CYTIDINE DEAMINASE INHIBITORS FOR THE TREATMENT OF PANCREATIC CANCER

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for use in the treatment of pancreatic cancer in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Pancreatic ductal carcinoma (PDA) is the most common type of pancreatic cancer ^l . Despite decades of intense efforts from researchers and clinicians, PDA remains a challenge to treat, with 5 year rates survival lower than 6% for patients with cancers of all stages ^l . Most PDA is identified at a late stage, when surgical intervention is not possible. Even with complete resection and negative results from analyses of tumour margins, long-term survival after surgery is poor; tumors recur in virtually all patients. To put this into perspective, PDA is estimated to become one of the top three leading cause of cancer-related death by 2030 ² .

Progress in the treatment of PDA has been incremental. Combination cytotoxic therapies such as FOLFIPJNOX ³ , along with gemcitabine ⁴ and albumin-bound paclitaxel ⁵ , have provided meaningful gains, but there is lot of needs for improvement. The only targeted agents approved in the treatment of PDA is the EGFR inhibitor Erlotinib (Tarceva), which given in combination with gemcitabine, only slightly increases overall survival time compared with gemcitabine alone ⁶ . Taken together, the current treatment approaches for PDA increase survival times of patients in weeks to months. In this dismal context, the inventors have elected cancer gene therapy as a promising approach for PDA management ¹ . The inventors conducted the first-in-human clinical trial, based on the use of non-viral vectors to transfer anticancer genes that sensitize PDA to gemcitabine ⁸ . This early phase clinical trial demonstrates that intratumoral gene delivery is safe and feasible in patients with unresectable PDA. In addition, a population of patients with locally advanced tumors benefited from this treatment, with two patients surviving for up to two years following gene therapy ⁸ . A phase II clinical trial is under preparation.

While leading-edge, this trial also highlights the need to further characterize the molecular mechanisms involved in the resistance to treatment. Accordingly, the inventors have interrogated their clinical samples for the expression of key proteins involved in resistance of cancer cells to gemcitabine. The inventors found that cytidine deaminase (CDA) was the only gene (i) upregulated in resected PDA samples compared to normal parenchyma, (ii) overexpressed in microbiopsies from locally advanced and metastatic PDA resisting to therapy and (iii) detectable in microbiopsies of PDA patients treated by gene therapy (Figure 1).

Cytidine deaminase (CDA) is a key enzyme of the pyrimidine salvage pathway that catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively ⁹ . In PDA, gemcitabine is inactivated primarily by CDA-mediated conversion to difluorodeoxyuridine. Experimental evidences demonstrate that CDA expression is high in gemcitabine-resistant cells ¹⁰ ' ^u , while tetrahydro uridine (THU), a nonspecific CDA inhibitor ¹² , increases the sensitivity to gemcitabine ¹³ . Macrophages were found to mediate gemcitabine resistance of PDA by upregulating CDA in cancer cells ¹⁴ and wflb-Paclitaxel potentiates gemcitabine activity by reducing CDA levels in a mouse model of PDA ¹⁵ . Thus, there are evidences lending credence to CDA as a key protein involved in the resistance of PDA cells to treatment. Accordingly, the inventors generated CDA-null human PDA-derived cell lines using lentiviral vectors encoding specific shR As. The inventors found that targeting CDA strongly sensitizes PDA-derived cells to chemotherapy, both in vitro and in vivo, and induces apoptosis (data not shown).

However, the genetic depletion of CDA per se, in the absence of chemotherapy, unexpectedly inhibited PDA-derived cells proliferation, altered cell cycle progression, with a prolonged S phase, a hallmark of DNA replication stress, and impaired tumor growth, as half of the mice engrafted with Mia PACA-2-null CDA were free of tumors (Figure 2).

There is no disclosure in the art of the role of CDA in PDA in the absence of chemotherapy, and the use of CDA inhibitors in the treatment of PDA in the absence of chemotherapy.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for use in the treatment of pancreatic cancer in a subject in need thereof. DETAILED DESCRIPTION OF THE INVENTION:

The inventors investigated molecular mechanisms involved in the resistance of PDA cells to treatment, the role of cytidine deaminase (CDA) in the resistance of PDA cells to treatment and in PDA in absence of treatment.

