nude mice, which is usually not the case, and clinical studies are often made directly in patients. The combination therapy, applying two medications from the beginning of treatment, considerably reduces risk of early resistance. In principle, there is no time to experimentally verify tumor of the patient against various effective combinations of medications as such verification is extremely time demanding, tumor keeps growing inside the patient and prognosis worsens. According to the literature, only double combinations of medications were exceptionally tested as the first treatment (11-15) (with better effect than mono-treatment) for the so-called targeted treatment of tumors or experimentally; nevertheless, triple-combination therapy has never been tested. A classical chemotherapy will naturally always apply combination of non-targeted, commonly used antineoplastic agents.

Citation from sources:

1. Davies H, Bignell GR, Cox C et al.: Mutations of the BRAF gene in human cancer. Nature. 2002, 4l7(6892):949-54.

2. Yan J, Wu X, Yu J et al.: Analysis of NRAS gain in 657 patients with melanoma and evaluation of its sensitivity to a MEK inhibitor. Eur J Cancer. 2018, 89:90- 101.

3. Kong X, Kuilman T, Shahrabi A et al.: Cancer drug addiction is relayed by an ERK2- dependent phenotype switch. Nature. 2017, 550(7675):270-274. 4. Hong A, Moriceau G, Sun L et al: Exploiting Drug Addiction Mechanisms to Select against MAPKi-Resistant Melanoma. Cancer Discov. 2018, 8(l):74-93.

5. Sidaway P: Targeted therapies: Drug addiction revealed in BRAF and MEK inhibitor- resistant melanoma cells. Nat Rev Clin Oncol. 2015, 12(4): 189.

6. Obenauf AC, Zou Y, Ji AL: Therapy-induced tumour secretomes promote resistance and tumour progression. Nature. 2015, 520(7547):368-72.

7. Vandamme N, Berx G: Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity. Front Oncol. 2014, 4:352.

8. Tulchinsky E, Pringle JH, Caramel J et al.: Plasticity of melanoma and EMT-TF reprogramming. Oncotarget. 2014, 5(1): 1-2.

9. Meacham CE, Morrison SJ: Tumour heterogeneity and cancer cell plasticity. Nature. 2013, 501(7467):328-37.

10. Blakely CM, Watkins TBK, Wu W et al.: Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. Nat Genet. 2017, 49(12): 1693- 1704.

11. Watanabe M, Umezawa K, Higashihara M et al. Combined inhibition of NF-kB and Bcl-2 triggers synergistic reduction of viability and induces apoptosis in melanoma cells. Oncol Res. 2013, 21(4): 173-80.

12. Vlckova K, Reda J, Ondrusova L et al.: GLI inhibitor GANT61 kills melanoma cells and acts in synergy with obatoclax. Int J Oncol. 2016, 49(3):953-60.

13. Korkut A, Wang W, Demir E et al.: Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells. Elife. 2015, l8;4.

14. Boshuizen J, Koopman LA, Krijgsman O et al.: Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat Med. 2018, 24(2):203-2l2.

15. Dinavahi SS, Noory MA, Gowda R: Moving Synergistically Acting Drug Combinations to the Clinic by Comparing Sequential versus Simultaneous Drug Administrations. Mol Pharmacol. 2018, 93(3):190-l96.

16. Santini R, Vinci MC, Pandolfi S et al.: Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells. Stem Cells. 2012, 30(9): 1808-18.

17. Faiao-Flores F, Alves-Femandes DK, Pennacchi PC et al.: Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. Oncogene. 2017, 36(13).T849-1861 18. Stecca B, Mas C, Clement V et al.: Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways. Proc Natl Acad Sci U S A. 2007, l04(14):5895-900.

19. Vlckova K, Ondrusova L, Vachtenheim J et al: Survivin, a novel target of the Hedgehog/GLI signaling pathway in human tumor cells. Cell Death Dis. 2016, 7:e2048.

20. Haq R, Yokoyama S, Hawryluk EB et al.: BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition. Proc Natl Acad Sci U S A. 2013, 110(1 l):432l-6.

21. Nguyen M, Marcellus RC, Roulston A et al. Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1 -mediated resistance to apoptosis. Proc Natl Acad Sci U S A. 2007, l04(49):19512-7.

22. Segura MF, Fontanals-Cirera B, Gaziel-Sovran A et al.: BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy. Cancer Res. 2013, 73(20):6264-76.

