WO2020193740A1 - New strategy for treating pancreatic cancer - Google Patents

Abstract

The present invention relates to the treatment of pancreatic cancer. In this work, the inventors assess whether mitochondrial features heterogeneity in between PDAC patients could be used to refine their stratification and develop novel targeted cancer therapies. Through a functional analysis (Seahorse), they demonstrate that human pancreatic cancer cells have fully functional mitochondrial respiration (OXPHOS). Furthermore, they show that OXPHOS in PDAC cells is able to generate most of their ATP, thus refuting the well-established belief that ATP production by glycolysis predominates over OXPHOS in PDAC. Importantly, their study provides the demonstration that PDAC tumors can be stratified according to their energetic metabolism. Moreover, they show a correlation between OXPHOS rate and abundance of mitochondrial respiratory Complex I both at mRNA and protein level. Finally, they show a synergy of Phenformin treatment with standard chemotherapy (Gemcitabine) specifically for high OXPHOS patient cells. Thus the invention relates to an inhibitor of the mitochondrial respiration for use in the treatment of pancreatic cancer in a patient in need thereof. Particularly the patient has a high OXPHOS.

Description

NEW STRATEGY FOR TREATING PANCREATIC CANCER

FIELD OF THE INVENTION:

The present invention relates to an inhibitor of the mitochondrial respiration for use in the treatment of pancreatic cancer in a patient in need thereof.

BACKGROUND OF THE INVENTION:

Metabolic reprogramming is a feature of cancer cells which recently gained considerable interest as a promising targetable pathway to improve therapy of currently untreatable cancers (1-5). Among refractory cancers, Pancreatic Ductal AdenoCarcinoma (PD AC) still represents important challenges for oncologists. PD AC is the most common form of pancreatic cancer and one of the most deadly human cancers with a survival median of only six months from diagnosis and a 5-year relative survival rate of less than 4%. PD AC is the fourth leading cause of death by cancer in the Western world and projected to become the second by 2030 in the USA (6). Because it is asymptomatic, PDAC is often diagnosed late and at an advanced stage, when patients are no longer eligible for surgical resection, which is possible in less than 15% of the cases and increases the 5-year survival rate to only 20%. Chemotherapy (Gemcitabine, FOLFIRINOX, Nab-Paclitaxel) and radiation only allow a marginal increase in survival (7). New strategies are urgently needed to develop effective treatment options. Recent progress has been made in our understanding of the physiopathology of the PDAC, such as the characterization of its large genetic heterogeneity and the presence of an important stroma which can support cancer cell growth and impede accessibility to drugs (7, 8). Targeted therapies have not yet had a real impact on this disease, in particular targeting oncogenic Kras (mutated in most of PDAC) proved to be very disappointing. Recent efforts have demonstrated an extensive reprogramming of metabolism in pancreatic cancer, as is the case in all cancers (9).

Alterations of cancer cell metabolism both derive from and drive carcinogenesis, and metabolic reprogramming is an absolute requirement for cancer cell survival and proliferation within their hostile environment exhibiting hypoxia and nutrient deprivation. Active metabolism is essential for: (i) the generation of energy to fuel biochemical reactions; (ii) the generation of biochemical building blocks required for cell growth and division; and (iii) the maintenance of biochemical homeostasis, in particular redox potential. A better knowledge of the key aspects of tumor metabolism in different types of cancers will allow the identification of vulnerabilities, opening new therapeutic opportunities to improve the clinical management of cancers.

Metabolic changes in mitochondria in primary cancer cells are still incompletely defined. Mitochondria are organelles playing a crucial role in cell physiology. They are involved in the production of energy in the form of ATP by the respiratory chain in the presence of oxygen (therefore the synonym “oxidative phosphorylation” or OXPHOS). They can produce high levels of Reactive Oxygen Species (ROS), the superoxide anion being a by product of respiration generated mostly at Complexes I and III of the electron transfer chain (ETC) due to the incomplete reduction of molecular oxygen (10, 11). Mitochondria are also involved in the metabolism of carbohydrates, amino acids, and lipids (Fatty Acid Oxidation (FAO), also called beta oxidation), and in apoptosis. Their role in cancer cells has been neglected for many years, because the Warburg effect (aerobic glycolysis) suggested that high glycolysis rate in cancer cells even in the presence of oxygen is compulsory for production of ATP because of impaired OXPHOS-driven ATP production and thus defective mitochondria. However, this concept has recently been challenged. Indeed, recent studies showed that mitochondria are still functional in many subsets of cancer cells and necessary for cell proliferation (12-14), and more importantly that this function is targetable in clinical applications (diagnostic, prognostic, and therapy) (14). Recent reviews recapitulate the state- of-the-art in the numerous mitochondrial alterations in cancer cells and their primary role in carcinogenesis (15-20). Despite these efforts, more work remains to be done to decipher alterations of mitochondrial metabolism in cancer. This knowledge should pave the way towards the development of innovative clinical strategies for better treatment of cancers.

Recently, PDAC tumoral metabolism was characterized using metabolomics in combination with other“omics”, and described as taking up increased amounts of glucose to fuel biosynthetic processes (21), displaying elevated glutaminolysis to maintain redox balance (22), and scavenging fatty acids as well as amino acids from extracellular space to synthesize macromolecules such as lipids and proteins (23). This suggests that mitochondria remain functional in PDAC despite metabolic reprogramming due to biochemical pathway alterations such as mitochondrial tricarboxylic acid (TCA) cycle (also called Krebs cycle) enzymes disruption. Recent publications also reported the pro-survival role of mitochondria in pancreatic cancer stem cells or dormant cells, supporting the importance of mitochondria in PDAC (24, 25). Notwithstanding, the role of mitochondria in proliferating tumoral pancreatic cells as well as the variability of metabolic adaptations between patients has not yet been investigated. SUMMARY OF THE INVENTION:

In this work, the inventors assess whether mitochondrial features heterogeneity in between PD AC patients could be used to refine their stratification and develop novel targeted cancer therapies. Through a functional analysis (Seahorse device monitoring mitochondrial respiration and glycolysis), they demonstrate that human pancreatic cancer cells, both PD AC cell lines and primary cells from patients, have fully functional mitochondrial respiration (OXPHOS). Furthermore, they show that OXPHOS in PD AC cells is able to generate most of their ATP, thus refuting the well-established belief that ATP production by glycolysis predominates over OXPHOS in PDAC. Importantly, their study provides the demonstration that PDAC tumors can be stratified according to their energetic metabolism (OXPHOS and glycolysis rates). Moreover, they show a correlation between OXPHOS rate and abundance of mitochondrial respiratory Complex I both at mRNA and protein level. This demonstration of a correlation between functional and transcriptional OXPHOS status in PDAC opens the possibility to stratify patients in clinics according to mitochondrial respiration gene expression. Finally, they show a synergy of Phenformin (targeting mitochondrial Complex I) treatment with standard chemotherapy (Gemcitabine) specifically for high OXPHOS patient cells, suggesting that Phenformin should be clinically tested as an anticancer agent in pancreatic cancer with appropriate patient selection.

Thus, the present invention relates an inhibitor of the mitochondrial respiration for use in the treatment of pancreatic cancer in a patient in need thereof. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

Predictive method

The inventors showed a correlation between OXPHOS rate and abundance of genes of mitochondrial respiratory Complexes both at mRNA and protein level. This demonstration of a correlation between functional and transcriptional OXPHOS status in PDAC opens the possibility to stratify patients in clinics according to mitochondrial respiration genes expression. Thus, in a particular embodiment, the method of the invention can be used to stratify patients suffering from a pancreatic cancer and select patients with a high expression of genes encoding the mitochondrial respiratory chain proteins. Particularly, these genes are genes of the mitochondrial respiratory Complexes I, II, III, IV or V or a combination of these genes. These patients will then be treated specifically with a combination of therapeutic compound already used to treat pancreatic cancer, like gemcitabine and/or with an inhibitor of the mitochondrial respiration and particularly an inhibitor of the Complexes I, II, III, IV or V of the oxidative phosphorylation chain in the mitochondria or an inhibitor of the genes of the Complexes I to V.

Thus, a first object of the invention relates to an in vitro method for predicting the survival time of a patient suffering from a pancreatic cancer comprising: i) determining, in a sample obtained from the patient, the level of OXPHOS; ii) comparing the level of OXPHOS determined at step i) with a predetermined reference value and iii) providing a bad prognosis when the level of OXPHOS determined at step i) is higher than its predetermined reference values, or providing a good prognosis when the level of OXPHOS determined at step i) is lower than its predetermined reference value.

As used herein, the term“OXPHOS” denotes the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy which is used to produce adenosine triphosphate (ATP). In most eukaryotes, this takes place inside mitochondria.

According to the invention, a patient with a high level of OXPHOS will have a bad prognosis.