From patient cohorts, the inventors identified cytidine deaminase (CDA), which catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively, as overexpressed (i) in cohorts of patients with PDA resisting to gemcitabine, (ii) in PDA tissue as compared to normal parenchyma, and (iii) in patients with PDA receiving gene therapy. As expected, targeting CDA at the genetic level sensitizes cancer cells to chemotherapy (gemcitabine dFdC) both in vitro and in vivo in experimental models of PDA, with very high efficacy. To their surprise, CDA targeting in the absence of chemotherapy strongly alters cell proliferation and tumor progression, when more than half of mice engrafted with CDA-null human PDA cells remained free of tumors. Using high throughput transcriptomic, proteomic and metabolomic studies, the inventors identified massive concomitant changes in tumor cell biology following CDA ablation that can broadly be categorized into alterations of both energetic and intermediate metabolism: ATP levels are severely curtailed and imbalanced, mitochondrial respiratory chain is inhibited, total ROS level are increased (Figure 3), TCA is altered as cellular pyrimidine and purine levels (Figure 4)·

Accordingly, the present invention relates to a method for treating pancreatic cancer in a subject in need thereof comprising the steps of administering to said subject a cytidine deaminase inhibitor compound. As used herein, the term "subject" denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with pancreatic cancer. In a particular embodiment, the term "subject" refers to any subject (preferably human) afflicted with Pancreatic ductal adenocarcinoma (PDAC). The method of the invention may be performed for any type of pancreatic cancer. The term "pancreatic cancer" refers to pancreatic cancer such as revised in the World Health Organisation Classification C25. The term "pancreatic cancer" also refers to Pancreatic ductal adenocarcinoma (PDAC) (31-35). The term "pancreatic cancer" also refers to metastatic pancreatic cancer, exocrine pancreatic cancer and locally advanced PDAC. 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 subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "cytidine deaminase" and "CD A" has its general meaning in the art and refers to cytidine deaminase "CDA", a key enzyme of the pyrimidine salvage pathway that catalyzes the hydro lytic deamination of cytidine and deoxy cytidine to uridine and deoxyuridine, respectively ⁹ . Because of the structural similarity to cytidine, several nucleoside -based drugs are also subject to deamination by CDA (Ferraris et al, 2014). In Pancreatic ductal adenocarcinoma, cytidine deaminase "CDA" inactivates gemcitabine via CDA-mediated conversion to difluorodeoxyuridine. The term "cytidine deaminase inhibitor" or "CDA inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the cytidine deaminase. The term "cytidine deaminase inhibitor" also refers to a compound that selectively blocks or inactivates hydrolytic deamination mediated by the cytidine deaminase. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates CDA with a greater affinity and potency, respectively, than its interaction with the other sub-types of the deaminase family. Compounds that block or inactivate CDA, but that may also block or inactivate other deaminase sub-types, as partial or full inhibitors, are contemplated. The term "CDA inhibitor" also refers to a compound that inhibits CDA expression. Typically, a CDA inhibitor compound is a small organic molecule, a polypeptide, an aptamer, an antibody, an intra-antibody, an oligonucleotide or a ribozyme.

Tests and assays for determining whether a compound is a CDA inhibitor are well known by the skilled person in the art such as described in Ferraris et al, 2014; US 6,136,791; WO2009/052287.

CDA inhibitors are well-known in the art such as illustrated by Ferraris et al, 2014; US 6,136,791; WO2009/052287.

In one embodiment of the invention, CDA inhibitors include but are not limited to Tetrahydrouridine (THU); Fluorinated Tetrahydrouridines and derivatives thereof such as 2'- fluorinated tetrahydrouridine derivatives;

2 ' -Deoxy-2 ' ,2 ' -difluoro-5 ,6-dihydrouridine;

(4R)-2 '-Deoxy-2 ' ,2 ' -difluoro-3 ,4,5 ,6-tetrahydrouridine;

(4S)-2 ' -Deoxy-2 ' ,2 ' -difluoro-3 ,4,5 ,6-tetrahydrouridine;

l-(2-Deoxy-2,2-difluoro-P-D-erythro-pentofuranosyl)-tetrahyd ro-2(lH)-pyrimidinone; 2 ' -Deoxy-2 ' -fluoro-5 ,6-dihydrouridine;

(4R)-2 ' -Deoxy-2 ' -fluoro-3 ,4,5 ,6-tetrahydrouridine;

