WO2009117769A1 - Inhibition of c-kit cancers - Google Patents

Abstract

The invention relates to a method for inhibiting c-KIT in a cancer cell, comprising treating the cancer cell with an agent for up regulating protein phosphatase 2A (PP2A) activity in the cancer cell. The agent can be an activator of PP2A such as FTY720. The inhibition of c-KIT can inhibit growth of cancer cells and has application in the prophylaxis or treatment of c-KIT cancers. Typically, the cancer is a non- leukemic cancer.

Description

INHIBITION OF c-KIT CANCERS

FIELD OF THE INVENTION

The invention relates to the use of an agent for up regulating protein phosphatase 2A (PP2A) activity for inhibiting the growth of c-KIT cancer cells. The invention also provides methods of screening for agents useful m the methods of the invention.

BACKGROUND OF THE INVENTION

The c-KIT proto-oncogene encodes a receptor tyrosine kinase whose ligand is stem cell factor (SCF). c-KIT is expressed by, and is critical for the development and growth of, mast cells, melanocytes, hematopoetic stem cells, and the interstitial cells of Cajal The structural features of c-KIT include an N-terminal signal peptide, an extracellular region comprised of five immunogloblm (Ig)-like loops which function in ligand binding and receptor dimerisation, a single hydrophobic transmembrane segment, and an intracellular region. The intracellular region contains a juxtamembrane (JMD) and tyrosine kinase domain (KD) split by an 80 amino acid interkinase sequence into the ATP binding pocket and the phosphotransferase catalytic site. SCF binding induces c-KIT dimenzation and auto-phosphorylation on tyrosines m the intracellular domain of the receptor This auto-phosphorylation creates docking sites for signal transduction molecules and induces substrate binding and phosphorylation [I]. Over expression of wild-type (WT) c-KIT induces hyper-phosphorylation and constitutive activation of downstream signalling pathways, resulting in uncontrolled cell proliferation. Such inappropriate expression and/or autocrine SCF secretion has been reported in acute myeloid luekamiea (AML), non small cell lung carcinomas, colorectal carcinoma, breast carcinoma, gynecological tumours, and neuroblastomas [2]. For example, c-KIT protein expression is found in the neoplastic cells of over 60% of de novo AML patients, and 95% of relapsed AML patients [3], and some studies have shown an association of c-KIT expression and poor outcome [4] [5]. In addition, specific mutations within the c-KIT gene can induce constitutive activation of c-KIT, and hence activation of downstream signalling pathways and tumourigenesis. Indeed, c-KIT mutations have been detected, and in some cases are directly involved in the pathogenesis of gastrointestinal stromal tumors (GISTs), AML, mastocytosis, testicular seminomas, sino-nasal lymphomas and melanoma, making c-KIT a target for a broad range of cancer therapies [6]. The most common activating mutations are within the kinase domain (KD) (e.g. D816V), the extracellular domain (ECD) (e.g. ΔTYD417- 419) and the juxtamembrane domain (JMD) (e.g. V560G). For example, mutations within c-KIT are detected on approximately 85% of GIST specimens [7], with the most common mutation being the JMD V560G. Activating mutations in c-KIT have also been identified in up to 46% of core-binding factor (CBF) AML patients, a subgroup which constitute 15-20% of adults with de novo AML [8]. The c-KIT mutations occur at high frequencies, often involve the amino acid D816, and are associated with increased risk of relapse and reduced survival in CBF-AML patients characterized by t(8;21) and inv(16) chromosomal abnormalities. Importantly, while cells expressing V560G mutant c-KIT are sensitive to tyrosine kinase inhibition with imatinib, the D816V mutant is highly resistant. Thus, this mutation is also found commonly as a secondary mutation in relapsed GIST patients.

Tmalinib is an ATP analogue that competitively binds and inhibits c-KΪT as well as the related tyrosine kinases BCR/ ABL and platelet derived growth factor (PDGFR). Drug binding results in prevention of tyrosine phosphorylation of proteins involved in signal transduction, leading to growth arrest or apoptosis of cells dependent on these tyrosine kinases for growth. The introduction of imatinib has shown remarkable success in CML (BCR/ABL⁺) [9] and GIST (C-KIT⁺) patients [10, H]. Limited clinical trials in AML patients have shown some favourable responses [12]. However, resistance to imatinib is emerging as a major clinical problem. In particular, many patients acquire secondary mutations in the target molecule (e.g., T315I for BCR/ ABL, and D816V for c-KIT) that render imatinib unable to bind, leading to reactivation of downstream signalling pathways [13]. Furthermore, as the most common intrinsic mutations in AML patients are within KD and are intrinsically resistant to imatinib, the majority of C-KIT⁺ AML patients will not respond to imatinib. Thus a greater understanding of the signalling pathways activated downstream of c-KIT is required in order to identify novel targets for the treatment of C-KIT⁺ cancers. Several oncogenic signalling molecules are activated by over expressed or mutant c-KIT, and are therefore important for tumoungenesis. These include the Ras/ERK, PI3K/Akt and JAK/STAT pathways [1], which result m enhanced transcriptional activity, inhibition of apoptosis, increased cell proliferation and oncogenic transformation. In vitro studies have shown positive effects of small molecules aimed at Ras, Src, and PI3K inhibition, in C-KIT⁺ cells, both alone and in combination with imatimb [14-16].

In normal cells, c-KIT activity is controlled by negative regulation. As with activation of downstream signalling pathways, negative regulation of c-KIT occurs via similar signalling pathways to other oncogenic tyrosine kinases, such as BCR/ ABL. For example, both c-KIT and BCR/ ABL are negatively regulated by SHP-I, a tyrosine phosphatase that dephosphorylates the oncogenic kinase, resulting in decreased activity and subsequent degradation [17]. BCR/ ABL is also negatively regulated by the tumour suppressor protein phosphatase 2A (PP2A). BCR/ ABL inactivates PP2A, and reduced PP2A activity is essential for BCR/ ABL leukaemogenesis in CML blast crisis progenitors [18]. Molecular or pharmacological reactivation of PP2A results in growth suppression, enhanced apoptosis, restored differentiation, and decreased in vivo leukaemogenesis of imatinib-sensitive and -resistant B CR/ AB L⁺ cell lines and primary CML blast crisis cells [18, 19] PP2A is a major serine/threonine phosphatase implicated m the negative regulation of numerous signal-transduction pathways that are involved in cell cycle progression, DNA replication and apoptosis [20]. PP2A is a ubiquitous multimeric enzyme composed of a structural subunit (Aa and Aβ), a catalytic subunit (Ca and Cβ), and a variable regulatory B subunit categorised under four unrelated families named B, B', B", and B'", each with several isoforms. The regulator}' B subunits bind to the AC dimer in a mutually exclusive manner to form distinct holoenzymes. Importantly, this association directs substrate specificity and targets PP2A enzyme activity to distinct sub-cellular localisations [20, 21].

PP2A has been reported to be mutated or down-regulated in a number of cancers, including breast, lung, colon, and others [22] The endogenous PP2A inhibitor protein, SET, is also over expressed or activated due to translocation m at least some leukaemias [23], and down regulation of PP2A-C has been reported in drug resistant leukaemia cells [24]. PP2A regulates the Ras/ERK and PI3K pathways, and is also a negative regulator of the JNK and JAK/STAT pathways [25]. Regulation of these pathways is dependent on different regulatory B subunits, but the precise regulation of PP2A in these processes is not clearly understood. PP2A inhibition had previously been proposed as a potential anticancer therapy [26]. However, the toxicity and lack of specificity associated with PP2A inhibitors has thus far proven to be unsuitable for cancer treatment.

