FIELD

The present invention concerns miRNAs for treatment and in vitro diagnosis of drug resistant tumors. In particular, the present invention concerns miRNAs for in vitro diagnosis of resistance of tumors to BRAF/MEK pathway (also named as MAPK pathway) inhibiting drugs and for treatment of tumors which are treated with said drugs, such as melanoma, by stimulating or inhibiting the expression of down-regulated or up-regulated miRNAs, respectively.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e). The name of the ASCII text file for the Sequence Listing is 33667855_1.txt, the date of creation of the ASCII text file is Oct. 9, 2020, and the size of the ASCII text file is 4.46 KB.

BACKGROUND

The natural history of metastatic melanoma has recently changed thanks to the development of novel immunotherapy and targeted therapy approaches, which have significantly improved patients' survival (1). The first type of these therapies is based on immune checkpoint inhibitors targeting CTLA4 and PD1/PDL1 interaction which have entered in the routine clinical practice and on other immune-modulators which are currently in advanced clinical trials (2). The second type is principally represented by small targeting inhibitors of kinases (KIs) of the MAPK pathway which were developed following the initial discovery that BRAF V600 mutations are among the major oncogenic drivers of melanoma proliferation and survival (1,3). Indeed, near 50% of patients harbor v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600 mutations, which is responsible for the uncontrolled activation of the mitogen-activated protein kinase (MAPK) signaling pathway (3).

This evidence led to the clinical development of BRAF inhibitors, firstly used as mono-therapies and more recently in combination with MEK inhibitors following the discovery that BRAFi resistance is frequently characterized by the reactivation of the MAPK signaling and the involvement of the MEK kinase (4). However, drug resistance virtually frustrates both mono-therapies with BRAF inhibitors and dual-therapies with combinations of a BRAF inhibitor plus a MEK inhibitor.

The majority of BRAF-mutated melanoma patients initially respond to KIs, until the development of de novo drug resistance, which creates an intractable clinical condition, especially in the cases of BRAF and MEK inhibitors acquired resistance (5). Furthermore, approximately 10% to 15% of melanoma patients harboring BRAF-mutations do not initially respond to first line targeted therapies, and even 40% to 50% of patients show only partial responses (6). These evidences strongly suggest that both ab initio and acquired resistance are a major hurdle to achieve durable control of metastatic disease.

A challenging issue is, therefore, in the field of melanoma, to generate powerful diagnostics capable to predict patients' response to therapies and to conceive combination therapies capable to block or revert development of drug resistance.

During last years, several studies directed to understand the molecular basis of resistance to KIs have identified both genetic and non-genetic (or otherwise called) phenotypic mechanisms (7).

Interestingly, the same genetic alterations have been identified both in BRAFi mono-therapy resistance as well as in combinations of BRAF inhibitors with MEK inhibitors. In most cases these secondary genetic alterations cause reactivation of MAPK signaling (5).

In contrast, non genetic/phenotypic mechanisms are linked to the activation of a highly heterogeneous and dynamic set of adaptive responses fueled by tumor cell plasticity (7). These adaptive responses involve a variety of redundant and often interchangeable intracellular pathways which contribute to cell resistance to cell death in the presence of MAPK signaling inhibitors. Blockade of a single pathway is in most cases insufficient to fully counteract these adaptive mechanisms because of the activation of bypass pathways. Therefore, there is the need of an alternative approach.

A novel miR-579-3p as a regulator of melanoma development and drug resistance (8) has been recently discovered. This miRNA is down-regulated in BRAF-mutated melanomas and correlated to worse patients' prognosis. In addition, miR-579-3p relevant target genes have been identified: the oncogenic BRAF itself and the MDM2 oncoprotein (8). These evidences strongly explain its oncosuppressive role in metastatic melanomas bearing BRAF V600 mutations. Most importantly, miR-579-3p is able to impair the development of resistance to MAPK inhibitors in vitro and its deregulation was confirmed in patients who developed resistance to targeted therapies in contrast to its target genes, which are up-regulated (8).

However, the great heterogeneity of human melanoma samples makes this single miRNA per se not suitable for providing an effective in vitro diagnosis of drug resistance.

