WO2025219330A1

WO2025219330A1 - Detection of ppix for use in methods for melanoma ferroptosis sensitivity and targeted therapy resistance prediction

Info

Publication number: WO2025219330A1
Application number: PCT/EP2025/060238
Authority: WO
Inventors: Frédéric LARBRET; Marcel DECKERT; Sophie TARTARE - DECKERT
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Nice Sophia Antipolis UNSA
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Nice Sophia Antipolis UNSA
Priority date: 2024-04-15
Filing date: 2025-04-14
Publication date: 2025-10-23
Anticipated expiration: 2026-10-15

Abstract

Here, the inventors have developed a spectral flow cytometry assay to measure heme biosynthesis. By using this new assay on melanoma cell lines, they observed a direct correlation between high PPIX levels in differentiated MITF^high cells and a protection against ferroptosis. Conversely, cell lines with low PPIX levels were associated with a dedifferentiated MITF^low phenotype enriched in stem cell markers and more sensitive to ferroptosis inducers. They also found that inhibition of PPIX biosynthesis in differentiated melanoma cells synergizes with iron overload to induce lipid peroxidation and tumor cell death. Finally, they show that decreased levels of PPIX linked to increased ferroptosis sensitivity can be acquired in vivo in melanoma tumors that relapse after anti-MAPK targeted therapies. The present invention relates to a method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therapy resistance comprising i) determining the level of protoporphyrin IX (PPIX) in a biological sample obtained from the subject and ii) comparing the level determined at step i) with a predetermined reference value: wherein if the level of the PPIX determined at step (i) is lower than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis sensitivity and targeted therapy resistance or wherein if the level of the PPIX determined at step (i) is higher than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis resistance and targeted therapy sensitivity. The present invention also relates to a method for treating resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a ferroptosis inducer.

Description

DETECTION OF PPIX FOR USE IN METHODS FOR MELANOMA FERROPTOSIS SENSITIVITY AND TARGETED THERAPY RESISTANCE PREDICTION

FIELD OF THE INVENTION:

The invention is in the field of oncology, more particularly the invention relates to PPIX for use in methods for melanoma ferroptosis sensitivity and targeted therapy resistance prediction.

BACKGROUND OF THE INVENTION:

Melanoma is a highly aggressive type of skin cancer that develops from melanocytes. Approximately half of all melanomas are due to BRAF gene mutations. Mutant BRAF proteins drive the constitutive activation of the MAPK signaling pathway, promoting tumor cell growth and survival. Targeted therapies with BRAF inhibitors (such as vemurafenib) and MEK inhibitors (such as trametinib) have been developed to inhibit the oncogenic BRAF pathway and their combination is used as first-line treatments for metastatic melanoma (1). Despite clear clinical success, the effectiveness of targeted therapies remains limited due to intrinsic resistance and to the development of acquired resistance by cancer cells, leading to metastatic relapse. Novel therapeutic strategies are thus warranted. Consequently, Recent research into cell death has revealed the existence of several types of cell death that could be clinically exploited (2). In this context, a new type of cell death called ferroptosis could represent the Achilles heel of cancer cells with innate resistance to treatment and of cancer cells with acquired resistance to treatment. Ferroptosis is characterized by iron-dependent lipid peroxidation (3). Unlike apoptosis, ferroptosis is based on the accumulation of lipid hydroperoxides derived from polyunsaturated fatty acids in cellular membranes.

It has been shown that sensitivity to ferroptosis evolves in melanoma according to the tumor cell differentiation trajectory (4). Ferroptosis sensitivity is associated with a dedifferentiated cell phenotype that is also characterized by intrinsic resistance to targeted therapies and aggressiveness. Therefore, ferroptosis-inducing drugs are promising new agents to overcome drug resistance and tumor relapse. Several types of inhibitors have been developed for this purpose. One the most promising drug family are those targeting GPX4 (5). GPX4 is unique among the glutathione peroxidase family in that it can directly reduce lipid hydroperoxides within cellular membranes. Therefore, this enzyme plays a crucial role in protecting cells from ferroptosis. Targeting iron metabolism also represents a potential approach since iron participates in Fenton reactions, generating highly reactive oxygen species like hydroxyl radicals, which in turn, contribute to lipid peroxidation (6).

In this context, the inventors have focused their research on the different actors that play a major role in iron metabolism in tumor cells. They were especially interested in heme biosynthesis which has been shown to be involved in therapy resistance of leukemic cells (7).

SUMMARY OF THE INVENTION:

The invention relates to a method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therapy resistance comprising i) determining the level of protoporphyrin IX (PPIX) in a biological sample obtained from the subject and ii) comparing the level determined at step i) with a predetermined reference value: wherein if the level of the PPIX determined at step (i) is lower than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis sensitivity and targeted therapy resistance or; wherein if the level of the PPIX determined at step (i) is higher than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis resistance and targeted therapy sensitivity. The invention also relates to a method for treating resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a ferroptosis inducer. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Here, the inventors have first developed a spectral flow cytometry assay to measure heme biosynthesis by using the iron chelating agent Deferoxamine (DFX). The principle of this assay is based on measuring the autofluorescence of cells generated by the presence of protoporphyrin IX (PPIX), the last intermediary of heme synthesis. PPIX exhibits a typical red fluorescence (from 635 nm to 700 nm) under violet light (405 nm) excitation wavelength but quenched by Fe²⁺ ions binding to form heme. Treatment of cells with the iron chelating agent Deferoxamine (DFX) reduces the iron contained in the cytoplasm inhibiting the conversion of PPIX to heme resulting in greater PPIX accumulation and fluorescence emission. It allows to assess the level of PPIX in cells according to fluorescence intensity, an event that is detectable by imaging methods such as flow cytometry (8). By using this new assay on melanoma cell lines, they observed a direct correlation between high PPIX levels in differentiated MITF^hlgh cells and a protection against ferroptosis. Conversely, cell lines with low PPIX levels were associated with a dedifferentiated MITF^low phenotype enriched in stem cell markers and more sensitive to ferroptosis inducers. They also found that inhibition of PPIX biosynthesis in differentiated melanoma cells synergizes with iron overload to induce lipid peroxidation and tumor cell death. Finally, they show that decreased levels of PPIX linked to increased ferroptosis sensitivity can be acquired in vivo in melanoma tumors that relapse after anti-MAPK targeted therapies. In conclusion the study provides a comprehensive understanding of the interplay between PPIX levels and sensitivity to ferroptosis in melanoma cells, shedding light on potential markers for predicting resistance to targeted therapies and alternative strategies targeting iron metabolism in cancer cells.

