WO2018078083A1 - New method for treating multiple myeloma - Google Patents

Abstract

The present invention relates to a method of treating multiple myeloma. The inventors analyzed two sets of 18 and 40 samples from symptomatic t(4; 14) MM at presentation by exome or RNA sequencing, respectively. They confirm the high mutational rates in the NRAS, KRAS, BRAF and FGFR3 genes which have been previously described, and strongly suggests that these events are mutually exclusive in t(4;14) MM. Mutations in ATM/ATR, MAPK and MYCBP2 occur at relatively high frequencies (11.4%, 14% and 8% respectively), while very few t(4;14) patients carry alterations in FAM46C or CCND1. Mutations in PRKD2, the gene coding for the PKD2 serine/threonine kinase, affect around 11.4% of the cases and this alteration is associated with progression to symptomatic myeloma in one patient. The inventors also tested the inhibition of PKD2 activity by kb NB 142-70 and the inhibition of PKD 1, PKD2 and PKD3 by CRT0066101 and observed that these agents induced cell growth arrest and apoptosis of tumor plasma cells in vitro. These results show that PKD2 and others members of the PKD family is a therapeutic target in patients with MM. Thus, the present invention relates to a PKD inhibitor for use in the treatment of multiple myeloma.

Description

NEW METHOD FOR TREATING MULTIPLE MYELOMA

FIELD OF THE INVENTION:

The present invention relates to a PKD inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Multiple myeloma (MM) is an incurable hematological disorder that arises from the transformation of a plasma cell through a stepwise genetic mutational process. MM is a heterogeneous disease and despite recent therapeutic progress, while some patients show long term survival, many others respond poorly to treatments and have short overall survival. There are two major cytogenetic events which are known to correlate with these variable clinical outcomes, namely copy number abnormalities (CNAs) and translocations involving the immunoglobulin heavy chain locus on chromosome 14 (Avet-Loiseau et al., 2007). Hyperdiploidy is the most frequent CNA, which is observed in more than 50% of cases and is associated with favorable prognosis (Avet-Loiseau et al, 2009). Conversely, other CNAs such as the common del(17p), del(lp) or gain(lq), which are found in approximatively 10%>, 9% and 38%o of patients respectively, are associated with adverse prognosis. In addition, the t( 11 ; 14) translocation, which affects around 15% of the patients, indicates a favorable prognosis, while the translocations t(4;14), t(14;16) and t(14;20), which respectively account for 15%, 4% and 1%) of cases, are associated with poor outcomes (Avet-Loiseau et al, 2007; Avet-Loiseau et al, 2009; Fonseca et al, 2003). However, these CNAs and translocations are also detected in patients with monoclonal gammopathy of undetermined significance (MGUS) which is regarded as the pre -tumor stage of MM, indicating that additional events likely cooperate with these initial genetic lesions to induce the development of the disease. This is demonstrated clearly by the subgroup of MM patients bearing the t(4; 14) translocation. Although this subgroup is characterized by an overall poor prognosis, within the group there is still considerable heterogeneity, with both "high risk" and "standard risk" patients (Moreau et al., 2007). In addition, the t(4; 14) translocation has been seen in some cases of asymptomatic MM as well as in patients with MGUS (Karlin et al, 2011), suggesting that, beside the translocation itself, each patient undergoes specific oncogenic processes. Unlike other translocations recurrently affecting tumor plasma cells, the t(4;14) deregulates two genes, namely FGFR3 and MMSET. Although FGFR3 was initially suggested to be the driver oncogene in t(4;14) MM, it is lost by deletion of the der(14) chromosome in about 30%> of cases, pointing towards a dominant role of MMSET during transformation. Recently, we have shown that del(17p), when occurring in patients expressing a truncated MMSET protein from the MB4-2 breakpoint translocated allele indicates an especially poor prognosis. However, the genetic abnormalities specific for t(4;14) MM or those associated with their adverse outcomes are yet to be characterized.

The recent development of next generation sequencing has led to the identification of numerous somatic genetic aberrations affecting tumor plasma cells. Activating mutations in NRAS, KRAS and BRAF frequently occur and together affect more than 43% of MM patients independently of the initial genetic lesions that characterize MM subgroups. Other genes such as TRAF3, CYLD, DIS3 or FAM46C are also mutated, however, none of these lesions appear to have an impact on the survival of patients. In contrast, mutations in CCND1, which regulates cell cycle, and alterations in the TP53, ATM or ATR genes, which are involved in the response to DNA damage, all indicate adverse prognosis. Similarly, deregulation of MYC, which occurs via translocation or amplification in all subgroups of MM, is associated with shorter overall survival. Thus, genome/exome sequencing is a powerful approach to establish correlations between specific genetic alterations and patients outcomes.

SUMMARY OF THE INVENTION:

The present invention relates to a PKD inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors analyzed two sets of 18 and 40 samples from symptomatic t(4; 14) MM at presentation by exome or RNA sequencing, respectively. Their work confirms the high mutational rates in the NRAS, KRAS, BRAF and FGFR3 genes which have been previously described, and strongly suggests that these events are mutually exclusive in t(4;14) MM. Mutations in ATM/ATR, MAPK and MYCBP2 occur at relatively high frequencies (11.4%, 14% and 8% respectively), while very few t(4;14) patients carry alterations in FAM46C or CCND1. Mutations in PRKD2, the gene coding for the PKD2 serine/threonine kinase, affect around 11.4% of the cases and this alteration is associated with progression to symptomatic myeloma in one patient. Thus, the inventors test the inhibition of PKD2 activity by kb NB 142- 70 and the inhibition of PKD1, PKD2 and PKD3 by CRT0066101 and observed that these agents induced cell growth arrest and apoptosis of tumor plasma cells in vitro. Thus, these results show that PKD2 and others members of the PKD family is a therapeutic target in patients with MM. Accordingly, the present invention relates to a PKD inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

As used herein the term, "PKD" for "protein kinase D" has its general meaning in the art and denotes a family of three enzymes: PKD1, PKD2 and PKD3.

