WO2012130720A2 - PREDICTION OF RESPONSIVENESS TO PIK3/mTOR INHIBITORS - Google Patents

Abstract

A method for determining the responsiveness of a mammalian tumor cell to treatment with an inhibitor of the phosphoinosite 3-kinase (PI3K) and/or mammalian target of rapamycin (mTOR) pathway, said method comprising determining the c-MYC and/or NOTCH1 level in said tumor cell, wherein said level is indicative of whether the cell is likely to respond or is responsive to the treatment. It further refers to an in vitro method for the identification of a resistance to treatment or of predicting or monitoring the efficacy of treatment, of solid tumor cancer with an inhibitor of the PI3K and/or mTOR pathway in a patient suffering from said cancer. It further refers to a combination comprising an amount of an inhibitor of the PI3K and/or mTOR pathway, and an amount of a c-MYC and/or NOTCH 1 inhibitor.

Description

PREDICTION OF RESPONSIVENESS TO PIK3/mTOR INHIBITORS

TECHNICAL FIELD OF THE INVENTION The invention is related to the area of cancer therapy and management. In particular, it relates to providing therapies that are tailored to a patient's own tumor. In accordance therewith, it relates to a method for determining responsiveness of a tumor cell to treatment with an inhibitor of the phosphoinosite 3-kinase (PI3K) and/or mammalian target of rapamycin (mTOR) pathway.

BACKGROUND OF THE INVENTION

Many factors contribute to patients' response to anti-cancer therapy, including pharmacogenetics, tumor microenvironment, vascularity and genetic aberrations.

Identifying the molecular mechanisms that influence response to anti-cancer drugs can improve therapy by identifying those individuals who will benefit most while avoiding unnecessary treatment. However, due partly to the heterogeneity between tumors, it has been challenging to identify robust biomarkers and functionally link cancer genes to drug sensitivity. Nonetheless, catalogues describing the molecular changes in the major tumor types, currently emerging from sequencing efforts, will theoretically enable systematic studies into the molecular aberrations underpinning treatment response.

Another important objective of cancer research is to develop new anti-cancer treatments with increased specificity for cancer cells. For example, the monoclonal antibody Trastuzumab directly targets HER2/NEU positive breast cancer and BRAF kinase inhibitors have recently shown promise in melanoma carrying BRAF mutations.

The molecular changes that occur in cancer cells can result in a dependency on gene products that are not essential in normal cells. Inhibition of these proteins would thus result in cell cycle arrest or death of the cancer cell but would not affect fitness of their normal counterparts. This notion, which has been termed synthetic

sickness/lethality or non-oncogene addiction, has provided a framework to identify drugs that do not target the cancer gene directly yet are specific for cells that contain the aberration. Indeed, the observation that BRCA mutant cells are hypersensitive to inhibition of the enzyme PARP has recently found its way into the clinic and represents the paradigm for synthetic lethality-based therapy. However, there are currently only a few cancer-relevant synthetic lethal interactions that have been identified. Thus, a systematic analysis of the effect of individual cancer genes on the cellular response to existing and experimental drugs may identify new targeted anti-cancer therapies directly relevant for the clinic.

The challenge of such a systematic approach is the large number of

combinations between drugs and genes that would require experimental analysis. The promise of insight into drug actions as exemplified by similar screens in model organisms, most notably yeast, warrants development of suitable methods in human cells.

The ability to predict patients' response to treatment is of paramount importance for patient stratified medicine and increasingly recognized as an integral part of cancer drug discovery.

In breast cancer the size, stage, rate of growth, and other characteristics of the tumor determine the kinds of treatment. Treatment may include surgery, drugs

(hormonal therapy and chemotherapy), radiation and/or immunotherapy. Surgical removal of the tumor provides the single largest benefit, with surgery alone being capable of producing a cure in many cases. To somewhat increase the likelihood of long-term disease-free survival, several chemotherapy regimens are commonly given in addition to surgery. Some breast cancers are sensitive to hormones such as estrogen and/or progesterone, which makes it possible to treat them by blocking the effects of these hormones.

The mammalian target of rapamycin (mTOR) is a critical regulator of several normal cell processes in numerous cell types, including cells of the breast. Several proteins, including PI3-kinase (PI3K), Akt, and PTEN impinge on mTOR signaling. PI3K is an enzyme that phosphorylates certain components of the cell membrane. Once these components become phosphorylated, they bind to a protein called Akt. Akt then becomes phosphorylated and activated. This triggers activation of several downstream signaling pathways, which increase cell survival, proliferation, and cell growth. One important player in the growth and proliferation pathways is mTOR. When activated by Akt, mTOR promotes cell growth and proliferation by stimulating protein synthesis. In addition to receiving signals from Akt, mTOR monitors the cell's environment for the presence of growth factors and nutrients. If the cell needs additional resources, mTOR can increase nutrient uptake and promote angiogenesis. mTOR can also increase the activity of Akt, thus enhancing the other downstream effects of this protein. One of the regulators of this pathway is PTEN, it removes the phosphate groups added to membrane phospholipids by PI3K. This prevents activation of Akt and its downstream pathways.