23. Muller J, Rrijgsman O, Tsoi J et al.: Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Com un. 2014, 5:5712

24. Tiffen JC, Gunatilake D, Gallagher SJ et al.: Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes. Oncotarget. 2015, 6(29):27023-36.

25. Cerezo M, Lehraiki A, Millet A et al.: Compounds Triggering ER Stress Exert Anti- Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell. 2016, 29(6):805- 819.

Summary of the Invention:

The summary of the invention is to provide five different combinations of three agents - effective antineoplastic medications in one preparation on cells in the cell culture, specifically targeted against human malignant melanoma (advanced stages II - IV). In these stages, follow up treatment is largely ineffective (except for immunotherapy, which however works only in small percentage of cases). The state of the art indicates that tumor cells must be eliminated in as short time as possible to make treatment effective. We have found out that all five effective combinations of medications can completely eradicate tumor cells in no more than 5 days (see Fig. 2 below), i.e., the effect of tested combinations of agents has been extraordinary strong. In ti _l ls patent application, we enumerate triple-combinations of chemicals (5 different triple- combinations); always two chemicals constitute the base and only the third one differs in all combinations. Their effect on cells in malignant melanoma is powerful, though varies according to the applied cell line and is very quick as cells are eradicated no later than 7 days even though lower concentrations of chemicals are used. We tested only combinations of targeted medications (low-molecular inhibitors), not (for instance) a combination of administration of one chemical and immunology therapy, phototherapy, radiotherapy etc.

I.e., our objective was to attack tumor cells with effective chemicals, each of them included in another area of cell biology and not targeting at“driver” mutations of oncogenes, when it is known that resistance will always appear. To make treatment effective, it is important to eradicate also subpopulations of cells, which are highly invasive, and the so-called cancer stem cells (cancer stem cells, CSC) (16) and metastatic cells recruit from them.

GANT61 and Obatoclax constituted the base of our triple-combination agent. GANT61 is a potent agent, specifically inhibiting the activity of GLI factors, which are effectors of the Hedgehog signaling pathway (17-19), significant for progression of melanoma. Obatoclax is a very strong inhibitor of the anti-apoptotic BCL2 family of genes and also inhibits anti- apoptotic protein Mcl-l (20, 21). In each case, one of the following five chemical was added to the referred base of two agents. JQ1, which is a very strong inhibitor of activity of epigenetic factor BRIM, significant for progression of melanoma (22). Other agents were SGI-7079, AXL kinase inhibitor, present in subpopulations of invasive cells of melanoma (23). Inhibitors GSK323 and GSK126 act similarly and are strong inhibitors of methyltransferase EZH2 (the part of“polycomb repression 2” complex). EZH2 was described as an important epigenetic factor of melanoma development (24). HA15 is an agent described as very strongly and specifically acting on melanoma cells by the mechanism of endoplasmic reticulum stress and mitochondria (25).

It was noted that concentration of active ingredients in the given triple-combination necessary to eradicate tumor cells is as follows: a) GANT61 from 10 to 20 pmol/l; b) Obatoclax from 150 to 300 nmol/l; cl) (+)-JQl from 125 to 500 nmol/l; c2) SGI-7079 from 125 to 500 nmol/l; c3) GSK343 from 0.25 to 1.0 pmol/l; c4) GSK126 from 25 to 100 nmol/l; and c5) HA15 from 2.5 to 10 pmol/l.

Six defined cell lines of tumors were tested for the highest concentrations of active ingredients and four cell lines for two lower concentrations of active ingredients. Totally, tests included three concentrations of agents in all five combinations. I.e., 15 results were achieved. Moreover, tests on control, non-melanoma lines were carried-out (cells of pancreatic tumors) applying one, the highest, concentration in all five combinations.

The objective of the solution was to find appropriate combinations of medications, file most effective to quickly eradicate tumor cells, a pre-requisite for successful targeted treatment. Concentrations of agents, used in the patent application, are most likely usable also for human application to the tumor tissue (verification demands further pharmacological studies already in the clinical testing stage). We carried-out two additional experiments with concentrations of agents lower than the highest; we found that by reducing concentration we eradicated cells only in two cell lines; in case of other two lines, the lowest concentrations were already ineffective (Fig. 5. and Tab. 2), effective was only the last triple-combination (cocktail no. V).

Another objective of the invention was the requirement so that the combination of three agents would eradicate all molecular types of malignant melanoma, for example with mutations and without mutations of oncogenes BRAF, NRAS, RAC1, and others. Similar approach is original to the melanoma as well as all five applied combinations of targeted medications are original.