Determining a high or low OXPHOS status is well known in the art. For example and according to the invention, the measurement of the“level of OXPHOS” (or“OXPHOS status”) can be done by measurement of the cellular oxygen consumption rate (OCR). This kind of measurement can be easily done directly on a sample, for example pancreatic cancerous cells obtained from a patient, thanks to a device like Seahorse (see for example: https://www.agilent.com/en/products/cell-analysis/seahorse-analyzers). Then, determining a high or a low“level of OXPHOS” will be easily obtained by comparing the obtained level to threshold (reference value) obtained with control (like with patients with no pancreatic cancer for example).

The inventors also determined a correlation between the expression levels of the genes of the different Complex of the mitochondria with the OXPHOS status. A high expression of these genes is correlated with a high OXPHOS.

Thus, the expression level of at least one gene expressed in the mitochondrial respiratory Complexes (Complexes I to V) of the mitochondria can be measured to determine the OXPHOS status.

The genes for the Complex I are selected in the group consisting in ND1, ND2, ND3, ND4, ND4L, ND5, ND6, NDUFB2, NDUFAF4, NDUFAF6, NDUFAF2, NDUFA12, NDAUFAF3, NDUFAF5, NDUFA5, NDUFB6, NDUFS4, NDUFA 13, NDUFV2, NUBPL, NDUFB4, NDUFS7, NDUFB8, NDUFAF7, NDUFB3, NDUFS5, NDUFA 1 1 , NDUFC 1 , NDUFB 10, NDUFS8, NDUFA10, NDUFB9, NDUFA7, NDUFA 1 , NDUFB5, NDUFB7, NDUFA2, NDUFB 1 , TMEM 126B, NDUFB 1 1 , NDUFS6, NDUFV3, NDUFS3, NDUFS2, NDUFA8, NDUFV 1 , NDUFAB 1 , PARK7 / DJ I , ND6, NDUFA6, NDUFS 1 , NDUFA9, TIMMDCl .

The genes for the Complex II are selected in the group consisting in SDHAF1, SDHB, SDHAF2, SDHAF3, SDHD, SDHC, SDHA.

The genes for the Complex III are selected in the group consisting in cytochrome b, LYRM7, UQCRB, UQCRQ, BCS1L, UQCR11, TTC19, UQCRFS1, UQCRC1, UQCR10, CYC1, UQCRC2, AARS2, UQCC3 , UQCC2, UQCC1, TTC19, BCS1L, MAIP1, IMMP1L, SAMM50, IMMP2L, SLC25A33.

The genes for the Complex IV are selected in the group consisting in COX1, COX2, COX3, COX4, COX5A, COX5B, COX6A, COX6B, COX6C, COX7A, COX7B, COX7C, COX8, PET 100, TIMM21, SELRC1, NDUFA4, COX20, SURF1, CAV3, COX18, ANK3, SERPl, SCOl, COX15, MT-C03, A4GALT, SPTBN1, COX11, COX10, COA6.

The genes for the Complex V are selected in the group consisting in A6, A8, ATP5F1 A, ATP5A, ATP5F1B, ATP5F1C, ATP5F1D, ATP5F1E, ATP5MC1, ATP5MC2, ATP5MC3, ATP5MD, ATP5ME, ATP5MF, ATP5MG, ATP5MPL, MT-ATP6, MT-ATP8, ATP5PB, ATP5PD, ATP5PF, ATP5PO, ATP5IF1.

More particularly, the expression level of one gene by Complexes (thus a total of five genes) can be measured.

More particularly, the expression level of at least one gene selected in the group consisting in ATP5A, UQCRC2, SDHB, COX2 and NDUFB8 can be used to determine the level of OXPHOS.

More particularly, the expression level of the gene NDUFB8 can be used to evaluate the level of activity of the Complex I and thus the level of OXPHOS.

More particularly, the expression level of the gene SDHB can be used to evaluate the level of activity of the Complex II and thus the level of OXPHOS.

More particularly, the expression level of the gene UQCRC2 can be used to evaluate the level of activity of the Complex III and thus the level of OXPHOS.

More particularly, the expression level of the gene COX2 can be used to evaluate the level of activity of the Complex IV and thus the level of OXPHOS. More particularly, the expression level of the gene ATP5A can be used to evaluate the level of activity of the Complex V and thus the level of OXPHOS.

As used herein, the term“NDUFB8” for“NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial” denotes an enzyme that in humans is encoded by the NDUFB8 gene. NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8 is an accessory subunit of the NADH dehydrogenase (ubiquinone) Complex, located in the mitochondrial inner membrane. It is also known as Complex I and is the largest of the five Complexes of the electron transport chain. The Entrez reference number is 4714.

As used herein, the term“SDHB” for“Succinate dehydrogenase [ubiquinone] iron- sulfur subunit, mitochondrial” also known as iron-sulfur subunit of Complex II (Ip) denotes a protein that in humans is encoded by the SDHB gene. The succinate dehydrogenase (also called SDH or Complex II) protein Complex catalyzes the oxidation of succinate (succinate + ubiquinone => fumarate + ubiquinol). SDHB is one of four protein subunits forming succinate dehydrogenase, the other three being SDHA, SDHC and SDHD. The SDHB subunit is connected to the SDHA subunit on the hydrophilic, catalytic end of the SDH Complex. It is also connected to the SDHC/SDHD subunits on the hydrophobic end of the Complex anchored in the mitochondrial membrane. The Entrez reference number is 6390.

As used herein, the term“UQCRC2” for“Cytochrome b-cl Complex subunit 2, mitochondrial” also known as QCR2, UQCR2, or MC3DN5 denotes a protein that in humans is encoded by the UQCRC2 gene. The product of UQCRC2 is a subunit of the respiratory chain protein Ubiquinol Cytochrome c Reductase (UQCR, Complex III or Cytochrome bcl Complex), which consists of the products of one mitochondrially encoded gene, MTCYTB (mitochondrial cytochrome b) and ten nuclear genes: UQCRC1, UQCRC2, Cytochrome cl, UQCRFS1 (Rieske protein), UQCRB, " l lkDa protein", UQCRH (cyt cl Hinge protein), Rieske Protein presequence, "cyt. cl associated protein", and "Rieske-associated protein." Defects in UQCRC2 are associated with mitochondrial Complex III deficiency, nuclear, type 5. The Entrez reference number is 7385.

As used herein, the term“COX2” for“cytochrome c oxidase subunit 2” also known as cytochrome c oxidase polypeptide II denotes a protein that in humans is encoded by the MT- C02 gene. [4] Cytochrome c oxidase subunit II, abbreviated COX2, COXII, COII, or MT-C02, is the second subunit of cytochrome c oxidase (Complex IV). The Entrez reference number is 4513. As used herein, the term“ATP5A” for“synthase-coupling factor 6, mitochondrial” denotes an enzyme that in humans is encoded by the ATP5PF gene. It’s a subunit of mitochondrial ATP synthase (Complex V). The Entrez reference number is 522.

In a particular embodiment the OXPHOS level of a patient can be determined by determining by measurement of the cellular oxygen consumption rate (OCR) and/or of the expression level of at least one gene expressed in the mitochondrial respiratory Complexes (Complexes I to V) of the mitochondria and especially the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8. In a particular embodiment the level of OXPHOS is determined by both methods.

As used herein the term "sample" in the context of the present invention is a biological sample isolated from a patient and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a patient. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, bone marrow aspirate, urine, saliva or any other bodily secretion or derivative thereof. As used herein "blood" includes whole blood, plasma, serum, circulating cells, constituents, or any derivative of blood. In a particular embodiment, the sample is a blood sample, more particularly a biological sample comprising circulating white blood cells (WBC). In a particular embodiment the sample is a pancreatic cancer surgery piece or biopsy. These surgery pieces or biopsy obtained from patients can be used to make Patient-Derived Xenografts (PDXs). The later were maintained in cell culture to produce primary cells of PD AC patients.

Measuring the expression level of the genes listed above can be done by measuring the gene expression level of these genes or by measuring the level of the protein of the corresponding genes and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR). Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A“detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and / or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDaminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (puhlished May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes. Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670, 113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avi din-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl. Acad. Sci. 83 :2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are“specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and passing the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test patient, optionally first passed by a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of Complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized Complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et al., Hepatology 2014, 60: 1674-1685 or Ueno A et al., Journal of hepatology 2014, 61 : 1080-1087).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABLE This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

According to the invention, the level of the proteins of the genes listed above may also be measured and can be performed by a variety of techniques well known in the art. For measuring these proteins, techniques like ELISA (see below) allowing to measure the level of the soluble proteins are particularly suitable.

In the present application, the“level of protein” or the“protein level expression” or the “protein concentration” means the quantity or concentration of said protein. In another embodiment, the“level of protein” means the level of the proteins fragments. In still another embodiment, the “level of protein” means the quantitative measurement of the proteins expression relative to a negative control.

According to the invention, the protein level of the proteins may be measured at the surface of the tumor cells or in an extracellular context (for example in blood or plasma).

Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.

Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a Complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody Complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen Complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art. Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins. Thus, in a particular embodiment, fragment of the proteins may also be measured.

Predetermined reference values used for comparison may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) value for the genes’ expression may be predetermined by carrying out a method comprising the steps of

a) providing a collection of samples from patients suffering a pancreatic cancer (after diagnosis of the cancer for example);

b) determining the expression level of the genes or proteins for each sample contained in the collection provided at step a);

c) ranking the tumor tissue samples according to said gene/protein expression level and determining a threshold value above which the expression level is said to be“high” and below which the expression level is said to be“low”;

d) quantitatively defining the threshold/cut-off/reference value by determining the number of copies of the said gene/protein corresponding to the threshold/cut-off/reference value; to be done by constructing a calibration curve using known input quantities of cDNA or protein for the said gene/protein;

e) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

f) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient (i.e. the duration of the overall survival (OS));

g) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

h) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets

i) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest. For example the expression level of the genes/proteins has been assessed for 100 pancreatic cancer samples from 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the highest expression level and sample 100 has the lowest expression level. 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 pancreatic cancer patient, 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.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate pancreatic cancer samples and therefore the corresponding patients. Thank to this reference value, the level of expression will be compared and the level of OXPHOS will be determined.

Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the person skilled in the art.

The person skilled in the art also understands that the same technique of assessment of the expression level of a gene should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a gene of a patient according to the method of the invention.

Such predetermined reference values of expression level may be determined for any gene defined above.

The present invention also relates to kits for predicting the survival time of a patient suffering from pancreatic cancer comprising means for determining, in a biological sample from the patient the expression level of the gene of the invention.

Therapeutic method A second object of the invention relates to an inhibitor of the mitochondrial respiration for use in the treatment of pancreatic cancer in a patient in need thereof.

The invention also relates to a method for treating a pancreatic cancer in a patient in need thereof by administrating to said patient an inhibitor of the mitochondrial respiration.

As used herein, the term“mitochondrial respiration” or“oxidative phosphorylation” or “OXPHOS” has its general meaning in the art and denotes, in eukaryotes, the electron transport chain that establishes a proton gradient (chemiosmotic potential) across the boundary of inner membrane by oxidizing NADH and FADH2 produced from the Krebs cycle. ATP is synthesized by the ATP synthase enzyme when the chemiosmotic gradient is used to drive the phosphorylation of ADP. The electrons are finally transferred to exogenous oxygen and, with the addition of two protons, water is formed. This process occurs in the mitochondrial cristae and uses four protein Complexes counted from I to IV. The resulting transmembrane proton gradient is used to make ATP via ATP synthase sometimes described as Complex V of the electron transport chain.

Thus, particularly, the invention relates to an inhibitor of the oxidative phosphorylation (OXPHOS) for use in the treatment of pancreatic cancer in a patient in need thereof.

In a particular embodiment, the invention relates to an inhibitor of the Complexes I, II, II, IV and/or V for use in the treatment of pancreatic cancer in a patient in need thereof.

More, the inventors show that an inhibitor of the mitochondrial respiration could be very suitable to sensitize pancreatic cancerous cells to therapeutic compounds already used to treat pancreatic cancer. These therapeutic compounds already used to treat pancreatic cancer are for example the gemcitabine, 5-fluorouracil (5-FU), Capecitabine, oxaliplatine, cisplatine, the irinotecan, FOLFIRINOX (a chemotherapy regimen made up of the following four drugs: FOL for folinic acid, F for fluorouracil (5-FU), IRIN for irinotecan and OX for oxaliplatin) or Nab- Paclitaxel.

Thus the invention also relates to an inhibitor of the mitochondrial respiration to sensitize pancreatic cancerous cells to therapeutic compounds used to treat pancreatic cancer.

In other words, the invention relates to an inhibitor of the mitochondrial respiration to sensitize pancreatic cancerous cells to therapeutic compounds used to treat pancreatic cancer.

In another particular embodiment, the invention relates to an i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer or use in the sensitization of pancreatic cancerous cells. Particularly, the compound is gemcitabine, the 5-fluorouracil (5-FU), the Capecitabine, the oxaliplatine, the cisplatine, the irinotecan, FOLFIRINOX (a chemotherapy regimen made up of the following four drugs: FOL for folinic acid, F for fluorouracil (5-FU), IRIN for irinotecan and OX for oxaliplatin) or Nab-Paclitaxel.

According to the invention, the pancreatic cancer can be a Pancreatic Ductal AdenoCarcinoma (PD AC).

As used herein, the term“Pancreatic Ductal AdenoCarcinoma” or“PDAC” has its general meaning in the art and refers to pancreatic ductal adenocarcinoma such as revised in the World Health Organisation Classification C25.

As used herein, the term“patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the patient according to the invention is a human. More particularly, the patient is a human suffering of a pancreatic cancer.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient 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 patient 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 patient during treatment of an illness, e.g., to keep the patient 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., disease manifestation, etc.]).

The term“inhibitor of the mitochondrial respiration” or“inhibitor of the oxidative phosphorylation (OXPHOS)” denotes molecules or compounds which can inhibit the activity of the mitochondria to produce ATP or a molecule or compound which destabilizes the mitochondria production of ATP. Particularly, the inhibitors of the invention can inhibit or destabilize the Complexes I, II, III, IV or V of the oxidative phosphorylation chain in the mitochondria.

Particularly, the invention relates to an inhibitor of the Complexes I, II, III, IV or V of the oxidative phosphorylation chain for use in the treatment of pancreatic cancer in a patient in need thereof. Particularly, the inhibitor is an inhibitor of the Complexes I or III.

More particularly, the inhibitor of the mitochondrial respiration can be an inhibitor or an antagonist of genes of the mitochondrial respiratory Complexes I, II, III, IV or V and particularly the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8 (see above).

The invention also relates to a method for treating a patient suffering from a pancreatic cancer comprising the step of:

1. determining if the patient as a good or a bad prognosis according to the invention and;

2. administrating to said patient a compound useful for the treatment of a pancreatic cancer as defined in the present invention when the prognosis of the patient is bad as determined by methods of the invention.

Particularly, the compound useful for the treatment of a pancreatic cancer can be the gemcitabine, 5-fluorouracil (5-FU), Capecitabine, oxaliplatine, cisplatine, the irinotecan, FOLFIRINOX (a chemotherapy regimen made up of the following four drugs: FOL for folinic acid, F for fluorouracil (5-FU), IRIN for irinotecan and OX for oxaliplatin) or Nab-Paclitaxel.

The compound can be also an inhibitor of the mitochondrial respiration as described above (like an inhibitor or an antagonist of genes of the mitochondrial respiratory Complexes I, II, III, IV or V and particularly the genes ATP 5 A, UQCRC2, SDHB, COX2 and NDUFB8).

In particular embodiment, a combination of compound useful for the treatment of a pancreatic cancer and an inhibitor of the mitochondrial respiration can be done to the patient with a bad prognosis or with a high OXPHOS. Thus, the invention also relates to an inhibitor of the mitochondrial respiration for use in the treatment of a pancreatic cancer in a patient in need thereof wherein said patient has a bad prognosis as determined in the method above.

In another particular embodiment, the invention relates to an i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer in a patient in need thereof wherein said patient has a bad prognosis as determined in the method above. In a particular embodiment, the patient has a high OXPHOS.

In one embodiment, the inhibitors according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

Compounds useful for inhibiting the mitochondrial respiration are well known in the art (see for example SE Weinberg, NS Chandel - Nature chemical biology, 2015).

As explained, inhibitors of the mitochondrial respiration can be inhibitors of the Complexes I, II, III, IV or V of the oxidative phosphorylation chain.

Thus, inhibitors which can be used to inhibit the Complex I of the oxidative phosphorylation chain can be but are not limited to biguanide compounds like metformin, phenformin or buformin or compounds like rotenone.

Thus, inhibitors which can be used to inhibit the Complex II of the oxidative phosphorylation chain can be but are not limited to pyrvinium pamoate, carboxin, thenoyltrifluoroacetone, atpenin A5, Malonate or oxaloacetate.

Thus, inhibitors which can be used to inhibit the Complex III of the oxidative phosphorylation chain can be but are not limited to antimycin A, Myxothiazol or stigmatellin.

Thus, inhibitors which can be used to inhibit the Complex IV of the oxidative phosphorylation chain can be but are not limited to potassium cyanide or azide.

Thus, inhibitors which can be used to inhibit the Complex V of the oxidative phosphorylation chain can be but are not limited to oligomycin A, venturicidin or DCC (rebastinib). In a particular embodiment, the inhibitor of the mitochondrial respiration can be the Tigecycline which inhibits mitochondrial protein synthesis.

In a particular embodiment, the inhibitor of the mitochondria respiration according to the invention can also be an inhibitor of the genes or protein expressed in the Complex (Complexes I to V) of the mitochondria. Particularly, the inhibitor can be an inhibitor of the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8 or of the corresponding proteins.

