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WO2016123679A1 - Méthode de traitement - Google Patents

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WO2016123679A1
WO2016123679A1 PCT/AU2016/050075 AU2016050075W WO2016123679A1 WO 2016123679 A1 WO2016123679 A1 WO 2016123679A1 AU 2016050075 W AU2016050075 W AU 2016050075W WO 2016123679 A1 WO2016123679 A1 WO 2016123679A1
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WIPO (PCT)
Prior art keywords
inhibitor
jun
mapk pathway
treatment
resistance
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English (en)
Inventor
Petranel Theresa Christine FERRAO
Robert Nicholas Jorissen
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Priority claimed from AU2015900371A external-priority patent/AU2015900371A0/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present specification relates generally to a method of treatment.
  • the present invention provides a method for treating or preventing drug resistance in a subject with melanoma.
  • Mutation of codon 600 (V600E) of the serine/threonine kinase BRAF is the most common genetic aberration that occurs in melanoma, with mutations identified in approximately 50% of patients with advanced melanoma (Davies et al. (2002) Nature 417: 949). This mutation results in the constitutive activation of the BRAF protein, which drives cellular proliferation and survival through the MAPK pathway via the activation of downstream kinases MEK and ERK.
  • Other mutations of the MAPK pathway in melanoma include mutation of NRAS, occurring in about 10-30% of melanomas and the genetic aberration of HRAS by mutation and gene amplification during melanoma tumourigenesis.
  • MAPK pathway members such as BRAF and MEK
  • BRAF and MEK are highly effective in achieving clinical responses in patients with BRAFV600-mutant metastatic melanoma, with clinical benefit observed in 80-90% of patients (Flaherty et al. (2010) New England Journal of Medicine 363: 809).
  • MAPK inhibitors the majority only display partial responses. These patients eventually develop adaptive or acquired resistance and progress with drug resistant disease.
  • an additional 10-20% of patients exhibit inherent resistance to MAPK inhibition and do not respond to targeted therapy.
  • the present inventors have shown that increased c-JUN expression and activity mediates cell survival associated with inherent and adaptive drug resistance to MAPK pathway inhibition in BRAFV600-mutant melanoma.
  • the identification of factors that contribute to the survival of cells during drug treatment can be used to improve therapeutic regimens to achieve more complete and durable responses to targeted therapeutics.
  • the present specification provides a method for treating or preventing resistance to an inhibitor of the MAPK pathway in a subject, the method comprising administering to the subject an effective amount of an inhibitor of c-JUN.
  • Another aspect of the invention is the use of an inhibitor of c-JUN for the preparation of a medicament for the treatment or prevention of resistance to an inhibitor of the MAPK pathway.
  • the present specification provides a method for treating
  • BRAFV600mutant melanoma in a subject comprising administering to the subject an effective amount of an inhibitor of the MAPK pathway in combination with an inhibitor of c-JUN.
  • the present invention also relates to the use of an inhibitor of the MAPK pathway in combination with an inhibitor of c-JUN for the preparation of a medicament for the treatment of BRAFV600-mutant melanoma.
  • a method of treating recurrent BRAFV600-mutant melanoma comprising administering to a subject who has previously presented with BRAFV600-mutant melanoma an effective amount of an inhibitor of c-JUN.
  • the invention also relates to the use of an inhibitor of c-JUN for the preparation of a medicament for the treatment of recurrent BRAFV600-mutant melanoma.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of c-JUN for the treatment or prevention of resistance to an inhibitor of the MAPK pathway in a subject and a pharmaceutically-acceptable carrier.
  • the present invention also provides a kit when used for the treatment or prevention of resistance to an inhibitor of the MAPK pathway in a subject, comprising an inhibitor of c- JUN and a pharmaceutically-acceptable carrier, together with instructions for use.
  • Figure 1 is a photographic representation of western blots that show elevated c- JUN and P-c-JUN expression is strongly associated with resistance to vemurafenib.
  • (A) Vemurafenib resistant cell lines (IC50 > 10 6 M; LOXIMVI, C057-M1, HS294T and RPMI7951) have significantly elevated protein levels of key modulators of the JNK signalling pathway.
  • the representative Tubulin blot shows comparable protein loaded for each cell line across the panel. The sizes (kDa) of protein markers are shown on the left of the blots.
  • (B) Vemurafenib resistant cell have significantly high gene expression levels of c-JUN and CD274 (the gene that encodes for the PD-L1 protein).
  • the heatmap shows relative gene expression with respect to samples. Red indicates high relative gene expression and blue represents low gene expression levels.
  • Figure 2 shows that inhibition of c-JUN or JNK treatment in combination with vemurafenib enhances response in drug resistant cell lines.
  • A A graphical representation of concentration of vemurafenib (loglOM; x-axis) against cell number (% of control; y-axis) showing that combination treatment with JNK-IN-8 and vemurafenib results in synergistic activity to re-sensitise resistant cell lines to vemurafenib treatment.
  • D A graphical representation of concentration of vemurafenib (loglOM; x-axis) against cell number (% of control; y-axis) that show the targeted knock down of JUN using siRNA prior to vemurafenib treatment also enhances the response to vemurafenib treatment in resistant cell lines.
  • Figure 3 shows that increased c-JUN expression and activity is associated with development of acquired resistance to vemurafenib.
  • A A graphical representation of time (h; x-axis) against fold change relative to GAPDH (y-axis) showing that c-JUN gene expression increases following a time course of treatment with vemurafenib in sensitive cell lines.
  • B A photographic representation of a western blot analysis of c-JUN expression (C-JUN) and activity (P-C-JUN) across the same time course demonstrating that the increase in gene expression was correlated with an increase in the protein expression and activity of c-JUN.
  • the representative Tubulin blot shows comparable protein loaded for each cell line across the panel.
  • C A graphical representation of c-JUN gene expression relative to GAPDH (y-axis) from A375 xenografts from animals treated with control chow (green) or chow complexed with PLX4720 (a precursor drug compound to vemurafenib used for in vivo studies) (red). Each symbol represents the average of triplicate data for each xenograft tumour and the horizontal bar of the same colour indicates the average +/-the SEM for the three mice on each plot.
  • E A graphical representation of gene expression of c-JUN relative to GAPDH (y-axis) as a fold change to DMSO treatment in A375 cells treated with either 500 nM vemurafenib (V), 500 nM MEK inhibitor selumetinib (M), 100 nM ERK inhibitor SCH772984 (E) or combinations of V, M and E as indicated (x-axis) shown directly above the corresponding photographic representation of a western analysis of the same conditions following 48 h of drug treatment.
  • the representative Tubulin blot shows comparable protein loaded for each cell line across the panel.
  • Figure 4 shows that the drug induced increase in c-JUN mediates cell survival during the development of early adaptive resistance.
  • A A photographic representation of a western blot analysis demonstrating the efficient knock-down of c-JUN expression and activity following pre-treatment with siRNA targeting c-JUN. The representative Tubulin blot shows comparable protein loaded for each cell line across the panel.
  • B A graphical representation of vemurafenib-associated cell death comparing the proportion of dead cells (% cells PI positive; y-axis) with pre-treatment with siRNA targeting c-JUN.