Activation of the mTOR pathway is associated with poor prognosis in many cancers, including breast cancer, and is linked to resistance to many types of therapy.

In an effort to inhibit PI3K/Akt/mTOR signalling, drugs targeting PI3K, Akt, and mTOR are being tested in preclinical models and clinical trials.

One mTOR inhibitor is a small molecule called rapamycin. Rapamycin enters the cell and binds to a protein called FKBP12. This complex binds to and inhibits mTOR. Inhibition of mTOR with rapamycin has been found to slow cancer cell proliferation. Small molecule mTOR inhibitors, including rapamycin (also called

Sirolimus), CCI-779 (Temsirolimus), RAD-001 (Everolimus), Akt inhibitors, including KRX-0401 (Perifosine, Perifosineare), and PI3K inhibitors are being tested in clinical trials for breast cancer.

Crosstalk between signaling pathways has been shown to be a potential mechanism for drug resistance for many anti-cancer drugs. However, the complexity of signaling networks hampers a priori prediction of which pathways could play a role. The mechanisms that possibly confer resistance to inhibitor molecules designed for cancer therapy and in particular the PI3K/mTOR have not been fully explored. The understanding of the mechanism is of particular importance given the large fraction of solid tumors with activating mutations in PI3K pathway (Liu, P.et al. Nature reviews. Drug discovery 8, 627-644 (2009)).

Currently, only the activation status of the PI3K pathway itself is considered a predictor of sensitity. Specific PI3K mutations are considered to be a predictor of resistance to inhibiting therapy.

WO2006/122053A2 describes indicators useful for predicting the likelihood that a tumor is sensitive to an mTOR inhibitor, which are FKBP, HIF-1alpha, VHL and RHEB. As additional biomarkers there is disclosed (individually or in conjunction with one another) a variety of upstream and downstream indicators, inter alia Myc. A higher level of such indicator is described to be indicative that the tumor is sensitive to an mTOR inhibitor. There is a need to identify factors that can be used to predict responsiveness or resistance to therapeutic targets, such as those involved in the PI3K/mTOR pathway.

SUMMARY OF THE INVENTION

It is an objective of the invention to provide a method for determining the responsiveness of a tumor to treatment with an inhibitor of the PI3K/mTOR pathway, specifically to allow appropriate patient stratification and improved methods of treating such tumor.

The objective is solved by the subject matter of the invention.

According to the invention there is provided a method for determining the responsiveness of a mammalian tumor cell to treatment with an inhibitor of the phosphoinosite 3-kinase (PI3K) and/or mammalian target of rapamycin (mTOR) pathway, said method comprising determining the c-MYC level and/or NOTCH 1 in said tumor cell, wherein said level is indicative of whether the cell is likely to respond or is responsive to the treatment. The level of either of the marker genes c-MYC or

NOTCH1 may be determined, or both of them, as described below in more detail. Specifically the c-MYC and/or NOTCH 1 upregulation is indicative for the resistance of said tumor cell to the treatment. This was particularly suprising, since the prior art disclosure of WO2006/122053A2 teaches a series of indicators, wherein a higher level of an indicator was supposed to be indicative that a tumor is sensitive to an mTOR inhibitor.

Specifically the tumor cell is a cancer cell or cells from solid tumors, such as epithelial tumors, including malignant or benign tumors, and specifically solid tumors, including a breast tumor or breast cancer. Specifically the present invention has utility for the improved treatment of patients suffering from cancer, in particular those suffering from a solid tumor or a malignant solid tumor. Cancer associated with solid tumor is also referred to as "solid cancer", also referred to as "solid tumor cancer". The mammalian tumor is specifically derived from a subject that is either a human or non- human animal. Specifically the tumor cells are from biological samples of those subjects, who have been diagnosed with a disease associated with said tumor, including cancer. Such subjects are hereinafter also referred to as "patients". It is preferred that the cell or sample is obtained from a tumor of a patient, including tumor derived cells, e.g. from tumor tissue, distinct metastases or disseminated tumor cells circulating in bodily fluids.

In specific cases, the solid tumor is a breast cancer and the patients are suffering from breast cancer, in particular late stage or metastatic disease.

In a preferred embodiment the method according to the invention further comprises that the level of NOTCH1 is additionally determined.

In accordance with the invention, there is further provided an in vitro method for the identification of a resistance to treatment of solid tumor cancer, specifically breast cancer, with an inhibitor of the PI3K and/or mTOR pathway in a patient suffering from said cancer, said method comprising the following steps:

(a) obtaining a sample from a patient suffering from solid tumor cancer, and

(b) evaluating in said sample the level of c-MYC and/or NOTCH1 expression, preferably the NOTCH 1 activity, whereby the c-MYC and/or NOTCH 1 upregulation is indicative for the resistance of said patient to the inhibitor.