Thus, in one embodiment, the inhibitor according to the invention is an antibody. Antibodies directed against the proteins of the invention (like the proteins ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against the proteins of the invention (like the proteins ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et ah, 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies. Coumpounds useful in practicing the present invention also include antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the proteins of the invention (like the proteins ATP5A, UQCRC2, SDHB, COX2 and NDUFB8).

Humanized antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies are selected.

In another embodiment, the antibody according to the invention is a single domain antibody against the proteins of the invention (like the proteins ATP5A, UQCRC2, SDHB, COX2 and NDUFB8). The term“single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called“nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term“VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term“complementarity determining region” or“CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the“Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The“Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the compound according to the invention 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. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et ah, 1996).

Then, for this invention, neutralizing aptamers are selected.

In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is an antagonist of the proteins of the invention (like the proteins ATP5A, UQCRC2, SDHB, COX and NDUFB8) and is capable to prevent their functions.

In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell.

The term“cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group Complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half- life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery. In another embodiment, the inhibitor according to the invention is an inhibitor of the gene expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX and NDUFB8).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of the genes expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) for use in the present invention. Genes expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that the genes expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) are specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of the genes expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of the genes expression of the invention (like the genes ATP5A, UQCRC2, SDHB, COX2 and NDUFB8) can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencapsul ati on .

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In a particular embodiment, an endonuclease can be used to reduce or abolish the expression of the genes expression of the invention (like the genes ATP5 A, UQCRC2, SDHB, COX and NDUFB8).

Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al, 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e267T), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al, 2014 Cell Res. doi: 10.1038/cr.2014.1 T), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e267L), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.113.160713), monkeys (Niu et al, 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.1 L), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In order to test the functionality of a putative inhibitor of the mitochondrial respiration a test is necessary. For that purpose, to identify inhibitors of the mitochondrial respiration, we can follow mitochondrial respiration by measuring oxygen consumption rate with Seahorse after acute or chronic treatment of cells with the putative drug. An inhibition of the oxygen consumption will be the demonstration that the tested drug is an inhibitor of the mitochondrial respiration.

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising an inhibitor of the mitochondrial respiration according to the invention for use in the treatment of pancreatic cancer in a patient in need thereof.

In another particular embodiment, the invention relates to a therapeutic composition comprising an inhibitor of the mitochondrial respiration according to the invention for use in the treatment of a pancreatic cancer in a patient with a bad prognosis as determined in the method above.

In another particular embodiment, the invention relates to a therapeutic composition comprising an inhibitor of the mitochondrial respiration according to the invention for use in the treatment of pancreatic cancer in a patient with a bad prognosis.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers 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 form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration.

Preferably, 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 doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.

For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).

Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum Complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol s, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or non opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofmac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.

Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.

In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.

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. Targeting mitochondrial respiratory Complex I with Phenformin and drugs targeting respiratory Complex I in combination with Gemcitabine treatment sensitizes Panc-1 cells to cell death. (A and D), and different concentrations of Gemcitabine alone and Gemcitabine in combination with 0.5 mM Phenformin for 72h. Cell viability is indicated as a % of the control (vehicle treated). Data are means of triplicates ± SEM. (B-C) Dose response curves to treatment with Metformin and Rotenone, respectively. Live cells are indicated as a % of the control (vehicle treated). Data are means of triplicates ± SEM. Figure 2: Complex I is more abundant in high OXPHOS PDAC patients compared to low OXPHOS. (A) Mitochondrial Complexes protein levels (ATP5A, UQCR2, SDHB, COXII, NDUFB8) belonging to mitochondrial Complexes (V, III, II, IV, I), respectively quantified from WB by Image J. P value from Mann- Whitney test. A.U. = Arbitrary unit. (B) Mitochondrial NDUFB8 protein level belonging to mitochondrial Complex I quantified from WB by Image J. P value from Mann- Whitney test. A.U. = Arbitrary unit.

Figure 3. Targeting mitochondrial Complex I with Phenformin counters resistance to Gemcitabine in high OXPHOS patients. (A) Survival curves for two patient groups: high and low OXPHOS. (B) Representative dose-response curves for 6 primary PDAC cancer cells derived from PDX, 3 high OXPHOS (PDAC027T, PDAC074T, PDAC084T, depicted as 27, 74, and 84, respectively) and 3 low OXPHOS (PDAC022T, PDAC032T, PDAC085T, depicted as 22, 32, and 85, respectively). Cells were treated during 72 h with different concentrations of Gemcitabine alone (blue curves) and in combination with 0.5 mM Phenformin (red curves). Cell viability is indicated as the % of the control (vehicle treated). Data are means of triplicates ± SEM.

Figure 4. OXPHOS shift experiments demonstrating that Gemcitabine sensitivity is a feature of OXPHOS low cells. (A). Panc-1 cells from high OXPHOS to low OXPHOS by treatment with Tigecycline (Tig) enhances Gemcitabine (Gem) cytotoxicity efficacy. Cell viability was monitored after 72h of treatment with or without Gem (Left: 1 nM; Right: 4 nM) and Tig (50 mM). Live cells are indicated as a % of the control (vehicle treated). Data are means of triplicates ± SEM (*, ** and *** p<0.05, p<0.01 and p<0.001 respectively; symbol over the bar = versus non-treated control; ns = not significant). Data are representative of three independent experiments. (B) MIA PaCa-2 and BxPC-3 cells shift from low OXPHOS to high OXPHOS by culture in Galactose instead of Glucose induces Gem resistance. Cell viability was monitored after 72h of treatment with Gem (4 nM). Live cells are indicated as % of the control (vehicle treated). Data are means of triplicates ± SEM (***p<0.001 versus non-treated; ns = not significant). Data are representative of three independent experiments.

Figure 5. Targeting mitochondrial respiratory Complex I with Phenformin enhances Gemcitabine antitumoral activity in high OXPHOS tumors in two preclinical mouse models. Experimental schematic representation of othotopic xenografts. One (A. for MIA-PaCa-2) or two (B. for Panc-1) millions of cells were surgically implanted into the pancreas of 6-week-old female Swiss nude mice. The presence of a pancreatic tumor was confirmed by exploratory laparotomy at day 16 (A. MIA PaCa-2) or day 33 (B. Panc-1). After surgery recovery, treatments were started during 4 weeks. Weight of orthotopic xenografts was measured. Vehicle (Control) n = 6; Gemcitabine (Gem) alone n = 5 and 9 for A. MIA- PaCa-2 and B. Panc-1, respectively; Combo Gemcitabine + Phenformin (Gem + Phen) n = 8 and 11 for A. MIA-PaCa-2 and B. Panc-1, respectively; Phenformin (Phen) alone n = 5. The mean in each group is shown as a horizontal line p values from Mann Whitney test. *, ** and *** p<0.05, p<0.01, and p<0.001 respectively; ns = not significant. C. Experimental scheme of orthotopic syngeneic allografts; one million of KPC luc2 cells were intraperitoneally injected into 6-week-old female C57BL/6 mice. The presence of a pancreatic tumor was confirmed by bioluminescence (day 10), and the treatments were started at day 11 during 6 days. Weight of orthotopic allografts was measured. Vehicle (Control) n = 6; Gemcitabine (Gem) alone n = 5; Combo Gemcitabine + Phenformin (Gem + Phen) n = 6; Phenformin (Phen) alone n = 6. The mean in each group is shown as a horizontal line p values from Mann Whitney test. * and ** p<0.05 and p<0.01, respectively.

EXAMPLE:

Material & Methods

Cell culture

The classical PD AC cell lines (BxPC-3, Capan-1, Capan-2, MIA PaCa-2 and Panc-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BxPC-3, Capan-1 and Capan-2 cells were maintained in RPMI (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Lonza) and MIA PaCa-2 and Panc-1 were maintained in DMEM supplemented with 10% FBS. All cell lines were routinely maintained at 37°C with 5% C02 in a humidified atmosphere. The SOJ-6 cell line was kindly provided by Dr. Eric Mas (INSERM U1068, Marseille, France) and cultured in RPMI supplemented with 10% FBS in similar conditions as the other cell lines. The authentication was performed by the ATCC for all cell lines except SOJ-6. The primary cells from PD AC patients were from Patient- Derived Xenografts (PDX) of the PaCaOmics biobank, in which patients were included under the Paoli- Calmettes Institute clinical trial number 2011-A01439-32. Consent forms of informed patients were collected and registered in a central database. The tumor tissues used for xenograft generation were deemed excess to that required for the patient’s diagnosis. Primary cells derived from PDX were obtained and maintained in serum-free defined media (SFDM) medium as described (26). The murine PDAC cell line KPC luc2 (LSL-Kras^G12D/+; Trp53^R172H/+; Elas-CreER, transfected with a luciferase-encoding vector) was a gift from Dr. Nathalie Auphan-Anezin (CIML, Marseille, France). The KPC1 cell line was originally obtained by S.F. Konieczny (West Lafayette, USA) from a pancreatic tumor in a KPC mouse on C57BL/6 background. KPC luc2 cells were maintained in RPMI medium supplemented with 10% FBS and 600 pg/ml Hygromycine B (ThermoFisher Scientific) for selection of cells containing the luciferase-encoding vector. All lines were regularly controlled for mycoplasma contamination and found to be negative.