  • Figure 5 shows that JNK inhibition reverses the changes associated with adaptive resistance to vemurafenib.
  • B A graphical representation of cell death following treatment with the various combinations comparing the proportion of dead cells (% cells PI positive; y-axis) with combination treatment in A375 cells following 48 h treatment.
  • C A graphical representation of cell migration comparing cell index (x-axis) against time (h; y-axis) as quantified using the Xcelligence system and cells cultures in CIM plates.
  • D A photographic representation of a western blot analysis comparing the activity of c-JUN in A375 cells treated for 48 h with various combinations. The representative Tubulin blot shows comparable protein loaded for each cell line across the panel.
  • Figure 6 shows Bliss analysis of cell death.
  • A Percentage of cells PI+ (dead) following treatment with various combinations of vemurafenib and JNK-In-8 in A375 cells following 2 days treatment.
  • the A375 cells were pre-treated with either DMSO or 500nM
  • B Bliss analysis of cell death data shown in Figure 6A.
  • the highest single activity (HSA) excess was calculated as the difference between the observed fractional activity and the highest of the fractional activities from single doses of each drug.
  • the percentage Bliss excess showing the combined drug activity for each of the various drug dose combinations.
  • the red colour indicates synergistic activity.
  • a mutation includes a single mutation, as well as two or more mutations
  • an inhibitor includes a single inhibitor, as well as two or more inhibitors
  • reference to “the disclosure” includes single and multiple aspects taught by the disclosure; and so forth.
  • the present invention is predicated on the inventors' surprising findings that both inherent and early adaptive resistance to MAPK inhibitor therapy is mediated through increased c- JUN expression and activity in BRAFV600-mutant melanoma. Accordingly, the present specification teaches a method of treating or preventing resistance to an inhibitor of the MAPK pathway in a subject, the method comprising administering to the subject an effective amount of an inhibitor of c-JUN. Use of an inhibitor of c-JUN may also be used in the preparation of a medicament for the treatment or prevention of resistance to an inhibitor of the MAPK pathway.
  • Melanoma is a cancer arising from the malignant transformation of melanocytes, pigment producing cells of the skin, eye, mucosal epithelia and meninges. It is among the most aggressive and treatment-resistant cancers.
  • novel targeted therapies and immunotherapies has significantly improved patient outcomes, particularly for those with advanced disease.
  • the incidence of melanoma continues to rise.
  • Melanoma is generally detected by physical examination of skin lesions after symptoms have developed. The presence of melanoma is typically confirmed by microscopic analysis of skin tissue obtained by biopsy.
  • Symptoms of melanoma include a change in an existing mole, the development of a new pigmented or unusual- looking growth on the skin, irregular shaped moles, moles with irregular, notched or scalloped borders, growths with many colours or an uneven distribution of colour, new growth in a mole, itching in a mole and blood or other discharges from a mole.
  • melanomas are classified into five distinct stages: (i) common acquired and congenital nevi without dysplastic changes; (ii) dysplastic nevi with structural and architectural atypia; (iii) radial-growth phase (RGP) melanoma; (iv) vertical-growth phase (VGP) melanoma; and (v) metastatic melanoma.
  • Benign and dysplastic nevi are characterised by the disruption of the epidermal melanin unit, leading to an increased number of melanocytes in relation to keratinocytes.
  • RGP melanoma, or in situ melanoma grow laterally and remain confined to the epidermis.
  • VGP melanoma invades the upper layer of the epidermis and beyond, and penetrates into the underlying dermis and subcutaneous tissue through the basement membrane, forming expansive nodules of malignant cells.
  • Metastatic melanoma represents the final stage of disease progression whereby the cancer has spread from the primary site to nearby tissues and more distant lymph nodes, or has metastasised to other organs such as the lungs and brain.
  • the melanoma contemplated by the methods of the present specification is metastatic melanoma. In another embodiment, the metastatic melanoma is
  • TNM is often employed for this purpose, where (T) denotes the thickness and ulceration of the melanoma, (N) denotes the spread of the melanoma to the lymph nodes and (M) denotes the spread of the melanoma to different parts of the body.
  • Stage 0 is the earliest stage of melanoma and is limited to in situ tumours with no detectable cancer cells in the regional lymph nodes or metastases at distant sites.
  • Stage I tumours are localised melanomas that are classified into two distinct sub-stages, stage IA tumours are less than 1 mm thick without ulceration; and stage IB tumours may range from less than 1 mm to 2 mm thick both with and without ulceration.
  • Stage II tumours are also localised melanomas that are classified into three distinct sub-stages, stage IIA may range from 1.01 to 4.00 mm thick both with and without ulceration; stage IIB tumours may range from 2.01 to > 4.00 mm thick both with and without ulceration; and stage IIC tumours are > 4.00 mm thick with ulceration.
  • Stage III tumours are of any thickness or ulceration with macro-or micro-metastatic burden in the lymph nodes.
  • stage IV tumours represent the most advanced stage of disease and are characterised by a primary tumour of any thickness or ulceration with macro-or micro-metastatic burden in the lymph nodes and metastases in the skin, subcutaneous tissue, nodal region, lung, other visceral metastases and distant metastases.
  • the therapeutic regimen for the treatment of melanoma can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the age, weight, family history and general health of the subject in addition to the type, size, stage and molecular characteristics of the melanoma, for example, the mutation status of BRAF.
  • prevention refer to any and all uses which remedy a condition or symptom, prevent the establishment of a condition of a disease, or otherwise prevent, hinder, retard, abrogate or reverse the onset or progression of a condition or disease or other undesirable symptoms in any way whatsoever.
  • treating does not necessarily imply that a subject is treated until total recovery or cure.
  • the treatment or prevention need not necessarily remedy, prevent, hinder, retard, abrogate or reverse all of said symptoms, but may remedy, prevent, hinder, retard, abrogate or reverse one or more of said symptoms.
  • the agents, uses ,methods and protocols of the present disclosure that involve treatment or prevention may prevent, reduce, ameliorate or otherwise delay the resistance to treatment, or of a highly undesirable event associated with resistance to treatment or an irreversible outcome of resistance to treatment, but may not itself prevent resistance to treatment in melanoma or an outcome associated therewith (e.g. a symptom associated with melanoma). Accordingly, treatment and/or prevention include amelioration of the symptoms of resistance to treatment in melanoma or preventing or otherwise reducing the risk of resistance to treatment.
  • inhibitors do not necessarily imply the complete inhibition of the specified event, activity or function. Rather, the inhibition may be to an extent, and/or for a time, sufficient to produce the desired effect. Inhibition may be prevention, retardation, reduction, abrogation downregulation or otherwise hindrance of an event, activity or function. Such inhibition may be in magnitude and/or be temporal in nature. In particular contexts, the terms “inhibit” and “prevent”, and variations thereof may be used interchangeably.
  • the inhibition of a specified event, activity or function can be a result of inhibition, either by reducing absolute levels of the specified event, activity or function or by antagonising the specified event, activity or function such that effectiveness is decreased. Even the partial antagonism of the specified event, activity or function may act to reduce, although not necessarily eliminate, effectiveness.