Mains definitions of the present invention

As used herein, the term “heme” refers to is a ring-shaped iron-containing molecular component of hemoglobin, which is necessary to bind oxygen in the bloodstream. It is composed of four pyrrole rings with 2 vinyl and 2 propionic acid side chains. Heme is biosynthesized in many kind of cells like melanocytes, and in tissues including bone marrow or the liver . Heme plays a critical role in multiple different redox reactions in mammal, due to its ability to carry the oxygen moiety. Reactions include oxidative metabolism (cytochrome c oxidase, succinate dehydrogenase), xenobiotic detoxification via cytochrome P450 pathways (including metabolism of some drugs), gas sensing (guanyl cyclases, nitric oxide synthase), and microRNA processing (DGCR8). Heme is a coordination complex "consisting of an iron ion coordinated to a tetra-porphyrin acting as a tetradentate ligand, and to one or two axial ligands". Hemes are most commonly recognized as components of hemoglobin, the red pigment in blood, but are also found in a number of other biologically important hemoproteins such as myoglobin, cytochromes, catalases, heme peroxidase, and endothelial nitric oxide synthase.

As used herein, the term “heme biosynthesis” refers to production of heme properly called porphyrin synthesis, as all the intermediates are tetrapyrroles that are chemically classified as porphyrins. The pathway is initiated by the synthesis of 5-aminolevulinic acid (dALA or SALA) from the amino acid glycine and succinyl-CoA from the citric acid cycle (TCA cycle). The ratelimiting enzyme responsible for this reaction, ALA synthase, is negatively regulated by glucose and heme concentration. Mechanism of inhibition of ALAs by heme or hemin is by decreasing stability of mRNA synthesis and by decreasing the intake of mRNA in the mitochondria. This mechanism is of therapeutic importance: infusion of heme arginate or hematin and glucose can abort attacks of acute intermittent porphyria in patients with an inborn error of metabolism of this process, by reducing transcription of ALA synthase.

As used herein, “ferroptosis” means regulated cell death that is iron-dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. Ferroptosis is distinct from apoptosis, necrosis, and autophagy.

As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or is susceptible to have melanoma. In particular embodiment, the subject has or is susceptible to have cutaneous melanoma. More particularly, the subject according to the invention has or susceptible to have melanoma resistant. In a particular embodiment, the subject has or susceptible to have melanoma resistant to at least one of the treatments as described above. The subject having a melanoma resistant is identified by standard criteria. The standard criteria for resistance, for example, are Response Evaluation Criteria In Solid Tumors (RECIST) criteria, published by an international consortium including NCI.

As used herein, the term “melanoma” also known as malignant melanoma, refers to a type of cancer that develops from the pigment-containing cells, called melanocytes. There are three general categories of melanoma: 1) cutaneous melanoma which corresponds to melanoma of the skin; it is the most common type of melanoma; 2) mucosal melanoma which can occur in any mucous membrane of the body, including the nasal passages, the throat, the vagina, the anus, or in the mouth; and 3) ocular melanoma also known as uveal melanoma or choroidal melanoma, is a rare form of melanoma that occurs in the eye. In a particular embodiment, the melanoma is cutaneous melanoma.

As used herein, the term “microphthalmia-associated transcription factor” (MITF) refers to a basic helix-loop-helix leucine zipper transcription factor involved in lineage-specific pathway regulation of many types of cells and in particular in the regulation of melanocytes. MITF has the following human UniProt number 075030 and the following Gene ID: 4286.

In some embodiment, a MITF^hlgh cells refers to a high level of MITF in the cells and a MITF^low cells refers to a low level of MITF in the cells. As used herein, the term “differentiation” refers to the process in which a stem cell changes from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types.

As used herein, the term “dedifferentiation” is a transient process by which cells become less specialized and return to an earlier cell state within the same lineage. This suggests an increase in cell potency, meaning that, following dedifferentiation, a cell may possess the ability to redifferentiate into more cell types than it did before dedifferentiation. This is in contrast to differentiation, where differences in gene expression, morphology, or physiology arise in a cell, making its function increasingly specialized. Cellular dedifferentiation expresses the total or partial loss of the characteristics of a cell that it had acquired during its development (cell growth and cell division) or differentiation.

Method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therap resistance

In a first aspect, the invention relates to a method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therapy resistance comprising i) determining the level of protoporphyrin IX (PPIX) in a biological sample obtained from the subject and ii) comparing the level determined at step i) with a predetermined reference value:

- wherein if the level of the PPIX determined at step (i) is lower than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis sensitivity and targeted therapy resistance or;

- wherein if the level of the PPIX determined at step (i) is higher than the predetermined reference value is indicative that the said patient is having melanoma ferroptosis resistance and targeted therapy sensitivity.

As used herein, the terms “melanoma ferroptosis sensitivity” refer to the ability of the subject to respond to the cell death triggered by iron-dependent phospholipid peroxidation.

As used herein, the terms “melanoma ferroptosis resistance” refer to the ability of the subject of having melanoma resistance to the cell death triggered by iron-dependent phospholipid peroxidation. As used herein, the terms “targeted therapy sensitivity” refer to the ability of the subject to respond to targeted therapy.

As used herein, the terms “targeted therapy resistance” refer to the ability of the subject of having resistance to targeted therapy.

In some embodiment, the present invention indicates that a high level of PPIX is associated with differentiated cells. In particular, the present invention indicates that a high level of PPIX is associated with differentiated MITF^hlgh cells.

In some embodiment, the present invention indicates that a low level of PPIX is associated with dedifferentiated cells. In particular, the present invention indicates that a low level of PPIX is associated with dedifferentiated MITF^low cells.

In some embodiment, the present invention indicates that the dedifferentiated cells are resistant to targeted therapies chosen from BRAF inhibitors or MEK inhibitors. In particular, the present invention indicates that the dedifferentiated MITF^low cells are resistant to targeted therapies.

As used herein, the terms "aggressive" and "invasive" are used herein interchangeably. When used herein to characterize a melanoma, they refer to the proclivity of a tumor for expanding beyond its boundaries into adjacent tissue. Invasive melanoma can be contrasted with organ- confined cancer wherein the tumor is confined to a particular organ or to a particular location in an organ. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, that degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located.

As used herein, the term “metastatic melanoma” refers to the spread of melanoma tumor cells from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A "metastatic cancer" is a cancer that spreads from its original, or primary, location, and may also be referred to as a "secondary cancer" or "secondary tumor" . Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate. In one embodiment, the melanoma is resistant melanoma.