Further to the evidence that individual miRNAs can affect sensitivity to target therapies in melanoma, it is also known a study concerning a comprehensive analysis of the changes affecting the entire miRNome during the development of drug resistance to MAPK inhibitors (9). This study identifies specific miRNAs from specific cell lines as possible factors responsible for drug resistance to BRAF kinase inhibitors. However, it does not provide any experimental data in order to validate the diagnostic and/or therapeutic function of these miRNAs, and in particular of combination of miRNAs as diagnostics or therapeutics.

In the light of the above, it is therefore apparent the need to provide methods for in vitro diagnosis of tumors which are resistant to BRAF/MEK pathway BRAF-mutated melanoma patients and of their resistance to inhibiting drugs, and for treating said resistance.

SUMMARY

According to the present invention, a population of miRNAs which is deregulated during the development of drug resistance has been identified by a large study of the entire miRNAome in vitro. Briefly, through the analysis of changes in the expression of the whole miRnome of BRAF-mutated melanoma cells before and after the establishment of resistance to a BRAFi, a set of several deregulated miRNAs, which have been divided in miRNAs facilitators or antagonists of drug resistance, has been identified. Several data were obtained mostly pointing out to the therapeutic and diagnostic features of these miRNA and were later reported in detail also in Fattore et al (10).

Interestingly, a set of intracellular pathways affected by these miRNAs with a prominent involvement of pro-inflammatory and pro-angiogenetic genes was also identified through bioinformatic and experimental approaches.

All together, these data show that miRNA deregulation in concert with cytokine aberrant expression is responsible for the establishment of resistance to targeted therapies in metastatic melanoma.

In particular, it has been found that miR-4443 and miR-4488 are up-regulated, wherein miR-204-5p and miR-199b-5p are down-regulated in BRAF-mutated melanoma patients, i.e. in patients who show resistance to BRAF/MEK pathway inhibiting drugs.

As for the diagnostic application, liquid biopsy of circulating nucleic acids is a highly sensitive and specific non-invasive diagnostic modality to monitor disease burden and to define biomarkers predictive of drug response or resistance. MicroRNAs (miRs) are ideal biomarkers since they are actively released by tumor cells and cells of the tumor microenvironment, and can be easily detected in the circulation (11). In this context, the above mentioned two BRAFi-resistant up-regulated miRs (miR-4443 and miR-4488) and two downregulated (miR-199b-5p and miR-204-5p;) were further tracked in plasma samples derived from BRAF mutated melanoma patients before initiation of target therapy and at Disease Progression (PD). It has been observed that miRs deregulation is associated with therapeutic resistance with significant AUC predictive values.

The experiments reported below show that the above mentioned miRNAs are suitable as markers of resistance to BRAF/MEK pathway inhibiting drugs with high sensitivity, specificity and accuracy.

As for the therapeutic application, the experiments reported below show that the inhibition of the expression of the up-regulated miRNAs or the stimulation of the expression of the down-regulated miRNAs is effective in reducing drug resistance.

Cancer is one of the main applications of potential miRNA-based therapies (12). Importantly, since a single miRNA is capable to bind simultaneously several different mRNAs, the use of miRNAs offers the possibility to target simultaneously multiple pathways involved in tumor development and progression, providing the opportunity to develop new powerful drugs for the therapy of cancer (13). However, the use of miRNA-based drugs is hampered by the rapid degradation by nucleases and the poor and unspecific cellular uptake (14). Nanotechnology can overcome these biopharmaceutical issues, because of its potential to preserve RNA stability and to enhance intracellular uptake. In detail, stable nucleic acid lipid particles (SNALPs) have been previously developed to deliver miRNAs in an experimental model of multiple myeloma (15). These carriers, previously proposed by Semple et al. (16) are characterized by a high RNA encapsulation, stability in presence of serum, ability to protect miRNA against enzymatic degradation and ability to increase oligonucleotide uptake into the target cells.

It is therefore specific object of the present invention a method for in vitro diagnosis of resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in tumors, said method comprising measuring the expression, in a biological sample, of at least two, three or all of the following microRNAs:

miR-199b-5p:(SEQ ID NO: 1)cccaguguuuagacuaucuguuc,miR-204-5p:(SEQ ID NO: 2)uucccuuugucauccuaugccu,miR-4443:(SEQ ID NO: 10)uuggaggcguggguuuu,miR-4488:(SEQ ID NO: 11)agggggcgggcuccggcg,

wherein miR-199b-5p and miR-204-5p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-4443, miR-4488 are over-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in comparison with their expression in controls which do not present said resistance.