Thus, in one embodiment, the invention relates to a PKD1, or a PKD2 or a PKD3 inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

In a particular embodiment, the invention relates to a PKD2 inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

As used herein, the term "subject" denotes a mammal. In a preferred embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted with multiple myeloma.

As used herein, "multiple myeloma" or "MM" has is general meaning in the art. MM is classically subdivided into subtypes based on recurrent chromosomal changes, including copy number abnormalities and chromosomal translocations. The main sub-groups are characterized by copy number abnormalities, including hyperdiploidy, gain of chromosome 1 q21 and deletion of chromosome 17p, or by chromosomal translocations, including the t(l l;14) (ql3;q32), the t(4;14) (pl6;q32), the t(14;16) (q32;q23), the t(14;20) (q32;ql l) and, more rarely, the t(6;14) (p21;q32). Some of these abnormalities have been shown to dictate patient outcome. For example, hyperdiploidy, which affects 50% to 60% of patients and the t(l 1 ; 14) translocation (20%) of cases) are associated with good prognosis. Conversely, gain of lq (30% to 35%), deletion of 17p and t(4;14) or t(14;16) translocations determine poorer outcomes.

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, "PKD1" for "protein kinase Dl" has its general meaning in the art and denotes an enzyme that in humans is encoded by the PRKD1 gene (NCBI sequence number of the protein: NP 002733.2).

As used herein the term, "PKD2" for "serine/threonine-protein kinase D2" has its general meaning in the art and denotes an enzyme that in humans is encoded by the PRKD2 gene (UniProtKB/Swiss-Prot sequence number: Q9BZL6.2 or NCBI numbers of the different isoforms NP 001073349.1, ΝΡ ΟΟΙ 073350.1, ΝΡ ΟΟ 1073351.1 or NP 057541.2).

As used herein the term, "PKD3" for "serine/threonine-protein kinase D2" has its general meaning in the art and denotes an enzyme that in humans is encoded by the PRKD2 gene (NCBI sequence number of the protein: NP 005804.1).

The term "PKD inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PKD family. The term "PKD inhibitor" also refers to a compound that selectively blocks or inactivates the kinase activity of the PKD enzyme family that is to say the transfer of a phosphate group to another protein. The term "PKD inhibitor" also refers to a compound that inhibits PKD family expression. The term "PKD2 inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PKD2. The term "PKD2 inhibitor" also refers to a compound that selectively blocks or inactivates the kinase activity of the enzyme that is to say the transfer of a phosphate group to a protein. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PKD2 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the PKD family (PKD1 or PKD3 for example). Compounds that block or inactivate PKD2, but that may also block or inactivate other PKD sub-types, as partial or full inhibitors, are contemplated. The term "PKD2 inhibitor" also refers to a compound that inhibits PKD2 expression.

The term "PKD1 inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PKD1. The term "PKD1 inhibitor" also refers to a compound that selectively blocks or inactivates the kinase activity of the enzyme that is to say the transfer of a phosphate group to a protein. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PKD1 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the PKD family (PKD2 or PKD3 for example). Compounds that block or inactivate PKD1, but that may also block or inactivate other PKD sub-types, as partial or full inhibitors, are contemplated. The term "PKDl inhibitor" also refers to a compound that inhibits PKDl expression.

The term "PKD2 inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PKD2. The term "PKD2 inhibitor" also refers to a compound that selectively blocks or inactivates the kinase activity of the enzyme that is to say the transfer of a phosphate group to a protein. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PKD2 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the PKD family (PKDl or PKD3 for example). Compounds that block or inactivate PKD2, but that may also block or inactivate other PKD sub-types, as partial or full inhibitors, are contemplated. The term "PKD2 inhibitor" also refers to a compound that inhibits PKD2 expression.

The term "PKD3 inhibitor" has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PKD3. The term "PKD3 inhibitor" also refers to a compound that selectively blocks or inactivates the kinase activity of the enzyme that is to say the transfer of a phosphate group to a protein. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PKD3 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the PKD family (PKDl or PKD2 for example). Compounds that block or inactivate PKD3, but that may also block or inactivate other PKD sub-types, as partial or full inhibitors, are contemplated. The term "PKD3 inhibitor" also refers to a compound that inhibits PKD3 expression. Typically, a PKD inhibitor or a PKD1 inhibitor or a PKD2 inhibitor or a PKD3 inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, an intra-antibody (or intrabody), an oligonucleotide or a ribozyme.

In one embodiment, the compound 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.

Inhibitors of PKD are well known in the art (see for example LaVallea CR et al, 2010).

In one embodiment, the PKD inhibitor and particularly the PKD2 inhibitor according to the invention is the kb NB 142-70 compound as described in Bravo-Altamirano K et al., 2011.

In another embodiment, PKD inhibitor according to the invention are the indolocarbazole G66976 compound, the CID755673 compound, the CRT006610 compound, the CRT0066101 compound as described in LaVallea CR et al, 2010.

In one embodiment, the PKD inhibitor according to the invention is an antibody. Antibodies or directed against PKD 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 PKD 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 al., 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 anti- PKD single chain antibodies. Coumpounds useful in practicing the present invention also include anti- PKD 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 PKD. Humanized anti-PKD 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 of PKD are selected.

In a particular embodiment, neutralizing antibodies of PK1 are selected.

In a particular embodiment, neutralizing antibodies of PKD2 are selected.

In a particular embodiment, neutralizing antibodies of PKD3 are selected.

In a particular embodiment, the antibody according to the invention may be the D1A7 sell by Cell Signaling Technology.

In another embodiment, the antibody according to the invention is a single domain antibody against PKD and particularly against PKD2. 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 PKD inhibitor according to the invention is an intra-antibody that is to say antibody that works within the cell to bind to an intracellular protein and thus bind to a PKD in the cell.