  • the proteinaceous molecules described above may be derived from any suitable source such as natural, recombinant or synthetic sources and includes fusion proteins or molecules which have been identified following, for example, screening.
  • the reference to non- proteinaceous molecules may be, for example, a reference to a nucleic acid molecule or it may be a molecule derived from natural sources, such as for example, by screening, or may be a chemically synthesised molecule.
  • the present invention contemplates small molecules capable of acting as antagonists.
  • Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing c-JUN from carrying out its normal biological function.
  • Antagonists include monoclonal antibodies and antisense nucleic acids which prevent transcription or translation of activin genes or mRNA in mammalian cells. Modulation of expression may also be achieved utilising antigens, RNA, ribosomes, DNAzymes, aptamers, antibodies or molecules suitable for use in cosuppression. Suitable antisense oligonucleotide sequences (single stranded DNA fragments) of activin may be created or identified by their ability to suppress the expression of activin.
  • Antagonists also include any molecule that prevents c-JUN activity/function.
  • expression refers to the transcription and translation of a nucleic acid molecule.
  • Reference to “expression product” is a reference to the product produced from the transcription and translation of a nucleic acid molecule.
  • Derivatives of proteinaceous or non-proteinaceous agents include fragments, parts, portions or variants from either natural or non-natural sources. Non-natural sources include, for example, recombinant or synthetic sources. By “recombinant sources” is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source. Parts or fragments include, for example, active regions of the molecule.
  • Derivatives may be derived from insertion, deletion or substitution of amino acids.
  • Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place.
  • Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above. Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-pro teinaceous molecules. Analogues of the molecules contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
  • nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
  • the derivatives of the nucleic acid molecules utilised in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules.
  • Derivatives of nucleic acid sequences also include degenerate variants.
  • a “variant” or “mutant” should be understood to mean molecules which exhibit at least some of the functional activity of the form of molecule (e.g. follistatin) of which it is a variant or mutant.
  • a variation or mutation may take any form and may be naturally or non-naturally occurring.
  • Chemical and functional equivalents should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening.
  • chemical or functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening (see further below).
  • Antagonistic agents can also be screened for utilising such methods.
  • Suitable therapeutic regimens would be known to persons skilled in the art. In the context of melanoma, most subjects will have surgery to remove the cancerous tissue. For early stage melanomas, surgery will generally be sufficient to cure the disease. However, for more advanced disease, other treatments such as chemotherapy, radiotherapy,
  • immunotherapy and targeted molecular therapy may also be required.
  • Reference to “surgery” includes wide local excision, Moh's micrographic surgery, lymph node dissection and other surgical interventions to remove metastatic tumour tissue from distant sites.
  • Radiotherapy includes both external radiation therapy and internal radiation therapy used to damage cancer cells and inhibit their proliferation.
  • external radiation is commonly delivered to patients in the form of x-rays directed to the tumour site by a machine.
  • internal radiation is generally delivered to patients using multiple small tubes and/or catheters inserted at the tumour site.
  • Radiotherapy is generally used in combination with other treatment modalities, commonly surgery and chemotherapy.
  • chemotherapy means any agent that is administered to inhibit the growth of cancer cells or induce cancer cell death.
  • Chemotherapeutic treatments for melanoma include darcarbazine, temozolomide, nab-paclitaxel, paclitaxel, carmustine, cisplatin, carboplatin and vinblastine.
  • Chemotherapeutic agents may be administered as single agents or in combination with one or more agents.
  • Reference to “targeted therapy” includes any targeted therapeutic agent that is specifically designed to interfere with molecular alterations that are specific to cancer cells.
  • Targeted therapeutic agents may include, but are not limited to, monoclonal antibodies and small molecule inhibitors. For example, vemurafenib, and dabrafenib are small molecule inhibitors that inhibit the function of BRAF; and trametinib is a small molecule inhibitor that inhibits the function of MEK.
  • modulatory agents The proteinaceous and non-pro teinaceous molecules referred to, above, are herein collectively referred to as “modulatory agents".
  • modulatory agent may be used interchangeably with the e terms “inhibitor”, “drug” “composition”, “agent”, “therapeutic agent”, “medicament” and “active”.
  • a “modulatory agent” encompasses a chemical compound or biological molecule or cellular composition that induces a desired
  • a target molecule for example, c-JUN.
  • Any of the aforesaid terms encompass pharmaceutically acceptable and pharmacologically active ingredients including, but not limited to, salts, esters, amides, pro-drugs, active metabolites, analogues and the like.
  • the term includes genetic, protein or lipid molecules or analogues thereof, in addition to the cellular compositions previously mentioned.
  • Modulatory agents may be targeted therapeutic agents that may be administered as single agents or in combination with one or more agents.
  • dabrafenib and trametinib vemurafenib and trametinib
  • vemurafenib and cobimetinib and encorafenib e.g., dabrafenib and trametinib
  • vemurafenib and trametinib vemurafenib and cobimetinib and encorafenib and
  • binimetinib are combinations of targeted therapeutic agents that may be used in the treatment of melanoma.
  • the agents which are utilised in accordance with the method of the present invention may take any suitable form.
  • proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins.
  • the subject non-pro teinaceous molecules may also take any suitable form. Both the proteinaceous and non-pro teinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-pro teinaceous molecules.
  • said agent is associated with a molecule which permits its targeting to a localised region.
  • the subject proteinaceous or non-pro teinaceous molecule may act either directly or indirectly to downregulate/inhibit gene expression or activity of the gene product.
  • Said molecule acts directly if it associates with the protein-encoding nucleic acid molecule or its expression product to modulate expression or activity, respectively.
  • Said molecule acts indirectly if it associates with a molecule other than the protein-encoding nucleic acid molecule or expression product which other molecule either directly or indirectly downregulates/inhibits the expression or activity of the protein-encoding nucleic acid molecule or expression product, respectively.
  • Screening for the modulatory agents can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising the a gene of interest (e.g. the gene encoding c-JUN) or functional equivalent or derivative thereof with an agent and screening for the downregulation/inhibition of protein production or functional activity, downregulation/inhibition of the expression of a nucleic acid molecule encoding the protein of interest, or downregulation/inhibition of the activity or expression of a downstream activin cellular target. Detecting such downregulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of activin activity such as luciferases, CAT and the like.
  • the gene of interest or functional equivalent or derivative thereof may be naturally occurring in the cell which is the subject of testing or it may have been transfected into a host cell for the purpose of testing. Further, the naturally occurring or transfected gene may be constitutively expressed - thereby providing a model useful for, inter alia, screening for agents which down regulate activin activity, at either the nucleic acid or expression product levels, or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which up-regulate activin expression.
  • an activin nucleic acid molecule may comprise the entire activin gene or it may merely comprise a portion of the gene such as the portion which regulates expression of the activin product.
  • the promoter region of the gene of interest e.g. the gene encoding c-JUN
  • detecting modulation of the activity of the promoter can be achieved, for example, by ligating the promoter to a reporter gene.
  • the promoter may be ligated to lucif erase or a CAT reporter, the downregulation of expression of which gene can be detected via modulation of fluorescence intensity or CAT reporter activity, respectively.