Justification for patentability of the admitted patent application: based on originality of referred experimental results, it provides a unique opportunity to use mentioned agents (selected on the grounds of long-term molecular-biological research on melanoma and possible goals of its treatment) in the therapy for melanoma. Described triple-combinations of biologically active chemical inhibitors were never tested for the mentioned purpose (or treatment of any other tumors) (according to the available professional literature). We justify introduction of all five triple-combinations included in one invention by chemical similarity between individual agents, their similarity from the point of biologically negative effect on cellular growth, and their extraordinary effectiveness in all combinations, especially in the highest described concentrations. Conveniently, if one triple-combination would seem less efficient for treatment, a switch to another triple-combination is possible during treatment.

Effects of the Invention

Decrease in cells containing either already published double-combination GANT61 + Obatoclax (less effective combination) (Fig. 1) or all five triple-combinations is evident from the graphical section of the patent application. For comparison, Fig. 3 shows four types of tumor cells of pancreatic cancers (application of the highest concentration of agents, i.e., experiments No. 16-20, see Tab. 2), affected by agents only slightly less than melanoma cells, only one pancreatic tumor line (PANC-l) is resistant to substances. The lowest concentrations of agents applied to melanoma cells (Tab. 3 exp. 6-10, and Fig. 4) increased their resistance, with the lowest concentration of agents (Tab. 2. exp. 11-15, and Fig. 5) already two lines of melanoma (Me Wo and SK-MEL-28) were resistant even on the 7 ^th day (except for the 5 ^th triple-combination, effective on all types of cells), but also acted on two other lines of triple combination and cells were eradicated already on the 5 ^th day (Fig. 5).

Justification for selection of two basic described agents: the first two agents (compound 1 and 2), i.e., GANT61 and Obatoclax, act in synergy and exhibit the so-called synthetic lethality in their combined application on melanoma cells (Vlckova K, Reda J, Ondrusova L et al.: GLI inhibitor GANT61 kills melanoma cells and acts in synergy with Obatoclax. Int J Oncol. 2016, 49(3):953-60). Therefore, GANT61 and Obatoclax were selected as the base of each of five triple-combinations. Additional one agent was added to these two compounds. Five additional active ingredients were used (one in each triple-combination) thus, five different mixtures (“cocktails”) were made, tested in various concentrations as to their speed of eradication of melanoma cell.

Justification for selection of other five used agents: Selection of agents stems from our long-standing experience, experiments and their results on cells of malignant melanoma, and available literature data:

The compound 3 ((+)-JQl): It is an inhibitor of the so-called BET bromodomain, which strongly and specifically inhibits BET domain, which is contained in the epigenetic factor BRD4. Generally, JQ1 acts as inhibitor of many tumors, including melanoma. Its extremely powerful effect assumes the importance of BRD4 factor in oncogenesis. Therefore, substance 3 ((+)-JQl) was selected for its very strong and specific effect on many types of tumor cells including melanoma. BRD4 is an oncogene protein with recurrent translocation in many tumors and competitive binding by JQ1 displaces the BRD4 oncoprotein from chromatin.

The compound 4 (SGI-7079): It is a strong inhibitor to Axl kinase protein which, recently, has been shown as a marker of invasive and oncogene properties of melanoma cells (Sensi M, Catani M, Castellano G, et al.: Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase. J Invest Dermatol. 2011, 13 l(l2):2448-57). Its level is increased in subpopulations with high invasiveness and its increase is accompanied with drop of the key transcription factor of melanoma MITF (Muller J, Krijgsman O, Tsoi J. et al.: Low MITF/Axl ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Common. 2014, 5:5712).

The compound 5 (GSK343): GSK343 is a potent and selective EZH2 inhibitor (an enhancer of zeste homolog 2). It is strongly specific to EZH2 with 60-fold higher selectivity of inhibition compared to the related protein EZH1 and shows >1, 000-fold higher selectivity compared to other histone methyltransferases. EZH2 is a histone methyltransferase, causing trimethylation of histone 3 at lysine 27 (H3K27me3), thus representing a typical epigenetic modulator. EZH2 is a subunit of the so-called“polycomb repressive complex 2 (PRC2)”. High level of EZH2 and occurrence of H3K27me3 are found in tumors, including melanoma; EZH2 inhibition has significant influence on reduction of tumor progression in melanoma (Gelato KA, Schockel L, Klingbeil O et al.: Super-enhancers define a proliferative PGC- la-expressing melanoma subgroup sensitive to BET inhibition. Oncogene 2018, 37(4):512-521).