In a particular embodiment, the intra-antibody is against PKD1.

In a particular embodiment, the intra-antibody is against PKD2.

In a particular embodiment, the intra-antibody is against PKD3.

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 al., 1996).

Then, for this invention, neutralizing aptamers of PKD are selected.

In a particular embodiment, neutralizing aptamers of PKD 1 are selected.

In a particular embodiment, neutralizing aptamers of PKD2 are selected.

In a particular embodiment, neutralizing aptamers of PKD3 are selected.

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

In a particular embodiment the polypeptide is an inhibitor of PKD capable to prevent the function of PKD and particularly PKD2.

In one embodiment, the polypeptide of the invention lay 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 PKD 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 bio distribution, 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 PKD inhibitor according to the invention is an inhibitor of PKD gene expression. In a particular embodiment, the PKD inhibitor is an inhibitor of PKD2 gene expression.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of PKD expression for use in the present invention. PKD gene expression can be reduced by contacting a 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 PKD gene 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 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 PKD gene expression 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 PKD mR A sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential R A 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 PKD gene expression 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 PKD. 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 nonessential 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 microencapsulation.

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 order to test the functionality of putative PKD2 inhibitor on multiple myeloma, an in vivo test is necessary. For that purpose, we developed a xenograft mouse model by injecting luciferase (luc) expressing human MM cell lines (NCI-H929, U266, JIM3 and RPMI) into immuno-deficient mice (NOD/SCID/Gamma-c-/-) as previously described (Bono et al, 2012). Engrafted mice may be treated (or not) with the inhibitor and the growth of multiple myeloma cells may be monitored in vivo by whole-body photon emission.

Another object of the invention relates to a method for treating multiple myeloma comprising administering to a subject in need thereof a therapeutically effective amount of a PKD inhibitor.

In a particular embodiment, the invention relates to a method for treating multiple myeloma comprising administering to a subject in need thereof a therapeutically effective amount of a PKD1 inhibitor.

In a particular embodiment, the invention relates to a method for treating multiple myeloma comprising administering to a subject in need thereof a therapeutically effective amount of a PKD2 inhibitor. In a particular embodiment, the invention relates to a method for treating multiple myeloma comprising administering to a subject in need thereof a therapeutically effective amount of a PKD3 inhibitor.

In another embodiment, the invention relates to a PKD inhibitor for use in the treatment of multiple myeloma in a subject diagnosed with a t(l 1; 14) translocation.

In a particular embodiment, the invention relates to a PKDl inhibitor for use in the treatment of multiple myeloma in a subject diagnosed with a t(l 1 ; 14) translocation.

In a particular embodiment, the invention relates to a PKD2 inhibitor for use in the treatment of multiple myeloma in a subject diagnosed with a t(l 1 ; 14) translocation.

In a particular embodiment, the invention relates to a PKD3 inhibitor for use in the treatment of multiple myeloma in a subject diagnosed with a t(l 1 ; 14) translocation.

According to the invention, the subject diagnosed with a t(l 1 ; 14) translocation suffers from a multiple myeloma.

In another embodiment, another compound used to usually treat multiple myeloma can be used in combination with the PKD inhibitor and particularly the PKD2 inhibitor.

Thus, the invention also relates to i) a PKD inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma in a subject in need thereof.

In a particular, the invention also relates to i) a PKD 1 inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma in a subject in need thereof.

In a particular, the invention also relates to i) a PKD2 inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma in a subject in need thereof.

In a particular, the invention also relates to i) a PKD3 inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma in a subject in need thereof.

In another embodiment, the invention also relates to i) a PKD inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma diagnosed with a t(l 1 ; 14) translocation in a subject in need thereof.

In another particular embodiment, the invention also relates to i) a PKDl inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma diagnosed with a t(l l;14) translocation in a subject in need thereof.

In another particular embodiment, the invention also relates to i) a PKD2 inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma diagnosed with a t(l l;14) translocation in a subject in need thereof.

In another particular embodiment, the invention also relates to i) a PKD3 inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma diagnosed with a t(l l;14) translocation in a subject in need thereof.

According to the invention, compounds usually use to treat multiple myeloma may be selected in the group consisting of Bortezomib (Velcade, PubChem number 387447), Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil, and Bendamustine (Treanda), dexamethasone, thalidomide analogues and antibodies like Daratumumab (Darzalex).

Therapeutic composition

Another object of the invention relates to a therapeutic composition comprising a PKD inhibitor according to the invention for use in the treatment of multiple myeloma.

In a particular embodiment, the invention relates to a therapeutic composition comprising a PKDl inhibitor, a PKD2 inhibitor or a PKD3 inhibitor according to the invention for use in the treatment of multiple myeloma.

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 and the like.

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 a PKD inhibitor 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, etoposide, 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, tetrahydrocannabinols, 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 an 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.

Screening method

In a further aspect, the present invention relates to a method of screening a candidate PKD inhibitor for use in the treatment of multiple myeloma, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that inhibit PKD.

In a particular embodiment the invention is suitable to screen PKD1 inhibitor, PKD2 inhibitor or PKD3 inhibitor.

In a further aspect, the present invention relates to a method of screening a candidate

PKD inhibitor for use in the treatment multiple myeloma) in a subject in need thereof, wherein the method comprises the steps of:

- providing a cell, tissue sample or organism expressing PKD that is to say PKD 1 ,

PKD2 or PKD3;

providing a candidate compound such as small organic molecule, antibodies, intra-bodies or polypeptide,

measuring the activity of PKD,

- and selecting positively candidate compounds that inhibit PKD that is to say

PKD1, PKD2 or PKD3.