  • the subject of detection could be a downstream regulatory target of the gene of interest, rather than the gene itself.
  • These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as the proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the activin nucleic acid molecule or expression product itself or which modulate the expression of an upstream molecule, which upstream molecule subsequently downregulates/inhibits expression or expression product activity. Accordingly, these methods provide a mechanism of detecting agents which either directly or indirectly modulate gene expression and/or activity.
  • oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above.
  • a selected biological agent such as a biomolecule, a macromolecule complex, or cell
  • each member of the library is screened for its ability to interact specifically with the selected agent.
  • a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction.
  • the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.
  • the present invention envisages the use of any suitable form of antibody including catalytic antibodies or derivatives, homologues, analogues or mimetics of said antibodies.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or subunits or may be specifically raised to target antigen. In the case of the latter, the antigen may first need to be associated with a carrier molecule.
  • fragments of antibodies may be used such as Fab fragments or Fab '2 fragments.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the antigen can also be used to screen for naturally occurring antibodies.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the antigen or derivative, homologue, analogue, mutant, or mimetic thereof, and either type is utilizable therapeutically.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the antigen, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable, they are generally less favoured because of the potential heterogeneity of the product.
  • monoclonal antibodies is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman 1981, Basic Facts about Hybridomas, in Compendium of
  • an antibody within the scope of the present invention specifically binds its target antigen.
  • “specifically binds” is meant high avidity and/or high affinity binding of an antibody to a specific antigen.
  • Antibody binding to its epitope on this specific antigen is stronger than binding of the same antibody to any other epitope, particularly those that may be present in molecules in association with, or in the same sample, as the specific antigen of interest.
  • Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g. 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the polypeptide of interest, e.g. by use of appropriate controls.
  • an aptamer is a compound that is selected in vitro to bind preferentially to another compound (in this case the identified proteins), in one aspect, aptamers are nucleic acids or peptides.
  • Random sequences can be readily generated from nucleotides or amino acids (naturally occurring and/or synthetically made) in large numbers but of course they need not be limited to these.
  • the nucleic acid aptamers are short strands of DNA that bind protein targets, such as oligonucleotide aptamers.
  • Oligonucleotide aptamers are oligonucleotides which can bind to a specific protein sequence of interest.
  • a general method of identifying aptamers is to start with partially degenerate oligonucleotides, and then simultaneously screen the many thousands of oligonucleotides for the ability to bind to a desired protein.
  • RNA inhibiting agents can be eluted from the protein and sequenced to identify the specific recognition sequence. Transfer of large amounts of a chemically stabilized aptamer into cells can result in specific binding to a polypeptide of interest, thereby blocking its function.
  • RNA inhibiting agents Klug et al. 1994, Mol Biol Rep 20:97-107; Wallis et al. 1995, Chem Biol 2:543-552; Ellington 1994, Curr Biol 4:427-429; Lato et al. 1995, Chem Biol 2:291-303; Conrad et al. 1995, Mol Divers 1:69-78; and Uphoff et al. 1996, Curr Opin Struct Biol 6:281-287].
  • RNA inhibiting agents may be utilized to inhibit the expression or translation of messenger RNA (“mRNA”) that is associated with a phenotype of interest.
  • an inhibitor may be a molecule that functions as a mediator of RNA interference.
  • RNA interference or "RNAi” describes a mechanism of gene silencing that is based on degrading or otherwise preventing the translation of mRNA in a sequence specific manner that is dependent on small, non-coding RNA approximately 18 to 30- nucleotide (nt) in length.
  • RNA-induced silencing complex RISC
  • RISC RNA-induced silencing complex
  • siRNAs are endogenously expressed from the genome, whereas siRNAs may be endogenous or arise from viral infection or other exogenous sources.
  • siRNA duplexes feature perfect base- pairing, while miRNA helices contain mismatches and more extended terminal loops. In the cytoplasm, the processing pathways converge for endogenous miRNAs and for typically exogenous siRNAs. Both types of RNAi precursors are cleaved down by a Dicer enzyme to a dsRNA duplex of the appropriate size for loading onto an Argonaute protein.
  • the resulting dsRNA is a duplex of 21 -to 25-nt strands, with a 2-nt overhang at each 3' terminus and a phosphate group at each recessed 5' terminus.
  • the bound duplex and Argonaute protein are subsequently loaded into the RISC complex in a strand dependent manner.
  • One strand, the guide strand, of the duplex is bound to Argonaute to direct silencing and the other strand, the passenger strand, is discarded.
  • the RISC performs cellular surveillance, binding single- stranded RNA (ssRNA) such as mRNA with complementarity to the guide strand.
  • Guide strand nucleotides 2-6 constitute the seed sequence and initialize binding to the target.
  • piRNAs are produced and processed by a completely distinct pathway, known as the 'ping pong cycle'. Briefly, piRNA genomic clusters are transcribed to produce the piRNA precursors. In the cytoplasm, these precursors are cleaved into short 23-29-nt antisense piRNAs. These short, single stranded RNAs (ssRNAs) are loaded into PIWI family Argonaute proteins AUB and PIWI. The loaded AUB or PrWI proteins then target the mRNA of active transposons for cleavage to produce sense piRNAs. The sense piRNAs are loaded into the PrWI- specific Argonaut protein AG03, which then directs cleavage of primary piRNA precursors and the subsequent production of more antisense piRNAs, completing the 'ping pong cycle'.
  • ssRNAs single stranded RNAs
  • RNAi molecules contemplated by the present invention should be understood to encompass all RNAi gene silencing mechanisms.
  • the induction of RNAi to inhibit a target gene could be achieved by administering, in accordance with the method or use of the present invention, exogenous RNA oligonucleotides that can induce an RNAi mechanism.
  • Reference to a "RNAi molecule” should therefore be understood as a reference to an RNA nucleic acid molecule that is double stranded or single stranded and is capable of effecting the induction of an RNAi mechanism to knock down the expression of a gene targeted or down regulating or preventing the onset of such a mechanism.
  • RNAi molecule may be capable of directly mediating an RNAi mechanism, or it may require further processing.
  • the subject RNAi molecule may be double stranded or single stranded.
  • RNAi molecules that are suitable for use in the present application include, but are not limited to, long double stranded RNA (dsRNA), hairpin double stranded RNA (hairpin dsRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA); microRNA (miRNA); and small temporal RNA (stRNA).
  • RNAi molecule for use in any given situation.
  • the subject RNAi molecule may nevertheless exhibit some degree of mismatch to the extent that hybridization sufficient to induce an RNAi response in a sequence specific manner can be effected.
  • the RNAi molecule of the present invention comprises at least 70%- 100% sequence complementarity.
  • thermodynamic rules see, e.g., Schwarz, et al. 2003, Cell 115: 199-208; Reynolds et al. 2004, Nat Biotechnol. 22:326-330; Khvorova et al. 2003, Cell 115:209-216).
  • Many computer programs are available for selecting regions of a sequence that are suitable target sites. These include programs available through commercial sources such as Ambion, Dharmacon, Promega, Invitrogen, Ziagen, and GenScript as well as non-commercial sources such as EMBOSS, The Wistar Institute, Whitehead Institute, and others.