(4S)-2 ' -Deoxy-2 ' -fluoro-3 ,4,5 ,6-tetrahydrouridine;

l-(2-Deoxy-2-fluoro-P-D-ribofuranosyl)tetrahydro-2(lH)-pyrim idinone;

1 -(2-Deoxy-2-fluoro-P-D-arabinofuranosyl)dihydro-2,4-( 1 H,3H)-pyrimidinedione; (4R)- 1 -(2-Deoxy-2-fluoro-P-D-arabinofuranosyl)tetrahydro-4-hydroxy -2( 1 H)- pyrimidinone; (4S)-l-(2-Deoxy-2-fluoro-P-D-arabinofuranosyl)tetrahydro-4-h ydroxy-2(lH)- pyrimidinone; and compounds described in Ferraris et al, 2014.

In one embodiment of the invention, CDA inhibitors include but are not limited to difluorotetrahydrouridine derivatives; 2'-fluoro-2'-deoxytetrahydrouridines;

2 ^* 2 ^, -DiFluoro-DiHydro-Uridine (DFDHU) ;

2 ^* 2 ^, -DiFluoro-TetraHydroUridine (DFTHU) ;

2'(R)-fluoro-2'deoxy-tetrahydrouridines;

2 ^* (R)-Fluoro-2 ^, deoxy-DiHydroUridine ((R)-FDHU) ;

2'(S)-fluoro-2'deoxy-tetrahydrouridines;

2 ^, (S)-fluoro-2 ^, deoxy-dihydrouridine ((S)-FDHU);

2'(S)-fluoro-2'deoxy-tetrahydrouridine ((S)-FTHU); and compounds described in WO2009/052287. In one embodiment of the invention, CDA inhibitors include but are not limited to

ASTX727 (E7727); S-methyl^'^-dideoxy-S'-azidocytidine (5mAZC); 5-methyl-2',3 ^* - dideoxycytidine; 5-ethyl-2',3'dideoxy-3'-azidocytidine; 5-propyl-2',3'-dideoxycytidine; 5- propyl-2',3'-dideoxy-3'-azidocytidine; 5-propene-2',3'-dideoxy-3'-azidocytidine; 5-propyne- 2',3'-dideoxy-3'-azidocytidine; and 5-propyne-2',3'-dideoxy-3'-azidocytidine; analogues thereof or a pharmaceutically effective salt thereof, and compounds described in US 6,136,791; and Zebularine (l-(P-D-Ribofuranosyl)-2(lH)-pyrimidinone) (Lemaire et al, 2009; Marquez et al, 2005).

In another embodiment, the CDA inhibitor of 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 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 al., 1996). Then after raising aptamers directed against CDA of the invention as above described, the skilled man in the art can easily select those blocking or inactivating CDA.

In another embodiment, the CDA inhibitor of the invention is an antibody (the term including "antibody portion").

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of CDA. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in CDA. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology W lley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. In a preferred embodiment, the CDA inhibitor of the invention is a Human IgG4. In another embodiment, the antibody according to the invention is a single domain antibody. 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): CDRl, 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 came lid 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 CDA inhibitor of the invention is a CDA expression inhibitor.

The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., CDA) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. CDA expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of CDA mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of CDA proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the m NA transcript sequence encoding CDA can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory R As (siR As) and small hairpin R A or short hairpin R A (shR A) can also function as CDA expression inhibitors for use in the present invention. CDA gene expression can be reduced by contacting the subject 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 CDA expression is 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 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 CDA expression inhibitors 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 CDA 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 a CDA expression inhibitors 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 and preferably cells expressing CDA. 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 (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 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.

In one embodiment, viral vectors are oncolytic viruses. Oncolytic virus refers to any virus capable of replicating in and killing tumor cells. Preferably, the virus is engineered e.g. to increase tumor cell selectivity. Representative examples of oncolytic virus include without limitation, adenovirus, reovirus, herpes simplex virus (HSV), Newcastle disease virus, poxvirus, myxoma virus, rhabdovirus, picornavirus, influenza virus, coxsackievirus and parvovirus. In preferred embodiments, the oncolytic virus is a vaccinia virus (e.g. Copenhagen, Western Reserve, Wyeth strain), rhabdovirus (e.g. vesicular stomatitis virus (VSV)), or adenovirus (e.g. ONYX-015, Delta-24-RGD). In a particularly embodiment, the oncolytic virus is an adenovirus such as Delta-24-RGD (Fueyo J et al, Oncogene, 19:2-12 (2000)). Oncolytic viruses include adenovirus, vaccinia virus, herpes virus, herpes simplex virus, reovirus, Seneca valley virus coxsackievirus, measles virus, poliovirus, VSV/rhabdovirus, parvovirus, retroviruses and viruses described in Kaufman et al, 2015; Chioccal and Rabkin, 2014.