Chemograms

Cells were seeded in 96-well plates (5,000 cells per well). Twenty-four hours later, the medium was supplemented with increasing concentrations of selected drugs in triplicates: UK5099, BPTES, Phenformin, Menadione, Rotenone, Metformin (all provided by Sigma- Aldrich, Saint-Quentin Fallavier, France), and Gemcitabine. Drugs that have to be dissolved in DMSO (UK5099 and BPTES) or chloroform (Rotenone) were prepared as 200x stock solutions. Cell viability was determined 72h later by Crystal violet viability assay. Briefly, cells were fixed in 1% Glutaraldehyde, washed twice with PBS, stained with crystal violet 0.1% for 10 min, then washed three times with PBS. Crystals were solubilized in SDS 1%, and absorbance was measured at 600 nm with Epoch-Biotek spectrophotometer.

Combination index (Cl) values were calculated for all tested drug concentrations according to the Chou and Talalay method (54, 55) using the following equation: Cl = (D)i / (Dx)i + (D)₂ / (Dx)₂ where (D)i and (D)₂ represent the dose of agent 1 and 2 used in combination to induce X% growth inhibition, and (Dx)i and (Dx)₂ represent the dose of agent 1 and 2 required to reach X% growth inhibition when used alone. The Cl theorem then provides quantitative definition for additive effects (0.8<CI<1.2), synergism (CI<0.8) and antagonism (CI>1.2) in drug combinations. Calculations were done with Graphpad Prism software (GraphPad Software Inc., La Jolla, CA).

Real-time metabolic analysis

Measurements were performed using a Seahorse Bioscience XF24 Extracellular Flux Analyzer (Agilent). This device allows the measurement of the cellular oxygen consumption rate (OCR in pmoles/min) and of the extracellular acidification rate (ECAR in mpH/min). Sixteen hours before the assay, cells at exponential growth were seeded into Seahorse 24-well plates and cultured at 37°C with 5% C02. The number of seeded cells was optimized to ensure 70-80% confluence the day of analysis (data not shown).

XF Cell Mito Stress Test (OXPHOS experiment)

OCR was measured using the Seahorse XF Cell Mito Stress Test Kit. Culture medium was replaced with OXPHOS assay medium (DMEM without phenol red (Sigma-Aldrich reference D5030), 143 mM NaCl, 2 mM glutamine, 1 mM sodium pyruvate and 10 mM glucose, pH 7.4) and the plate was pre-incubated for lh at 37°C in a non-C02 incubator. OCR was measured under basal conditions, then after sequential injections of 1 mM oligomycin (inhibitor of respiratory Complex V allowing calculation of ATP production by mitochondrion), FCCP (uncoupling agent allowing determination of the maximal respiration and the spare capacity; the concentration was optimized for each cell line, data not shown), and finally 0.5 mM rotenone (Complex I inhibitor) + 0.5 mM antimycin A (Complex III inhibitor) to stop mitochondrial respiration enabling the calculation of the background (i.e. non- 'mitochondrial respiration driven by processes outside the mitochondria). Levels of OCR were normalized to 10,000 seeded cells, which we found as the most accurate way to normalize when comparing different cell types.

XF Glycolysis Stress Test (Glycolysis experiment)

ECAR was measured using the Seahorse XF Glycolysis Stress Test Kit. Culture medium was replaced with glycolysis assay medium (DMEM without phenol red, 143 mM NaCl, 2 mM glutamine, pH 7.4) and the plate was pre-incubated for lh at 37°C in a non-C02 incubator. ECAR was measured which is corresponding to the non-glycolytic acidification used for background. Then, 10 mM glucose was injected which allows the calculation of basal glycolysis. Glycolytic capacity was then calculated following the injection of 1 pM oligomycin. Glycolytic reserve was calculated as the difference between glycolytic capacity and basal glycolysis. At the end of the experiment, glycolysis was stopped by adding 2-Deoxyglucose (100 mM). Levels of ECAR were normalized to 10,000 seeded cells. The contribution of OXPHOS and glycolysis to ATP production was calculated using the OCR and proton production rate (PPR) as previously described (56). Glycolysis was also measured using the recently developed XF Glycolytic Rate Assay (no glucose starvation required for background determination) with the same outcomes, suggesting that glucose starvation has no impact on glycolysis in PD AC cell lines.

XF Mito Fuel Flex Test

The Seahorse XF Mito Fuel Flex Test Kit was used to determine dependency and flexibility of cells to oxidize three critical mitochondrial fuels: glucose, glutamine and fatty acids. Culture medium was replaced by OXPHOS assay medium and the plate was pre- incubated for lh at 37°C in a non-C02 incubator. Inhibitors of mitochondrial pyruvate carrier (UK5099 2 pM), glutaminase (BPTES 3 pM), and carnitine palmitoyl-transferase 1A (Etoxomir 4 pM) were used. The rate of oxidation of each fuel was determined by measuring OCR in the presence or absence of fuel pathway inhibitors according to the manufacturers' instructions.

'H HRMAS NMR spectroscopy For each sample, ten microliters of D20 were added to the cell pellet (5x106 cells), mixed and placed into a 30 mΐ disposable insert. The insert was then placed into a 4 mm Zr02 HRMAS rotor. All NMR experiments were carried out on a Bruker Advance III spectrometer operating at 400 MHz for the Ή frequency equipped with a Ή/¹³ C / ¹³P HRMAS probe. Spectra were recorded at 277K with a spin rate of 4 kHz. A Carr-Purcell-Meiboom-Gill (CPMG) NMR spin echo sequence [90°— (t— 180°— t)h] with an effective spin echo time of 37.5 ms, preceded by a water presaturation pulse during a relaxation time of 2 s to reduce the signal intensities of lipids and macromolecules. For each spectrum, 380 free induction decays (FID) of 26624 Complex data points were collected using a spectral width of 8000 Hz. Each FID was then multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz and zero-filled prior to Fourier transformation. Subsequently, each spectrum was phased and referenced to the alanine signal (d = 1.46 ppm). Assignments of the NMR signals were performed using 1H-1H TOCSY spectrum (57), 1H-13C HSQC spectrum (58), in-house and online databases (59). Ή HRMAS NMR spectra were exported to NMRprocflow online software (10.1007/sl 1306-017-1178-y) to be baseline and signal shift corrected, and divided into 0.005 ppm-width buckets. To remove the effect of water suppression, the region between 4.70 and 5.27 ppm was discarded. The dataset was then normalized to the number of cells. Finally, the matrix was exported to the SIMCA-P + v.14 software (Umetrics, Umea, Sweden) for multivariate statistical analysis. First, Principal Component Analysis (PCA) was performed in order to check the homogeneity of the dataset. Orthogonalized Projections on Latent Structure Discriminant Analysis (OPLSDA) was then applied to a subset of the data matrix composed of Pane- 1, BxPC-3 and MIA PaCa-2 samples in order to target metabolic differences between OXPHOS and glycolytic samples. The resulting score and loading plots were used to visualize the discriminant features. A leave-one out internal cross-validation was performed in order to calculate Q2 and R2Y values representing, respectively, the predictive capability and the sensitivity of the model, as well as the CV-ANOVA p-value and to ensure the robustness of the statistical model. The integration of each discriminant signal, which represents the relative concentration of the corresponding metabolites, was then exported to Metaboanalyst online software (doi: 10.1093/nar/gky310) and a heatmap was calculated based on autoscaled features.

Immunoblotting

Cells at exponential growth were resuspended in lysis buffer (HEPES 50 mM, NaCl 150 mM, Triton-X100 1 %, EDTA ImM, EGTA 1 mM, glycerol 10%, NaF 25 mM, ZnC12 10 mM) with a cocktail of protease and phosphatase inhibitors added freshly. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad laboratories, France). Proteins (50 pg) were resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Then membranes were blocked lh at room temperature with TBS, 5% milk, and incubated overnight in TBS, 5% milk, 0.1% Tween containing appropriate primary antibodies: CPT1A (1 :500; Abeam #abl28569), Nrf2 (1 : 1000; Abeam #Ab62352), Total OXPHOS Human WB Antibody Cocktail (1 :200; Abeam #abl 10411), NDUFB8 (1 :2000; Abeam #abl 10242). After extensive washes in TBS, 0.1% Tween, membranes were incubated lh at RT with a HRP-conjugated secondary antibody at 1 :5000 before being revealed with ECL. Acquisition was performed with a Fusion FX7 imager (Vilber-Lourmat, France).