United States Patent Application No. 10/513,804 (US 2005/0215531) describes the use of a sphingosine-1 -phosphate (SlP) receptor agonist to treat solid tumours by inhibiting angiogenesis, and indicates that FTY720 may be employed as such a SlP receptor agonist. FTY720 is a known immunosuppressant drug that causes lymphopenia by preventing egress of lymphocytes from lymph nodes, and can induce apoptosis in activated lymphocytes.

United States Patent No. 6,998,391 describes the use of a DNA methylation inhibitor in combination with a protein kinase inhibitor for the treatment of cancer associated with abnormal activity of the kinase (e.g., such as c-KIT). The DNA methylation inhibitor is reported to exert its therapeutic effect via re-establishment of transcriptional activity of disease suppressing genes which may then further inhibit activity of the protein kinase.

International Patent Application No. PCT/US2007/012921 (WO 2007/143081) describes the use of FTY720 to increase PP2A activity in lymphoid cancers as a treatment for the cancers.

SUMMARY OF THE INVENTION

Broadly stated, the invention stems from the observation that protein phoshatase

2A (PP2A) is inactivated in cancer cells with aberrant/inappropriate expression of active c-KIT and further, that reactivation of PP2A or more generally up regulating PP2A activity in the cancer cells, can significantly inhibit the growth and cancer potential of the cells. This finding has significant application in the prophylaxis or treatment of cancers in which the cancer cells have aberrant expression of active c-KIT, and lends itself to screening agents for their capacity to inhibit c-KIT. In an aspect of the invention there is provided a method for inhibiting c-KIT in a cancer cell, comprising treating the cancer cell with an agent for up regulating PP2A activity in the cancer cell.

In at least some embodiments, the cancer is a non-leukemic c-KIT cancer. In another aspect of the invention there is provided a method for inhibiting growth of a cancer cell of a non-leukemic c-KIT cancer, comprising treating the cancer cell with an effective amount of an agent for up regulating PP2A activity.

In another aspect of the invention there is provided a method for prophylaxis or treatment of a non-leukemic c-KIT cancer in a mammal, comprising administering an effective amount of an agent for up regulating PP2A activity to the mammal.

The invention also extends to a method of screening agents that up regulate PP2A activity for use of the agent in treating a c-KIT cancer.

Hence, in another aspect of the invention there is provided a method of screening an agent for capacity to inhibit c-KIT, comprising: providing a sample of c-KIT cancer cells; selecting an agent for up regulating PP2A activity in the cancer cells; treating the cells with the agent; and determining whether growth of the cells is inhibited by the agent, the inhibition of growth of the cells by the agent being indicative the agent inhibits c-KIT. In another aspect of the invention there is provided a method of screening an agent for up regulating PP2A activity for capacity of the agent to inhibit c-KIT. comprising: providing a sample of c-KIT cancer cells; providing the agent for up regulating PP2A activity m the cancer cells; treating the cells with the agent; and determining whether growth of the cells is inhibited by the agent, the inhibition of growth of the cells by the agent being indicative the agent inhibits c-KIT.

Typically, the sample of c-KIT cancer cells is obtained from a cancer patient, and the screening in accordance with a method embodied by the invention can further involve comparing different agents for up regulating PP2A inactivity in the cancer cells. The agent which is most effective in inhibiting the growth of the c-KIT cells can then be selected for administration to the cancer patient for treatment of the cancer. Thus, a method of screening in accordance with the invention may provide a means for optimising the drug treatment for the patient's particular c-KIT cancer.

In another aspect of the invention there is provided the use of an agent for up regulating PP2A activity in the prophylaxis or treatment of a non- leukemic c-KIT cancer m a mammal.

In another aspect of the invention there is provided the use of an agent for up regulating PP2A activity in the manufacture of a medicament for prophylaxis or treatment of a non-leukemic c-KIT cancer in a mammal.

In yet another aspect of the invention there is provided the use of an agent for up regulating PP2A activity to inhibit growth of a non-leukemic c-KIT cancer cell.

Typically, the agent for up regulating PP2A activity will be an activator of PP2A.

As used herein, the term "c-KIT cancer" is to be taken to mean a cancer arising from, or associated with, aberrant or over expression, of activated c-KIT. Similarly, the term "c-KIT cancer cell" is to be taken to mean a cancer cell arising from, or associated with, aberrant or over expression of activated c-KIT by the cell, or cells engineered to express exongenous c-KIT. Aberrant expression of activated c-KIT includes expression of form(s) of c-KIT including wildtype (WT) and c-KIT having one or more mutations such as, but not limited to, the JMD V560G mutation, ΔTYD417-419, and mutations involving amino acid D816 (e.g., D816V) that result in inappropriate or up regulated activity of this tyrosine kinase.

In at least some forms of the screening method described herein the cancer can be a leukaemic cancer such as acute myeloid leukaemia (AML) although embodiments of the invention extends to other c-KIT cancers. Moreover, in one or more embodiments of the invention, the cancer may be a cancer that is resistant to treatment with an inhibitor of c-KIT such as imatinib.

For the purposes of the present invention, the term "non-leukemic cancer" is to be taken to mean a cancer that is not a haematological malignancy or neoplasm, or is otherwise not haematologically derived, but which includes mastocytosis, and cancers of mastocytes (mast cells) and progenitors of mastocytes. As such, the term excludes erythroid, lymphoid and myeloid cell cancers (e.g., lymphomas, and myelomas that are not cancers of mastocytes), cancers of progenitors of erythroid, lymphoid and myeloid cells (other than progenitors of mastocytes), and tumours and metastases of these cancers.

The mammal treated by a method embodied by the invention may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, or a primate or human being. Typically, the mammal will be a human being.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of this application

The features and advantages of the present invention will become further apparent from the following detailed description of non-limiting embodiments.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Graph showing active c-KIT inhibits PP2A. PP2A activity was determined on PP2A-C immunoprecipitates using a PP2A-specific phosphopeptide as substrate. PP2A activity was determined in FDC-Pl myeloid cells stably expressing empty vector (EV). or WT, V560G or D816V c-KIT, and the % PP2A activity normalised relative to empty vector (EV) control cells. Error bars represent standard error of the mean (SEM) n=3; **p<0.01 Students t-test compared to EV.

Figure 2: Graph showing PP2A structural subunit is decreased m C-KIT⁺ cells. Total protein was isolated from FDCPl empty vector (EV), WT c-KIT, V560G c-KIT and D816V c-KIT cells separated by SDS-PAGE and immunoblotted for the catalytic subunit, PP2A-C; catalytic subunit phosphorylation on tyrosine 307, pY³⁰⁷ PP2A-C; catalytic subunit methylation on leucine 309, mL309-PP2A-C; and the structural subunit, PP2A-A. Equal protein loading was confirmed by immunoblotting for actin. Gel image is representative of 3 independent experiments

Figure 3: Graph showing PP2A regulatory subunits are decreased in C-KIT⁺ cells. Total protein was isolated from FDCPl empty vector (EV), WT c-KIT, V560G c-KIT and D816V c-KIT cells separated by SDS-PAGE and immunoblotted for PP2A regulatory subunits Bδ, B'α, B'γ, and B'δ. Equal protein loading was confirmed by immunoblotting for actin. Gel image is representative of 3 independent experiments.