According to the present invention the tumors are those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs. For example, the tumors that can be resistant according to the present invention are melanoma, Colorectal cancer, papillary thyroid carcinoma, non small cell lung cancer, brain tumors, non-Hodgkin lymphoma. Specifically, the above-mentioned tumors are those which are BRAF mutated.

According to an embodiment of the present invention, the method can comprise measuring the expression of the following combinations of miRNAs listed from the most preferred: miR-199b-5p and miR-4488; miR-199b-5p and miR-4443; miR-4488 and miR-4443; miR-199b-5p, miR-4443 and miR-4488; miR-199b-5p and miR-204-5p.

In addition, the method according to the present invention can further comprise measuring the expression of at least one of the following miRNAs:

miR-145-5p:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p:(SEQ ID NO: 9)gcgacccauacuugguuucag,miR-1234:(SEQ ID NO: 12)ucggccugaccacccaccccac,miR-9-5p:(SEQ ID NO: 13)ucuuugguuaucuagcuguauga,miR-1915-5p:(SEQ ID NO: 14)accuugccuugcugcccgggcc,miR-4286:(SEQ ID NO: 15)accccacuccugguacc,miR-575:(SEQ ID NO: 16)gagccaguuggacaggagc,miR-630:(SEQ ID NO: 17)aguauucuguaccagggaaggu,

wherein miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-15b-5p, miR-221-3p, miR-551b-3p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 are over-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in comparison with their expression in controls which do not present said resistance.

The biological sample can be a liquid biological sample such as blood, serum, plasma, urine.

The method according to the present invention can be carried out for example by Real Time PCR, Droplet Digital PCR, Microarray, RNA Hybridization Methods such as Northern Blot or Dot Blot, RNA Next Generation Sequencing.

The present invention concerns also the use of at least two, three or all of the following microRNAs:

miR-199b-5p:(SEQ ID NO: 1)cccaguguuuagacuaucuguuc,miR-204-5p:(SEQ ID NO: 2)uucccuuugucauccuaugccu,miR-4443:(SEQ ID NO: 10)uuggaggcguggguuuu,miR-4488:(SEQ ID NO: 11)agggggcgggcuccggcg,

as biomarkers for the in vitro diagnosis of the resistance of tumors to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein miR-199b-5p and miR-204-5p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-4443, miR-4488 are over-expressed. As mentioned above the tumors which are resistant to the drugs are those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs. For example, the tumors are chosen from the group consisting of melanoma, Colorectal cancer, papillary thyroid carcinoma, non small cell lung cancer, brain tumors, non-Hodgkin lymphoma. Specifically, the above-mentioned tumors are those which are BRAF mutated.

According to an embodiment of the present invention, said two or three of the microRNAs can be the following combinations of miRNAs listed from the most preferred: miR-199b-5p and miR-4488; miR-199b-5p and miR-4443; miR-4488 and miR-4443; miR-199b-5p, miR-4443 and miR-4488; miR-199b-5p and miR-204-5p.

In addition, the use according to the present invention can further comprise the use of at least one of the following miRNAs as biomarkers for the in vitro diagnosis of the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs:

miR-145-5p:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p:(SEQ ID NO: 9)gcgacccauacuugguuucag,miR-1234:(SEQ ID NO: 12)ucggccugaccacccaccccac,miR-9-5p:(SEQ ID NO: 13)ucuuugguuaucuagcuguauga,miR-1915-5p:(SEQ ID NO: 14)accuugccuugcugcccgggcc,miR-4286:(SEQ ID NO: 15)accccacuccugguacc,miR-575:(SEQ ID NO: 16)gagccaguuggacaggagc,miR-630:(SEQ ID NO: 17)aguauucuguaccagggaaggu,
wherein miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-15b-5p, miR-221-3p, miR-551b-3p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 are over-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in comparison with their expression in controls which do not present said resistance.