As used herein, the term “resistant melanoma” refers to melanoma, which does not respond to a classical treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic of melanoma. The resistance of cancer for the medication is caused by mutations in the gene, which are involved in the proliferation, divisions or differentiation of cells. In the context of the invention, the resistance of melanoma can be caused by non-genetic events or by the mutations (single or double) in the following genes: BRAF, MEK, NRAS or PTEN. The term “PTEN” refers to Phosphatase and TENsin homolog, it is one of the most frequently inactivated tumor suppressor genes in sporadic cancers. Inactivating mutations and deletions of the PTEN gene are found in many types of cancers, including melanoma. Accordingly, such resistance is against to the treatments as described above.

In a particular embodiment, the melanoma has BRAF mutation.

As used herein, the term “BRAF” is a member of the Raf kinase family of serine/threonine- specific protein kinases. This protein plays a role in regulating the MAP kinase / ERKs signaling pathway, which affects cell division, differentiation, and survival. A number of mutations in BRAF are known. In particular, the V600E mutation is prominent. Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, K600E, A727V, and most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions. In a particular embodiment, the BRAF mutation is V600E/K in the context of the invention.

In a particular embodiment, the melanoma has MEK mutation.

As used herein, the term “MEK” refers to Mitogen-activated protein kinase, also known as MAP2K, MEK, MAPKK. It is a kinase enzyme, which phosphorylates mitogen-activated protein kinase (MAPK).

In a particular embodiment, the melanoma has NRAS mutation. As used herein, the term “NRAS” refers to neuroblastoma RAS viral oncogene homolog. It is a member of the Ras gene family. The naturally occurring human NRAS gene has a nucleotide sequence as shown in Genbank Accession number NM_002524 and the naturally occurring human NRAS protein has an aminoacid sequence as shown in Genbank Accession number NP_002515. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_010937, NM_001368638). The NRAS gene is in the Ras family of oncogene and involved in regulating cell division. NRAS mutations in codons 12, 13, and 61 arise in 15-20 % of all melanomas. The immune checkpoint inhibitor and the inhibitors of MEK (i.e. Trametinib) are used to treat the melanoma with NRAS mutations.

As used herein, the terms “targeted therapy for melanoma” or “targeted therapy” are used here interchangeably and refer to cancers treatment that identify and attack specific cell features of melanoma.

In some embodiment, the subject of the present invention has resistance to targeted therapy for melanoma.

In some embodiment, the targeted therapies of melanoma include but are not limited to BRAF inhibitors, MEK inhibitors or Immune checkpoint inhibitors.

In another embodiment, the melanoma is resistant to a treatment with the inhibitors of BRAF.

As used herein, the term “inhibitor of BRAF” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of BRAF. More particularly, such compound by inhibiting BRAF activity reduces cell division, differentiation, and secretion. In a particular embodiment, the inhibitor of BRAF is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of BRAF is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. The inhibitors of BRAF mutations are well known in the art. In a particular embodiment, the inhibitor of BRAF is Vemurafenib. Vemurafenib also known as PLX4032, RG7204 ou RO5185426 and commercialized by Roche as zelboraf. In a particular embodiment, the inhibitor of BRAF is Dabrafenib also known as tafinlar, which is commercialized by Novartis.

In another embodiment, the melanoma is resistant to a treatment with the inhibitors of MEK.

As used herein, the term “inhibitor of MEK” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of MEK. More particularly, such compound by inhibiting MEK activity reduces phosphorylation of MAPK. In a particular embodiment, the inhibitor of MEK is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The inhibitors of MEK are well known in the art. In a particular embodiment, the inhibitor of MEK is Trametinib also known as mekinist, which is commercialized by GSK. In a particular embodiment, the inhibitor of MEK Cobimetinib also known as cotellic commercialized by Genentech. In a particular embodiment, the inhibitor of MEK is Binimetinib also known as MEK162, ARRY-162 is developed by Array Biopharma.

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. In the context of the invention, the melanoma is resistant to a combined treatment characterized by using an inhibitor of BRAF mutation and an inhibitor of MEK as described above. For example, the combined treatment may be a combination of Vemurafenib and Cobimetinib.

In a further embodiment, the melanoma is resistant to a treatment with an immune checkpoint inhibitor.

As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD- 1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response. In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

In a particular embodiment, the immune checkpoint inhibitor is an antibody.

Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.

In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490. In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2, 3 -di oxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), 0- (3-benzofuranyl)-alanine, 0-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a 0- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, 0-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and 0-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :

In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

In some embodiments, the immune checkpoint inhibitor is an aptamer.

Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015. In some embodiment, the inventors show, by using the spectral flow cytometry assay of the present invention on melanoma cell lines, a direct correlation between high PPIX levels in differentiated MITF^hlgh cells and a protection against ferroptosis. Conversely, cell lines with low PPIX levels were associated with a dedifferentiated MITF^low phenotype enriched in stem cell markers and more sensitive to ferroptosis inducers.

In some embodiment, the inventors also show a decreased level of protoporphyrin IX (PPIX) in the biological sample obtained from the subject suffered from melanoma, linked to increased ferroptosis sensitivity, can be acquired in vivo in melanoma tumors that relapse after anti- MAPK targeted therapies.

As used herein, the term “protoporphyrin IX” (PPIX) is an organic compound, classified as a porphyrin, that plays an important role in living organisms as a precursor to other critical compounds like heme (hemoglobin) and chlorophyll. The name is often abbreviated as PPIX. The compound is encountered in nature in the form of complexes where the two inner hydrogen atoms are replaced by a divalent metal cation. When complexed with an iron(II) (ferrous) cation Fe²⁺, the molecule is called heme. Hemes are prosthetic groups in some important proteins. These heme-containing proteins include hemoglobin, myoglobin, and cytochrome c. Complexes can also be formed with other metal ions, such as zinc. PPIX is having the following CAS number : 553-12-8 and the following chemical formula: C34H34N4O4.

As used herein, the term “biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy. In a particular embodiment, biological sample for the determination of a level include samples such as a tissue sample, or a biopsy.

In a particular embodiment, the biological sample is a blood sample, more particularly, peripheral blood mononuclear cells (PBMC). Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis, which will preferentially lyse red blood cells. Such procedures are known to the experts in the art. In a particular embodiment, the biological sample is a tumor tissue sample. More particularly, in a particular embodiment, the biological sample is normal, primary or melanoma tissue.

As used herein, the term “tumor tissue sample” has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection. In some embodiment, the tumor tissue sample of the present invention is a live cell.

As used herein, the term “determining” includes qualitative and/or quantitative detection (i.e. detecting and/or measuring the level) with or without reference to a control or a predetermined value.