  • design can be based on the following considerations. Typically, shorter sequences, less than about 30 nucleotides are selected. The coding region of the mRNA is usually targeted. The search for an appropriate target sequence optionally begins 50-100 nucleotides downstream of the start codon, as untranslated region binding proteins and/or translation initiation complexes may interfere with the binding of the siRNA endonuclease complex. Some algorithms, e.g., based on the work of Elbashir et al. 2000 ⁇ Methods 26: 199-213) search for a selected sequence motif and select hits, with approximately 50% G/C-content (30% to 70% has also worked). If no suitable sequences are found, the search is extended.
  • nucleic acids e.g., ribozymes, antisense
  • Sfold see, e.g., Ding, et al., Nucl Acids Res 32 Web Server issue, W135-W141; Ding & Lawrence 2003, Nucl Acids Res 31:7280-7301; and Ding &
  • immunotherapy includes any agent that is specifically designed to induce, enhance or suppress an immune response in a subject to destroy cancer cells.
  • Activation immunotherapies are immunotherapies designed to elicit or amplify an immune response.
  • suppression immunotherapies reduce or suppress an immune response.
  • Immunotherapeutic agents may include, but are not limited to, recombinant, synthetic and natural preparations of interleukins, cytokines, chemokines, cytosine phosphate-guanosine, oligodeoxynucleotides and glucans, cell-based immune therapies, autologous immune enhancement therapy, engineered T-cells, adoptive T-cells, vaccines and monoclonal antibodies.
  • IL-2 interleukin 2
  • adoptive cell transfer with autologous T cells and immune checkpoint inhibitors such as ipilimumab, a humanised CTLA-4 blocking monoclonal antibody and monoclonal antibodies that antagonize PD-1 or PD-L1 (e.g. pembrolizumab, nivolumab) have been successfully used to treat melanoma.
  • the present invention further contemplates a combination of treatments, such as the administration of inhibitory/downregulating agent together with other proteinaceous or non-pro teinaceous molecules which may facilitate the desired therapeutic or preventative outcome.
  • the inhibitory/downregulating agent may be administered as a single dose or may be administered as multiple sequential doses or it may be continuously infused. Where more than one molecule is administered, there may be simultaneous administration in the same formulation or in different formulations via the same or different routes or sequential administration via the same or different routes.
  • simultaneous administration is meant a time difference of from seconds, minutes, hours or days.
  • BRAFV600mutant melanoma comprising administering to a subject an effective amount of an inhibitor of the MAPK pathway in combination with an inhibitor of c-JUN.
  • an inhibitor of the MAPK pathway and an inhibitor of c-JUN for the preparation of a medicament for the treatment of BRAFV600-mutant melanoma.
  • a method of treating recurrent BRAFV600-mutant melanoma comprising administering to a subject that has previously presented with BRAFV600-mutant melanoma an effective amount of an inhibitor of c-JUN.
  • an inhibitor of c-JUN with one or more other immunotherapeutic agents, such as monoclonal antibody directed against PD-L1, for the preparation of a medicament for the treatment of BRAFV600-mutant melanoma.
  • a method of treating recurrent BRAFV600-mutant melanoma comprising administering to a subject that has previously presented with BRAFV600-mutant melanoma the combination of an inhibitor of c-JUN with one or more other immunotherapeutic agents, such as monoclonal antibody directed against PD-L1.
  • references to the term “recurrent” or “persistent” includes any relapse in the disease of a melanoma subject that occurs following treatment with a targeted therapy. This is inclusive of melanoma subjects that have inherent or acquired resistance to the targeted therapy.
  • the inhibitor of the MAPK pathway is selected from the group consisting of: BRAF, MEK, ERK, RTK and/or RAS inhibitors.
  • the inhibitor of the MAPK pathway is a BRAF inhibitor.
  • the BRAF inhibitor is vemurafenib.
  • the inhibitor of c-JUN is an inhibitor of the JNK pathway.
  • Melanoma is one of the most treatment-resistant tumours and generally, traditional treatment modalities such as chemotherapy and radiotherapy have not been particularly effective in treating melanoma. Therefore, the development of targeted therapeutics and immunotherapies has revolutionised treatment for melanoma patients.
  • the most successful targeted therapies used for the treatment of melanoma are inhibitors of the MAPK signalling pathway.
  • the MAPK pathway is a signal-transduction pathway that transmits mitogenic signals from activated cell surface growth factor receptors and is recognised to be a key regulator of cellular growth and survival.
  • growth factors bind to surface receptor tyrosine kinases (RTKs) that transduce their growth promoting signals through the activation of the small G protein RAS, which leads to the activation of the serine/threonine kinase RAF, and then to the activation of MEK.
  • RTKs surface receptor tyrosine kinases
  • MEK phosphorylates and activates MAPK, also known as ERK.
  • inhibitors of the MAPK pathway have been shown to be highly effective in achieving clinical responses in patients with BRAF mutant melanoma.
  • inhibitors of the MAPK pathway include, but are not limited to, RAF inhibitors such as sorafenib,
  • Reference to "resistance” or “resistant” includes inherent, acquired and adaptive drug resistance. Cancer cells can exhibit inherent resistance to a drug which is present before treatment with a given drug begins as a result of inherited hereditary genetic
  • Adaptive drug resistance differs from inherent or acquired drug resistance as it is a transient state that usually reverts where the environmental trigger is removed.
  • the present inventors have made the surprising discovery that up-regulation of c-JUN expression and activity is responsible for mediating resistance to inhibitors of the MAPK pathway.
  • Gene expression, in vitro, in vivo and patient analyses resulted in the
  • c-JUN identification of c-JUN as a key molecule in resistance to inhibitors of the MAPK pathway. Therefore, c-JUN, or up-stream activators of c-JUN, such as the JNK pathway, which potentiates the transcriptional activity of c-JUN by phosphorylation of serine's 63 and 73, may be targeted for the treatment or prevention of resistance to treatment with inhibitors of the MAPK pathway in melanoma.
  • junction proto-oncogene refers to the protein or polypeptide that is encoded by the JUN gene.
  • c-JUN may be used herein to refer to either or both c-JUN polypeptide or a gene (polynucleotide) encoding a c-JUN polypeptide, interchangeably.
  • c-JUN is a component of the API transcription factor complex, comprising 331 amino acids with a molecular weight of approximately 35 kDa.
  • c-JUN consists of a basic leucine zipper domain that is essential for DNA binding.
  • c-JUN is the mammalian counterpart of v-JUN retroviral oncogene, and is recognised to act as a proto-oncogene in various mammalian cancers.
  • Aberrant activity and expression of c-JUN is responsible for the induction of a number of target genes that are involved in regulating cell cycle progression, migration and survival during tumourigenesis.
  • inhibition of c-JUN in malignant melanoma can treat or prevent resistance to vemurafenib by overcoming the re-activation of ERK signalling.
  • c-JUN includes vertebrate and non-vertebrate c-JUN.