Methods for measuring the inhibition of PKD and specifically pPKD2 are well known in the art. For example, myeloma-cell growth can be monitored by measuring incorporation of radio labeled Thymidine ([methyl-3H] thymidine (ΙμΟΛνεΙΙ)) in cells (including cell lines MM. IS, RPMI 8226, U266, NCI-H929 and JIM3, or plasma cells isolated from MM patients) cultured with various concentrations of the inhibitor for up to 24 hours in vitro. Measurement of apoptosis can also be carried out using Apoptosis Detection Kits, (Miltenyi Biotec GmbH). Briefly, MM cells are treated without and with PKD Inhibitor for 48 hours. Cells are harvested, washed with cold PBS and stained with Annexin V-APC and 7-AAD in binding buffer at room temperature for 15 minutes in the dark. Fluoresce is measured by flow cytometry.

Diagnostic method

The invention also relates to a method for diagnosing multiple myeloma in a patient comprising determining in a tumor sample obtained from said patient any substitution in the coding region of PRKD2. In one embodiment, the patient has a t(4; 14) translocation.

In another embodiment, the patient has at least one substitution selected in the group consisting of I582T, V385E and V385E in the coding region of PRKD2.

As used herein and according to all aspects of the invention, the term "sample" denotes tumor sample, blood, serum or plasma.

Actually numerous strategies for genotype analysis and mutations detection are available (Antonarakis et al, 1989 ; Cooper et al, 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct enzymatic test for the mutation. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC) (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology (see e.g. Little et al, 1996); and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base substitution mutations. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized (Nickerson et al., 1990).

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. PRKD2 is expressed in tumor plasma cells. Expression of PRKD2 (probe 209282_at, Affymetrix U133Plus2.0 array) in CD138+ plasma cells from bone marrow of patients with newly diagnosed multiple myeloma from the data set of Zhan and colleagues (Zhan et al., 2006) (GEO accession GSE2658). Data was extracted from a total of 252 patients distributed across 6 myeloma disease subtypes based on transcriptomic clustering : the group overexpressing Cyclin D genes (CCND1 or CCND3) (CD group, n=64), the group with a hyperdiploid signature (HY group, n=65), the group characterized by signature of: low bone disease (LB group, n=30), overexpression of c-MAF or MAFB (MF group, n=21), overexpression of MMSET (MS group, n=43), and by a proliferative signature (PR group, n=29). The graph shows log2 -transformed values of normalized expression (Affymetrix Microarray Suite GCOS1.1 softwaresuite).

Figure 2. PKD2 inhibitor kb NB 142-70 inhibits growth of MM plasma cells. (A)

Dose-related effects of kb NB 142-70 on proliferation of MM cell lines. Cells were cultured with indicated concentrations of kb NB 142-70. Uptake of [3H]-thymidine was measured during the last 10 hours of 48-hour cultures. (B) Survival of CD138+ plasma cells from bone marrow of MM patients in the presence or absence of kb NB 142-70 (10μΜ). MTT cleavage was measured during the last 4 hours of 48-hour cultures. (C) Apoptosis of MM cell lines and plasma cells from patients. Cells were culture with or without kb NB 142-70 for 48h and analyzed for AnnexinV and 7AAD staining by flow cytometry. Histograms show the percentage of AnnexinV/7AAD negative live cells. Data shown are the mean ± SD of experiments performed in triplicate (except for apoptosis of MM cells from patients).

Figure 3. PKD inhibitor CRT0066101 inhibits growth of MM cell lines. (A.) Dose- related effects of CRT0066101 on proliferation of MM cell lines. Cells were cultured with the indicated concentrations of CRT0066101. Proliferation was measured with the Cell Proliferation Kit 1 (MTT) from Sigma-Aldrich after a 48-hour culture. Data shown are the meant SD of experiments performed in triplicate.

EXAMPLE:

Material & Methods

Patient Samples

Samples were taken, after informed consent, from patients newly diagnosed with myeloma. Patients with a suboptimal response were randomly assigned to pretransplant treatment with a proteasome inhibitor triplet. Older or less fit patients did not receive an autologous stem-cell transplant. We sequenced the protein-coding exome of 18 tumour samples from 17 patients with MM. Two serial samples were available for one patient at diagnosis of asymptomatic MM and at progression to symptomatic MM. Fluorescence in situ hybridization (FISH) was available for all patients. The 17 patients [10 males and 7 females; median age 61.4 years (range 44-78) with a median follow-up since diagnosis of 16 months] was combined with 6 t(4;14) MM patients published (Bolli et al, 2014). 40 patients, from the IFM-DFCI 2009 cohort (NCT01191060), had a proteasome inhibitor triplet with autologous stem-cell transplant. t(4;14) was detected by classical FISH as described previously, in 40 samples (13,6% of all samples). 19/40 t(4;14) patients were treated with ASCT in first line. The t(4;14) was also confirmed by RT-PCR in 35 samples (87.5%). In total, we sequenced 27 samples from patients with t(4;14) by whole exome sequencing and 40 patients by RNA-seq. (31 males, 27 females and 21 NA; median age 58 years (range 30-65) with a median follow-up since diagnosis of 29 months).

Exome Sequencing and Variants Calling

Plasma cells from BM were CD138-selected using a magnetic-activated cell sorting system (Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany; http://www.miltenyibiotec.com). For each patient, DNA from both tumor and non-tumor samples were used for the exome capture and libraries were generated with the Nextera DNA Library Preparation Kits (Illumina). Samples were sequenced on Illumina HiSeq 2500 sequencer (paired end lOObp reads) and data were further analyzed on the Galaxy platform at (https://galaxy-public.curie.fr/). Alignment to the GRCh37/hgl9 genome was performed with Bowtie2 (v.2.1.0) and BAM files were piled up using Samtools (Mpileup). Single nucleotide variants (SNVs) and short indels for paired tumor and normal files were called using VarScan Somatic (v.2.3.5) with minimum coverage for normal and tumor samples of 8 and 6 respectively. Variants were annotated using ANNOVAR. All SNV and Indels were validated by direct analysis on IGV (v.2.3.66, Integrative Genomics Viewer, Broad Institute).