Figure 4: Graph showing reactivation of PP2A in C-KIT⁺ cells. (A) PP2A activity was determined as for Fig. 1 in cells pre-treated with 2.5μM FTY720 for 24h. (B) Cells in the absence or presence of 2.5μM FTY720 for 24h were stained with

Annexin V and the % positive cells assessed by flow cytometry. (C) FDCPl cells were grown in methylcellulose for 6 days in the presence or absence of lμM or 2.5μM FTY720 and the number of colonies counted. Error bars represent SEM n=3; *p<0.05, **p<0.01 Students t-test FTY720 treated versus untreated cells. Figure 5: Graph showing dephosphorylation of c-KIT upon PP2A reactivation. c-KIT was immunoprecipitated (IP) from FDC-Pl cells treated with 2.5μM FTY720 for 24h, and the total and phospho-tyrosine containing c-KIT determined by immunoblotting (IB) with a total phospho-tyrosine (pY) or c-KIT antibody. Gel is representative of 3 independent experiments. Figure 6: Graph showing delay of mutant c-KIT tumour growth in vivo with

PP2A activation by FTY720 FDCPl cells expressing V560G c-KIT (A and C) or D816V-C-KIT (B and D) were injected s.c. into DBA.2J mice and tumour volume and survival measured m mice untreated (saline control) or treated daily with 10mg/Kg FTY720 or 50mg/Kg imatinib. (A and B) The survival of mice was determined using the Kaplein Meier method. (C and D) Tumour weight at day 14 in saline or FTY720 treated mice.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

c-KIT cancers include, but are not limited to, acute myeloid leukaemia (AML), mesenchymal cancers including gastrointestinal stromal tumours (GISTs), non small cell lung carcinomas, colorectal carcinoma, breast carcinoma, ovarian cancer, neuroblastomas, mastocytosis, testicular seminomas, sino-nasal lymphomas and melanoma.

Acute myeloid leukaemia (AML) is a proliferative disorder involving the myeloid lineage of haematopoietic stem cells. ΛML is the most common type of leukaemia in aduits, yet has the lowest survival rale of all lcukemias. More than 50% of all AML cases affect patients over age 60, and long term overall survival in these patients is dismal, at only 5% to 20% [27]. As outlined above, c-KIT protein expression is found in the neoplastic cells of over 60% of de novo AML patients, and 95% of relapsed AML patients [3], and some studies have shown an association of c-KIT expression and poor outcome [4] [5].

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumour of the gastrointestinal tract. Mutations within c-KIT are detected on approximately 85% of GIST specimens [7]. The median age of adults at diagnosis of GIST ranges from 66 to 69 years [28]. The incidence of GIST is not known for all populations. However, an incidence rate of 10 per million persons has been suggested by the National Comprehensive Cancer Network, USA, which converts to approximately 3000 new GIST diagnoses in the United States per year [28, 29]. Reports also suggest that the incidence of GISTs is rising. Despite rising GIST incidence rates, there has been a marked improvement in survival since 2000, coinciding with the introduction of imatinib into clinical practice [29]. However, the development of resistance to imatinib is emerging as a major clinical problem.

Melanoma, a tumour arising from melanocytes, has become a major public health problem in many countries. Australia has the world's highest incidence rate for melanoma. Melanoma is the fourth most common cancer, with nearly 9500 cases diagnosed annually and more than 1200 deaths from melanoma each year (Cancer

Council Australia). Although early melanoma is curable through surgical excision, the prognosis of advanced melanoma is very poor (<15% survival) because it is resistant to most chemotherapeutic agents. A subset of melanoma has been found to maintain c-Kit overexpression or mutations. In a study of 102 primary melanomas mutations and/or copy number increases of c-KIT were found in 39% of mucosal melanomas, 36% of acral melanomas, and 28% of melanomas on chronically sun-damaged skin, but not in any (0%) melanomas on skin without chronic sun damage. Seventy-nine percent of tumours with mutations and 53% of tumors with multiple copies of KIT demonstrated increased KIT protein levels [30]. Eleven (69%) of 16 KIT mutations were predicted to affect the juxta-membrane domain, presumably resulting m a constitutive activation of KIT. Initial clinical trials of imatinib in melanoma were disappointing but the patients were not stratified according to c-KIT status [31]. A phase II clinical trial of imatinib is currently in process in patients affected by acral or mucosal melanoma, which often carry a c-Kit mutation.

Testicular cancer is the second most common cancer in young men aged 18 to 39. In New South Wales, Australia m 2003, 231 men were diagnosed with the disease, accounting for 1.3% of all cancers in men. The number of men diagnosed with testicular cancer has grown by 34% over the past decade, but the reason for this is not known. Seminomal testicular cancer is the most common form of the disease, and is associated with c-KIT mutations. Mutations in the c-KIT occur in approximately 8% of all testicular germ cell tumors (TGCT) and up to 93% of patients with bilateral disease [32]. In mastocytosis, a heterogeneous disorder characterised by the expansion and accumulation of mast cells in different organs and tissues, D816V c-KIT mutations have been identified in up to 93% of seminal mastocytosis patients, and a further 2.5% of patients harbour other c-KIT mutations [33]. Other cancers that have been shown to be associated with increased expression, autocrine activation, or mutations in c-KIT include non small cell lung carcinomas, colorectal carcinoma, breast carcinoma, ovarian cancer, neuroblastomas, and sino-nasal lymphomas, however the precise role of c-KIT in these cancers is not currently well defined.

In at least some embodiments of the invention, the c-KIT cancer is a non-leukemic cancer. That is, the cancer is not a blood cell cancer or cancer of a progenitor of a blood cell (e.g., a cancer of a progenitor of an erythroid, myeloid or lymphoid cell, such as hematopoietic stem cells (HSC), multipotent progenitor cells (MPPs) and lineage-restricted progenitor cells (LRPs)). Examples of lymphoid cells include B lymphocytes and T lymphocytes and their respective sub-types (e.g., CD4⁺ and CD8⁺ T cells), and natural killer (NK) cells. Examples of myeloid cells include granulocytes, megakaryocytes, macrophages, monocytes, monoblasts, eosinophils, and basophils. However, it will be understood that the invention extends to screening agents for up regulating PP2A activity for the inhibition of c-KIT cancers in all of the above cell types that is, including leukemic and non-leukemic cancer cell types. Leukemic cancers include, but are not limited to, acute lymphoblastic leukaemia (ALL), acute myelogenous leukaemia (AML), chronic myelogenous leukaemia (CML), chronic lymphocytic leukaemia (CLL), hairy cell leukaemia (HCL), lymphomas such as Hodgkin's disease and non-Hodgkin lymphoma and their subtypes, and multiple myeloma. It will also be understood that non-leukemic cancers include, but are not limited to, sarcomas, mesenchymal cancers including gastrointestinal stromal tumours (GISTs), mastocytosis, cancers of mastocytes (mast cells) and progenitors of mastocytes, carcinomas such as non small cell lung carcinomas, colon cancer, gastric cancers, colorectal carcinoma, breast carcinoma, gynaecological cancers including ovarian cancers and cancer of the uterus, vulva, vagma or cervix), prostate cancers, neuronal cancers, neuroblastomas, testicular cancers including testicular seminomas, pancreatic cancers and melanomas. Hence, the invention extends generally to the inhibition of the growth of non-leukemic cancers and in particular to cancers of cells that are not haematopoietic stem cell derived. Examples of haematopoietic stem cell derived cells include mastocytes and progenitors of mastocytes.