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., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 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, 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 microencapsulation.

Typically the inhibitors according to the invention as described above are administered to the subject in a therapeutically effective amount.

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

In a particular embodiment, the inhibitor according to the invention may be used in a concentration between 0.01 μΜ and 20 μΜ, particularly, the inhibitor of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10.0, 15.0, 20.0 μΜ.

In a further aspect, the method of the invention comprises the step of administering the subject with the CD A inhibitor according to the invention in combination with anti-pancreatic cancer treatment.

The term "pancreatic cancer treatment" has its general meaning in the art and refers to any type of pancreatic cancer therapy undergone by the pancreatic cancer subjects including surgical resection of pancreatic cancer, and any type of anti-pancreatic cancer compound such as fluorouracil, FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin, and leucovorin), nab- paclitaxel, inhibitors of programmed death 1 (PD-1), PD-1 ligand PD-L1, anti-CLA4 antibodies, EGFR inhibitors such as erlotinib, inhibitors of PARP, inhibitors of Sonic Hedgehog, gene therapy and radiotherapy.

The term "gene therapy" denotes the therapeutic gene transfer using expression vector coding for at least one gene selected from the group consisting of SSTR2, DCK and UMK to restore gene expression. The term "gene therapy" also refers to therapeutic gene transfer using non-viral vectors to restore expression of at least one gene selected from the group consisting of SSTR2, DCK and UMK such as described in WO 2009/056434. In a preferred embodiment of the invention, the term "gene therapy" refers to delivering DCK::UMK fusion gene, encoding for both DCK and UMK, using for example non-viral vectors. The term "SSTR2" has its general meaning in the art and refers to somatostatin receptor subtype 2 such as described in WO 2009/056434.

The term "DCK" has its general meaning in the art and refers to DeoxyCytidine Kinase such as described in WO 2009/056434.

The term "UMK" has its general meaning in the art and refers to Uridylate Monophosphate Kinase such as described in WO 2009/056434. In a further aspect, the method of the invention comprises the step of administering the subject with the CDA inhibitor according to the invention in combination with dihydroorotate dehydrogenase (DHODH) inhibitor such as leflunomide; Checkpoint kinase 1 (Chkl) inhibitor such as SCH900776 (MK-8776), PF-00477736, AZD7762, SAR-020106 and XL- 844; Ataxia telangiectasia and Rad3-related protein (ATR) inhibitor such as VE-821; WEE1 inhibitor such as AZD1775 (MK-1775), glutaminase (CB-839) and a-KG dehydrogenase (CPI-613).

According to the present invention, the inhibitor of the invention is administered sequentially or concomitantly with one or more anti-pancreatic cancer compound.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for treating pancreatic cancer in a subject in need thereof, wherein the method comprises the steps of:

- providing a CDA, providing a cell, tissue sample or organism expressing a CDA,

- providing a candidate compound such as a small organic molecule, a polypeptide, an aptamer, an antibody or an intra-antibody,

measuring the CDA activity,

and selecting positively candidate compounds that inhibit CDA activity.

Methods for measuring CDA activity are well known in the art (Ferraris et al., 2014). For example, measuring the CDA activity involves determining a Ki on the CDA cloned and transfected in a stable manner into a CHO cell line and human recombinant CDA, measuring CDA catalysed deamination of CDA substrates such as cytidine to uridine in the present or absence of the candidate compound.

Tests and assays for screening and determining whether a candidate compound is a CDA inhibitor are well known in the art (Ferraris et al., 2014). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit CDA activity.

Activities of the candidate compounds, their ability to bind CDA and their ability to inhibit CDA activity may be tested using CDA assay such as described for example in Ferraris et al., 2014, isolated pancreatic cancer cells or CHO cell line cloned and transfected in a stable manner by the human CDA or methods such as described in the Example.