Flow cytometry

ROS and mitochondrial mass measurements

Cells were seeded in 12-well plates (175,000 cells per well). The day after, CellRox Orange, MitoSOX Red, and MitoTracker Deep Red (Molecular Probes) were added in culture medium to a final concentration of 5 mM, 10 mM and 200 nM for 30 min, 20 min and 10 min, respectively. After incubation, cells were washed with warm PBS, detached with Accutase, and resuspended in HBSS (Gibco, Life Technologies) for flow cytometry. Ten thousand events per sample were acquired in a MACSQuant-VYB (Miltenyi Biotec) and data analysis was performed using FlowJo software.

Mitochondrial membrane potential

Measurement was performed using the MITO-ID Membrane potential detection kit (ENZ-51018) according to the manufacturer’s protocol. Briefly, cells were collected, washed, and preincubated in 500 pi of the Assay Solution containing 5 pi of MITO-ID MP Detection Reagent for 15 min. Then, 10,000 events per sample were acquired in a MACSQuant-VYB and orange fluorescence data analysis was performed using the FlowJo software.

Fluorescence microscopy

Mitochondrial network

Cells were seeded on coverslips in 12-well plates (200,000 cells per well). The day after, the mitochondrial network was analyzed by incubation of cells in the presence of MitoTracker DeepRed FM (200 nM, Molecular Probes) at 37°C for 30 min. Then, cells were washed twice with PBS and fixed with warm 4% paraformaldehyde. Finally, samples were mounted using the ProLong™ Gold antifade reagent with DAPI. Confocal images were acquired using an inverted microscope equipped with LSM 880 with Airyscan detector controlled by Zeiss Zen Black, 63x lens.

Nrf2 immunofluorescence Cells were seeded on coverslips in 12-well plates (300,000 cells per well). The day after, the cells were fixed in 4% formaldehyde then washed twice with PBS, 0.1% triton. Then, cells were permeabilized in PBS, 0.2% triton for 5 min at room temperature, labeled for lh with anti- Nrf2 primary antibody (1 :200; Abeam #ab62352), washed with PBS, and incubated with an anti-rabbit secondary antibody coupled to the fluorochrome Alexafluor 488 for 30 min at room temperature. After 3 washes with PBS, a drop of ProLongTM Gold antifade Reagent with DAPI (Life Technologies) was placed on the slide before mounting the coverslip. Image acquisition of mounted samples was carried out on a microscope Nikon Eclipse 90i fluorescence microscope.

Transmission Electron Microscopy (TEM)

The cells at exponential growth were fixed in 37°C-warm 4% paraformaldehyde and 2.5% Glutaraldehyde in PBS overnight. The next day, the plates were washed three times in PBS at RT. The cells were then scraped in PBS, transferred in a 2 ml microtube and pelleted by centrifugation for 5 min at 5000 rpm. The supernatant was removed, the cells were resuspended in 2% 37°C-warm low- melting-point agarose and transferred into microvettes (Sarstedt) and pelleted by centrifugation for 5 min at 5000 rpm. The microvettes were then placed on ice for 10 minutes. The microvettes tips were then removed with a razor blade and the agarose- embedded cell pellet was processed through a classical protocol for TEM. Briefly, the pellets were post-fixed in aqueous 1% Os04 for one hour, and left overnight in aqueous 1% uranyl acetate. The next day, the pellets were dehydrated in graded series of ethanol baths (10 minutes each) and infiltrated with epon resin in ethanol (1 :3, 2:2, 3 : 1, 2 hours each and pure resin overnight). The next day the pellets were embedded in fresh pure epon resin and cured for 48h at 60°C. Seventy nm-ultrathin sections were performed on a Leica UCT Ultramicrotome (Leica, Austria) and deposited on formvar-coated slot grids. The grids were contrasted using lead citrate and observed in an FEI Tecnai G2 at 200 KeV. Acquisition was performed on a Veleta camera (Olympus, Japan).

NADP+/NADPH quantification

Cells were seeded at a density of 5,000 cells per well in 96-well plates and allowed to attach overnight. Measurement was performed according to the manufacturer’s protocol. Briefly, 50 mΐ of NADP/NADPH-Glo™ Detection Reagent (Promega, G9081) was added to each well and incubated for 60 minutes at RT. Luminescence was recorded using Tristar LB 941 apparatus (Berthold technologies).

Glutathione quantification Cells were seeded at a density of 5,000 cells per well in 96-well plates and allowed to attach overnight. GSH + GSSG were quantified using a GSH/GSSG-Glo Assay kit (Promega, V6611) according to the manufacturers' instructions. Luminescence was recorded using Tristar LB 941 apparatus (Berthold technologies).

Canonical correlation analysis

The canonical correlation analysis between transcriptome of PDX (RNA-Seq) and OCR+ECAR data (from Seahorse analysis) was performed by PLS approach (Partial least square) using the 2 first component (lixOmics package) (60). To focus on genes more specifically correlated with OCR, we chose to focus on the component that shows opposite loading values of OCR and ECAR. For biological interpretation of the component of interest, we performed a gene set enrichment analysis on its loading values. The p-values were adjusted for false discovery rate (FDR).

Differential gene expression analysis

The differential expression analysis between High and Low OXPHOS groups was performed with Limma package (Bioconductor). GSEA analysis was performed to assess the enriched pathways using the logFold change statistic with the package fGSEA (Bioconductor).

OXPHOS shift assays

Panc-1 cells were forced to shift from high OXPHOS to low OXPHOS status by disruption of mitochondrial metabolism by treatment with Tigecycline (Sigma-Aldrich; inhibiting mitochondrial protein synthesis). Panc-1 cells were seeded in 96-well plates (5,000 cells per well). Twenty-four hours later, the medium was supplemented with Gemcitabine (1 or 4 nM) and Tig (50 mM). Cell viability was determined 72h later by Crystal violet viability assay.

MIA PaCa-2 and BxPC-3 cells were forced to shift from low OXPHOS to high OXPHOS status by culture in Galactose instead of Glucose. MIA PaCa-2 and BxPC-3 cells were cultivated for 3 weeks (equivalent to 6 consecutive passages) in DMEM (ThermoFisher Ref 11966025) supplemented with either Glucose (25 mM) or galactose (10 mM) as sole source of carbon. After this adaptation, the cells were seeded in 96-well plates (5,000 cells per well), Gemcitabine (4 nM) was added in the medium 24 hours later, and cell viability was determined 72h later by Crystal violet viability assay.

In vivo experiments

Orthotopic xenografts were made with the Panc-1 and MIA PaCa-2 cells (high and low OXPHOS, respectively). Two million Panc-1 or one million MIA PaCa-2 cells, were surgically implanted into the pancreas of anesthetized 6-week-old female Swiss nude mice CrkNu(lco)- Foxnlnu (Charles River, France). The presence of a pancreatic tumor was confirmed by exploratory laparotomy at day 16 (MIA PaCa-2) or day 33 (Panc-1). After surgery recovery, mice were randomly assigned to four groups and treated by intraperitoneal injection with PBS (vehicle), Gemcitabine (120 mg/kg twice a week), Phenformin (50 mg/kg daily), and combination Gemcitabine + Phenformin. Four weeks after the start of treatment, mice were sacrificed by cervical dislocation, necropsy was performed, and pancreatic tumors were weighed. Orthotopic syngeneic allografts were obtained by intraperitoneal injection of one million KPC luc2 cells in 6-week-old female C57BL/6 mice (Charles River, France). Tumoral growth was followed by bioluminescence upon injection of 3 mg luciferin-EF (Promega) using a Photon Imager device (Biospace Lab). Same treatments as done for xenografts were started 11 days after implantation. Mice were sacrificed when the non-treated mice (vehicle-injected as controls) were moribund (ethical limit point) at day 17 and pancreatic tumors were weighed.

All mice were kept under specific pathogen-free conditions and according to the current European regulation; the experimental protocol was approved by the Institutional Animal Care and Use Committee (#16711).

Statistical Analysis

Results are expressed as the mean ± SEM of triplicates, and at least three independent experiments were done for each analysis. Statistical analysis of data was performed by one-way analysis of variance (ANOVA) and only p values < 0.05 were considered statistically significant.