  • Suitable vertebrates include, but are not limited to, any member of the subphylum Chordata including primates, rodents (e.g. mice, rates, guinea pigs), lagomorphs (e.g. rabbits, hares), bovines (e.g. cattle), ovines (e.g. sheep), caprines (e.g. goats), porcines (e.g. pigs), equines (e.g. horses), canines (e.g. dogs), felines (e.g. cats), avians (e.g.
  • c-JUN is human c-JUN.
  • the human c-JUN is a protein encoded by the mRNA sequence represented by GenBank Accession numbers NM_002228.3. Reference to the term c-JUN also includes homologs thereof.
  • the term "homolog” typically refers to peptides with similar biological activity, although differing in amino acid sequence at one or more amino acid positions when the sequences are aligned.
  • the amino acid sequences of two homologous c-JUN peptides may differ only by one amino acid residue with in the aligned amino acid sequences of five to ten amino acids.
  • two homologous c-JUN peptides of fifteen to twenty or more amino acids can differ by up to three amino acid residues when aligned.
  • Homologous c-JUN peptides may also differ by up to approximately 5%, 10%, 20% or 25% of the amino acid residues when the amino acid sequences of the two peptide homologs are aligned.
  • Homologs of c-JUN may be found in the same species (i.e. between two or more individuals of the same species), in related species and/or sub-species, or in different species.
  • homologs include those found in non-human vertebrates and non-vertebrates. Suitable vertebrates that fall within the scope of the invention include, but are not limited to, any member of the subphylum Chordate including primates, rodents (e.g. mice, rates, guinea pigs), lagomorphs (e.g. rabbits, hares), bovines
  • a preferred homolog is one found in a primate (e.g. a human, ape, monkey, chimpanzee).
  • a c-JUN homolog may be from the same species (e.g. human).
  • homologs will have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a particular amino acid or nucleotide sequence, as determined, for example, by sequence alignment programs known in the art using default parameters.
  • JNK Jun N-terminal kinase
  • stress-activated protein kinase refers to a MAPK signalling pathway that is related to the MAPK pathway as described above.
  • the JNK pathway is a signalling pathway that is activated by environmental and genotoxic stresses to control cell proliferation, differentiation, survival and the migration of different cell types.
  • the JNK proteins are encoded by three genes, MAPK8 (which encodes JNK1), MAPK9 (which encodes JNK2) and MAPK10 (which encodes JNK3), which are alternatively spliced to produce at least ten different isoforms.
  • JNK1 and JNK2 are ubiquitously expressed throughout the body, whereas JNK3 is generally expressed in the brain.
  • JNKs are activated by upstream MKK4 and MKK7 kinases.
  • the major target of activated JNK is the transcription factor API, which comprises c-FOS and c-JUN. JNKs phosphorylate c-JUN, which then combine with FOS to form the API transcription factor.
  • inhibition of the JNK signalling pathway can prevent the phosphorylation of c-JUN, which is known to stabilise c-JUN levels, thereby providing an alternative therapeutic strategy for preventing or treating resistance mediated through c-JUN.
  • vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordate including primates, rodents (e.g. mice, rates, guinea pigs), lagomorphs (e.g. rabbits, hares), bovines (e.g. cattle), ovines (e.g. sheep), caprines (e.g. goats), porcines (e.g. pigs), equines (e.g. horses), canines (e.g.
  • rodents e.g. mice, rates, guinea pigs
  • lagomorphs e.g. rabbits, hares
  • bovines e.g. cattle
  • ovines e.g. sheep
  • caprines e.g. goats
  • porcines e.g. pigs
  • equines e.g. horses
  • canines e.g.
  • the subject is a primate (e.g. a human, ape, monkey, chimpanzee). In a preferred embodiment, the subject is a human.
  • the present specification teaches a method for treating or preventing resistance to an inhibitor of the MAPK pathway in a subject, said method comprising administering to the subject an effective amount of an inhibitor of c-JUN.
  • the present specification teaches the use of an inhibitor of c-JUN for the preparation of a medicament for the treatment or prevention of resistance to an inhibitor of the MAPK pathway.
  • Reference to an "effective amount” means the amount of an inhibitor of c-JUN when administered to a subject, in particular a human subject, in need of such treatment, is sufficient to effect treatment or prevent resistance to an inhibitor of the MAPK pathway.
  • the resistance to an inhibitor of the MAPK pathway is inherent resistance. In another embodiment, the resistance to an inhibitor of the MAPK pathway is acquired resistance. In yet another embodiment, the resistance to an inhibitor of the MAPK pathway is adaptive resistance.
  • the subject has been administered an inhibitor of the MAPK pathway for the treatment of cancer. In another embodiment, the subject has been administered an inhibitor of the MAPK pathway for the treatment of BRAFV600-mutant melanoma.
  • BRAFV600-mutant melanoma means melanoma with a V600E or V600K mutation in BRAF. Genomic analyses have identified activating BRAF mutations in as many as 60% of human melanomas. Importantly, the majority of these mutations (80%) cluster in the kinase-activation domain (V600E) of BRAF and result in a single
  • Activated BRAF phosphorylates and activates MEK proteins (MEK1 and MEK2), which then activates downstream MAP kinases.
  • the BRAF genotype of melanoma patients may be determined by routine methods well known to a person skilled in the art, for example, polymerase chain reaction (PCR) using appropriate primers or gene sequencing methods.
  • PCR polymerase chain reaction
  • the inhibitor of the MAPK pathway is selected from the group consisting of: RAF, MEK, ERK, RTK and RAS inhibitors.
  • RAF kinases are a family of serine/threonine- specific protein kinases that are related to retroviral oncogenes.
  • the three RAF kinase family members are ARAF, BRAF and CRAF.
  • MEK is a dual specificity protein kinase that is encoded by the MAP2K7 genes. This kinase specifically activates the JNK pathway to mediate cellular responses to
  • ERK is an extracellular signal related kinase that is encoded by MAPKl gene. This kinase acts as an integration point for the transduction of biochemical signals associated with the cellular processes of proliferation, differentiation, transcription regulation and
  • RTKs are high affinity surface receptors that transduce extracellular signals for many polypeptide growth factors, cytokines and hormones. They are regulators of normal cellular processes and also play a role in tumour development and progression. For example, ⁇ , ERBB2, the EPH and FGFR are frequently mutated in melanoma.
  • the RAS genes are the most frequently mutated oncogenes in human cancer. There are three RAS genes in humans, HRAS, KRAS and NRAS. Each encodes small GTPases that are
  • the inhibitor of the MAPK pathway is a BRAF inhibitor.
  • BRAF means "B-Raf proto-oncogene, serine/threonine kinase”, which is a RAF serine/threonine protein kinase that regulates the MAPK pathway.
  • BRAF B-Raf proto-oncogene, serine/threonine kinase
  • mutations in the BRAF gene are associated with oncogene-driven tumourigenesis in melanoma.
  • the BRAF inhibitor is vemurafenib.
  • vemurafenib refers to a reversible, ATP-competitive small molecule inhibitor of the kinase domain of mutant BRAF.
  • vemurafenib inhibits the phosphorylation of MEK by BRAF and induces growth-inhibition or apoptotic cell death.