A further object of the present invention is an antagonist of at least one of miR-4443 and miR-4488 and/or a miRNA mimic of at least one of miR-199b-5p and miR-204-5p for use in the treatment of tumors which are resistant to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein said antagonist is chosen from the group consisting of Locked Nucleic Acid (LNA) targeting miR-4443, Locked Nucleic Acid (LNA) targeting miR-4488, antimiR-4443: aaaacccacgcctccaa (SEQ ID NO:18), antimiR-4488: cgccggagcccgccccct (SEQ ID NO:19), whereas said miRNA mimic is chosen from the group consisting of miR-199b-5p mimic: cccaguguuuagacuaucuguuc (SEQ ID NO:1), miR-204-5p mimic: uucccuuugucauccuaugccu (SEQ ID NO:2). As mentioned above the tumors are those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs.

Antagonists and miRNA mimics, preferably miRNA mimics, according to the present invention can be administered and delivered by lipid nanoparticles since the use of naked RNA-based molecules in therapy is hampered by their rapid enzymatic degradation in biological fluids and poor efficiency in crossing cell membranes. For example antagonists and miRNA mimics according to the present invention can be administered and delivered by stable nucleic acid lipid particles (SNALPs). The route of administration can be intravenous administration (17).

According to the present invention, when a mixture of said antagonist and/or miRNA mimic is used (i.e. a mixture of more than one antagonist or a mixture of more than one miRNA mimic or a mixture of one or more antagonists with one or more miRNA mimics is used), said mixture can be: miR-199b-5p, miR-204-5p and miR-579-3p; miR-199b-5p and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and antimiR-4488 or LNA targeting miR-4488; antimiR-4488 or LNA targeting miR-4488 and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and miR-204-5p; miR-199b-5p and antimiR-4443 or LNA targeting miR-4443; miR-199b-5p and antimiR-4488 or LNA targeting miR-4488, wherein the mixtures are listed from the most preferable.

In addition, said antagonist and/or miRNA mimic can be in combination with at least one of the following antagonists and/or miRNA mimic:

antimiR-1234:(SEQ ID NO: 20)gtggggtgggtggtcaggccgaorLNA targeting miR-1234, antimiR-9-5p:(SEQ ID NO: 21)tcatacagctagataaccaaagaorLNA targeting miR-9-5p, antimiR-1915-5p:(SEQ ID NO: 22)ggcccgggcagcaaggcaaggtorLNA targeting miR-1915-5p, antimiR-4286:(SEQ ID NO: 23)ggtaccaggagtggggtorLNA targeting miR-4286, antimiR-575:(SEQ. ID. NO. 24)gctcctgtccaactggctcorLNA targeting miR-575, antimiR-630:(SEQ ID NO: 25)accttccctggtacagaatactorLNA targeting miR-630, miR145-5p mimic:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p mimic:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p mimic:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107 mimic:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p mimic:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p mimic:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p mimic:(SEQ ID NO: 9)gcgacccauacuugguuucag.

The present invention concerns also a combination of an antagonist of at least one of miR-4443 and miR-4488 and/or of a miRNA mimic of at least one of miR-199b-5p and miR-204-5p with at least one MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drug for the simultaneous, sequential or separate use in the treatment of tumors which are resistant to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein said antagonist is chosen from the group consisting of Locked Nucleic Acid (LNA) targeting miR-4443, Locked Nucleic Acid (LNA) targeting miR-4488, antimiR-4443: aaaacccacgcctccaa (SEQ ID NO:18), antimiR-4488: cgccggagcccgccccct (SEQ ID NO:19), whereas said miRNA mimic is chosen from the group consisting of miR-199b-5p mimic: cccaguguuuagacuaucuguuc (SEQ ID NO:1), miR-204-5p mimic: uucccuuugucauccuaugccu (SEQ ID NO:2), wherein said antagonist and/or miRNA mimic is used against the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs.

According to the present invention, the MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs can be for example vemurafenib, Trametinib(GSK1120212), dabrafenib, sorafenib (a Raf kinase inhibitor), other Raf inhibitors such as SB590885, PLX4720, XL281, RAF265, encorafenib, MEK inhibitors such as cobimetinib, CI-1040, PD0325901, Binimetinib (MEK162), selumetinib.

The term “simultaneous use” according to the present invention is understood as meaning the administration of at least one MAPK pathway inhibiting drug (first component of the combination) and the antagonist and/or mimic of miRNAs of the present invention (second component of the combination) in a single and identical pharmaceutical form.

The term “separate use” is understood as meaning the administration, at the same time, of the above mentioned first and second component of the combination according to the invention in distinct pharmaceutical forms.