The compound 6 (GSK126): Similarly to the compound 5, this compound is a potent inhibitor of EZH2. GSK126 is a potent and selective inhibitor of EZH2 methyltransferase, also more than 1, 000-fold more selective for EZH2 than for twenty other human methyltransferases. I.e., we used this agent as the second alternative with similar chemical and biological properties as the agent No. 5. (EZH2 acts as a transcription activator or repressor, depending on the cell context)

The compound 7 (HA15): This agent proved to be, in one published work, as extraordinary effective for melanoma cells (Cerezo M, Lehraiki A, Millet A. et al: Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell 2016, 29(6):805-819). The agent overcomes known resistance caused by application of BRAF inhibitor (mutated and activated oncogene in ca. 60% of cases of melanoma). The specific target of this agent is a chaperone complex BiP/GRP78/HSPA5 and activity of HA15 in the cell induces endoplasmic reticulum stress with subsequent cell death by autophagy and apoptosis (Cerezo M, Rocchi S.: New anti-cancer molecules targeting HSPA5/BIP to induce endoplasmic reticulum stress, autophagy and apoptosis. Autophagy. 2017, 13(1):216-217).

Description of used chemical compounds (active ingredients). (Citations represent only appropriate selection of 5 citations for each agent from the large quantity of existing citations on the relevant compound.) Compound 1:

Trivial name: GANT61, synonym: NSC136476

Chemical name: 2-[[3-[[2-(dimethylamino)phenyl]methyl]-2-pyridin-4-yl- 1 ,3-diazinan- 1 -

-yl]methyl]-N,N-dimethylaniline

Molecular formula: C ₂₇ H ₃ 5N ₅ , molecular weight: 429.6

Structural formula:

Pharmacological activity (according to selleckchem.com): GANT61 is an inhibitor for GLI1 as well as GLI2 -induced transcription by disabling the binding activity of mentioned transcription factors to DNA.

Examples of biological effect in vitro and/or in vivo (according to selleckchem.com): GANT61 is an inhibitor for GLI1 as well as GLI2-induced transcription, inhibits Hedgehog signalling pathway with IC50 of 5 mM in GLI1 -expressing HEK293T cells.

Citation (GANT61):

- Vlckova K, Reda J, Ondrusova L et al.: GLI inhibitor GANT61 kills melanoma cells and acts in synergy with obatoclax. Int J Oncol. 2016, 49(3):953-60 (12);

- Mazumdar T, DeVecchio J, Shi T et al.: Hedgehog signalling drives cellular survival in human colon carcinoma cells. Cancer Res. 2011, 7l(3):1092-102;

- Wickstrom M, Dyberg C, Shimokawa T, et al.: Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo. Int J Cancer. 2013, 132(7): 1516-24;

- Faiao-Flores F, Alves-Femandes DK, Pennacchi PC, et al.: Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. Oncogene. 2017, 36(l3):1849-1861(17);

- Geng L, Lu K, Li P, et al.: GLI1 inhibitor GANT61 exhibits antitumor efficacy in T-cell lymphoma cells through down-regulation of p-STAT3 and SOCS3. Oncotarget. 2017, 8(30):48701-487l0.

Compound 2: Trivial name: Obatoclax mesylate, synonym: GX15-070

Chemical name: 2-(2-((3 ,5 -Dimethyl-1 H-pyrrol-2-yl)methylene)-3 -methoxy-2H-pyrrol-5 -yl)- lH-indole methanesulfonate

Molecular formula: C ₂₀ Hi9N ₃ O.CH403S, molecular weight: 413.49

Structural formula:

Pharmacological activity (according to selleckchem.com): an antagonist of BCL-2 with Kj of 0.22 mM. Obatoclax, a BH3 mimetic, is capable of binding and inhibiting a broad spectrum of members of the BCL-2 family, i.e., BCL-2, Bcl-xL as well as Mcl-l.

Examples of biological effects in vitro or in vivo (according to selleckchem.com): Obatoclax completely inhibits reactivation of the function Mcl-l due to Bak in the concentration 0.5 mM in SK-MEL-5 cells and overcomes resistance to ABT-373-induced apoptosis caused by inhibition of Mcl-l with KB/Bcl-2 cells.

It is an antagonist to anti-apoptotic factor BCL-2 (K, is 0.22 mM in non-cell experiment).

Obatoclax is used as the mesylate salt, in the literature can also be found references to tartrate salt (tartaric acid salt) or hydrochloride salt.