Copy-number analysis

Copy number across the exome was determined using control-FREE copy number and allelic content caller Control-FREEC (v.6.7) (http://bioinfo-out.curie.fr/projects/freec/) and cancer clonal fraction calculated. Copy number across the exome was determined using Control-FREEC using 500 base pair bins, each overlapping with the subsequent and previous 250bp. A minimum average read depth of 50 was required in the control samples, with at least 2 neighboring bins required to show copy number aberration to call a region as gained or lost. To assess the CN state of mutated genes, genes mapping in CNA regions were retrieved using UCSC RefSeq transcript annotation track. The Circos plot represents somatic amplifications (red) and deletions (blue) found in each case, distributed across all chromosomes. Samples are displayed according to increasing case number from inner to outer track.

Somatic Signature analysis We performed a mutational signature analysis in order to determine the contributions of mutational signatures previously described in multiple myeloma (Bolli et al., 2015) in our series. The input data was the proportion of mutations belonging to the 96 possible mutation categories, taking into account the type of substitution and the surrounding bases at 5' and 3' of the mutated base, as previously described (Alexandrov et al, 2013). The sequence context of each mutation was retrieved using the R package SomaticSignatures (Gehring et al., 2015). We used non-negative matrix factorization (NMF) to decompose our mutation matrix M (n x r, where n is the 96 type mutation categories and r is the number of samples) as the product of a matrix P (n x p, where n is the 96 type mutation categories and p is the mutational processes) representing the signature of each mutagenic process and a matrix E representing the exposure of each tumor to each process. The P matrix comprised of a pre-defined set of signatures (COSMIC signatures 1, 2, 5 and 13) previously identified in multiple myeloma (Forbes et al, 2015). We implemented the fast combinatorial strategy approach from the NMF package (Gaujoux and Seoighe, 2010) in order to solve the following nonnegative least squared problem. min ||P - M*E||_F, s.t. E >=0 where |.|_F is the Frobenius norm. The resulting output E(r x p) gives the intensity of each process in each tumor. Signatures contributing less than 5% of mutations were excluded from the model.

RNA sequencing

RNA purification and preparation was performed as previously described (Rashid et al, 2014). Briefly, RNA was extracted from purifued CD 138 positive (purity >90%) cells. RNA quality was determined on the Bioanalyzer using the RNA Pico Kit (Agilent, Santa Clara, CA). We used 100 ng of total RNA for each sample. Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA). Libraries were analyzed by quantitative polymerase chain reaction (PCR) using the Universal Library Quantification Kit for Illumina (Kapa Biosystems, Wilmington, MA) and run on the 7900HT Fast quantitative PCR machine (ABI, Grand Island, NY). Libraries passing QC were diluted to 2 nM using sterile water, and then sequenced on the HiSequation 2000 (Illumina, San Diego, CA) at a final concentration of 12 pM, following all manufacturers' protocols.

RNA sequencing data analysis

We followed a computational workflow for the analysis of mutational profiles on RNA- seq data using a set of possibly unmatched normal samples from the same study, as reported previously (Mosen-Ansorena et al., ASH, 2015). Briefly, we first ran FASTQC to check the quality of the paired-end 50bp reads in all samples and then used the spliced aligner MapSplice (version 2.1.7) (Wang et al., 2010) in order to map the reads to the transcriptome of the human reference genome hgl9 (GRCh37). We subsequently followed GATK's best practices for RNA-seq variant detection. We used the computational algorithms VEP (Cunningham et al., 2015), Sorting intolerant from tolerant (SIFT, http://sift.jcvi.org/www/SIFT_dbSNP.html) and Polymorphism pheno-typing (Polyphen, http://genetics.bwh.harvard.edu/pph2/dbsearch.shtml) to perform mutation annotations. A number of filters were finally applied to reduce the initial set of variants to a collection where most of the variants are the more likely to be somatic mutations, as opposed to having a germline or artefactual origin. In order to minimize potential batch effects, we also removed recurrent variants associated with certain batches.

Correlation Studies

Correlation between mutated genes and cytogenetic abnormalities was determined using

Pearson product-moment correlation coefficient (PCC) performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com). Correlation coefficients were plotted using corrplot (http://cran.r- project.org/web/packages/corrplot/index.html).

Antibody and chemicals

kb NB 142-70 ( R&D systems (TOCRIS)) and Bortezomib (formerly PS-341; Velcade;JANSSEN-CILAG) were dissolved in DMSO. Other reagents were obtained as follows: PMA (phorbol-12-myristate- 13 -acetate, Sigma Aldich). Antibodies raised against PRKD2, phospho-PRKD2(Ser876) were purchased from Merck Millipore (Merck); Antibodies raised against Aktl , phospho-Akt(Ser308 ), p44/42 MAPK were purchased from Cell Signaling Technology) and phospho-ERK (E-4) was purchased from Santa CruzBiotechnology.

Cell culture

Cell lines JIM3,NCIH929MM.1 S, RPMI8226 and U266 were maintained in RPMI 1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 10 μg/ml streptomycin, and 2 mM L-glutamine. The LP1 cell lines was maintained in DMEM medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 10 μg/ml streptomycin, and 2 mM L-glutamine. For inhibition assays, cells were plated at 2 x 105 cells/mL, and grown with (or without) 20μΜ kb NB 142-70 for 24 hours. For activation, cells were treated with PMA (0Λμg/mL) for 20 minutes.