The activator of PP2A can be any drag or compound that activates the phosphatase, such as a pharmacologic chemical species, a complex (e.g., a metal complex), peptide agent, fusion protein, or oligonucleotide. In particular, the activator may be selected from the group consisting of, but not limited to, FTY720 (also called fingolimod), forskolin, 1,9-dideoxyforskolin, ceramides (also called sphingosines) such as C2-ceramide, topoisomerase inhibitors such as etoposide (Eposin , Etopophos , Vepesid™, VP- 16™), tubulin polymerisers such as methyl-3,5-diiodo-4-(4'- methoxypropoxy)benzoate (DIME or DIPE), fatty acids such as palmitate, and thiol alkylating agents such as N-ethylmaleimide (NEM). Other agents for up regulating PP2A activity for prophylaxis or treatment of c-KIT cancers as described herein include genetic molecules such as over expression constructs for the endogenous PP2A activator PTPA, PP2A or individual PP2A gene subunits. Similarly, such agents may also take the form of DNA/RNA inhibition molecules such as shRNA or antisense sequences, including those specific to the endogenous PP2A inhibitor SET, or to an individual PP2A gene subunit or specific region of the PP2A gene (e.g., a transcriptional regulatory control subunit such as a promoter). FTY720 is particularly suitable for use in methods as described herein. An expression vector or construct will typically include transcriptional regulatory control sequences to which an inserted nucleic acid sequence encoding the agent for effecting up regulation of PP2A activity is operably linked. By "operably linked" is meant the nucleic acid insert is linked to the transcriptional regulatory control sequences for permitting transcription of the inserted sequence without a shift in the reading frame of the insert Such transcriptional regulatory control sequences include promoters for facilitating binding of RNA polymerase to initiate transcription, expression control elements for enabling binding of ribosomes to transcribed mRNA, and enhancers for modulating promoter activity. The expression vector or construct employed will depend on the host cell to be transfected, the mode of transfection and the required level of transcription of the nucleic acid insert.

Numerous expression vectors suitable for transfection eukaryotic (e g , mammalian cells) are known in the art. Expression vectors suitable for transfection of eukaryotic cells include pSV2neo, pEF.PGK.puro, pTk2, pRc/CNV, pcDNAI/neo, adenoviral vectors and pAdEasy based expression vectors most preferably incorporating a cytomegalovirus (CMV) promoter although as will be understood, any suitable such regulatory control sequences may be utilised. Any means for achieving the introduction of nucleic acid into cells for expression of the encoded agent for up regulating PP2A activity can be used. Transfer methods known in the art include viral and non-viral transfer methods. Suitable virus into which appropriate viral expression vectors may be packaged for delivery to target cells include adenovirus, vaccinia virus, retroviruses of avian, murine and human origin, herpes viruses including Herpes Simplex Virus (HSV) and EBV, papovaviruses such as SV40, adeno-associated virus and genetically engineered and attenuated forms thereof. Particularly preferred virus are replication deficient recombinant adenovirus. Suitable expression vectors and constructs are for instance described in Molecular Cloning. A Laboratory Manual., Sambrook et al., 2nd Ed. Cold Spring Harbour Laboratory., 1989, and subsequent editions thereof.

Rather than utilising viral mediated transfection of cells, constructs for expression of an agent for up regulating PP2A activity can be introduced into target cancer cells in vitro or in vivo by liposome mediated transfection. The liposomes can carry targeting or binding molecules for maximising efficiency of delivery to the target cells. Such targeting molecules include antibodies or binding fragments thereof (eg., Fab and (Fab')₂), ligands and cell surface receptors for facilitating fusion of liposomes to the target cancer cells. Alternatively, nucleic acid sequences and the like may be intracellularly delivered in vitro using conventional cold or heat shock techniques, DNA guns or for instance, calcium phosphate co-precipitation or electroporation protocols as are known m the art. Yet another strategy is to design the agent for up regulating PP2A activity to have the inherent ability to pass across the lipid bilayer of a cell such as by coupling the agent to a suitable hydrophobic moiety, and all such methods are expressly encompassed.

A method embodied by the invention for evaluating the suitability or capacity of an agent for up regulating PP2A activity to treat a c-KIT cancer can employ any suitable assay system via which the inhibition of growth of the cancer cells as a result of treatment with the agent can be evaluated. Suitable such assay formats include evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (e g., cell counts, ³H-thymidine uptake and MTT assays) all of which are well known to the skilled addressee. Typically, the level of inhibition of the c-KIT cancer cells treated with the agent is compared to control cells which have not been exposed to the activator or other suitable reference data. As will be understood, the results can be used to determine whether the particular activator assayed should be used for treatment of the cancer or whether another agent for up regulating PP2A activity should be used. Indeed, a number of different such activators can be assayed on different aliquots of test c-KIT cells obtained from a cancer patient or c-KIT cell lines, and the most effective at inhibiting growth of the cells selected for treating the c-KIT cancer. Such methods have particular application in evaluating whether a particular agent for up regulating PP2A activity is suitable for use in treating c-KIT cancer cells which express a particular mutation in c-KIT or whether a different such agent would be more suitable.

As will also be understood, the term "growth" of cancer cells is to be taken to encompass proliferation of the cancer cells. Similarly, "inhibition of growth" of cancer cells includes partial or total inhibition of growth of the cells, including death of the cells.

Up regulating PP2A activity of c-KIT cancer cells as described herein may also render drug resistant c-KIT cancers more susceptible to other anti-cancer treatments. Accordingly, the agent for up regulating PP2A activity can be administered to the individual alone or be co-administered with one or more other drugs conventionally used for the treatment of cancer. For example, the agent may be co-administered in combination with one or more tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib etc) and/or with conventional anti-cancer drugs such as platinum complexes (e.g., cisplatin, oxaliplatin etc) By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations administered by the same or different routes, or by sequential administration by the same or different routes, wherein the agent for up regulating PP2A activity and other therapeutic compound(s) or drug(s) exert their physiological effect during overlapping periods. The agent for up regulating PP2A activity will generally be formulated into a pharmaceutical composition comprising the agent(s) and a pharmaceutically acceptable carrier. Pharmaceutical compositions include sterile aqueous solutions suitable for injection and sterile powders for the extemporaneous preparation of injectable solutions. Such injectable compositions will be fluid to the extent that syringability exists. Injectable solutions will typically be prepared by incorporating the activator(s) in the selected carrier prior to sterilising the solution by filtration. In the case of sterile powders, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the activator and any additional desired ingredient from previously sterile filtered solutions thereof. For oral administration, the agent can be formulated into any orally acceptable carrier deemed suitable. In particular, the agent can be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule. Moreover, the agent(s) can be provided m the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions or syrups. Agents for up regulating PP2A activity as described herein can also be formulated into topically acceptable preparations including creams, lotions or ointments for internal or external application. Topically acceptable compositions can be applied directly to the site of treatment including by way of dressings and the like impregnated with the preparation.