Citation (Obatoclax):

- Huang S, Okumura K, Sinicrope FA.: BH3 mimetic obatoclax enhances TRAIL-mediated apoptosis in human pancreatic cancer cells. Clin Cancer Res. 2009, 15(1): 150-9;

- Vlckova K, Reda J, Ondrusova L et al.: GLI inhibitor GANT61 kills melanoma cells and acts in synergy with obatoclax. Int J Oncol. 2016, 49(3):953-60 (12);

- Nguyen M, Marcellus RC, Roulston A, et al.: Small molecule obatoclax (GX15-070) antagonizes MCL-l and overcomes MCL-l -mediated resistance to apoptosis. Proc Natl Acad Sci U S A. 2007, 104(49): 19512-7 (21);

- Yazbeck YY, Li C, Grandis JR, et al.: Single-agent obatoclax (GX15-070) potently induces apoptosis and pro-survival autophagy in head and neck squamous cell carcinoma cells. Oral Oncol. 2014, 50(2): 120-7;

- Koehler BC, Scherr AL, Lorenz S, et al.: Pan-Bcl-2 inhibitor obatoclax delays cell cycle progression and blocks migration of colorectal cancer cells. PLoS One. 2014, 9(9):el0657l . Compound 3:

Trivial name: (+)-JQl, synonym: —

Chemical name: (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4 ]tri- azolo [4,3 -a] [1 ,4]diazepin-6-yl)acetate

Molecular formula: C ₂₃ H ₂₅ C1N ₄ 0 ₂ S, molecular weight: 456.99

Structural formula:

Pharmacological activity (according to selleckchem.com): (+)-JQl is a BET bromodomain inhibitor, IC50 of 77 nM/33 nM for BRD4(l/2) protein in vitro, binding is specific only to the BET family of proteins.

Examples of biological effects in vitro or in vivo (according to selleckchem.com): (+)-JQl (500 nM) decreases the rapid proliferation of NMC 797 and Per403 cells in the culture. (+)- JQ1 (50 mg/kg) inhibits growth of tumors from NMC 797 cells in nude mice.

The agent is a BET bromodomain inhibitor, with IC50 of 77 nM and 33 nM for BRD4(l), resp. BRD4(2) in the non-cell experiment in vitro.

Citation (7011:

- Shao Q, Kannan A, Lin Z, et al.: BET protein inhibitor JQ1 attenuates Myc-amplified MCC tumor growth in vivo. Cancer Res. 2014, 74(23):7090-l02;

- Korkut A, Wang W, Demir E. et al.: Perturbation biology nominates upstream-downstream drug combinations in RAF inhibitor resistant melanoma cells. Elife. 2015, l8;4;

- De Raedt T, Beert E, Pasmant E, et al.: PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature. 2014, 5l4(752l):247-5l;

- Segura MF, Fontanals-Cirera B, Gaziel-Sovran A et al.: BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy. Cancer Res. 2013, 73(20):6264-76 (22);

- Filippakopoulos P, Qi J, Picaud S et al.: Selective inhibition of BET bromodomains. Nature. 2010, 468(7327): 1067-73. Compound 4:

Trivial name: SGI-7079, synonym: CHEMBL3809908;

Chemical name: 3-(2-((3-Fhioro-4-(4-methyl-l-piperazinyl)phenyl)amino)-5-me thyl-7H- pyrrolo[2,3-d]pyrimidin-4-yl)phenylacetonitrile

Molecular formula: C ₂₆ H ₂₆ F ₇ , molecular weight: 455.53

Structural formula:

Pharmacological activity (according to selleckchem.com): a new selective inhibitor of tyrosine kinase Axl (Ki = 5.7 nM).

Example of biological effects in vitro or in vivo (according to selleckchem.com): is effective (Ki=5.7 nM) in inhibition of activity of kinase Axl in vitro and inhibits Gas6 ligand-induced tyrosine phosphorylation of human Axl expressed in HEK293T cells (EC50=T00 nM), inhibits growth of tumors in dependency on concentration.