Activities of the candidate compounds and their ability to bind to the CDA, or their ability to inhibit CDA activity may be assessed by the determination of a Ki on the CDA cloned and transfected in a stable manner into a CHO cell line, human recombinant CDA, measuring pancreatic cancer cells apoptosis induction, pancreatic cancer cells proliferation inhibition, alteration of pancreatic cancer cell cycle progression, tumor growth inhibition in the present or absence of the candidate compound. The ability of the candidate compounds to inhibit CDA activity may be assessed by measuring CDA catalysed deamination of CDA substrates, total ATP level, mitochondrial ROS production, and reduced GSH/oxidized GSSG such as described in the example.

Cells expressing another deaminase than CDA or mutated CDA may be used to assess selectivity of the candidate compounds.

The inhibitors of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the inhibitor of the invention and a pharmaceutical acceptable carrier for use in treating pancreatic cancer in a subject of need thereof.

Typically, the inhibitor 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" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, intramuscular, intravenous, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, intraperitoneal, intramuscular, intravenous and intranasal administration forms and rectal administration forms.

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, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising inhibitors of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active inhibitors in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the inhibitors of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of pancreatic cancer.

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in treating pancreatic cancer in a subject in need thereof.

The invention also provides kits comprising the inhibitor of the invention. Kits containing the inhibitor of the invention find use in therapeutic methods.

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. (A) CDA is overexpressed in tumors (n=20, matched pairs), (B) in patients treated with gemcitabine (n=50) and (C) in patients receiving gene therapy (n=14).

Figure 2. CDA targeting alters cell cycle (A) induces cell death by apoptosis (B), inhibits cell proliferation (C) and tumor growth (D) in the absence of chemotherapy.

Figure 3. CDA targeting ATP production (A) inhibits oxygen consumption (B), induces cellular ROS (C) and decreases GSH/GSSG ratio (D) in human PDA cells. Figure 4. CDA targeting alters purine and PRPP synthesis (A) pyrimidine synthesis (B); and TCA cycle (C), in human PDA cells.

Figure 5. CDA mRNA expression in normal pancreatic cells expressing hTERT, HPV E6/E7, SV40 small antigen and oncogenic KRAS.

EXAMPLE:

Pancreatic ductal carcinoma (PDA) is the most common type of pancreatic cancer ^l . Despite decades of intense efforts from researchers and clinicians, PDA remains a challenge to treat, with 5 year rates survival lower than 6% for patients with cancers of all stages ^l . Most PDA is identified at a late stage, when surgical intervention is not possible. Even with complete resection and negative results from analyses of tumour margins, long-term survival after surgery is poor; tumors recur in virtually all patients. To put this into perspective, PDA is estimated to become one of the top three leading cause of cancer-related death by 2030 ² .

Progress in the treatment of PDA has been incremental. Arguably, combination cytotoxic therapies such as FOLFIRINOX ³ , along with gemcitabine ⁴ and albumin-bound paclitaxel ⁵ , have provided meaningful gains, but there is lot of needs for improvement. The only targeted agents approved in the treatment of PDA is the EGFR inhibitor Erlotinib (Tarceva), which given in combination with gemcitabine, only slightly increases overall survival time compared with gemcitabine alone ⁶ . Taken together, the current treatment approaches for PDA increase survival times of patients in weeks to months.

In this dismal context, the inventors have elected cancer gene therapy as a promising approach for PDA management ¹ . The inventors conducted the first-in-human clinical trial, based on the use of non-viral vectors to transfer anticancer genes that sensitize PDA to gemcitabine ⁸ . This early phase clinical trial performed in Toulouse (collaboration IUCT- CRCT Team 1) demonstrates that intratumoral gene delivery is safe and feasible in patients with unresectable PDA. In addition, a population of patients with locally advanced tumors benefited from this treatment, with two patients surviving for up to two years following gene therapy ⁸ . A phase II clinical trial is underway. While groundbreaking, this trial also highlights the need to further characterize the molecular mechanisms involved in the resistance to treatment. Accordingly, the inventors have interrogated our clinical samples for the expression of key proteins involved in resistance of cancer cells to gemcitabine. The inventors found that cytidine deaminase (CDA) was the only gene (i) upregulated in resected PDA samples compared to normal parenchyma, (ii) overexpressed in microbiopsies from locally advanced and metastatic PDA resisting to therapy and (iii) detectable in microbiopsies of PDA patients treated by gene therapy (Figure 1). CDA is a key enzyme of the pyrimidine salvage pathway that catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively ⁹ . In PDA, gemcitabine is inactivated primarily by CDA-mediated conversion to difluorodeoxyuridine. Experimental evidences demonstrate that CDA expression is high in gemcitabine- resistant cells ¹⁰ ' ^u , while tetrahydro uridine (THU), a non-specific CDA inhibitor ¹² , increases the sensitivity to gemcitabine ¹³ . Macrophages were found to mediate gemcitabine resistance of PDA by upregulating CDA in cancer cells ¹⁴ and wab-Paclitaxel potentiates gemcitabine activity by reducing CDA levels in a mouse model of PDA ¹⁵ . Thus, there are mounting evidences lending credence to CDA as a key protein involved in the resistance of PDA cells to treatment. Accordingly, the inventors generated CDA-null human PDA-derived cell lines using lentiviral vectors encoding specific shRNAs. The inventors found that targeting CDA strongly sensitizes PDA-derived cells to chemotherapy, both in vitro and in vivo, and induces apoptosis (data not shown).