  • the inhibitor of the MAPK pathway is administered prior to the inhibitor of c-JUN.
  • the MAPK pathway may be administered 2-10 days prior to the inhibitor of c-JUN.
  • Reference to "2-10 days prior" means 2, 3, 4, 5, 6, 7, 8, 9 or 10 days prior.
  • Inhibitors of c-JUN include, but are not limited to, c-JUN peptide inhibitors.
  • Inhibitors of the JNK pathway include, but are not limited to SP6000125, JNK-IN-8, CC-401, SU3327, AS 601245, PGL5001 and BI78D3.
  • any inhibitor of the JNK pathway and c-JUN is within the scope of the present specification.
  • an inhibitor of c-JUN may be administered in combination with chemotherapy, radiotherapy, targeted therapy and/or immunotherapy.
  • an inhibitor of c-JUN may be administered in combination with two or more treatment modalities (i.e. chemotherapy, radiotherapy, targeted therapy and/or
  • Treatment modalities will typically be selected with a view to treating melanoma and/or melanoma recurrence and/or the development of resistance to MAPK inhibitors.
  • an inhibitor of c-JUN may be administered in combination with an immunotherapy, such as a monoclonal antibody directed against PD-L1.
  • composition contemplated by the present specification comprises an inhibitor of c-JUN, which may be prepared in a manner known in the art and are those suitable for enteral, such as oral or rectal, and parental administration to a subject, particularly a human subject, comprising an effective amount of an inhibitor of c-JUN alone, or in combination with one or more pharmaceutically acceptable carriers or diluents, especially suitable for enteral or parental application.
  • compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical modulatory pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context, patient or organ tolerance, etc. The amount of agent adequate to accomplish this is defined as a "therapeutically effective dose”.
  • the dosage schedule and amounts effective for this use, i.e., the "dosing regimen” will depend upon a variety of factors, including the pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the
  • pharmacokinetics i.e., the pharmaceutical composition's rate of absorption
  • Suitable pharmaceutical compositions contain from about 0.1% to about 99.9% of the active ingredient.
  • Reference to "about 0.1% to about 99.9%” means 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • compositions of the invention may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents.
  • compositions include that suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parental (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the composition is suitable for parental administration. In another embodiment, the composition is suitable for intravenous administration. In a further embodiment, the composition is suitable for subcutaneous administration.
  • compositions suitable for parental administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • Preferred unit dosage compositions are those containing a daily dose or unit, daily sub- dose, or an appropriate fraction thereof, of the active ingredient.
  • the pharmaceutical composition further comprises an inhibitor of the MAPK pathway.
  • the inhibitor of the MAPK pathway is selected from the group consisting of RAF, MEK, ERK, RTK and/or RAS inhibitors.
  • the inhibitor of the MAPK pathway is a BRAF inhibitor.
  • the BRAF inhibitor is vemurafenib.
  • the pharmaceutical composition comprises an inhibitor of c-JUN that is an inhibitor of the JNK pathway.
  • the present invention contemplates a kit when used for the treatment or prevention of resistance to an inhibitor of the MAPK pathway in a subject comprising an inhibitor of c- JUN and a pharmaceutically-acceptable carrier, together with instructions for use.
  • the inhibitor of c-JUN is an inhibitor of the JNK pathway.
  • kits may optionally include appropriate therapeutic agents to be administered in combination with an inhibitor of c-JUN, including, but not limited to an inhibitor of the MAPK pathway.
  • the inhibitor of the MAPK pathway is selected from the group consisting of RAF, MEK, ERK, RTK and/or RAS inhibitors.
  • the inhibitor of the MAPK pathway is a BRAF inhibitor.
  • the BRAF inhibitor is vemurafenib.
  • STR genotyping using 6 STR loci was performed to confirm the 15 identity of each cell line.
  • IC 50 values shown in Table 1 are representative of average IC50 values from multiple experiments. An average IC 50 value of 1000 nM (1 ⁇ ) was used to separate the sensitive and resistant cell lines.
  • transfection reagent Lipid and siRNA were each diluted separately in non- supplemented media for 5 mins and then complexed for 15 mins prior to addition to cells.
  • SRB Sulphorhodamine B
  • Cell lines were seeded into 96-well microtitre plates for 48 hrs prior to the addition of drug. Cells were treated for 72 hrs with drug ranging in concentration from 0.01 nM to 30 ⁇ and analysed for total cell number according to SRB absorbance. Briefly, cells were fixed in situ by the addition of cold trichloroacetic acid (TCA) and stained using 0.4% (w/v) SRB/ 1% (v/v) acetic acid. SRB absorbance was measured at 515 nM in 10 mM tris(hydroxymethyl)aminomethane (TRIS) to generate dose response curves. IC50 values for each cell line were calculated as the drug dose resulting in 50% reduction in SRB absorbance relative to solvent treated control cells.
  • Membranes were then blocked with 0.1% TRIS buffered saline-Tween-20 v/v; TBST) containing 5% (w/v) skim milk powder and probed with antibodies. Bound antibodies were detected using horseradish peroxidase conjugated secondary antibodies incubated with enhanced chemoluminescence (ECL) or ECL-Plus Western Blotting Detection Reagents (GE Healthcare), which were exposed to X-Ray film (Fugifilm) and developed by autoradiography.
  • ECL enhanced chemoluminescence
  • GE Healthcare ECL-Plus Western Blotting Detection Reagents
  • Fresh frozen tumour samples were obtained prior, during and on progression, from patients participating in clinical trials involving vemurafenib, dabrafenib, trametinib or a combination of both dabrafenib and trametinib undertaken at the Melanoma Institute Australia and Westmead Hospital (NSW, Australia). Studies had local institutional review board approval and all patients provided written informed consent.
  • A375 cells 4 x 10 6 A375 cells were mixed in a 1: 1 ratio with high concentration Matrigel and injected subcutaneously into the right flank of female NOD-SCID-ILy mice. Tumours were monitored every 2-3 days and tumour volume measurements taken according to the length and width of tumours.
  • Treatment commended 10 days post- injection of A375 cells and the average volume of tumours observed was 183.4 mm3 (range: 39.4 - 401 mm3).
  • PI Propidium Iodide
  • JNKIN-8 result in an enhanced response in resistant cell lines when compared to resistant cell lines treated with vemurafenib alone ( Figure 2). Furthermore, JNK-IN-8 was also able to inhibit c-JUN phosphorylation in all of the resistant cell lines, which was associated with a reduction in vemurafenib-associated induction of p-ERK ( Figure 2).
  • Enforced constitutive expression of c-JUN by retroviral transduction of A375 cells is sufficient to induce cellular changes associated with the development of resistance in vemurafenib sensitive cell lines.
  • Increased c-JUN expression resulted in increased activity, as demonstrated by an increase in the levels of p-c-JUN ( Figure 4).
  • the constitutive expression of c-JUN results in an increase in the levels of p- ERK ( Figure 4), suggesting that c-JUN could contribute to the p-ERK rebound detected following drug treatment, which previous studies have reported to be a key component of the development of resistance (Lito et al. (2012) Cancer Cell, 22: 668).