Citation fSGI-7079):

- Byers LA, Diao L, Wang J, et al.: An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res. 2013, 19(l):279-90;

- Myers SH, Brunton VG, Unciti-Broceta A: AXL Inhibitors in Cancer: A Medicinal Chemistry Perspective. J Med Chem. 2016 Apr 28;59(8):3593-608;

- Sensi M, Catani M, Castellano G, et al.: Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase. J Invest Dermatol. 2011, 131(12):2448-57;

- Muller J, Krijgsman O, Tsoi J. et al.: Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Com un. 2014, 5:5712 (23);

- Miller MA, Oudin MJ, Sullivan RJ. Et al.: Reduced Proteolytic Shedding of Receptor Tyrosine Kinases Is a Post-Translational Mechanism of Kinase Inhibitor Resistance. Cancer Discov. 2016, 6(4):382-99.

Compound 5:

Trivial name: GSK343, synonym: 1346704-33-3 Chemical name: N- [(6-methyl-2-oxo-4-propyl- 1 H-pyridin-3 -yl)methyl] -6-[2-(4-methyl- piperazin- 1 -yl)pyridin-4-yl]-l -propan-2-ylindazol-4-carboxamide

Molecular formula: C _3i H ₃₉ N ₇ 0 ₂ , molecular weight: 541.69

Structural formula:

Pharmacological activity (according to selleckchem.com): a potent and selective EZH2 protein inhibitor with IC50 of 4 nM in the in vitro reaction, showing 60-fold higher selectivity compared to EZH1, and 1,000-fold more selective for EZH2 also in comparison with twenty other human methyltransferases. Example of biological effect in vitro or in vivo (according to selleckchem.com): inhibits trimethylation of lysine H3K27 (H3K27me3) with IC50 of 174 nM in tumor cells of mammary gland HCC1806, strongly inhibits cell proliferation in tumor cells in mammary gland and prostate gland (tumor line of the prostate gland LNCaP is the most sensitive to GSK343, with IC50 of 2.9 mM).

Citation (GSK343T

- Yu H, Ma M, Yan J. et al.: Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy. J Tran si Med. 2017, 15(1):243;

- Poirier JT, Gardner EE, Connis N. et al.: DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2. Oncogene. 2015, 34(48):5869-78;

- Fane ME, Chhabra Y, Hollingsworth DEJ et al.: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF. EBioMedicine. 2017, 16:63-75;

- Souroullas GP, Jeck WR, Parker JS et al.: An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat Med. 2016, 22(6):632-

40;

- Mahmoud F, Shields B, Makhoul I et al.: Role of EZH2 histone methyltrasferase in melanoma progression and metastasis. Cancer Biol Ther. 2016, 17(6):579-91.

Compound 6: Trivial name: GSK126, synonym: 1346574-57-9

Chemical name: 1 -[(2S)-butan-2-yl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)me thyl]-3- methyl-6-(6-piperazin- 1 -ylpyridin-3 -yl)indole-4-carboxamide

Molecular formula: C _3i H ₃₈ N ₆ 0 ₂ , molecular weight: 526.87

Structural formula:

Pharmacological activity (according to selleckchem.com): a potent, highly selective EZH2 methyltransferase inhibitor with IC50 of 9.9 nM, >1, 000-fold more selective for EZH2 compared with twenty other human methyltransferases.

Example of biological effects in vitro or in vivo (according to selleckchem.com): potently inhibits methylation to H3K27me3 (histone H3 lysine 27 trimethylation) and H3K27me3 in both EZH2 wild-type and mutant DLBCL cells. Effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and induces transcriptional activation of EZH2 target genes in sensitive cell lines. In H2087 cells inhibits expression of VEGF-A and phosphorylase kinase Ser(473)-AKT, thus triggering inhibition of cell proliferation, migration, and metastases.

Citation (GSK126V.

- McCabe MT, Ott HM, Ganji G at al.: EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 2012, 492(7427):108-12;

- Tiffen J, Wilson S, Gallagher SJ et al.: Somatic Copy Number Amplification and Hyperactivating Somatic Mutations of EZH2 Correlate With DNA Methylation and Drive Epigenetic Silencing of Genes Involved in Tumor Suppression and Immune Responses in Melanoma. Neoplasia. 2016, 18(2):121-32;

- Fisher ML, Adhikary G, Grun D et al.: The Ezh2 polycomb group protein drives an aggressive phenotype in melanoma cancer stem cells and is a target of diet derived sulforaphane. Mol Carcinog. 2016, 55(l2):2024-2036;

- Tiffen JC, Gunatilake D, Gallagher SJ et al.: Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes. Oncotarget. 2015, 6(29):27023-36 (24); - Ott HM, Graves AP, Pappalardi MB et al: A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation. Mol Cancer Ther. 2014, 13(12):3062-73.