Cell lysis and Western blotting

For western blot, cells were harvested and lysed on ice for 30 minutes with 30 of a modified NP40 lysis buffer (1% NP-40, 1% SDS, 50 mM Tris (Ph-7.5), 150 mM NaCl, deuterium depleted water, 10 % CompleteTM Protease Inhibitor Cocktail (Roche), 1 % phosphatase inhibitor and 2mM phenylmethylsulfonyl fluoride (Sigma Aldrich). Cell lysates (3(^g/lane) were separated by 8% SDS-PAGE prior to electrophoretic transfer onto AmershamTM ProtranTM nitrocellulose membranes (Amersham Biosciences, Inc.). After blocking with 5% nonfat milk in phosphate-buffered saline-Tween 20 buffer at room temperature for 1 h, membranes were sequentially blotted with the indicated specific primary Abs and then with horseradish peroxidase-conjugated secondary mouse or rabbit Abs and were developed using chemiluminescence (Amersham Biosciences, Inc.).

DNA synthesis and cell-proliferation assay

Cells were plated at 2.0 x 105 cells/mL in a volume of ΙΟΟμΙ in 96 well plates in triplicate. All cells were treated with kb NB 142-70 for 48 hours at concentrations of Ο.ΙμΜ, 0.3μΜ, ΙμΜ, and 3μΜ, then labeled with [methyl-3H] thymidine (^Ci/well) overnight at 37°C. Thymidine incorporation was analyzed using a liquid scintillation counter (Wallace, PerkinElmer).

Colorimetric assays were also performed to assay drug activity. Cells from 48-hour cultures were pulsed with 10 μΐ, of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT; Sigma- Aldrich) to each well for 4 hours, followed by 100 μΐ, isopropanol that contained 0.04 HC1. Absorbance readings at a wavelength of 570 nm were taken on a spectrophotometer (Bio-Tek, Inc.).

Annexin/7-AAD stain

Measurement of apoptosis was carried out using the Apoptosis Detection Kit, Miltenyi

Biotec GmbH. Briefly, MM cell lines were treated without and with PKD Inhibitor, kb NB 142- 70 (ΙμΜ and 3μΜ) for 48 hours. Cells were harvested, washed with cold PBS and stained with Annexin V-APC and 7-AAD (eBioscience) in binding buffer at room temperature for 15 minutes in the dark. Stained cells were fixed in binding buffer and acquired on a BD FACSCanto II (Becton Dickinson) 20 min later using Diva Software (v.8.0.1). The flow cytometric data was analyzed by Flowjo (v7.6.5).

Results

Landscape of genetic alterations in t(4;14) MM.

We analyzed 24 tumor-normal paired samples from 23 patients with t(4;14) MM using whole exome sequencing (WES), copy number variation and cytogenetics. Among those, 6 samples were collected from asymptomatic patients and 18 were collected from symptomatic myeloma at diagnosis. The 3 molecular breakpoints (MB) of the translocation within the WHSC1/MMSET gene, referred to as MB4-1, -2 and -3, were determined in 17 samples. Nine symptomatic patients displayed a del(17p) by FISH, while none of the asymptomatic cases had this aberration. Copy-number alterations, which were analyzed from the exome sequencing data identified known recurrent aberrations occurring in t(4;14) MM such as del(lp), (+lq), del(13), del(14q) and del(17p). Gain of chromosome 3, 7 and 15 was observed in 1 of the 6 asymptomatic patients but was not detected in symptomatic MM (data not shown). Exome sequencing of the 24 samples led to identification of 641 non- silent coding mutations, 337 silent variants in coding sequences and 51 insertion/deletion (indels) for a total of 1029 lesions (Mean 42.88 variants per sample ± 18.19) (data not shown), here was no correlations between the number of variants and the molecular breakpoint or the stage of disease, however patients with del(17p) displayed higher numbers of both nonsynonymous and total mutations (Mean 34.74 and 55.75 variants per sample respectively) compared to the none del(17p) cases (Mean 22.69 and 36.44 variants per sample respectively) (data not shown).

To explore whether t(4; 14) MM experienced specific mutational processes, we extracted the mutational signatures using context dependent base substitutions from the exome sequencing data available from 24 samples. In line with previous studies (Alexandrov et al., 2013; Bolli et al., 2014), signatures corresponding to a generic mutation pattern with enrichment of C>T transitions at CpG dinucleotides (Signature 1) or characterized by T>C mutations (Signature 5), corresponded to the majority of variants in t(4; 14) patients. Two additional signatures (Signature 2 and 13), characterized by C>T, C>G and C>A mutations in a TpC context, which are thought to arise from over-activity of members of the APOBEC family of cytidine deaminases, also contributed to the mutational spectrum but affected a minority of variants ((data not shown). Overall, we found that there was a bias towards C>T transition, independently of the transcribed strand, in all t(4; 14) MM, including asymptomatic casesWe next analyzed our exome data set to identify genes that were mutated in at least two of the 18 symptomatic patients and could therefore represent driver events in t(4; 14) MM data not shown). Non silent mutations in the FGFR3 gene were found in 4 cases (22%) and affected both extra- and intra-cellular domains of the receptor (data not shown). Activating mutations in KRAS or NRAS (affecting codons 12, 13, and 61) were present in 5 (27%) and 2 (11%) patients, respectively, while BRAF (V600E) was mutated in a single case. Of note, alterations in FGFR3, NRAS, KRAS and BRAF appeared all mutually exclusive. However, analysis at the reads level revealed that in most cases, the tumor contained a small proportion (<0.5%) of reads bearing an activating mutation in a second one of these genes, suggesting the presence of minor subclones carrying this variant (data not shown). In addition, we found mutations in genes encoding members of the mitogen-associated protein kinase (MAPK) family in 2 patients (9.5%), including MAP2K7 (S311L) and MAP4K4 (S758F). Altogether, mutations of genes involved in the FGFR3/RAS/BRAF/MAPK pathway were the most prevalent (14/18 patients). Interestingly, PRKD2, a gene coding for the PKD2 serine/threonine kinase, appeared mutated in 4 patients (22%). We also identified mutations in the DNA damage response genes TP53 and ATM in 1 and 3 cases respectively, with TP53 being impaired in a del(17p) patients. Furthermore, the gene expressing the long non-coding R A, MALAT1 was mutated in 4 cases, including one patient (P6) who carried two independent lesions at the asymptomatic stage as well as after progression to a symptomatic stage (data not shown). Beside these relatively frequent alterations, we found mutations in other genes, namely DIS3 (2/18), DENND3 (2/18), and MAX (2/18). Of note, mutations in FGFR3 were observed in 2 asymptomatic cases, while KRAS, MAPK and EZH2 mutations were in a single patient each (data not shown).