However, to our surprise, the genetic depletion of CDA per se, in the absence of chemotherapy, profoundly inhibited PDA-derived cells proliferation, altered cell cycle progression, with a prolonged S phase, a hallmark of DNA replication stress, and impaired tumor growth, as half of the mice engrafted with Mia PACA-2-null CDA were free of tumors (Figure 2).

In order to identify the key pathways altered by CDA deficiency in PDA cells, the inventors then performed unbiased 2D-DIGE proteomic studies. The inventors found that the expression/stability of a number of proteins involved in mitochondrial function was altered in response to CDA deficiency. Accordingly, the inventors identified a decrease of the total ATP level and a switch in ATP production from mitochondrial to glycolytic source, so-called Pasteur effect, as the inventors observed when inhibiting mitochondrial oxidative phosphorylation with hypoxia or mitochondrial inhibitors eg. rotenone, antimycin A or metformin in human glioblastoma and acute myeloid cells ^16,17 . In addition, mitochondrial oxygen consumption in CDA-depleted cells is decreased compared to controls, with elevated mitochondrial ROS production and decreased reduced GSH/ oxidized GSSG, suggesting an impairment of the redox balance leading to an oxidative stress (Figure 3). Finally, using a global LC/MS-based metabolomic approach, CDA deficiency resulted in a profound decrease in both purine and pyrimidine productions, as well as multiple alterations in TCA cycle with elevated lactate production (Figure 4).

Inventor's aforementioned results demonstrate for the first time that targeting CDA, an enzyme involved in pyrimidine salvage pathway, strongly alters PDA proliferation and tumor progression, via massive modification of cancer cell metabolism and alteration of cell cycle, in the absence of chemotherapy.

Expression and role of CDA during PDA oncogenesis

As described above, the inventors found that PDA tumors expressed high levels of

CDA compared to normal parenchyma and that targeting CDA in human PDA-derived cells strongly alters cell proliferation and tumor progression. Invasive PDA arises through multistage genetic and histologic progression from microscopic precursor lesions designated as pancreatic intraepithelial neoplasia (PanIN) that are believed to develop and progress asymptomatically over several decades ¹⁸ ' ¹⁹ . An early event during malignant transformation is the acquisition of activating mutations in the KRAS oncogene which occurs in >90% of patients with PDA ²⁰ . PDA are highly "addicted" to this oncogene for multiple parameters influencing tumour initiation, progression and maintenance as demonstrated using genetically engineered mouse (GEM) models ²¹ . The inventors obtained preliminary results showing that CDA is upregulated at the mRNA level in human pancreatic cells transformed by the KRAS oncogene (Figure 6).

The inventors obtained preliminary data strongly suggesting that CDA is essential for PDA tumor initiation (figure 2).

Molecular mechanisms following CDA ablation in PDA cells.

Our aforementioned results demonstrate that targeting CDA in PDA cells results in alterations in both S-phase progression and energetic metabolism. Analysis of the DNA replication program in CDA-depleted PDA cells.