As used herein, the term “detecting” means determining if the PPIX is present or not in a biological sample and the term “measuring” means determining the amount of PPIX in a biological sample. Typically the level may be determined for example by immunoassays such as an ELISA performed on a biological sample, such as a tumor sample obtained from the patient. In some embodiment, the level of PPIX can also be determined by mass spectrometry and label free methods by using flow cytometry or imagery.

As used herein, the term “level” refers to the level of protoporphyrin IX (PPIX). Typically, the level of the PPIX from the tumor cell may be determined by a spectral flow cytometry assay.

In some embodiment, the inventors develop a spectral flow cytometry assay to measure heme biosynthesis. The principle of this assay is based on measuring the autofluorescence of cells treated with the iron chelator Deferoxamine. Treatment of cells with the iron chelating agent Deferoxamine (DFX) reduce the iron contained in the cytoplasm inhibiting the conversion of PPIX to heme resulting in greater PPIX accumulation and fluorescence emission. It allows to assess the level of PPIX in cells according to fluorescence intensity, an event that is detectable by spectral flow cytometry.

As used herein, the term “deferoxamine” (DFX) also known as desferrioxamine B, desferoxamine B, MPO-B, DFOA, DFO, DFB or Desferal is a linear bacterial siderophore used as a medication for the treatment of iron overload. Deferoxamine acts by chelating iron present in the blood and allowing its elimination via the urine. By reducing excess iron in the body, this chelating agent reduces the damage done by iron to many organs or tissues such as the liver.

As used herein, the term “fluorescence” has its general meaning in the art and refers to an emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light.

In some embodiment, the level of protoporphyrin IX (PPIX) is compared with the level of a predetermined reference value.

As used herein, the term “the predetermined reference value” refers to a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of cell densities in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the cell density in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured densities in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is determined by carrying out a method comprising the steps of a) providing a collection of tumor tissue samples from subject suffering from melanoma; b) providing, for each tumor tissue sample provided at step a), information relating to the actual clinical outcome for the corresponding subject (i.e. the duration of the disease-free survival (DFS) and/or the overall survival (OS)); c) providing a serial of arbitrary quantification values; d) quantifying the cell density for each tumor tissue sample contained in the collection provided at step a); e) classifying said tumor tissue samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising tumor tissue samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising tumor tissue samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of tumor tissue samples are obtained for the said specific quantification value, wherein the tumor tissue samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the subjects from which tumor tissue samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant P-value obtained with a log-rank test, significance when P<0.05) has been calculated at step g).

For example the cell density has been assessed for 100 tumor tissue samples of 100 subjects. The 100 samples are ranked according to the cell density. Sample 1 has the highest density and sample 100 has the lowest density. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer subject, Kaplan-Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated (log-rank test). The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum P-value is the strongest. In other terms, the cell density corresponding to the boundary between both subsets for which the P-value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of cell densities. Thus in some embodiments, the predetermined reference value thus allows discrimination between a poor and a good prognosis with respect to DFS and OS for a subject. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P-value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the outcome can be determined by comparing the cell density with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e g. generally the minimum P-value which is found).

In a particular embodiment, the method according to the invention further comprises a step of classification of subject by an algorithm and determining whether a subject will have melanoma ferroptosis sensitivity and targeted therapy resistance.

Typically, the method of the present invention comprises a) quantifying the level of the PPIX in the biological sample; b) implementing a classification algorithm on data comprising the quantified of PPIX levels so as to obtain an algorithm output; c) determining the probability that the subject have melanoma ferroptosis sensitivity and targeted therapy resistance from the algorithm output of step b).

In some embodiments, the method according to the invention wherein the algorithm is selected from Linear Discriminant Analysis (LDA), Topological Data Analysis (TDA), Neural Networks, Support Vector Machine (SVM) algorithm and Random Forests algorithm (RF). selected from Linear Discriminant Analysis (LDA), Topological Data Analysis (TDA), Neural Networks, Support Vector Machine (SVM) algorithm and Random Forests algorithm (RF).

In some embodiments, the method of the invention comprises the step of determining the subject response using a classification algorithm. As used herein, the term "classification algorithm" has its general meaning in the art and refers to classification and regression tree methods and multivariate classification well known in the art such as described in US 8,126,690; WO2008/156617.

As used herein, the term “support vector machine (SVM)” is a universal learning machine useful for pattern recognition, whose decision surface is parameterized by a set of support vectors and a set of corresponding weights, refers to a method of not separately processing, but simultaneously processing a plurality of variables. Thus, the support vector machine is useful as a statistical tool for classification. The support vector machine non-linearly maps its n- dimensional input space into a high dimensional feature space and presents an optimal interface (optimal parting plane) between features. The support vector machine comprises two phases: a training phase and a testing phase. In the training phase, support vectors are produced, while estimation is performed according to a specific rule in the testing phase. In general, SVMs provide a model for use in classifying each of n subjects to two or more disease categories based on one k-dimensional vector (called a k-tuple) of biomarker measurements per subject. An SVM first transforms the k-tuples using a kernel function into a space of equal or higher dimension. The kernel function projects the data into a space where the categories can be better separated using hyperplanes than would be possible in the original data space. To determine the hyperplanes with which to discriminate between categories, a set of support vectors, which lie closest to the boundary between the disease categories, may be chosen. A hyperplane is then selected by known SVM techniques such that the distance between the support vectors and the hyperplane is maximal within the bounds of a cost function that penalizes incorrect predictions. This hyperplane is the one which optimally separates the data in terms of prediction (Vapnik, 1998 Statistical Learning Theory. New York: Wiley). Any new observation is then classified as belonging to any one of the categories of interest, based where the observation lies in relation to the hyperplane. When more than two categories are considered, the process is carried out pairwise for all of the categories and those results combined to create a rule to discriminate between all the categories. As used herein, the term "Random Forests algorithm" or "RF" has its general meaning in the art and refers to classification algorithm such as described in US 8,126,690; WO2008/156617. Random Forest is a decision-tree-based classifier that is constructed using an algorithm originally developed by Leo Breiman (Breiman L, "Random forests," Machine Learning 2001, 45:5-32). The classifier uses a large number of individual decision trees and decides the class by choosing the mode of the classes as determined by the individual trees. The individual trees are constructed using the following algorithm: (1) Assume that the number of cases in the training set is N, and that the number of variables in the classifier is M; (2) Select the number of input variables that will be used to determine the decision at a node of the tree; this number, m should be much less than M; (3) Choose a training set by choosing N samples from the training set with replacement; (4) For each node of the tree randomly select m of the M variables on which to base the decision at that node; (5) Calculate the best split based on these m variables in the training set. In some embodiments, the score is generated by a computer program.