Compound 7:

Trivial name: HA15, synonym: 1609402-14-3

Chemical name: N-[4-[3-[[5-(dimethylamino)naphtalene-l-yl]sulfonylamino]phe nyl]-l,3- thiazol-2-yl]acetamide

Molecular formula: C ₂₃ H ₂₂ N ₄ 0 ₃ S ₂ , molecular weight: 466.58

Structural formula:

Pharmacological activity (according to selleckche .com): HA15 is a molecule specifically inhibiting protein complex BiP/GRP78/HSPA5.

Example of biological effects in vitro or in vivo (according to selleckchem.com): HA15 induces endoplasmic reticulum stress leading to cell death in vitro and overcomes BRAF inhibitor resistance in melanoma cells. IC50 has not been determined yet.

Citation (HA 15):

- Cerezo M, Lehraiki A, Millet A. et al.: Compounds Triggering ER Stress Exert Anti- Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell 2016, 29(6):805-

819;

- Cerezo M, Rocchi S.: New anti-cancer molecules targeting HSPA5/BIP to induce endoplasmic reticulum stress, autophagy and apoptosis. Autophagy. 2017, 13(1):216-217;

- Cerezo M, Rocchi S: New anti-cancer molecules targeting HSPA5/BIP to induce endoplasmic reticulum stress, autophagy and apoptosis. Autophagy. 2017, 13(1):216-217;

- Ruggiero C, Doghman-Bouguerra M, Ronco C et al.: The GRP78/BiP inhibitor HA15 synergizes with mitotane action against adrenocortical carcinoma cells through convergent activation of ER stress pathways. Mol Cell Endocrinol. 201, pii: S0303-7207(18):30070-4;

- Moriya C, Taniguchi H, Nagatoishi S et al.: PRDM14 directly interacts with heat shock proteins HSP90a and glucose-regulated protein 78. Cancer Sci. 201, 109(2):373-383. Brief Description of the Drawings

Fig. 1 : Published (citation (12) experimental therapy for the cell culture by double- combination of compounds GANT61 and Obatoclax (in this experiment in the concentration of only 100 nM, contrary to triple-combinations, and showing hardly any effect on melanoma cells as an independently applied agent). The picture is borrowed from the publication (12).

Fig. 2: Experiments carried-out with the highest concentrations of agents (unpublished experiment). It can therefore be clearly concluded that all six types of melanoma cells in the culture were totally eradicated in a very short time (max. in five days) by all five triple- combinations of antineoplastic compounds applying the highest concentrations (Tab. 2, experiments No. 1-5). Identification C.I, C.II, etc. in Fig. 2 always indicates number of the applied triple-combination (C as a“combination” or“cocktail”). This is the main (and remarkably successful) result of experimental part of the application.

Fig. 3: Comparison of effectiveness of applied triple-combinations on tumor cells of the pancreas (unpublished experiment). These cells were slightly more resistant in the highest concentration of agents (other concentrations were not tested), PANC-l cell line was totally resistant, eradicated only by the first triple-combination as late as on the 7 day after its application on the cells. Identification C.I, C.II, etc. in Fig. 3 always indicates number of the applied triple combination.

Fig. 4: Experiments carried-out with the medium concentrations of agents (unpublished experiment). It can therefore be concluded that the medium concentrations of applied agents (Tab. 2, exp. 6-10) eradicated the cells and period of their survival extends (except for the line SK-MEL-3, which is highly sensitive). Nevertheless, except for the cell line SK-MEL-28, all cells are eradicated as late as on the 7 ^th day of the experiment also with these concentrations, applying all five types of combinations. Identification C.I, C.II, etc. in Fig. 4 always indicates number of the applied triple combination.

Fig. 5: Experiments carried-out with the lowest concentrations (unpublished experiment). It can therefore be clearly concluded that with the lowest applied concentrations of agents (Tab. 2, exp. 11-15) by the triple-combination of antineoplastic compounds two cell lines of melanoma cells (50lmel and SK-MEL-3) were completely eradicated in a very short period of time (max. after five days) when applying the triple-combination of antineoplastic compounds, specifically all cells in all five“cocktails”. On the contrary, two cell lines (MeWo and SK- MEL-28) were resistant to the application of medications as late as on the 7 ^th day, except for the combination No. 5, where cells were eradicated already on the 3 ^rd , resp. 5 ^th day of the experiment. Identification C.I, C.II etc. in Fig. 5 always indicates number of the applied triple combination. It appeared again that the cell line SK-MEL-3 was highly sensitive also to low concentrations of agents.