  • Drug-induced increase in c-JUN mediates cell survival during the development of resistance
  • nucleic acid sequences which may be targetable by inhibitory RNA and proteins targetable by small molecule inhibitors. Targeting such
  • sequences/proteins is expected to inhibit or reduce c-JUN activity.
  • NM_ codes NCBI RefSeq nucleotide sequence codes (mRNA sequences)
  • NM_001278548.1 (GI:513788280) -> NP_001265477.1 mitogen-activated protein kinase 8 isoform 5
  • NM_002750.3 GL513788275
  • NP_002741.1 mitogen-activated protein kinase 8 isoform alpha 1
  • NM_139046.2 (GI:513788276) -> NP_620634.1 mitogen-activated protein kinase 8 isoform betal
  • NM_139049.2 (GI:513788277) -> NP_620637.1 mitogen-activated protein kinase 8 isoform alpha2
  • MAPK9 mitogen-activated protein kinase 9 [ Homo sapiens (human) ]
  • NM_001308244.1 (GL815891107) -> NP_001295173.1 mitogen-activated protein kinase 9 isoform gamma2
  • NM_002752.4 (GL205277404) -> NP_002743.3 mitogen-activated protein kinase 9 isoform alpha2
  • NM_139068.2 (GL205277406) -> NP_620707.1 mitogen-activated protein kinase 9 isoform alpha 1
  • NM_139069.2 (GL205277408) -> NP_620708.1 mitogen-activated protein kinase 9 isoform betal
  • NM_139070.2 (GL205277410) -> NP_620709.1 mitogen-activated protein kinase 9 isoform beta2
  • MAPK10 mitogen-activated protein kinase 10 [ Homo sapiens (human) ]
  • NM_001318067.1 (GL969536251) -> NP_001304996.1 mitogen-activated protein kinase 10 isoform 5
  • NM_001318068.1 (GL969536253) -> NP_001304997.1 mitogen-activated protein kinase 10 isoform 6
  • NM_001318069.1 (GL969536247) -> NP_001304998.1 mitogen-activated protein kinase 10 isoform lx
  • NM_002753.4 (GL969536249) -> NP_002744.1 mitogen-activated protein kinase 10 isoform 2
  • NM_138980.3 (GL969536250) -> NP_620446.1 mitogen-activated protein kinase 10 isoform 3
  • MAP4K4 mitogen-activated protein kinase kinase kinase kinase kinase 4 [Homo sapiens (human)] Gene ID: 9448 Also known as
  • NM_001242559.1 (GL336020357) -> NP_001229488.1 mitogen-activated protein kinase kinase kinase kinase 4 isoform 4
  • NM_001242560.1 (GL336020359) -> NP_001229489.1 mitogen-activated protein kinase kinase kinase kinase 4 isoform 5
  • NM_004834.4 (GI: 336020352) -> NP_004825.3 mitogen-activated protein kinase kinase kinase kinase 4 isoform 1
  • NM_145686.3 (GL336020354) -> NP_663719.2 mitogen-activated protein kinase kinase kinase kinase 4 isoform 2
  • NM_145687.3 (GL336020356) -> NP_663720.1 mitogen-activated protein kinase kinase kinase kinase 4 isoform 3 MAP2K7 mitogen-activated protein kinase kinase 7 [ Homo sapiens (human) ]
  • NM_001297555.1 (GL662033892) -> NP_001284484.1 dual specificity mitogen- activated protein kinase kinase 7 isoform 1
  • NM_001297556.1 GL662033894
  • NP_001284485.1 dual specificity mitogen- activated protein kinase kinase 7 isoform 2
  • NM_145185.3 (GL662033896) -> NP_660186.1 dual specificity mitogen-activated protein kinase kinase 7 isoform 3
  • API Also known as API; AP-1; c-Jun NM_002228.3 (GL44890066) -> NP_002219.1 transcription factor AP-1 REFERENCES

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Abstract

La présente description concerne de manière générale une méthode de traitement. En particulier, mais pas exclusivement, la présente invention concerne une méthode permettant de traiter ou de prévenir la pharmacorésistance chez un sujet atteint de mélanome.
PCT/AU2016/050075 2015-02-06 2016-02-08 Méthode de traitement Ceased WO2016123679A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019032717A1 (fr) * 2017-08-08 2019-02-14 Memorial Sloan Kettering Cancer Center Utilisation d'inhibiteurs de braf pour traiter des réactions cutanées provoquées par un traitement avec un inhibiteur de mek
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
CN118490684A (zh) * 2020-07-29 2024-08-16 陈洪亮 C-jun n末端激酶抑制剂su3327的用途

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WO2013152038A1 (fr) * 2012-04-02 2013-10-10 Buck Institute For Research On Aging Ciblage de cellules sénescentes et de cellules cancéreuses par l'interférence avec jnk et/ou foxo4

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US20120301878A1 (en) * 2011-05-25 2012-11-29 Neal Rosen Methods and compositions for the detection of drug resistant braf isoforms
WO2013152038A1 (fr) * 2012-04-02 2013-10-10 Buck Institute For Research On Aging Ciblage de cellules sénescentes et de cellules cancéreuses par l'interférence avec jnk et/ou foxo4

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LOGRASSO, P. ET AL.: "Inhibitors of c-JUN-N-Terminal Kinase (JNK).", MINI-REVIEWS IN MEDICINAL CHEMISTRY, vol. 8, no. 8, 2008, pages 755 - 766 *
NGUYEN, T. V. ET AL.: "Sorafenib resistance and JNK singnaling in carcinoma during extracellular matrix stiffening.", BIOMATERIALS, vol. 35, no. 22, 2014, pages 5749 - 5759 *
RAMSDALE, R. ET AL.: "The transcription cofactor c-JUN mediates phenotype switching and BRAF inhibitor resistance in melanoma.", SCIENCE SIGNALLING, vol. 8, no. 390, 18 August 2015 (2015-08-18) *
TAKASHIMA, A. ET AL.: "Protein Kinase Cdelta is a Therapeutic Target in Malignant Melanoma with NRAS Mutation.", ACS CHEMICAL BIOLOGY, vol. 9, no. 4, 2014, pages 1003 - 1014 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
WO2019032717A1 (fr) * 2017-08-08 2019-02-14 Memorial Sloan Kettering Cancer Center Utilisation d'inhibiteurs de braf pour traiter des réactions cutanées provoquées par un traitement avec un inhibiteur de mek
US11458139B2 (en) 2017-08-08 2022-10-04 Memorial Sloan Kettering Cancer Center Use of BRAF inhibitors for treating cutaneous reactions caused by treatment with a MEK inhibitor
IL272510B1 (en) * 2017-08-08 2024-10-01 Memorial Sloan Kettering Cancer Center Use of braf inhibitors for treating cutaneous reactions caused by treatment with a mek inhibitor
IL272510B2 (en) * 2017-08-08 2025-02-01 Memorial Sloan Kettering Cancer Center Use of BRAF inhibitors to treat skin reactions induced by MEK inhibitor therapy
CN118490684A (zh) * 2020-07-29 2024-08-16 陈洪亮 C-jun n末端激酶抑制剂su3327的用途

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