A pharmaceutical composition containing one or more agents for up regulating PP2A activity as described herein can also incorporate one or more preservatives such as parabens, chlorobutanol, phenol, and sorbic acid. In addition, prolonged absorption of the composition may be brought about by the inclusion of ingredients for delaying absorption such as aluminium monosterate. Tablets, troches, pills, capsules and like can also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; a disintegrating agent such as corn starch, potato starch or alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; and a flavouring agent. Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations and solutions including for instance, physiological saline. Use of such ingredients and media for pharmaceutically active substances is well known. Except insofar as any conventional media or agent is incompatible with the activator, use thereof is expressly encompassed. Parenteral compositions in dosage unit form for ease of administration and uniformity of dosage can also be provided. Dosage unit form as used herein is to be taken to mean physically discrete units, each containing a predetermined quantity of the agent for up regulating PP2A activity which is calculated to produce a therapeutic or prophylactic effect When the dosage unit form is a capsule, it can contain the activator in a liquid carrier. Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with enteric coatings, or shellac, sugars or both.

A pharmaceutical composition will generally contain about from at least about 0.1% by weight of the agent for up regulating PP2A activity up to about 80% w/w of the composition. The amount of the activator will be such that a suitable effective dosage will be delivered to the individual taking into account the proposed mode of administration. Typically, pharmaceutical compositions will contain from about 0.1 μg to about 4000 mg of the agent. More usually, the agent will be present in a range of from about 500 μg to about 20 mg and most usually, in a range of from about 0. lmg to lOmg. The dosage of the agent will depend on a number of factors including whether the agent is to be administered for prophylactic or therapeutic use, the cancer for which the activator is intended to be administered, the severity of the condition, the age of the individual, and related factors including weight and general health of the individual as may be determined in accordance with accepted medical principles. For instance, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the individual's response. Similarly, frequency of administration can be determined in the same way that is, by continuously monitoring the individual's response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.

Typically, the agent for up regulating PP2A activity will be administered in accordance with a method embodied by the invention at a dosage up to about 50 mg/kg body weight and usually, in a range of from about 5 μg/kg to about 100 μg/kg body weight, more usually in a range of from about 10 μg/kg to 80 μg/kg and most usually, in a range of from about 10 μg/kg to about 50 μg/kg.

Suitable routes of administration of the activator include but are not limited to intravenously, mtrapeπtonealy, subcutaneously, intramuscularly, by infusion, orally, rectally, topically and by implant. With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply a tumour or target tissues to be treated. The activator can also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue. Suitable pharmaceutically acceptable carriers and formulations useful in compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co.. 1995)", the contents of which is incorporated herein in its entirety by reference.

The invention is described herein after with reference to non-limiting Examples.

EXAMPLES

1. Materials and methods 1.1 Cell culture The FDC-Pl mouse factor-dependent early myeloid cell line [34] expressing empty vector (EV). WT, and the oncogenic V560G and D816V mutant forms of human c-KIT were described previously [35]. Infected cell lines were maintained in DMEM/10% FCS supplemented with 25 U/ml mouse granulocyte-macrophage colony- stimulating factor (GM-CSF) (FDC-Pl EV), 100 ng/ml recombinant human stem cell factor (SCF; Peprotech, Rocky Hill, NJ) (FDC-Pl WT c-KIT), or the absence of factor (FDC-Pl V560G and D816V c-KIT). 1.2 Phosphatase activity assay

Protein phosphatase activity was determined using the PP2A-C immunoprecipitation phosphatase assay kit as per manufacturer's instructions (Millipore, Billerica, MA) [18]. Briefly, cells were untreated or treated with 2.5 μM FTY720 for 12 hours and lysed in 20 mM imidazole-HCL, 2 mM EDTA, 2 mM EGTA for 30 minutes. Protein lysate (250 μg) was added to 4 μg anti-PP2A-C 1D6 and 40 μl of Protein-A agarose beads for 2 hours at 4°C. Immune complexes were incubated with a phospho-threonine peptide at 32⁰C for 10 minutes, and the release of free phosphate was measured colourimetrically (620 nm). The percentage of phosphatase activity was determined by dividing the free phosphate of the test cells by that of the control untreated WT c-KIT cells. Three independent assays were averaged to determine PP2A activity.

1.3 Immunoblottiiig Cells (2 x 10⁷) were lysed in RIPA Buffer (1% nonidet P-40 (NP40), 150 mM

NaCl, 50 mM Tris [pH 8.0], 1% SDS) supplemented with 5 mM sodium fluoride, 1 mM sodium vanadate, 1 mM β-glycerol-phosphate, 1 mM phenylmethyl sulphonyl fluoride (PMSF) and complete protease inhibitor cocktail (Roche Diagnostics) for 30 minutes on ice. Whole cell lysates were clarified (12 000 x g for 15 minutes), denatured and subjected to SDS-PAGE before immunoblotting. The antibodies used were anti-PP2A A (Calbiochem); anti-PP2A B'α, anti-PP2A B'δ, anti PP2A B'ε (Novus); anti-PP2A C^Y207 (Epitomics). Anti-PP2A Ba and anti-PP2A B'γ were a kind gift from William Hahn (Dana Farber Cancer Institute, Boston, MA) [36]. An affmity-purified polyclonal antibody was raised against a peptide derived from the C-terminal of PP2A [28]. Anti- actin (Sigma) was used as a loading control.

1.4 Immunoprecipitation - Phosphorylation assay

Immunoprecipitation of c-KIT has been described previously [52]. Where indicated, FDC-Pl cells were treated with 2.5 μM FTY720 (Cayman Chemicals), and harvested at the indicated time points. Briefly, 1 mg of modified RIPA lysates was incubated with 4 μg anti human KIT4 monoclonal antibody and 40 μl protein-A sepharose beads (Millipore) for 2 hours at 4⁰C. The immunoprecipitated complex was washed and subjected to SDS-PAGE. c-KIT phosphorylation was determined by anti- phosphotyrosine 4G10 (Millipore) and total c-KIT levels were evaluated using anti-c- KIT (M- 14) (Santa Cruz Biotechnology).

1.5 Cell proliferation assay

To evaluate cell viability after 48-hour treatment with FTY720, the Cell Titer96 Aqueous Cell Proliferation Assay (Promega) was used as previously described [52]. The concentration of drug that kills 50% (IC50) of the cell population was analysed using fit-spline lowess regression [38] The relative sensitivity was determined by dividing the IC50 of the c-KIT cells by that of the EV cells. Assays were plated m quadruplicate and repeated 10 times.

1.6 Apoptosis assay

The induction of apoptosis by FTY720 was measured using the Annexin V- FITC apoptosis detection kit as per manufacturer's instructions (BD Biosciences). Briefly, untreated and 2.5 μM FTY720-treated cells were harvested at 24 hours, resuspended in binding buffer (10 mM HEPES [pH 7.4], 140 mM NaCl, 2.5 mM CaCl₂) then stained with Annexin V-FITC and PI for 15 minutes at room temperature m the dark. Samples were run on a FACSCalibur flow cytometer (BD Biosciences), and the data was analysed using CellQuest software (BD Biosciences). Four independent assays were averaged to determine the percentage of annexin-V positive cells.

1.7 Clonogenic assay

Methylcellulose colony formation assays were carried out by plating 2 x 10² FDC-Pl cells into 1% MethoCult H4230 (Stem Cell technologies Inc.). Where indicated, cells were plated in the presence of appropriate factors and 2 5 μM FTY720. Colonies (>125 μM) were scored 7 days later. Assays were performed in triplicate and repeated three times.