The algorithm of the present invention can be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The algorithm can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a processor for performing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. However, a computer need not have such devices. Moreover, a computer can be embedded in another device. Computer-readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry. To provide for interaction with a user, embodiments of the invention can be implemented on a computer having a display device, e.g., in non-limiting examples, a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, for displaying information to the user and a keyboard and a pointing device, e.g., a mouse or a trackball, by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback, e.g., visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including acoustic, speech, or tactile input. Accordingly, in some embodiments, the algorithm can be implemented in a computing system that includes a back-end component, e.g., as a data server, or that includes a middleware component, e.g., an application server, or that includes a front-end component, e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the invention, or any combination of one or more such back-end, middleware, or front-end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communication network. Examples of communication networks include a local area network (“LAN”) and a wide area network (“WAN”), e.g., the Internet. The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

Method of treatins melanoma in a patient in need thereof

Inventors show that inhibition of PPIX biosynthesis in differentiated melanoma cells synergizes with iron overload to induce lipid peroxidation and tumor cell death.

Accordingly, in a second aspect, the invention relates to a method of treating melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant in a patient in need thereof comprising administering to the patient a therapeutically effective amount of ferroptosis inducer.

In particular, the invention relates to a method for treating resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a ferroptosis inducer.

In particular, the invention relates to a method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therapy resistance comprising i) determining the level of protoporphyrin IX (PPIX) in a biological sample obtained from the subject and ii) comparing the level determined at step i) with a predetermined reference value, and administered a ferroptosis inducer if the level of the PPIX determined at step (i) is lower than the predetermined reference value.

As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term “ferroptosis inducer” denotes a compound able to increase ferroptosis occurrence. As example, the ferroptosis inducer may be ferric ammonium citrate (FAC), APR- 246, Ras Synthetic Lethal 3 (RSL3), ML162, ML210, acrolein, Erastin, Imidazole Ketone Erastin (IKE), Piperazine Erastin (PE), sulfasalazine, sorafenib, Ferroptosis Inducer 56 (FIN56), Ferroptosis inducer endoperoxide (FIN02), Caspase-Independent Lethal 56 (CIL56), mevalonate-derived coenzyme Q10, buthionine sulfoximine (BSO), amentoflavone, dihydroartemisinin (DHA), typhaneoside, artesunate, Withaferin A (WA).

As used herein, the term “RSL3” refers to a ferroptosis activator in a VD AC -independent manner, exhibiting selectivity for tumor cells bearing oncogenic RAS. RSL3 binds, inactivates GPX4 and thus mediates GPX4-regulated ferroptosis. RSL3 has the following CAS number: CAS: 1219810-16-8 and the following chemical formula:

As used herein, the term “FIN56” refers to a specific inducer of ferroptosis. FIN56 induces ferroptosis by inducing degradation of GPX4. FIN56 also binds to and activates squalene synthase. FIN56 has the following CAS number: CAS: 1083162-61-1 and the following chemical formula:

In a particular embodiment, the ferroptosis inducer is FAC.

As used herein, the term “ferric ammonium citrate” (FAC) refers to a specific inducer of ferroptosis. FAC has the following CAS number: CAS: 1185-57-5 and the following chemical formula:

In a particular embodiment, the ferroptosis inducer is Erastin.

As used herein, the term “Erastin” refers to a cell-permeable compound that binds to VDAC to alter its gating. Induces non-apoptotic cell death in tumor cells harboring activating mutations in RAS-RAF-MEK signaling. Erastin has the following CAS number: CAS: 571203-78-6 and the following chemical formula:

In some embodiment, the subject of the present invention is administered with an ferroptosis inducer.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g ferroptosis inducer) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

By a "therapeutically effective amount" is meant a sufficient amount of ferroptosis inducer for use in a method for the treatment of melanoma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The ferroptosis inducer as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Combined preparation

In a third aspect, the invention relates to i) ferroptosis inducer and ii) a classical treatment as a combined preparation for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, i) a ferroptosis inducer for use according to the invention, and ii) a classical treatment as a combined preparation for use in the treatment of resistant melanoma.

In some embodiment, i) a ferroptosis inducer for use according to the invention, and ii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, i) a ferroptosis inducer for use according to the invention, and ii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of resistant melanoma.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

The ferroptosis inducer can be used alone as a single inducer or in combination with other a classical treatment. When several inhibitors are used, a mixture of inhibitors is obtained.

As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat melanoma. In the context of the invention, the classical treatment refers to radiation therapy, immunotherapy or chemotherapy.

In a particular embodiment, the invention relates i) a ferroptosis inducer and ii) a chemotherapy used as a combined preparation for use in the treatment melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, i) a ferroptosis inducer and ii) chemotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use in the use in the treatment of melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In a particular embodiment, the invention relates i) a ferroptosis inducer and ii) a radiotherapy used as a combined preparation for use in the treatment of melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, i) a ferroptosis inducer and ii) radiotherapy as a combined preparation according to the invention for simultaneous, separate or sequential use in the use in the treatment of melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

As used herein, the term “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

In a particular embodiment, the invention relates i) a ferroptosis inducer and ii) an immune checkpoint inhibitor used as a combined preparation for the treatment melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, the ferroptosis inducer and an immune checkpoint inhibitor as a combined preparation according to the invention, wherein the immune checkpoint inhibitor is selected from the group consisting of but not limited to: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK). Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS). In particular embodiment, immune checkpoint inhibitor is described above.

In a particular embodiment, i) a ferroptosis inducer and ii) a BRAF inhibitor as a combined preparation for use in the treatment melanoma or an aggressive/invasive melanoma, metastatic melanoma or resistant melanoma.

In a particular embodiment, i) a ferroptosis inducer and ii) a BRAF inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

In a particular embodiment, i) a ferroptosis inducer and ii) a BRAF inhibitor as a combined preparation according to the invention, wherein the BRAF inhibitor is selected from the group consisting of but not limited to: Vemurafenib, Encorafenib, or Dabrafenib. In a particular embodiment, i) a ferroptosis inducer and ii) a MEK inhibitor as a combined preparation for use in the treatment of melanoma, an aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

In a particular embodiment, i) a ferroptosis inducer and ii) a MEK inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

In a particular embodiment, i) a ferroptosis inducer and ii) a MEK inhibitor as a combined preparation according to the invention, wherein the MEK inhibitor is selected from the group consisting of but not limited to: Trametinib, Cobimetinib or Binimetinib.