The term “sequential use” is understood as meaning the successive administration of the above mentioned first and second component or second and first component of the combination according to the invention, each in a distinct pharmaceutical form.

According to the combination of the present invention for the above mentioned use, when more than one of said antagonist and/or miRNA mimic is used (i.e. a mixture of more than one antagonist or a mixture of more than one miRNA mimic or a mixture of one or more antagonists with one or more miRNA mimics is used), the following mixtures of said antagonist and/or miRNA mimic can be used: miR-199b-5p, miR-204-5p and miR-579-3p; miR-199b-5p and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and antimiR-4488 or LNA targeting miR-4488; antimiR-4488 or LNA targeting miR-4488 and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and miR-204-5p; miR-199b-5p and antimiR-4443 or LNA targeting miR-4443; miR-199b-5p and antimiR-4488 or LNA targeting miR-4488, wherein the mixtures are listed from the most preferable.

In addition, the combination can further comprise at least one of the following antagonists and/or miRNA mimics:

antimiR-1234:(SEQ ID NO: 20)gtggggtgggtggtcaggccgaorLNA targeting miR-1234, antimiR-9-5p:(SEQ ID NO: 21)tcatacagctagataaccaaagaorLNA targeting miR-9-5p, antimiR-1915-5p:(SEQ ID NO: 22)ggcccgggcagcaaggcaaggtorLNA targeting miR-1915-5p, antimiR-4286:(SEQ ID NO: 23)ggtaccaggagtggggtorLNA targeting miR-4286, antimiR-575:(SEQ. ID. NO. 24)gctcctgtccaactggctcorLNA targeting miR-575, antimiR-630:(SEQ ID NO: 25)accttccctggtacagaatactorLNA targeting miR-630, miR145-5p mimic:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p mimic:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p mimic:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107 mimic:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p mimic:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p mimic:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p mimic:(SEQ ID NO: 9)gcgacccauacuugguuucag.

The present invention concerns also a pharmaceutical composition comprising or consisting of antagonist of at least one of miR-4443 and miR-4488 and/or miRNA mimic of at least one of miR-199b-5p and miR-204-5p, in association with one or more excipients and/or adjuvants, said pharmaceutical composition being for use in the treatment of tumors which are resistant to MAPK pathway inhibiting drugs, wherein said antagonist is chosen from the group consisting of Locked Nucleic Acid targeting miR-4443, Locked Nucleic Acid targeting miR-4488, antimiR-4443: aaaacccacgcctccaa (SEQ ID NO:18), antimiR-4488: cgccggagcccgccccct (SEQ ID NO:19), whereas said miRNA mimic is chosen from the group consisting of miR-199b-5p mimic: cccaguguuuagacuaucuguuc (SEQ ID NO:1), miR-204-5p mimic: uucccuuugucauccuaugccu (SEQ ID NO:2).

According to an embodiment, when a mixture of said antagonist and/or miRNA mimic is used in the pharmaceutical composition, said mixture can be: miR-199b-5p, miR-204-5p and miR-579-3p; miR-199b-5p and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and antimiR-4488 or LNA targeting miR-4488; antimiR-4488 or LNA targeting miR-4488 and miR-204-5p; antimiR-4443 or LNA targeting miR-4443 and miR-204-5p; miR-199b-5p and antimiR-4443 or LNA targeting miR-4443; miR-199b-5p and antimiR-4488 or LNA targeting miR-4488.

According to a further embodiment of the present invention, said antagonist and/or miRNA mimic which is in the pharmaceutical composition can be in combination with at least one of the following antagonists and/or miRNA mimics:

antimiR-1234:(SEQ ID NO: 20)gtggggtgggtggtcaggccgaorLNA targeting miR-1234, antimiR-9-5p:(SEQ ID NO: 21)tcatacagctagataaccaaagaorLNA targeting miR-9-5p, antimiR-1915-5p:(SEQ ID NO: 22)ggcccgggcagcaaggcaaggtorLNA targeting miR-1915-5p, antimiR-4286:(SEQ ID NO: 23)ggtaccaggagtggggtorLNA targeting miR-4286, antimiR-575:(SEQ. ID. NO. 24)gctcctgtccaactggctcorLNA targeting miR-575, antimiR-630:(SEQ ID NO: 25)accttccctggtacagaatactorLNA targeting miR-630, miR145-5p mimic:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p mimic:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p mimic:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107 mimic:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p mimic:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p mimic:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p mimic:(SEQ ID NO: 9)gcgacccauacuugguuucag.