To validate and extend the search for mutations, we performed RNA-seq on 40 additional t(4; 14) MM patients at diagnosis and analyzed the mutational profiles of transcribed genes (data not shown). In line with the exome sequencing data, we found mutations in FGFR3, KRAS, NRAS and BRAF in 8 (20%), 3 (7.5%), 4 (10%) and 2 (5%) patients, respectively. Again, these alterations were all mutually exclusive. Six cases (15%) carried a non- silent mutation in at least one member of the MAPK family. MAP3K4 was mutated in 5 patients including 3 cases with a G538R substitution. Mutations in ATM/ATR and PRKD2 were relatively frequent and were present in 4 (10%>) and 3 (7.5%) cases, respectively. Other genes such as MYCBP2, MKI67 and DNND3, which were altered in a single case in the cohort analyzed by WES, were found mutated by RNAseq in 4, 3 and 1 patients respectively. One patient, who had a del(17p), also carried a mutation in TP53. Interestingly, two genes, MYCBP2 and MKI67, were each independently mutated in 3 of the 40 patients (7.5%) screened by RNAseq (data not shown).

Compared to previously published series (Bolli et al, 2014), the pooled results of WES and RNA-seq reveal that NRAS and KRAS are less frequently mutated (13% and 8% respectively) in the symptomatic t(4; 14) MM subgroup than in total MM, while mutations in FGFR3 were more frequent (19%) (data not shown). Overall, in our study the FGFR3/RAS/BRAF/MAPK genes were mutated in 35 t(4;14) MM (57.3%). In addition, we found marked elevated frequency of mutations in PRKD2 (11.4%), ATM/ATR (11.4%) and MYCBP2 (8.2%), reduced frequency in FAM46C (1.6%) and no mutation in TRAF3 and CCND 1. Of note, among the 84 MM sequenced by Bolli and colleagues, none of the non t(4; 14) patients were mutated in PRKD2, further indicating that this genetic lesion is associated with t(4;14) MM. Altogether, our results define a specific mutational landscape for t(4; 14) MM, including specific transcription dependent C>A transitions, prevalence of mutation in members of the FGFR3/RAS/BRAF/MAPK pathway, elevated frequency of ATM/ATR or PRKD2 alterations and decreased mutation rates in FAM46C.

Non-silent mutations in PRKD2 correlates with MB4-1 or -3 breakpoints and is associated with disease progression in one t(4;14) patient.

We next investigated the correlation between the mutations occurring in our cohort of

67 t(4; 14) MM and several recurrent cytogenetic abnormalities. We found that mutation in MAX positively correlated with mutation in TP53 or del(17p) (data not shown). Mutations in ATM/ATR were strongly associated with the MB4-2 Bp and significantly correlated with mutations affecting genes coding for members of the MAPK family. As expected from the mutually exclusive occurrence of mutations in NRAS, KRAS, BRAF and FGFR3 (data not shown), there was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF (pooled together). Last, we observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or -3 Bp.

To get further insights into the mechanisms associated with progression of t(4;14) MM, we analyzed the evolution of the mutational landscape in one patient (P6) with serial samples collected at diagnosis and 33 months later, at time of progression from asymptomatic to symptomatic MM. From the exome sequencing data, we first analyzed the copy-number variation to characterize clonal evolution from large scale genomic aberrations over time. We found no major changes in copy-number, with similar loss within chromosome 8, 13, 18 and 22 in early and late samples (data not shown). In contrast, in addition to initial alterations, non- silent mutations in LRFN1, NOLI 2, ZCCHC4 and PRKD2 genes, which were barely (or not) detectable in the early sample, were each present in around 20% of the reads in the late sample (data not shown). From the percentages of mutated reads at each altered gene, and assuming that there was similar contamination of non-tumor cells in the early and late samples, we clustered point mutations based on clonality and plotted the fraction of tumor cells harboring each variant on the x axis for the early asymptomatic sample, and on the y axis for the late sample (data not shown). This representation clearly shows the linear evolution of the tumor during symptomatic disease progression with appearance of additional mutations. Thus, in the absence of copy -number variation, mutations in only 4 genes, including PRKD2, are associated with progression from asymptomatic to symptomatic MM in this t(4; 14) patient.

PRKD2 is expressed in tumor plasma cells and is mutated in t(4;14) MM cell lines.

Our exome and RNA sequencing results pointed towards a role of PKD2, the protein encoded by the PRKD2 gene, in the progression of a subset of t(4;14) MM. To get insights into the role of PKD2 in MM, we searched for PRKD2 mutations in the publicly available sequence data of a series of more than 60 MM cell lines accessible at the Translational Genomics Research Institute (https://myelomagenomics.tgen.org/). Three cell lines, namely NCIH929, JIM1 and JIM3, all carrying a t(4; 14) translocation, display missense mutations in PR D2, leading to I582T, V385E and V385E substitutions, respectively, which were confirmed by Sanger sequencing of cDNA (data not shown). A fourth t(4;14) bearing cell line, KMS 18, has a silent mutation in PRKD2, while JJN3 t(14,16) and JMW1 t(4;14) display a fusion of this gene with NCOA3 and POLD 1 , respectively. These observations are consistent with our results in MM patients and show that alterations in the coding sequence of PRKD2 affect preferentially t(4;14) tumor plasma cells.