In a particular embodiment, i) a ferroptosis inducer, ii) a BRAF inhibitor and iii) a MEK inhibitor as a combined preparation according to the invention for simultaneous, separate or sequential use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

In a particular embodiment, i) a ferroptosis inducer, ii) a BRAF inhibitor and iii) a MEK inhibitor as a combined preparation according to the invention, wherein the BRAF inhibitor is selected from the group consisting of but not limited to: Vemurafenib, Encorafenib, or Dabrafenib and wherein the MEK inhibitor is selected from the group consisting of but not limited to: Trametinib, Cobimetinib or Binimetinib.

Pharmaceutical composition

The ferroptosis inducer for use according to the invention alone and/or combined with classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a fourth aspect, the invention relates to a pharmaceutical composition comprising a ferroptosis inducer for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant. In a particular embodiment, the pharmaceutical composition according the invention comprising i) a ferroptosis inducer and ii) a classical treatment.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) a ferroptosis inducer and ii) a BRAF inhibitor.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) a ferroptosis inducer and ii) a MEK inhibitor.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) a ferroptosis inducer, ii) a BRAF inhibitor and iii) a MEK inhibitor.

The ferroptosis inducer and the combined preparation as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum - drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Method of screening

In another aspect, the present invention relates to a method of screening a drug that regulates the amount of PPIX. In some embodiment, the present invention relates to a method of screening PPIX synthesis.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Deferoxamine assay (above histograms) and Ferroptosis assay (heatmap) on melanoma cell lines. Histogram shows deferoxamine index (DFX index) of each cell line. Deferoxamine index is calculated according to the formula described in Material and Methods section. Dotted line shows the DFX index positivity limit fixed at 0.5. Graph is the mean of n=6 independent experiments. Heatmap shows melanoma cell sensitivity to the indicated ferroptosis inducer compounds. Cells were treated with Erastin (5pM), RSL3 (50nM), FIN56 (250nM) or FAC (5mM) for 48h and stained with propidium iodide for cell viability analysis by flow cytometry. Grey intensity heatmap shows the percentage of live cells. Cell viability is normalized to percentage of live cells treated with DMSO. Heatmap results are the mean of n=6 independent experiments

Figure 2. Pharmacologic inhibition of protoporphyrin IX production sensitizes differentiated melanoma cells to ferroptosis induced by iron overload. (A) UACC62P cells were treated with DMSO, DFX, Heptanoic acid (HA) or DFX plus HA and iron-free PPIX levels were measured by DFX assay as described above. Representative flow cytometry histograms show PPIX fluorescence emission. (B) UACC62P cells were treated with DMSO, HA (750pM), FAC (5mM) or HA plus FAC for 12h. Lipid peroxidation was assessed following cell staining with C11-BODIPY581/591 probe and flow cytometry analysis. Bar graph shows normalized fluorescence emission induced by oxidized Cl l-BODIPY. (C) UACC62P, Mel501 and SKMEL19 cells were treated with DMSO, HA (750pM), FAC (5mM) or HA plus FAC for 12h and stained with propidium iodide for cell viability analysis by flow cytometry. Cell viability is normalized to percentage of live cells treated with DMSO. Results are the mean of n=3 (UACC62P, SKMEL19) or n=6 (Mel501) independent experiments.

Figure 3. In vivo tumor relapse after targeted therapy is associated with a reduction of protoporphyrin IX expression, increased sensitivity to ferroptosis and melanoma cell dedifferentiation. (A) Diagram of the experimental syngeneic model of melanoma tumour relapse following BRAF/MEK inhibitors treatment. (B) Melanoma cells were isolated from control (n=12) and relapsed tumors (n=15) and subjected to the DFX assay. Scatter plot shows DFX index of each cell culture sample. The assay was performed in duplicate for each cell culture sample. (C) Isolated cells were tested for sensitivity to ferroptosis using Erastin (5pM), RSL3 (50nM), FIN56 (250nM) or FAC (5mM). Cells were treated for 48h, stained with propidium iodide and analyzed by flow cytometry. Cell viability was normalized to the percentage of live cells treated with DMSO. (D) Immunoblot analysis of protein extracts from control (n=2) and relapsed (n=2) ex vivo tumor cell cultures using antibodies against MITF and HSP90 as loading control.

Figure 4 : Deferoxamine and Ferroptosis Assays on NRAS-mutant Melanoma Cell Lines. (A) The bar graph displays the Deferoxamine Index (DFX index) for each cell line, calculated using the formula described in the Materials and Methods section. The dashed line marks the DFX index positivity threshold that is set at 0.5. (B) The heatmap depicts melanoma cell sensitivity to the indicated ferroptosis-inducing compounds. Cells were treated with Erastin (5 pM), RSL3 (50 nM), FIN56 (250 nM), or FAC (5 mM) for 48 hours, followed by propidium iodide staining to assess cell viability via flow cytometry. The heatmap’s grayscale intensity represents the percentage of live cells, with cell viability normalized to the percentage of live cells treated with DMSO. Data represent the mean of three independent experiments (n=3). The BRAF -mutant cell lines UACC62P and UACC62R were included as a control.

EXAMPLE:

Material & Methods

Cells, culture, antibodies and reagents

Isogenic pairs of vemurafenib-sensitive (P) and vemurafenib-resistant (R) cells (M229) were generated by R.S. Lo (9) and described before (10). The isogenic pair of vemurafenib-sensitive (UACC62P) and vemurafenib-resistant (UACC62R) cells was provided by R. Neubig (11). Short-term cultures of patient melanoma cells MM074 and MM099 were generated in the laboratory of Pr G. Ghanem. Other cell lines were previously described (12,13). YUMM1.7 mouse melanoma cells were obtained from M. Bosenberg (14) and cultured in Opti-MEMl medium supplemented with 3% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin solution. Human melanoma cell lines were cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 7% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin solution. Cells resistant to vemurafenib were continuously maintained in culture with IpM of vemurafenib. Culture reagents were purchased from ThermoFisher Scientific. Vemurafenib (PLX4032, BRAFi), and trametinib (GSK1120212, MEKi) were purchased from Tocris Bioscience.