Results

Mitochondrial metabolism is still efficient in pancreatic cancer cells to produce energy

Most of the pancreatic cancer patients face resistance to chemotherapy, which is primary based on an innate property of aggressive tumoral cells. This feature can be reproduced in the laboratory in in vitro settings (data not shown). Six classical PDAC cell lines treated with gemcitabine show differential sensitivity, with the Panc-1 cell line being the most resistant even at high concentrations of gemcitabine. We wondered whether the heterogeneity in response to chemotherapy could be linked to energetic metabolism differences. To test this, we analyzed mitochondrial respiration (OXPHOS) and glycolysis in these 6 PDAC cell lines using a Seahorse device. This analysis showed different levels of basal respiration, ATP production by mitochondria, and respiratory spare capacity between the 6 cell lines, the Panc-1 being the most efficient for mitochondrial respiration (data not shown). Furthermore, different levels of basal glycolysis as well as of glycolytic reserve were observed between the different cell lines (data not shown). ATP production was shown to rely mostly on mitochondrial respiration (OXPHOS) compared to glycolysis (data not shown). Thus the cell lines can be classified into three different groups with respect to their basal energetic features (data not shown): the Panc-1 is mostly respiratory (OXPHOS group), the MIA PaCa-2 and BxPC-3 are mostly glycolytic (Glycolytic group), and the Capan-1, Capan-2 and to a lesser extent the SOJ-6 have a relative lower energetic metabolism compared to the other cell lines (Less metabolic group). An alternative representation of the cell energetic preference is the ratio OXPHOS/Glycolysis (data not shown), further illustrating prominent OXPHOS in Panc-1 cells. Finally, this analysis showed that all cell lines are plastic (i.e. able to shift to the other energetic pathway if necessary) at different levels (data not shown), the Capan-1 and Panc-1 being the most plastic to OXPHOS or glycolysis, respectively. Finally, we addressed the dependency and flexibility of mitochondrial respiration towards the three main metabolic pathways feeding the tricarboxylic acid (TCA) cycle (glucose, glutamine and fatty acids) by Mito Fuel Flex Seahorse analysis. Respiration was shown to depend on the glucose and glutamine pathways in all cell lines except Panc-1 (data not shown). Moreover, dependency for the fatty acid pathway was observed in all cell lines, including Panc-1 which is the least dependent. Regarding flexibility, the Panc-1 cell line was the only one to be flexible towards the three TCA fuels. Taken together, these data demonstrate that mitochondria are fully able to produce energy in all the tested PD AC cell lines (at high level in one of these cell lines), and that all cell lines can increase the mitochondrial respiration or shift to glycolysis depending on their environment.

More importantly, we also analyzed OXPHOS and glycolysis in 21 PDAC primary cells of the recent PaCaOmics PDAC PDX biobank. This work led to the classification of these cells into four different groups with respect to their basal energetic features: OXPHOS, Glycolytic, Less metabolic, and Energetic (high OXPHOS and glycolysis) (data not shown). Taken together, these data illustrate the high energetic heterogeneity between PDAC tumors, most of them showing a hybrid OXPHOS/glycolysis phenotype. Importantly, this work distinguishes PDAC tumors with high OXPHOS rate.

Mitochondrial respiration shows different degrees of dependency and flexibility towards TCA fuels in pancreatic cancer cells

The mitochondrial respiratory chain depends on the electron donors NADH and FADH2 produced by the TCA, which is fed by three main metabolic fuels: glucose and glutamine and fatty acids. We thus addressed the dependency and flexibility of mitochondrial respiration towards these fuels in the 6 different PDAC cell lines, by the use of specific inhibitors of each of these pathways in Seahorse analysis: UK5099 (inhibitor of the Mitochondrial Pyruvate Carrier, MPC) and BPTES (inhibitor of the Glutaminase, GLS) (data not shown). Respiration was shown to depend on the glucose and glutamine pathways in all cell lines except the Panc- 1. Moreover, dependency for the fatty acids pathway was observed in all cell lines, including Panc-1 which is the least dependent. Regarding flexibility, the Panc-1 cell line was the only one to show flexibility towards all three main TCA fuels (data not shown). Moreover, a metabolomics analysis was done by HRMAS-NRM. Interestingly, the Panc-1 showed the highest level of metabolites including those feeding the TCA such as fatty acids and amino- acids (data not shown). Interestingly, Panc-1 cells also showed the highest level of the Carnitine palmitoyltransferase IA (CPT1A) protein involved in fatty acid metabolism in mitochondria (data not shown). These data suggest that high respiratory capacities of mitochondria in Panc-1 cells are supported by an active TCA functioning independently of fuel origin. We also assessed the effects of the inhibitors UK5099 and BPTES on cell viability (data not shown). The dose- response curves show that inhibition of GLS is insufficient to kill all cells even at high doses, whereas inhibiting MPC is able to induce cell death in all cell types, suggesting that all cell lines depend on glucose and not glutamine for survival. These data illustrate the metabolic specificities of Panc-1 cells, which seem to have a rich metabolic content making their mitochondrial respiration highly independent and very flexible for fuels.

The structure of mitochondria reflects respiration activity

To further investigate the high mitochondrial respiratory capacities of Panc-1 and to a less extent SOJ-6 cell line, we analyzed mitochondria by flow cytometry, fluorescence microscopy and Transmission Electron Microscopy (TEM). Mitochondrial mass was monitored by Mitotracker staining followed by flow cytometry analysis (data not shown), showing no difference between cells except a moderately weaker staining in Capan-2 cells (statistics are shown with regards to Panc-1 cells as the highest OXPHOS). Accordingly, Mitotracker staining followed by fluorescence microscopy analysis did not show any obvious differences of mitochondrial network between cell lines (data not shown). Similarly, no difference in mitochondrial membrane potential was detected (data not shown). TEM analysis was highly informative, showing distribution of mitochondria in all the cytoplasm except for Panc-1 cell line in which mitochondria are close to the nucleus (data not shown), and many figures of elongated mitochondria in Panc-1 and SOJ-6 cells contrary to the other cell lines showing only round mitochondria (data not shown). Cristae appeared normal in all cell lines except MIA PaCa-2 showing unstructured cristae in many swollen mitochondria. These observations suggest a nice dynamic of mitochondria in Panc-1 and SOJ-6 cells which could be at the basis of their active mitochondrial respiration.

The redox state is independent of OXPHOS status in pancreatic cancer cells As mitochondria are also involved in redox metabolism which is deregulated in cancer cells, we then monitored both reactive oxygen species (ROS) and antioxidant molecules in the pancreatic cancer cell lines. Total ROS levels were shown to be only slightly different between the cell lines, with the lowest level in Capan-2 and MIA Paca-2 cells and the highest in Capan- 1 (data not shown). Superoxide anions were quantified specifically in mitochondria, showing a much lower level in Panc-1 compared to the 5 other cell lines (data not shown), suggesting optimal ETC function towards respiration with poor leaky electrons to superoxide formation in the Panc-1 cells. Key antioxidant defenses known to accumulate in cancer cells were also monitored. Immunofluorescence microscopy (data not shown) and Western blotting (data not shown) showed less abundant level of the transcription factor Nrf2 in Capan-1 and Capan-2 cell lines. The main small antioxidant molecules NADPH and Glutathione were also found to be less abundant in these two cell lines, which are the less metabolic ones, suggesting a link between energetic metabolism activity and redox potential. Nonetheless, the ratio of reduced over oxidized glutathione (GSH/GSSG) is well balanced in all cell lines including the Capan-1 and Capan-2 (data not shown). Treatment with increasing doses of menadione, a ROS inducer, revealed the vulnerability of all cell lines to high oxidative stress, independently of their antioxidant content (data not shown). Taken together, these data suggest that the Panc-1 cell line is under oxidative stress, which is the case for all PDAC cell lines, but it generates less mitochondrial superoxide through a highly potent respiratory chain.

The mitochondrial respiratory Complex is a good target to counter resistance to

Gemcitabine in the high OXPHOS Panc-1 cell line

To investigate further the respiratory chain, we monitored the level of one protein of each of the five mitochondrial respiratory Complexes by Western blotting using OXPHOS antibodies cocktail (data not shown), demonstrating that Complex I is poorly abundant in all cell lines, but most abundant in Panc-1 and SOJ-6 cells which have the highest OXPHOS. This was confirmed using anti-NDUFB8 (protein belonging to mitochondrial Complex I) antibody alone (data not shown).

Interestingly, Complex III is least abundant in the Panc-1 cells, which could account for the lower production of superoxide anions in these cells. We then decided to treat the cell lines with molecules targeting Complex I: Phenformin (Fig. 1 A), Metformin (Fig. IB), and Rotenone (Fig. 1C). Our data show that these three drugs are able to induce pancreatic cancer cell death. More importantly, we showed that combining Phenformin at 0.5 mM (corresponding to a concentration close to the IC50) with increasing doses of Gemcitabine sensitizes specifically Panc-1 cells to cell death (Fig. ID). Specifically in the case of high OXPHOS Panc-1 cells, combination indexes lower than 0.5 for low gemcitabine concentration show a synergy of cytotoxicity between Phenformin and Gemcitabine (data not shown). Altogether, these data suggest that targeting Complex I in the high OXPHOS cells could be a good option to counter their intrinsic resistance to gemcitabine-induced cell death.