1.8 In vivo tumour growth - Xenograft mouse model

8 to 10-wk-old female DBA/2J mice (Animal Resources Centre, Canning Vale, WA, Australia) were s.c. injected on both flanks with either 5 x 10⁶FDC-Pl V560G c- KIT or 3.5 x 10⁶ FDC-P1-D816V c-Kit cells in 200 μl 1:1 PBS/Matrigel (Trevigen). Once the tumours reached a volume of -200 mm³ (day 5), mice were randomised into three groups that received daily i.p. injections of either saline, 50 mg/Kg imatinib (Novartis) or 10 mg/Kg FTY720. Tumour volume (TV) was measured every second day based on the formula: TV = [length (mm) x width² (mm)]/2. On day 14 and 21, 3 mice from FDC-Pl V560G c-KIT injected groups and 6 mice from FDC-P1-D816V c- Kit injected groups were sacrificed. The remaining mice were used for survival studies and culled when tumour volumes reached -2100 mm³. Peripheral blood was harvested via heart puncture and blood smears subject to Wrights/Giemsa staining. For pathological examination, tissue sections from formalin-fixed, paraffin-embedded bone- marrow , spleen and liver were stained with hematoxylin/eosin. All animal studies were performed with the approval of The University of Newcastle Animal Care and Ethics Committee, Newcastle, New South Wales, Australia.

1.9 Statistical Analysis

Statistical significance between untreated and FTY720-treated samples was assessed using an unpaired Student' s t-test. Survival probabilities between groups were determined by the Kaplan-Meier method and differences in survival distributions was evaluated by the log-rank test. All statistical analysis was performed using GraphPad Prism software (GraphPad Software).

2. Results

2.1 c-KIT inhibits PP2A activity and alters expression of PP2A subunits

FDC-Pl mouse myeloid cells expressing EV alone, WT c-KIT, the constitutively active imatinib-sensitive juxtamembrane V560G mutant or the oncogenic imatinib-resistant kinase domain D816V mutant [35] were used in this study. To explore whether PP2A is regulated by c-KIT, the activity of PP2A in whole cell lysates extracted from FDC-Pl cells (Fig. 1) was measured. Cells expressing V560G and D816V c-KIT displayed significantly reduced total cellular PP2A activity, compared to WT c-KIT cells. There was no significant difference between EV control cells and those expressing the WT c-KIT receptor This indicates that activating mutations of c-KIT inhibit PP2A activity. Furthermore, the data suggests that PP2A represents a novel therapeutic target in C-KIT⁺ cancers. The cellular localisation and substrate specificity of PP2A is regulated by post- translational modification of the catalytic subunit and binding of regulatory B subunits [21, 25]. To investigate the mechanism by which c-KIT activation inhibits PP2A levels of the catalytic (C) subunit by immunoblotting were examined. There was no change in the total expression of PP2A-C in cells expressing WT or mutant c-KIT (Fig 2). PP2A can be transiently phosphorylated on the catalytic subunit at amino acid Y307 or methylated on L309. c-KIT cells express slightly reduced levels of both phosphorylated and methylated PP2A-C.

Next, the protein expression of individual PP2A regulatory B subunits was investigated. The Bβ and Bγisoforms are neuronal specific and are not expressed m the FDC-Pl cell line (data not shown). Interestingly, WT, V560G and D816V c-KIT cells have markedly reduced expression of Bδ, B'α, and B'δ subunits (Fig. 3). In addition, the WT c-KIT cells express a lower molecular weight isoform of PP2A-B'γ, while no PP2A-B'γ was observed in the mutant c-KIT cells. Downregulation of this subunit by shRNA induces tumourigenicity in HEK293T cells [36], thus inhibition of this subunit by mutant c-KIT may be a contributing mechanism for c-KIT induced tumourigenicity.

2.2 Reactivation of PP2A inhibits cell proliferation in activating c-KIT mutants

To determine whether re-activation of PP2A is a potential strategy for the treatment of C-KIT+ malignancies, the pharmacological PP2A activator, FTY720 [40] was utilised. Treatment of FDC-Pl cells expressing V560G or D816V mutant c-KIT with 2.5μM FTY720 for 12h significantly increased PP2A activity, yet had no affect on PP2A activity in the EV or WT c-KIT cells. This suggests that cells expressing mutant c-KIT have heightened sensitivity to FTY720-induced PP2A activation. The effect of FTY720 on cellular proliferation using an MTS assay was then examined. The cells were treated with increasing doses of FTY720 (0.5 - 8 μM) for 48 hours and the concentration required to kill 50% of the cell population (IC₅₀) was determined (Table 1). This data indicates that c-KIT mutants more sensitive to inhibition by FTY720 than WT c-KIT and c-KIT^" cell lines, respectively. In this assay, we also tested the efficacy of another chemically distinct PP2A activator, forskolin, was also tested. Consistent with FTY720, cells expressing mutant c-KIT cells are more sensitive to activation of PP2A by forskolin than the control cells (Table 1). That FTY720 and forskolin have distinct mechanisms of action, yet both activate PP2A, indicates that the cytotoxicity observed was due to reactivation of PP2A.

Table 1: Cytotoxicity of PP2A activators in C-KIT⁺ cells

FTY720 Forskolin

Cell Line ID50 ±SEM¹ Sensitivity ID50 άSEM Sensitivity

EV 5.5 ±0.37 1 40.75 ±7.06 1

WT 4.4 ±0.40 1.25 37.46 ±12.87 1.09

V560G 2.8 ±0.34 1.96 19.40 ±6.68 2.10

D816V 2.5 ±0.20 2.20 24.10 ±10.12 1.69

ID50 is the concentration (μM) of drug required to kill 50% of cells. Calculated by fit cubic spline regression of cytotoxicity data. SEM = standard error of the mean. 2Sensitivity calculated by dividing the ID50 of the EV by the ID50 of the c-KIT cells lines

2.3 FTY720 induces apoptosis and inhibits clonogenic potential in cells expressing c-KIT activating mutants

The ability of FTY720 to induce apoptosis in FDC-Pl cell lines by staining with an apoptotic marker, annexin-V was investigated. Consistent with its ability to inhibit proliferation in cells expressing the juxtamembrane V560G c-KIT mutant, 2.5 uM FTY720 treatment for 24 hours markedly increased the percentage of annexin-V positive cells (24.1 ± 13.2%, n=2). Interestingly, FDC-Pl D816V c-KIT cells displayed an even greater percentage of annexin-V-positive cells (59.4 + 5.8%, n= 4), indicating rapid induction of apoptosis in the presence of a c-KIT kinase domain mutation. FDC- Pl expressing EV alone or the WT c-KIT receptor showed no difference in the percentage of annexin-V-positive cells with 2.5 uM FTY720 treatment up to 48 hours. No change in cell cycle distribution was observed (data not shown) These results highlight differential sensitivity to FTY720 between two categories of activating c-KIT mutations. The drastic increase m PP2A activity observed in FTY720 treated FDC-Pl D816V c-KIT cells (Fig. 4) could account for this Moreover, FTY720 concentrations which have minimal impact on WT c-KIT-dependent proliferation and SCF-induced phosphorylation can effectively induce apoptosis in cells expressing constitutively activated c-KIT receptors.

The effect of FTY720 on long term proliferation was evaluated using a colony formation assay. FDC-Pl expressing V560G or D816V c-KIT showed a dose- dependent decrease in clonogenic potential, with a single dose of 2.5 uM resulting m 53% and 43% colony formation, respectively, compared to untreated. This correlates with the IC50 value for these cell lines being 2.5 uM. In contrast, colony formation of FDC-Pl WT c-KIT cells in the presence of 2.5 uM FTY720 was 85% of untreated, indicating only a slight effect. Furthermore, there was no difference observed between untreated and FTY720-treated control cells transfected with EV alone.