Rapid and accurate DNA replication is critical for cell proliferation and for the faithful transmission of genetic information to daughter cells. Proliferating cells are continuously exposed to a variety of events impeding the progression of replication forks, commonly referred to as replication stress (RS). Sources of RS include DNA lesions of endogenous or exogenous origin and regions of the genome that are intrinsically difficult to replicate, such as highly-transcribed genes, secondary DNA structures and tightly-bound protein-DNA complexes ^40,41 . Arrested replication forks represent a major source of genomic instability and RS has been implicated in various aspects of the cancer process. Cells exposed to acute RS activate a DNA-Damage-Response (DRR) when the fork collapse, and an ATR/Chkl- dependent checkpoint pathway known as the DNA replication checkpoint ⁴² to arrest the cell cycle, limit fork collapse and delay origin firing until RS is relieved. Moreover, it coordinates fork repair processes and allows the completion of DNA replication ⁴³ ' ⁴⁴ The inventors have found that CDA depletion perturbs the dynamics of the S-phase in

PDA cells (Figure 3) and induces the phosphorylation of CHKl kinase, two hallmarks of oncogene-driven replicative stress. This is highly unusual in the context of KRAS-promoted malignancies that commonly do not show any evidences of replicative stress nor activated DNA damage repair pathways; accordingly, CHKl inhibitors fail to hamper tumor progression in experimental models of PDA ⁴⁵ . In addition, the inventors found that CDA depletion results in the inhibition of both purine and pyrimidine cellular pools (Figure 4). Recent reports also demonstrate that CDA deficiency leads to under replication of cellular DNA, due to the partial inhibition of activity of the DNA Repair enzyme PARP-1 ⁴⁶ . Consequences of CDA targeting on PDA cell metabolism

To fuel its elevated demand for energy and macromolecular biosynthesis, PDA show increased nutrient acquisition that is coupled to increased flux through downstream metabolic pathways ⁵³ . In PDA cells grown in vitro, glycolysis predominates over mitochondrial oxidative phosphorylation of pyruvate, regardless of oxygen tension (Warburg effect). In PDA, oncogenic KRAS plays a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis (HBP) and nonoxidative pentose phosphate pathways (PPP), thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control ⁵⁴ . Furthermore KRAS signaling drives increased glutamine metabolism for cytosolic NADPH production to maintain redox homeostasis and support cell division and proliferation in PDA cells. Accordingly, recent reports have shown preclinical and clinical relevance for targeting cell metabolism and especially glutamine metabolism to overcome chemoresistance or radioresistance in human PDA or NSCLC cancers, respectively. In CDA-null Mia PACA-2 cells, the inventors found that global ATP levels are severely curtailed, with glycolysis and mitochondria equally participating to ATP production as a consequence of the Pasteur effect induction (Figure 3). This massive drop in ATP levels, combined with the decrease of 6-phosphogluconate production, could account for the major impact on phosphoribosyl pyrophosphate (PRPP) production, resulting in the reduction of both purine and pyrimidine levels and cell cycle progression as well as in the induction of an energetic crisis in these cells.

Interestingly, CDA depletion does not perfectly mimics KRAS extinction leading to decrease glycolysis and lactate production ⁵⁴ , as fructose- 1 ,6-diphosphate, glycerate 2/3 phosphate and lactate are elevated following CDA deficiency (Figure 4). Accordingly, the inventors measure the glycolytic flux in CDA-deficient cells and the expression of key proteins involved in glycolysis in response to CDA targeting. The inventors also found that CDA inactivation is accompanied by significant alterations to TCA cycle intermediates (citrate, cis-aconitate, Figure 4), that could be explained by the decrease of pyruvate dehydrogenase (PDH) expression identified by 2D-DIGE in CDA-null cells. However, the levels a-ketoglutarate (a-KG), succinate, fumarate and malate are maintained in these cells, suggesting alternative anaplerotic pathways for TCA supply and the absence of glutamine metabolism as opposed to KRAS extinction. Interestingly, KRAS-driven PDA cells are known to utilize alternative carbon sources to fuel the TCA cycle, such as glutamine ⁵⁴ . The first step in glutamine catabolism involves its conversion to glutamate catalyzed via the glutaminase enzymes (GLS1 and GLS2). Glutamate is a source of a-KG generated by the function of glutamine dehydrogenase (GLUD1) or glutamate-oxaloacetate transaminase (GOT1, GOT2). Thus, the glutamine conversion to a-KG, oxaloacetate and aspartate could be essential to CDA-depleted cells to survive. Interestingly, the inventors identified by 2D-DIGE in CDA-null cells a decrease of one of the SDH subunit linked to O2 consumption decrease (Figure 3).