In vivo experiments

Animal housing was carried out in in our accredited animal housing facility in the “Centre Mediterraneen de Medecine Moleculaire”. Experiments were performed in accordance with the Institutional Animal Care and the local ethical committee and within the context of approved project applications (CIEPAL-Azur agreement NCE/2018-509). The syngeneic model of melanoma response and relapse to BRAF/MEK inhibitors was described before (15). Briefly, 5x 105 YUMM1.7 cells were subcutaneously (s.c.) inoculated in one flank of C57BL/6 mice. Tumors were measured with caliper and treatments were started when tumors reached a volume equivalent to 100 mm3, after randomization of mice into control and test groups. A combination of Vemurafenib (30 mg/kg p.o) and Trametinib (0.3 mg/kg p.o) was administered every 3 days in vehicle (90% corn oil, 10% DMSO). Control mice were treated with vehicle only. Mice were followed for up to 50 days and sacrificed when tumors reached a volume of >500mm3. Tumors were dissociated using the GentleMACS Octo Dissociator.and the tumor dissociation kit (Miltenyi Biotec ref 130-096-730). Isolated tumor cells were then grown in culture for 24h in OptiMEMl supplemented with 3% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin solution before performing deferoxamine and ferroptosis sensitivity assays.

Deferoxamine Assay

Cells were treated overnight with 200pM of Deferoxamine mesylate salt (DFX) (Sigma Aldrich ref D9533). The day of the experiment, cells were detached with 0.05 of Trypsin-EDTA and run on an Aurora (Cytek) spectral flow cytometer equipped with 4 lasers (488nm, 405nm, 633nm and 355nm). As a positive control for PPIX expression, cells were treated with 5- aminolevulinic acid (5 ALA) and as a negative control, cells were treated with DMSO. PPIX fluorescence spectrum was collected in channels from 630nm to 700nm under the 405nm excitation wavelength. For each condition, at least 5,000 cells that fell within the morphology gate were acquired. Deferoxamine index was calculated according to the following formula: (MFI DFX treated cells-MFI DMSO treated cells)/(rSD DFX treated cells + rSD DMSO treated cells). For the pharmacologic inhibition of protoporphyrin IX production, 4,6-Dioxoheptanoic acid (HA) (Sigma Aldrich ref D1415) was used at 750pM for 24h before deferoxamine and ferroptosis assays.

Ferroptosis sensitivity assay

Cells were treated separately with 4 different ferroptosis inducers: Erastin, RSL3, FIN56 and ferric ammonium citrate (FAC). All compounds were purchased from Sigma Aldrich. Erastin (ref E7781) was used at 5pM. RSL3 (ref SML-2234) was used at 50nM. FIN56 (ref SML- 1740) was used at 250nM and FAC (ref RES20400-A7) at 5mM. After 48h of treatments, supernatant and cells were recovery and stained with propidium iodide (Ipg/ml). Cells were then analyzed by using a BD FACScanto II flow cytometer equipped with 3 lasers (405nm, 488nm, 633nm). Percentage of live cells in ferroptosis-treated samples was normalized to the percentage of live cells in DMSO-treated samples.

Flow cytometry immunostaining Human melanoma cells were stained with the following antibody panel: CD44 BV421 (Biolegend, clone IM7, ref 103039), CD271 APC (Miltenyi clone REA648, ref 130-116-658) and PDL-1 PECY7 (Biolegend clone MH3, ref 374505). 100x105 cells were incubated with antibodies for Ih at 4°C. Cells were then washed twice with PBS-EDTA buffer supplemented with 0.5% BSA. Samples were analyzed on an Aurora spectral flow cytometer (Cytek) equipped with 4 lasers (355 nm, 405 nm, 488 nm and 633 nm). For lipid peroxidation detection, cells were stained with IpM Cl l-BODIPY 581/591 (ThermoFisher Scientific ref D3861) for 30 min at 37°C. Fluorescence emission from nonoxidized (581/591 nm) and oxidized (488/510 nm) forms of the probe was analyzed on a BD FACSCantoII cytometer.

Immunoblot

Cells were lysate in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Pierce, Fisher Scientific). Whole cell lysates were separated using SDS-PAGE and were transferred onto PVDF membranes (GE Healthcare Life Sciences) for immunoblot detection. Membranes were incubated with primary antibodies for 2h, then extensively washed and incubated with the peroxi dase-conjugated secondary antibody for Ih. Chemiluminescence system (GE Healthcare Life Science) was used to develop membranes. The following primary antibodies were used: HSP90 from Santa-Cruz (sc-13119), AXL from Cell Signaling (ref 4566), MITF from Sigma (HPA003259). Anti-rabbit IgG, HRP -linked antibody from Cell Signaling (ref 7074) was used as secondary antibody.

Statistical analysis

All data are presented as the mean ± SD of at least three independent experiments. P-values and linear correlation were determined by using the Prism V9.0.0 software (GraphPad, La Jolla, CA, USA). Data sets were tested for normality and T tests were performed to determine statistical significance. P-values of 0.01 (**), 0.001 (***) and 0.0001 (****) were considered statistically significant.

Results

Dedifferentiated melanoma cells with intrinsic or acquired resistance to inhibition of the BRAF oncogenic pathway expressed low levels of protoporphyrin IX To assess the level of heme biosynthesis in melanoma cells, we have developed an assay using deferoxamine (DFX). The principle of the assay is to treat cells with an iron chelator such as DFX (Data not shown). The use of a large excess (200uM) of DFX diverts the free iron contained in the cytoplasm from its endogenous storage proteins such as ferritin and protoporphyrin IX (PPIX). PPIX is the last intermediary in heme synthesis before iron incorporation by ferrochelatase. It fluoresces at red wavelengths (630nm to 700nm) when excited at 405nm. This fluorescence disappears after iron incorporation into PPIX, transforming it into heme. Deferoxamine-induced iron diversion allows PPIX to keep its fluorescent properties. Deferoxamine-treated cells producing PPIX can therefore be detected by spectral flow cytometry. The intensity of fluorescence emitted is then proportional to the quantity of PPIX expressed in cells and therefore indirectly to the level of heme synthesis. DFX assay results are expressed as the DFX index, which represents the mean fluorescence intensity of cells treated with DFX deducted from the mean fluorescence intensity of control cells treated with DMSO. For each tested melanoma cell line, an average deferoxamine index (n>6) is determined and is representative of the degree of PPIX expressed in these cells (Figure 1). Cells with a deferoxamine index greater than 0.5 were mainly differentiated MITF^hlgh/AXL^low melanoma cell lines described for their sensitivity to BRAF inhibitors (9,10,16). Conversely, cell lines with a DFX index inferior to 0.5 are dedifferentiated MITF^low/AXL^hlgh cells with intrinsic or acquired resistance to BRAF inhibitors. In contrast to the differentiated therapysensitive cell lines, the dedifferentiated resistant cells express high levels of the sternness marker CD44, the neural crest marker CD271 (NGFR) and PDL1 as shown by flow cytometry (Data not shown)