2.4 Reactivation of PP2A induces dephosphorylation of c-KIT c-KIT activity is regulated by tyrosine phosphorylation. Inhibition of c-KIT phosphorylation inactivates c-KIT, and hence inhibits activation of downstream signalling pathways. To determine if PP2A re-activation regulates c-KIT activation, c- KIT was immunoprecipitated from the FDCPl cells treated with or without 2.5μM FTY720 for 24h. As shown in Fig. 5, FTY720 reactivation of PP2A results in a marked reduction of c-KIT tyrosine phosphorylation, and by 24h results in decreased total c- KIT protein expression. Importantly, FTY720 induced the dephosphorylation of both WT and c-KIT mutants (Fig. 5). This suggests that PP2A inhibition is required for sustained c-KIT phosphorylation, and that reactivation of PP2A results in c-KIT dephosphorylation. Thus PP2A is a negative regulator of c-KIT activation.

2.5 FTY720 delays mutant c-KIT tumor growth in vivo The efficacy of FTY720 against established tumors expressing either the V560G or D816V mutant c-KIT in a xenograft mouse model was evaluated. In this model. FDC-Pl cells were s.c. injected into the left and right flanks of syngeneic DBA/2J mice. Once the tumours reached -200 mm³ on day 5, the mice received daily i.p. injections of either salme, imatinib (50 mg/Kg) or FTY720 (10mg/Kg). In preliminary toxicity studies an initial decline in animal weight after the first two FTY720 treatments was observed. However this stabilised at day 5 of treatment, and the mice continued to gain weight after this point. No further signs of toxicity were observed. Haematological data revealed no difference in the number of red blood cells compared to untreated controls. The only side effect noted was a decrease in lymphocytes As FTY720 is an immunosuppressant and is known to cause lymphopenia in animal models and humans, this finding was expected [37].

Compared to the saline treated mice, administration of FTY720 markedly delayed the growth of tumors expressing V560G or D816V mutant c-KIT. This effect was observed up to 20 days post-tumor injection. The estimated probabilities for survival were calculated using the Kaplan- Meier method, and the log-rank test was used to determine the differences among survival distributions. For tumors harbouring the V560G c-KIT mutant, the median survival for FTY720 treated mice was 24 days, a modest increase from 21 days for saline-treated mice (p < 0 05, n = 9) (Fig. 6A). Similarly for tumors expressing D816V c-KIT, FTY720 significantly prolonged the survival of mice compared to untreated controls (18 days vs 22 days, p < 0.001, n = 30) (Fig. 6B).

At day 14, three mice from the V560G c-KIT tumor groups were sacrificed, with the tumors and organs evaluated by visual inspection and light microscopy. The tumor mass of FTY720-treated mice was reduced compared to untreated control mice, (360.8 ± 27.01mg vs 195.3 ± 50.99, n = 6, p = 0.0167) (Fig. 6C). In the D816V c-KIT tumor groups, FTY720 mice also had significantly reduced tumor burden compared to saline-treated mice (734±83mg vs 442mg, p=0.0066, n=12) (Fig. 6D). It was noted that tumors expressing the D816V c-KIT mutation grew at a faster rate than those harbouring V560G c-KIT. As the kinase domain mutation elicits stronger receptor activation [39] and demonstrates a more transformed phenotype compared to the juxtamembrane mutation, this result was not surprising. Consistent with this observation, saline-treated mice from the FDC-Pl D816V c-KIT group developed splenomegaly. H & E stained sections of spleen revealed a disruption of splenic architecture caused by infiltration of tumor cells from the primary s.c site, (data not shown). Importantly, the spleen size and morphology of FTY720-treated mice resembled that of age-matched non-xenografted controls. These results show that tumors harbouring human c-KIT mutations, especially those located within the kinase domain, are sensitive to FTY720. This particular class of c-KIT mutations are resistant to inhibition with small molecular compounds (e.g., imatinib and dasatinib). Therefore, this data provides evidence that re-activation of PP2A is a valid alternative therapeutic approach for the treatment of drug-resistant C-KIT⁺ malignancies. 3. Discussion

This study has shown for the first time that PP2A inhibition is a crucial mediator of C-KIT⁺ myeloid leukaemogenesis. Constitutive activation of c-KIT via the kinase domain mutation, D816V, or juxtamembrane domain mutation, V560G, inhibits activity of the tumour suppressor, PP2A. The mechanism of PP2A inhibition is associated with decreased expression of PP2A regulatory subunits. Importantly, this study shows that reactivation of PP2A specifically inhibits the growth and clonogenic potential of both imatinib sensitive and imatinib resistant C-KIT⁺ myeloid cells. As c-KIT is the driving force behind a range of cancers, reactivation of PP2A can be a useful strategy for the treatment of a number of cancer types.

Regulation of PP2A activity and specificity is complex. Translation of the catalytic subunit is tightly controlled [20], and indeed, no changes in the total protein expression of PP2A-C in cells with or without c-KIT (Fig. 2) was found. The expression of the structural (A) and a number of regulatory PP2A subunits were however decreased in the c-KIT expressing cells. The principal role of the A subunit is that of a scaffold for the recruitment of B subunits and additional proteins into the PP2Λ complex. Binding of the A sυbunit to both C and B subunits occurs via a hydrophobic surface created by loops between 15 imperfect, 39 amino-acid repeats ('HEAT repeats') [40, 41]. Interestingly, suppression of PP2A A using shRNA results in activation of the PI3K/Akt pathway and induces tumourigenicity in transformed HEK293T cells [42, 43]. Rescue of WT PP2A-A expression inhibits the tumourigenesis. Furthermore, mutations in PP2A-A have been identified in breast, lung, colon, and other cancers [22]. These mutants inhibit binding of specific B regulatory subunits [44, 45], and cannot rescue the tumourigenic phenotype of HEK293T cells with suppressed WT [42, 43]. Thus PP2A-A mutations contribute to cancer development by inducing functional haploinsufficieny, disturbing PP2A holoenzyme composition, and altering the selective enzymatic activity of PP2A [42, 43]. Consistent with a role for decreased regulatory subunit binding in cancer development. shRNA downregulation of the B'γ subunit also reduces PP2A activity and induces tumourigenicity of HEK293T cells [36]. B'γ is reduced to undetectable levels in both the mutant c-KIT myeloid cells (Fig. 3). Down-regulation of PP2A subunits has also been observed in human tumours and cancer cell lines [22]. Thus down regulation of the structural and regulatory subunits in cells expressing active c-KIT would contribute to the reduced PP2A activity seen in these cells, and may be required for c-KIT induced leukaemogenesis