Next, we analyzed a publically available expression array to investigate PRKD2 expression in the tumor plasma cells from a large cohort of MM patients. We found that PRKD2 is transcribed in MM cells and that its expression is not restricted to a specific sub group of patients (Figure 1). In line with this, we found that the PKD2 protein is expressed in plasma cells isolated from 3 patients as well as in several MM cell lines, except LP1 (data not shown). The PKD serine/threonine kinases share two CI -domains, which bind diacylglycerol and phorbol esters (PMA) and an auto-inhibitory PH-domain. Upon stimulation, both PKC- dependent and -independent activation of PKDs occur at serine residues. Phosphorylation of both Ser707 and Ser711 in the activation loop of the kinase domain is followed by trans- or auto-phosphorylation at Ser876 in the C-terminal region which marks the activation of the protein (Fu and Rubin, 2011). To explore the activation status of PKD2 in MM cells, we performed western blot analysis of PKD2 phosphorylation at serine 876 (p-PKD2) in MM cells. We found low levels of p-PKD2 in tumor plasma cells from patients as well as in U266, MM.1 S and RPMI8226 cell lines (data not shown). In contrast, PKD2 was overexpressed in NCI-H929 where there was a marked elevated level of p-PKD2. Surprisingly, JIM3 cells, which like NCI- H929 carry a mutation in the PRKD2 gene, weakly expressed PKD2. Despite low PKD2 expression, JIM3 cells had similar levels of p-PKD2 as most MM cell lines (data not shown). Together, these results suggest that PKD2 is activated in NCI-H929 and to a lesser extent in JIM3 cell lines.

PKD inhibitor kb NB 142-70 induces growth arrest of tumor plasma cells and inhibits phosphorylation of PRKD2 in MM cell lines.

Given the potential role of PRKD2 in the pathophysiology of t(4;14) MM, we next examined the impact of PKD2 inhibition on MM-cell growth and survival, kb NB 142-70, an inhibitor of PKD proteins (Bravo-Altamirano et al., 2011), induced marked dose-dependent growth inhibition in all MM cell lines investigated including MM. IS, RPMI 8226 (RPMI), U266, that have wild type PRKD2, as well as NCI-H929 and JIM3, that express mutated PKD2 (Figure 2A). IC50 ranged from 0.3 μΜ for NCI-H929, which is mutated for PRKD2, to 1 μΜ for RPMI8226, which has markedly lower overall PKD2 expression than the other cell lines (Figure 2A). Importantly, kb NB 142-70 also induced growth inhibition of (CD 138+) MM cells isolated from 3 patients with drug-resistant disease and carrying un-mutated PR D2 (Figure 2B). We next sought to evaluate whether kb NB 142-70 induces apoptosis by staining MM cell lines with annexin V and 7-AAD. Our data demonstrate that NCI-H929 cells are more sensitive to kb NB 142-70 induced apoptosis than MM. IS or U266 cells (Figure 2C). Similar results were found with freshly isolated tumor plasma cells from 2 patients. To investigate the mechanism leading to kb NB 142-70 mediated MM-cell growth arrest, we performed western blot to analyze the impact of this drug on PKD2. As expected, phosphorylation of PKD2 on Ser876 was enhanced by treatment of U266, NCI-H929 and JIM3 cells by PMA, a classic activator of the PKC pathway (data not shown). Together, these data demonstrate that inhibition of PKD2 activity inhibits cell growth and induces apoptosis of MM cells in vitro.

PKD1, PKD2 and PKD3 inhibitor CRT0066101 inhibits growth of MM cell lines.

Given the potential role of PRKD1, PRKD2 and PRKD3 in the pathophysiology of t(4;14) MM, we next examined the impact of PKD1, PKD2 and PKD3 inhibition on MM-cell growth. CRT0066101, an inhibitor of PKD1, PKD2 and PKD3 proteins, induced marked dose- dependent growth inhibition in all MM cell lines investigated including NCI-H929, JIM3, MM.1 S and RPMI 8226 (Figure 3). Together, these data demonstrate that inhibition of PKD 1 , PKD2 and PKD3 activity inhibits cell growth of MM cells in vitro.

REFERENCES:

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

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Claims

CLAIMS:

1. A PKD inhibitor for use in the treatment of multiple myeloma in a subject in need thereof.

2. The PKD inhibitor for use according to claim 1 wherein the PKD is the PKD1, PKD2 or the PKD2.

3. The PKD inhibitor for use according to claim 2 wherein the PKD is the PKD2.

4. The PKD inhibitor for use according to claims 1 to 3 wherein the inhibitor is the kb- NB 142-70 or CRT0066101.

5. A method for treating multiple myeloma comprising administering to a subject in need thereof a therapeutically effective amount of a PKD inhibitor.

6. A PKD inhibitor for use in the treatment of multiple myeloma in a subject diagnosed with a t(l 1;14) translocation.

7. A i) PKD inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma in a subject in need thereof.

8. A i) PKD inhibitor and ii) a compound used to treat multiple myeloma, as a combined preparation for simultaneous, separate or sequential use in the treatment of multiple myeloma diagnosed with a t( 11 ; 14) translocation in a subject in need thereof.

9. The combined preparation according to claims 7 or 8 wherein the compound used to treat multiple myeloma are selected in the group consisting in Bortezomib (Velcade, PubChem number 387447), Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil, and Bendamustine (Treanda), dexamethasone, thalidomide analogues and antibodies like Daratumumab (Darzalex).

10. A therapeutic composition comprising a PKD inhibitor according to claims 1 to 4 for use in the treatment of multiple myeloma.

11. A method of screening a candidate PKD inhibitor for use in the treatment of multiple myeloma, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that inhibit PKD.

12. A method for diagnosing multiple myeloma in a patient comprising determining in a tumor sample obtained from said patient any substitution in the coding region of

PRKD2.