Methods of Testing

Day-by-day cell survival test used the method of the so-called“colony forming assay”. Cells were disseminated into wells; after their adhesion to the plastic surface (all types of cells represented adherent cells), the next day was considered as the day 0, when agent combinations were added. The next day was identified as the day 1 etc. Quantity of remaining cells was analysed on the days 1, 3, 5 and 7. Dark fields illustrate presence of cells, light fields illustrate absence of cells. Each cell line contains controlling segment, where only solvent was added (left column in Fig. 2-5, in Fig. 1 controls are in the upper line). Some agents were dissolved in dimethyl sulfoxide (DMSO), some in ethanol. I.e., only mixture of DMSO/ethanol 3:2 was added to the controlling segments. I.e., 1/1,000 of volume of the“stock” (concentrated storage solution) of each of triple-combination of agents was added to cells. Results were quantified by the software ImageJ (values are shown in the Tab. No. 3-6 and shows, identically to figures, the effect of agents quite illustratively). Quantification of Fig. No. 1 is shown directly in the picture (only main fields) and as it is the picture borrowed from the published publication, it has not been changed. The table clearly shows that in“empty” fields is the relative value 0 and/or quite imperceptibly above 0 (i.e., cells were destroyed by agents). Controls show, especially on the 7* day, very high relative values. At first sight, for example total eradication of all types of cells of melanoma already on the 5 ^th day (Fig. 2) is apparent. As the control, for comparison we used cells of pancreatic tumor, generally highly resistant to treatment (Fig. 3). These four types of pancreatic cancer were slightly more resistant than melanoma; however, on the 7 ^th day were also eradicated. PANC-1 line was an exception, eradicated after 7 days only by the first triple-combination of agents; these cells were resistant to other combination even after 7 days.

Description of the Preferred Embodiments:

This application deals with a unique embodiment of the invention, falling into the broad spectrum of solutions to an“appropriate combination of antineoplastic medicaments applicable from the beginning of tumor treatment”. 100% effectiveness of all five triple combinations of medicaments on cell cultures of melanoma cells in one (the highest) concentration was confirmed. Various, precisely defined, concentrations of agents were applied, and authentic results are displayed in the pictures. Applications of lower concentrations revealed varied sensitivity of melanoma lines to the individual triple-combinations; naturally, period of eradication of cells prolonged. In summary, melanoma cells were eradicated in all experiments (except for two extraordinary resistant cell lines with the lowest concentrations of compounds - Fig. 5) not later than 7 days from administration of the medicament.

Method of preparation of five triple-combinations of medications and their application to cells in the cell culture:

Tab. 1:

Table of agents used in five tested combinations (“cocktails”)

Preparation of agents before application on cells: control solution (without added inhibitors), used also to complete volume to 1,000-fold working concentration after appropriate dilution of all agents in ethanol or DMSO as instructed by the manufacturer (selleckchem.com) in all combinations. A solution (2 parts of ethanol (100%) + 3 parts of dimethyl sulfoxide (DMSO, 100%)) was used as a vehicle in control cells. Afterwards, all combinations in 1,000- fold working concentrations were 1,000-fold diluted to the final concentration lx in the appropriate cultivation media and applied on the cells. 1,000-fold diluted control solution, as already lx control solution, does not affect cell growth at all. Final (“working”) concentrations of all agents (applied on cells) are shown for each experiment in Tab. 2.

Tab. 2:

Table of final (“working”) concentrations of all agents used in combinations. Referred concentrations are present in the medium on the cells during the entire experiment (day 1-7).

Legend to Tab. 2: M = mol/1, Exp. = experiment, Tu. = tumor type, m. = melanoma, pa.t. = pancreatic tumor (experiment with these tumor cells were made for comparison only with the highest concentration of active ingredients, exp. 16-20).

Tab. 3:

Quantification of cell density to Fig. 2 (individual table cells comply with fields of the picture).

501mel Hbl

MeWo

SK-MEL-3

SK-MEL-5

SK-MEL-28

Tab. 4:

Quantification of cell density to Fig. 3 (individual table cells comply with fields of the picture).

BxPC-3

MiaPaca

PANC-1 PA-TU-8902

Tab. 5:

Quantification of cell density to Fig. 4 (individual table cells comply with fields of the picture).

501mel

MeWo

SK-MEL-3

S -MEL-28

Tab. 6:

Quantification of cell density to Fig. 5 (individual table cells comply with fields of the picture).

501mel

MeWo SK-MEL-3

SK-MEL-28