Targeting mitochondrial respiratory Complex synergizes with chemotherapy in high

OXPHOS PD AC primary cancer cells

To further assess the synergetic impact of Phenformin with Gemcitabine specifically in high OXPHOS PD AC cells, we analyzed primary cells obtained from freshly generated Patient- Derived Xenografts (PDX) of the PDAC PaCaOmics biobank (26). We first analyzed mitochondrial respiration (OXPHOS) and glycolysis in cells in culture derived from 21 PDAC patients using the Seahorse device, and classified them into four different groups with respect to their basal energetic features: OXPHOS, Glycolytic, Less metabolic, and Energetic (data not shown). Then, we performed a canonical PLS analysis between the metabolic variables (OCR, ECAR) and gene expression using PDX RNA-Seq data. The sample projection on the second dimensional space which is composed by the first two components enables us to distinguish high and low OXPHOS groups in red and blue respectively (data not shown). Our results clearly show a correlation between our functional metabolic study and the transcriptomic data. Interestingly, the GSEA analysis on the genes loading values of the second component showed enrichment in mitochondrial electron transport and Complex I assembly genes (NES= 2.03 and 2.01, respectively, adjusted-p-value= 3.7E-2) indicating a positive correlation between Complex I assembly and OCR level (data not shown). We also monitored the level of one protein of each of the five mitochondrial respiratory Complexes by Western blotting (Fig. 2A), demonstrating that Complex I is significantly more abundant in high OXPHOS tumors than in low OXPHOS. This was affirmed using anti-NDUFB8 antibody alone (Fig. 2B). Complex II and III are also in higher abundance in high OXPHOS compared to low OXPHOS patients, but not Complex IV and V (Fig. 2A). Metabolomic analysis by HRMAS-NMR was done for these 3 high OXPHOS patients compared to the 3 low OXPHOS patients, confirming that they segregate into two different groups in the OPLS-DA score plot (data not shown). Differential analysis highlighted the abundance of specific metabolites in each OXPHOS category: high OXPHOS cells were enriched in the metabolites Choline (precursor of Phosphocholine), Phosphocholine (major lipid in membranes), Glutamate (feeding the Krebs cycle), and Glutathione (antioxidant), whereas low OXPHOS cells showed accumulation of Glucose (data not shown). By examining the clinical data associated with each patient, we observed no link between the cell energetic preference (data not shown) and the differentiated status of tumors, or the classification as basal or classical tumors (27), or Kras and p53 mutations. In contrast, we observed that the overall survival of high OXPHOS patients was lower compared to low OXPHOS significance (Fig. 3A).

Altogether, these data show a correlation between mitochondrial respiratory genes expression and high OXPHOS status in PDAC, suggesting that high OXPHOS patients could be identified by transcriptomic analysis in the clinic. Moreover, these data point to mitochondrial respiratory Complex I as a vulnerability of high OXPHOS PDAC.

We then addressed the possibility that the combination Gemcitabine + Phenformin would be synergetic in high OXPHOS cells, as observed for Panc-1 cells. For this purpose, we chose 3 high OXPHOS patients (belonging to the Energetic group) compared to 3 low OXPHOS (belonging to the Less metabolic group). Noticeably, the OXPHOS/Glycolysis ratio determined for the six patients proved to be less informative regarding the cell energetic preference than the classification already talked. Transcriptomic data mining showed a highly significant enrichment in mitochondrial pathways (including Complex I genes) in the 3 high OXPHOS patients (27,74 and 84) compared to the 3 low OXPHOS (22, 32, and 85) (data not shown).

We then performed chemograms experiments. First, we treated the primary cells from these 3 patients with Phenformin, showing its ability to induce cell death for the six tested patients irrespective of their OXPHOS status (data not shown). More importantly, our data shown in Fig. 3B demonstrate that combining Phenformin at 0.5 mM with increasing doses of Gemcitabine specifically sensitizes high OXPHOS cells to Gemcitabine-induced cell death, with combination indexes lower than 0.5 (synergy) specifically for these high OXPHOS patients (data not shown). Altogether, these data suggest that targeting Complex I with Phenformin in the high OXPHOS cells potentiates their sensitivity to Gemcitabine-induced cell death.

The importance of the OXPHOS status in Gemcitabine response was also assessed by OXPHOS shift assays. Pharmacologic manipulation of high OXPHOS cell Panc-1 toward a low OXPHOS phenotype by inhibiting mitochondrial protein synthesis with Tigecy cline (61) was shown to increase cell sensitivity to Gemcitabine (Figure 4A). Conversely, culturing low OXPHOS cells (MIA PaCa-2 and BxPC-3) in a medium containing galactose instead of glucose as the sole sugar source, which shifts the energetic metabolism from glycolysis to mitochondrial OXPHOS (61), induced resistance to low concentrations of Gemcitabine (Figure 4B). Thus, manipulating the mitochondrial energetic status toward low OXPHOS or high OXPHOS confers sensitivity or resistance to Gemcitabine respectively, confirming the potential of inhibiting OXPHOS in combination with Gemcitabine to sensitize PD AC cells to chemotherapy.

Effect of Phenformin/Gemcitabine combination in high OXPHOS PD AC in vivo

We further assessed the synergistic impact of Phenformin with Gemcitabine in high OXPHOS PDAC cells in vivo in two different orthotopic mouse models: xenografts (Figures 5 A and 5B) and syngeneic allografts (Figure 5C). Panc-1 and MIA PaCa-2 cells were implanted in the pancreas of immunodeficient mice (xenografts). Treatments were administrated using either a combination of Phenformin and Gemcitabine, or each drug alone. Phenformin considerably increased the antitumoral effect of Gemcitabine in the high OXPHOS Panc-1 xenografts, whereas no impact of Phenformin on Gemcitabine antitumoral effect was observed in the low OXPHOS MIA PaCa-2 xenografts (Figures 5 A and 5B). For the syngeneic allografts, we used KPC luc2 cells, which showed a high OXPHOS status and moderate plasticity by Seahorse analysis (data not shown). In this immunocompetent context as well, the combination of the two drugs was more potent than Gemcitabine alone to induce tumor regression (Figure 5C). Altogether, these preclinical data demonstrate that targeting Complex I with Phenformin in high OXPHOS PDAC enhances Gemcitabine’ s anticancer effect.

Conclusion:

In conclusion, the work of the inventors shows that inhibitor of the mitochondrial respiration could be used for the treatment of pancreatic cancer and shows a correlation between functional (Seahorse experiments) and transcriptional (RNA-Seq) OXPHOS levels, opening the possibility to stratify patients in clinics according to mitochondrial respiration gene expression, which could be used for the identification of patients most likely to respond to the targeting of mitochondrial respiration in combination to standard chemotherapy.

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Claims

CLAIMS:

1. An inhibitor of the mitochondrial respiration for use in the treatment of pancreatic cancer in a patient in need thereof.

2. An inhibitor for use according to claim 1 wherein said inhibitor is an inhibitor of the Complexes I, II, III, IV or V of the oxidative phosphorylation chain.

3. An inhibitor for use according to claim 2 wherein said inhibitor is an inhibitor of the Complex I like the phenformin, the metformin, or the buformin.

4. An i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer or use in the sensitization of pancreatic cancerous cells.

5. An i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer for use according to claim 4 wherein the compound used to treat pancreatic cancer is gemcitabine, the 5-fluorouracil (5-FU), the Capecitabine, the oxaliplatine, the cisplatine, the irinotecan, FOLFIRINOX (a chemotherapy regimen made up of the following four drugs: FOL for folinic acid, F for fluorouracil (5-FU), IRIN for irinotecan and OX for oxaliplatin) or Nab-Paclitaxel.

6. An in vitro method for predicting the survival time of a patient suffering from a pancreatic cancer comprising: i) determining, in a sample obtained from the patient, the level of OXPHOS; ii) comparing the level of OXPHOS determined at step i) with a predetermined reference value and iii) providing a bad prognosis when the level of OXPHOS determined at step i) is higher than its predetermined reference values, or providing a good prognosis when the level of OXPHOS determined at step i) is lower than its predetermined reference value.

7. An in vitro method according to claim 6 wherein the OXPHOS level is determined by determining by measurement of the cellular oxygen consumption rate (OCR) and/or of the expression level of at least one gene expressed in the mitochondrial respiratory Complexes (Complexes I to V) of the mitochondria.

8. An in vitro method according to claim 7 wherein the genes are ATP5A, UQCRC2, SDHB, COX2 and NDUFB8.

9. An inhibitor of the mitochondrial respiration for use in the treatment of a pancreatic cancer in a patient in need thereof wherein said patient has a bad prognosis as determined in the claim 6.

10. An i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer in a patient in need thereof wherein said patient has a bad prognosis as determined in the claim 6.

11. An i) inhibitor of the mitochondrial respiration and a ii) therapeutic compound used to treat pancreatic cancer according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer in a patient in need thereof wherein said patient has a high OXPHOS.

12. A therapeutic composition comprising an inhibitor of the mitochondrial respiration according to the invention for use in the treatment of pancreatic cancer in a patient in need thereof.

13. A method for treating a pancreatic cancer in a patient in need thereof by administrating to said patient an inhibitor of the mitochondrial respiration.