Beside this, the pentose phosphate pathway (PPP) is important for tumorigenesis as it provides NADPH for macromolecules biosynthesis and ROS detoxification, as well as ribose 5-phosphate for DNA/RNA synthesis. RAS oncogene is known to promote resistance to oxidative stress trough GSH-based ROS scavenger pathways ⁵⁵ . Notably, the inventors found that the oxidative phase of the PPP was impaired in CDA-null cells (as measured following 6- phosphogluconate production), while PPP non-oxidative phase is unchanged.

Novel angles of therapeutic attack for PDA treatment

The inventors state that CDA, which is upregulated in cancer, is a regulatory master of

PDA oncogenesis that shares common features with KRAS oncogene but also presents unique opportunities to define new vulnerabilities for PDA treatment. Accordingly, the inventors define opportunity to replicative stress- and metabolic- based synthetic lethality strategies for PDA tumors depleted from CDA.

Drug Combinations to further boost replicative stress.

The inventors have already identified that CDA depletion induces the activation of CHK-1 that is highly evocative of replicative stress in PDA cells. Indeed, The ATR-CHK1 signaling cascade has a crucial role in limiting replicative stress and can be targeted by specific inhibitors such as the Chkl inhibitor SCH900776, to improve the effect of CDA inhibitors for their anti proliferative and anti tumoral activities on PDA experimental models. As CHKl activity is strongly enhanced by ATR-mediated phosphorylation, inhibition of ATR could produce similar responses to those observed with inhibition of CHKl, but with "added value". Indeed, ATR phosphorylates SMARCAL1 (a SWI/SNF family member that has annealing helicase activity), thereby limiting its fork regression activity and preventing the collapse of stalled replication forks ⁴² . Accordingly, specific ATR inhibitors, such as VE-821 could be interrogated during our program. Last, an alternative approach to infer with CHKl is to target WEEl . This kinase phosphorylates CDK1 and CDK2, rendering them less active. When WEEl is inhibited by drugs, CDK activity is enhanced and cells in S phase can be induced to enter mitosis prematurely, even before DNA replication is complete ⁵⁶ . Combined by the shortage of nucleotides provoked by CDA targeting, WEEl inhibition could not only reduce replication fork speed but also facilitate double strand breaks. Interestingly, WEEl inhibitors are currently under clinical evaluation for PDA. The inventors reveal alterations in the expression/activity of alternative DNA polymerases that could also be targeted together with CDA to maximize the replicative stress in PDA cells.

Drug Combinations to further block tumor metabolism.

The inventors have accumulated evidences that the tumor cell metabolism is massively altered following CDA targeting in PDA cells. This study reveals for the first time how essential pyrimidine salvaging is for this tumor type. Interestingly, other pathways are crucial for maintaining the cellular pyrimidine pool, such as the "Je novo" pathway driven by dihydroorotate dehydrogenase (DHODH). This pathway is present and functional, it may represent another therapeutic angle to target the newly described "addiction" of PDA cells to pyrimidine. Accordingly, the inventors evaluate the antiproliferative and antitumoral activity of leflunomide, a well-known inhibitor of DHODH, used in active moderate-to-severe rheumatoid arthritis and psoriatic arthritis ⁴⁹ , in combination with CDA inhibitors, in experimental models of PDA.

While complementary experiments are still needed, our results strongly suggest an exquisite addiction of CDA-null PDA cells to glutamine. This finding opens several other combinatory therapeutic options as glutaminase (CB-839) and a-KG dehydrogenase (CPI-613, a lipoate analog, inhibits mitochondrial enzymes pyruvate dehydrogenase (PDH) and a-KG dehydrogenase in NCI-H460 cell line, disrupts tumor cell mitochondrial metabolism) inhibitors are being evaluated clinically for PDA patients ⁵³ . The redox potential of CDA- depleted cells could be severely altered, as we measured a decrease in 0 ₂ consumption, an increase of mitochondrial ROS with concomitant decrease of GSH/GSSG ratio (Figure 4); remarkably, we identified adenosylhomocystemase (AHCY) as upregulated in CDA-null cells probably to compensate the deleterious effects of CDA depletion by upregulating GSH levels, offering new targeting possibilities using drugs against this enzyme (3-deazaneplanocine A, DZNe) in combination with small inhibitors against CDA.

REFERENCES:

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

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