Melanoma cells with low protoporphyrin IX levels are highly sensitive to ferroptosis

Melanoma cell lines tested with DFX were also tested for their sensitivity to ferroptosis. Four ferroptosis inducers were used: Erastin, RSL3, FIN56 and ferric ammonium citrate (FAC). After 48 h of incubation with these different compounds, cells were stained with propidium iodide to assess the percentage of living cells. It shows that dedifferentiated cells with intrinsic or acquired resistance to BRAF inhibitors (MM099, UACC62R, M238R and 1205Lu cells) are more sensitive to cell death induced by ferroptosis inducers than other cell lines. Sensitivity to ferroptosis is drastically increased by GPX4 inhibitors (RSL3 and FIN56) and by iron overload induced by FAC. In contrast, the effect of Erastin on cell death appears to be more moderate for all these cell lines except for 1205Lu cells. To confirm the efficiency of these compounds to induce ferroptosis, a fluorescent probe sensitive to lipid peroxidation (a hallmark of ferroptosis) was used: the C11-B0DIPY. When this probe is oxidized, it generates a green fluorescence that can be detected by flow cytometry. While the highest level of lipid peroxidation was induced by FAC, Erastin was not very effective and GPX4 inhibitors showed an intermediate effect. Finally, the correlation between the DFX index (determined by the average level of PPIX) and the ferroptosis resistance index (determined by the average viability after treatment with ferroptosis inducers) was estimated using a Pearson test (Data not shown) The Pearson correlation coefficient that was obtained (R=0.8434 with significance of the order of p>0.0005) demonstrates a positive correlation between the two variables. To examine the role of PPIX in cell resistance to ferroptosis inducers, we used a known inhibitor of heme synthesis: 4,6-Dioxoheptanoic acid (HA). UACC62P differentiated melanoma cells were pretreated for 24 h with HA before incubation with FAC for 48 h to induce iron overload. PPIX levels were then assessed following DFX treatment. Figure 2A shows that inhibition of heme synthesis strongly decreased the levels of PPIX that are detected using the deferoxamine assay. Cell staining with C11-B0DIPY confirms that heme depletion by HA synergizes with FAC- induced iron overload to induce massive lipid peroxidation (Figure 2B). As a consequence, the viability of differentiated melanoma cells (UACC62P, Mel501, SKMEL19) that were treated with the combination of HA and FAC is dramatically reduced compared to control and single agent treatments (Figure 2C). All together these results suggest that inhibition of PPIX biosynthesis sensitizes cells to ferroptosis induced by iron overload.

Protoporphyrin IX levels decrease in melanoma tumors escaping inhibition of the BRAF oncogenic pathway in vivo

To evaluate the modulation of PPIX expression and ferroptosis sensitivity in vivo, we established a syngeneic murine model of melanoma tumor relapse to BRAF/MEK inhibitors as described before (15)(Figure 3A). The murine melanoma cells YUMM1.7 were injected into C56BL/6 mice until tumors reach to size of 100 mm3. Mice were treated or not with a combination of BRAFI and MEKi until tumor relapse. Mice from the control group and from the tumor relapse group were sacrificed when tumors reached the size of 500 mm3 or more. Melanoma cells were isolated from control and relapsed tumors and ex vivo tumor cell cultures were subjected to DFX and ferroptosis sensitivity assays. Results from the DFX assay (Figure 3B) show that cells from control tumors had a significantly (p<0.0001) higher mean DFX index (0.98 +/- 0.27) than cells from relapsed tumors (0.55 +/- 0.16), indicating that in vivo escape from targeted therapies is associated with decreased PPIX expression. In parallel, tumor cells were tested for their sensitivity to ferroptosis inducers Erastin, RSL3, FIN56 and FAC) (Figure 3C). Cells extracted from relapsed tumors showed a higher sensitivity to Erastin and FIN56 compared to cells extracted from the control group. Finally, immunoblot analysis performed on two control and two relapsed tumors show that decreased PPIX level was associated with a decreased expression of the melanoma differentiation marker MITF (Figure 3D).

NRAS- mutant melanoma cells expressed low levels of protoporphyrin IX and are highly sensitive to ferroptosis inducers

To evaluate heme biosynthesis levels in NRAS-mutant melanoma cells, we performed the deferoxamine assay as previously described. These cell lines were also assessed for their sensitivity to ferroptosis using four ferroptosis inducers: Erastin, RSL3, FIN56, and Ferric Ammonium Citrate (FAC). The results of these experiments show that all NRAS-mutant cell lines (WM2032, HMVII, SBCL2, WM1361, and C8161) express low levels of protoporphyrin IX (PPIX) (Figure 4A) and exhibit sensitivity to ferroptosis inducers (Figure 4B). BRAF- mutant cell lines were included as positive (UACC62P) and negative (UACC62R) controls for PPIX expression and ferroptosis sensitivity.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS;

1. A method of determining whether a subject has or is at risk of having melanoma ferroptosis sensitivity and targeted therapy resistance comprising i) determining the level of protoporphyrin IX (PPIX) in a biological sample obtained from the subject and ii) comparing the level determined at step i) with a predetermined reference value:

2. The method according to claim 1 wherein a high level of PPIX is associated with differentiated cells.

3. The method according to claim 1 wherein a low level of PPIX is associated with a dedifferentiated cells.

4. The method according to claim 3 wherein the dedifferentiated cells are resistant to targeted therapies chosen from BRAF inhibitors or MEK inhibitors.

5. The method according to claims 1 to 4 wherein the biological sample is a tissue sample.

6. The method according to claim 5 wherein the tissue sample is a normal, primary or melanoma tissue.

7. The method according to 5 wherein the tissue sample has a BRAF mutation or a MEK mutation or a NRAS mutation.

8. The method of claims 1 to 6 wherein the level of PPIX is determined by spectral flow cytometry assay.

9. A method for treating resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a ferroptosis inducer.

10. The method according to claim 8 wherein the ferroptosis inducer is a ferric ammonium citrate (FAC) or Erastin or RSL3 or FIN56. i) A ferroptosis inducer and ii) a BRAF inhibitor as a combined preparation for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

11. i) A ferroptosis inducer and ii) a MEK inhibitor as a combined preparation for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

12. i) A ferroptosis inducer, ii) a BRAF inhibitor and iii) a MEK inhibitor as a combined preparation for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

13. A pharmaceutical composition comprising a ferroptosis inducer for use in the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma or melanoma resistant.

14. The pharmaceutical composition according to claim 14 comprising a BRAF inhibitor and a MEK inhibitor.