The pharmaceutical composition according to the present invention can further comprise at least one MAPK pathway inhibiting drug, wherein said antagonist and/or miRNA mimic is used against the resistance to MAPK pathway inhibiting drugs.

For example, MAPK pathway inhibiting drugs can be chosen from the group consisting of vemurafenib, Trametinib, dabrafenib, sorafenib, SB590885, PLX4720, XL281, RAF265, encorafenib, cobimetinib, Cl-1040, PD0325901, Binimetinib, selumetinib.

The present invention concerns also a method for in vitro diagnosis of resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in tumors, said method comprising measuring the expression, in a biological sample, of at least two, three or all of the following microRNAs:

miR-199b-5p:(SEQ ID NO: 1)cccaguguuuagacuaucuguuc,miR-204-5p:(SEQ ID NO: 2)uucccuuugucauccuaugccu,miR-4443:(SEQ ID NO: 10)uuggaggcguggguuuu,miR-4488:(SEQ ID NO: 11)agggggcgggcuccggcg,miR-145-5p:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p:(SEQ ID NO: 9)gcgacccauacuugguuucag,miR-1234:(SEQ ID NO: 12)ucggccugaccacccaccccac,miR-9-5p:(SEQ ID NO: 13)ucuuugguuaucuagcuguauga,miR-1915-5p:(SEQ ID NO: 14)accuugccuugcugcccgggcc,miR-4286:(SEQ ID NO: 15)accccacuccugguacc,miR-575(SEQ ID NO: 16)gagccaguuggacaggagc,miR-630(SEQ ID NO: 17)aguauucuguaccagggaaggu,

wherein miR-199b-5p, miR-204-5p, miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-15b-5p, miR-221-3p, miR-551b-3p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-4443, miR-4488, miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 are over-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in comparison with their expression in controls which do not present said resistance; preferably, said at least two microRNAs are different from miR-4443 and miR-18a-5p when the expression of only two microRNAs is measured. The tumors can be those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs.

It is a further object of the present invention, the use of at least two, three or all of the following microRNAs:

miR-199b-5p:(SEQ ID NO: 1)cccaguguuuagacuaucuguuc,miR-204-5p:(SEQ ID NO: 2)uucccuuugucauccuaugccu,miR-4443:(SEQ ID NO: 10)uuggaggcguggguuuu,miR-4488:(SEQ ID NO: 11)agggggcgggcuccggcg,miR-145-5p:(SEQ ID NO: 3)guccaguuuucccaggaaucccu,miR-18a-5p:(SEQ ID NO: 4)uaaggugcaucuagugcagauag,miR-455-3p:(SEQ ID NO: 5)gcaguccaugggcauauacac,miR-107:(SEQ ID NO: 6)agcagcauuguacagggcuauca,miR-15b-5p:(SEQ ID NO: 7)uagcagcacaucaugguuuaca,miR-221-3p:(SEQ ID NO: 8)agcuacauugucugcuggguuuc,miR-551b-3p:(SEQ ID NO: 9)gcgacccauacuugguuucag,miR-1234:(SEQ ID NO: 12)ucggccugaccacccaccccac,miR-9-5p:(SEQ ID NO: 13)ucuuugguuaucuagcuguauga,miR-1915-5p:(SEQ ID NO: 14)accuugccuugcugcccgggcc,miR-4286:(SEQ ID NO: 15)accccacuccugguacc,miR-575(SEQ ID NO: 16)gagccaguuggacaggagc,miR-630(SEQ ID NO: 17)aguauucuguaccagggaaggu,

as biomarkers for the in vitro diagnosis of the resistance of tumors to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein miR-199b-5p, miR-204-5p, miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-15b-5p, miR-221-3p, miR-551b-3p are down-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, whereas miR-4443, miR-4488, miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 are over-expressed in the resistance to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs in comparison with their expression in controls which do not present said resistance; preferably said at least two microRNAs are different from miR-4443 and miR-18a-5p when are used only two microRNAs. As mentioned above the tumors can be those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs.