Overall, specific inhibition of PP2A by c-KIT can provide a unique target for therapeutic intervention in C-KIT⁺ cancers. The present study shows that PP2A activity can be reactivated in the C-KIT⁺ FDCPl myeloid cells using the pharmacological agent, FTY720 (Fig. 4A). FTY720 (also called fingolimod), is a synthetic myriocin analog structurally similar to sphingosine. It is a water-soluble, non-toxic drug with high oral bioavailability that reversibly arrests lymphocyte trafficking (mainly of CD4+ T cells). It is currently used as an immunomodulator m Phase III trials for patients with multiple sclerosis [46] or undergoing renal transplantation [47]. FTY720- induced reactivation of PP2A has recently been shown to inhibit the in vitro and in vivo growth and survival of BCR/ABL⁺ CML [33]. FTY720 has also been shown to be effective in preclinical models of B -cell chronic lymphocytic leukaemia (B-CLL) [48]. Importantly, the toxicity and induction of apoptosis by FTY720 requires activation of PP2A [19, 48]. Inhibition of FTY720-induced PP2A activation by okadaic acid or SV40 small T antigen (a specific PP2A inhibitor), rescues BCR/ABL phosphorylation and the clonogenic potential of BCR/ ABL⁺ myeloid cells [19]. Okadaic acid also inhibits FTY720-induced apoptosis in CLL cells [48]. FTY720 is phosphorylated by sphingosine kinases, and the p-FTY720 antagonises sphingosine 1-phosphate receptors (SIPRs). It is this function that is thought to be important in its immunomodulatory role [49]. However, it has been shown that PP2A activation, BCR/ABL inactivation and inhibition of leukamogenesis does not require sphingosine kinase FTY720 phosphorylation nor the triggering of Gi protem-coupled SlPR mediated signalling [19]. In addition, FTY720 has been shown to activate purified PP2A AC dimers and ABC trimers in vitro [50], further suggesting that PP2A activation is not due to inhibition of SIPR signalling.

A chemically distinct PP2A activator, forskolin, was also found to inhibit the growth of C-KIT⁺ cells (Table 1) in the present study. Forskolin has traditionally been used as a tool to assess effects of c-AMP activation, however was recently found to also activate PP2A [51]. PP2A activation by forskolin has been shown to be independent of c-AMP activation, as 1,9-dideoxyforskolin, an analogue that does not affect cAMP, also activates PP2A [18]. The hypersensitivity C-KIT⁺ FDCPl cells to two distinct PP2A activators shows that reactivation of PP2A is the essential mechanism of action.

The mechanism by which PP2A reactivation impairs clonogenic potential and induces apoptosis is likely multi-factorial. PP2A is a negative regulator of PI3K, Ras/ERK and JAK/STAT pathways. Thus reactivation of PP2A may inhibit one or more of these essential growth and survival pathways. Furthermore, PP2A regulates apoptosis by dephosphorylation of the anti-apototic protein Bcl-2 and the pro-apoptotic protein Bax. Importantly, PP2A also activates the tyrosine phosphatase SHP-I [18], a negative regulator of c-KIT phosphorylation. Indeed, as found in the present study, reactivation of PP2A by FTY720 induces c-KIT tyrosine dephosphorylation of both imatinib sensitive and resistant activating mutants (Fig. 5).

This dephosphorylation, and hence inactivation of c-KIT would further inhibit the activation of downstream signalling pathways, leading to inhibition of cell growth and apoptosis. As D816V is the most common mutation in relapsed patients, and there is currently no kinase inhibitor that is active on this KD mutant, PP2A activation represents a unique and powerful strategy for treating such imatinib-resistant patients.

Although the invention has been described with reference to particular embodiments, it will be appreciated by those skilled in the art that numerous variations and/or modifications may be made departing from the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims

1. A method for inhibiting c-KIT in a cancer cell, comprising treating the cancer cell with an agent for up regulating protein phosphatase 2A (PP2A) activity in the cancer cell.

2. A method according to claim 1 wherein the cancer cell is a non-leukemic c-KIT cancer cell.

3. A method according to claim 2 wherein the agent is an activator of PP2A.

4. A method according to claim 3 wherein the activator is selected from the group consisting of FTY720, forskolin, 1,9-dideoxyforskolin, ceramides, C2-ceramide, topoisomerase inhibitors, etoposide, tubulin polymerisers, methyl-3,5-diiodo-4-(4'- methoxypropoxy)benzoate (DIME or DIPE), fatty acids, palmitate, thiol alkylating agents, and N-ethylmaleimide (NEM).

5. A method according to claim 4 wherein the activator is FTY720.

6. A method according to any one of claims 1 to 5 wherein the cancer cell is resistant to treatment with an inhibitor of c-KIT.

7. A method according to any one of claims 1 to 6 wherein the c-KIT is constitutively activated in the cancer cell.

8. A method according to claim 7 wherein the cancer cell expresses c-KIT with a mutation selected from the group consisting of V560G, ΔTYD417-419 and mutations involving amino acid D816.

9. A method according to any one of claims 1 to 8 wherein the cancer cell is a cell of a c-KIT cancer is selected from the group consisting of mastocytosis, mast cell cancers, mesenchymal cancers, gastrointestinal stromal tumours (GISTs), non small cell lung carcinomas, and melanomas.

10. A method for inhibiting growth of a non-leukemic c-KIT cancer cell, comprising treating the cell with an effective amount of an agent for up regulating protein phosphatase 2A (PP2A) activity in the cell.

11. A method according to claim 10 wherein the agent is an activator of PP2A.

12. A method according to claim 10 wherein the activator is selected from the group consisting of FTY720, forskolin, 1,9-dideoxyforskolin, ceramides, C2-ceramide, topoisomerase inhibitors, etoposide, tubulin polymerisers, methyl-3,5-diiodo-4-(4'- methoxypropoxy)benzoate (DIME or DIPE), fatty acids, palmitate, thiol alkylating agents, and N-ethylmaleimide (NEM).

13. A method according to claim 10 wherein the activator is FTY720.

14. A method according to any one of claims 10 to 13 wherein the cancer is resistant to treatment with an inhibitor of c-KIT.

15. A method for prophylaxis or treatment of a non-leukemic c-KIT cancer in a mammal, comprising administering an effective amount of an agent for up regulating protein phosphatase 2A (PP2A) activity to the mammal.

16. A method according to claim 15 wherein the agent is an activator of PP2A.

17. A method according to claim 15 or 16 wherein the cancer is resistant to treatment with an inhibitor of c-KTT.

18. A method of screening an agent for capacity to inhibit c-KIT, comprising: providing a sample of c-KIT cancer cells; selecting an agent for up regulating protein phosphatase 2A (PP2A) activity in the cancer cells; treating the cells with the agent; and determining whether growth of the cells is inhibited by the agent, the inhibition of growth of the cells by the agent being indicative the agent inhibits c-KIT.

19. A method according to claim 18 wherein the c-KIT cancer cells are cells of a non-leukemic cancer.

20. A method according to claim 18 or 19 wherein the agent is an activator of PP2A.

21. A method according to any one of claims 18 to 20 wherein the c-KIT cancer cells are cells of cancer resistant to treatment with an inhibitor of c-KIT.

22. Use of an agent for up regulating protein phosphatase 2A (PP2A) activity to inhibit c-KIT in a cancer cell.

23. Use of an agent for up regulating protein phosphatase 2 A (PP2A) activity in the prophylaxis or treatment of a non-leukemic c-KIT cancer in a mammal.

24. Use of an agent for up regulating protein phosphatase 2A (PP2A) activity in the manufacture of a medicament for prophylaxis or treatment of a non-leukemic c-KIT cancer m a mammal.

25. Use of an agent for up regulating protein phosphatase 2 A (PP2A) activity to inhibit growth of a non-leukemic c-KIT cancer cell.

26. The use according to any one of claims 22 to 25 wherein the agent for up regulating PP2A activity is an activator of PP2A.