The present invention concerns also an antagonist of at least one of miR-4443, miR-4488, miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 and/or a miRNA mimic of at least one of miR-199b-5p, miR-204-5p, miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-551b-3p, miR-221-3p, miR-15b-5p for use in the treatment of tumors which are resistant to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein said antagonist is chosen from the group consisting of antimiR-4443: aaaacccacgcctccaa (SEQ ID NO:18), antimiR-4488: cgccggagcccgccccct (SEQ ID NO:19), antimiR-1234: gtggggtgggtggtcaggccga (SEQ ID NO:20), antimiR-9-5p: tcatacagctagataaccaaaga (SEQ ID NO:21), antimiR-1915-5p: ggcccgggcagcaaggcaaggt (SEQ ID NO: 22), antimiR-4286: ggtaccaggagtggggt (SEQ ID NO:23), antimiR-575: gctcctgtccaactggctc (SEQ. ID. NO. 24), antimiR-630: accttccctggtacagaatact (SEQ ID NO:25), or the corresponding LNA of the above-mentioned antimiRNAs,

whereas said miRNA mimic is chosen from the group consisting of miR-199b-5p mimic: cccaguguuuagacuaucuguuc (SEQ ID NO:1), miR-204-5p mimic: uucccuuugucauccuaugccu (SEQ ID NO:2), miR145-5p mimic: guccaguuuucccaggaaucccu (SEQ ID NO:3), miR-18a-5p mimic: uaaggugcaucuagugcagauag (SEQ ID NO:4), miR-455-3p mimic: gcaguccaugggcauauacac (SEQ ID NO: 5), miR-107 mimic: agcagcauuguacagggcuauca (SEQ ID NO:6), miR-15b-5p mimic: uagcagcacaucaugguuuaca (SEQ ID NO:7), miR-221-3p mimic: agcuacauugucugcuggguuuc (SEQ ID NO:8), miR-551b-3p mimic: gcgacccauacuugguuucag (SEQ ID NO:9). The tumors can be those which are BRAF mutated tumors and are treated with MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs.

The present invention concerns also a combination of an antagonist of at least one of miR-4443, miR-4488, miR-1234, miR-9-5p, miR-1915-5p, miR-4286, miR-575, miR-630 and/or of a miRNA mimic of at least one of miR-199b-5p, miR-204-5p, miR-145-5p, miR-18a-5p, miR-455-3p, miR-107, miR-551b-3p, miR-221-3p, miR-15b-5p with at least one MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drug for the simultaneous, sequential or separate use in the treatment of tumors which are resistant to MAPK pathway (or more specifically BRAF/MEK pathway) inhibiting drugs, wherein said antagonist is chosen from the group consisting of antimiR-4443: aaaacccacgcctccaa (SEQ ID NO:18), antimiR-4488: cgccggagcccgccccct (SEQ ID NO:19), antimiR-1234: gtggggtgggtggtcaggccga (SEQ ID NO:20), antimiR-9-5p: tcatacagctagataaccaaaga (SEQ ID NO:21), antimiR-1915-5p: ggcccgggcagcaaggcaaggt (SEQ ID NO: 22), antimiR-4286: ggtaccaggagtggggt (SEQ ID NO:23), antimiR-575: gctcctgtccaactggctc (SEQ. ID. NO. 24), antimiR-630: accttccctggtacagaatact (SEQ ID NO:25), or the corresponding LNA of the above-mentioned antimiRNAs,

whereas said miRNA mimic is chosen from the group consisting of miR-199b-5p mimic: cccaguguuuagacuaucuguuc (SEQ ID NO:1), miR-204-5p mimic: uucccuuugucauccuaugccu (SEQ ID NO:2), miR145-5p mimic: guccaguuuucccaggaaucccu (SEQ ID NO:3), miR-18a-5p mimic: uaaggugcaucuagugcagauag (SEQ ID NO:4), miR-455-3p mimic: gcaguccaugggcauauacac (SEQ ID NO: 5), miR-107 mimic: agcagcauuguacagggcuauca (SEQ ID NO:6), miR-15b-5p mimic: uagcagcacaucaugguuuaca (SEQ ID NO:7), miR-221-3p mimic: agcuacauugucugcuggguuuc (SEQ ID NO:8), miR-551b-3p mimic: gcgacccauacuugguuucag (SEQ ID NO:9).