Specifically the method according to the invention refers to treatment with an inhibitor, which is a direct or indirect inhibitor of PI3K and/or Akt and/or mTOR.

Specifically said inhibitor is a kinase inhibitor.

Specific examples of said inhibitors are small molecule inhibitors, e.g. mTOR and/or Akt and/or PIK3 inhibitors, including BEZ235, PIK90, GDC-0941 , RAD001 (Everolimus), rapamycin (Sirolimus), CCI-779 (Temsirolimus), TORIN, Akt inhibitors, including KRX-0401 (Perifosine, Perifosineare).

According to a specific embodiment, the method provides for the determination of the levels of the gene(s) c-MYC and/or NOTCH1 by measuring the quantity and/or activity of the genes or gene expression products. Such gene expression products are either nucleic acids, oligonucleotides, or amplification products, or else polypeptides or proteins encoded by such genes, or reaction products, such as cleavage products or metabolites, specifically those which are indicative for an activated state of the gene(s).

According to another specific embodiment the level of the gene(s) is indirectly determined by analysing surrogate markers indicative for the level of c-MYC and/or NOTCH 1 and/or activation products of c-MYC and/or NOTCH 1 .

Specific surrogate markers are HES1 , HES2, HES3, HEY1 , HEY 2, elF4 family genes. According to a further specific embodiment the level of the gene(s) is

determined by measuring the level of NOTCH ligands, such as JAGGED1 , JAGGED2, DELTA-likel , Delta-like3 and Delta-like4,

The determination is specifically carried out to quantify the level, either in a semi-quantitative or quantitative way, e.g. employing reference or control levels, e.g. internal controls.

Specifically the method according to the invention provides for determining said level by a nucleic acid analysis, e.g. selected from the group consisting of methods which employ amplification methods, among them nucleic acid amplification methods, RT-PCR, microarrays, nuclear localization analysis, hybridization, NOTCH1 mutation analysis.

Specifically the method according to the invention provides for the determination of the MYC and/or NOTCH expression by measuring the respective gene amplification by PCR or in situ hybridization. A method of measuring the respective mRNA expression level may comprise the following steps:

a) extraction of the total RNAs of said biological sample, e.g. the tissue sample or cellular fraction of a body fluid, preferably from tumor cells, followed, where appropriate, by purification of the mRNAs,

b) reverse transcription of the RNAs extracted in step a), and

c) PCR amplification of the cDNAs obtained in step b) using at least a pair of primers specific for the mRNA to be quantified.

In a preferred embodiment, when the determination of the mRNA is carried out by a method comprising a PCR or RT-PCR amplification, primers and probe set specific for the mRNA to be quantified can be designed using the appropriate reference sequences.

According to a further specific embodiment, the level is determined by a protein analysis e.g. selected from the group consisting of immunoassays, such as ELISA, EIA, RIA, western blot, protein arrays, immunocytochemistry or immunohistochemistry methods.

MYC and NOTCH specific antibodies which may be used in an appropriate immunoassay are well known including, Cell signaling 94025/24215, Abeam

1 1917/83253, Pierce 82385, Santa Cruz sc-40/ sc-23307, Sigma-Aldrich M4439/ SAB4502019, Biolegend 626801/629101 . It is understood that the method according to the invention may also provide for the combination of means and methods for determination of such levels, including combination of the nucleic acid analysis and protein analysis.

According to a specific aspect of the invention there is provided an in vitro method of predicting the efficacy of a treatment of solid tumor cancer, specifically breast cancer, with an inhibitor of the PI3K and/or mTOR pathway, for a patient suffering from said cancer, comprising the steps of

a) determining in a cell or tissue sample obtained from said patient the level of c-MYC, and optionally the level of NOTCH 1 , and

b) comparing the activity or level of said marker gene(s) determined in a) with a reference level, wherein the extent of the difference between said level determined in a) and said reference level is indicative for the predicted efficacy of said treatment. Specifically an increase is indicative for the reduced predicted efficacy of the treatment or a reduced tumor response. More specifically, an increase of said marker activity or level is indicative for the reduced predicted efficacy of the treatment.

Such predicting is preferred before starting treatment, e.g. to stratify a patient population according to the likelihood of a response or chances of a successful treatment.

According to another specific aspect of the invention there is provided an in vitro method of monitoring the efficacy of a treatment of solid tumor or cancer, specifically breast cancer, with an inhibitor of the PI3K and/or mTOR pathway, in a patient suffering from said cancer comprising the steps

a) determining in a cell or tissue sample obtained from said patient the level of c-MYC, and optionally the level of NOTCH 1 , and

b) comparing the activity or level of said marker gene(s) determined in a) with a reference level, wherein the extent of the difference between said level determined in a) and said reference level is indicative for said efficacy. Specifically an increase is indicative for the reduced efficacy or a reduced tumor response in such treatment. More specifically an increase of said marker activity or level is indicative for the reduced efficacy.

Such monitoring is preferred in the course of a treatment schedule, e.g. during the first phase of treatment to verify the response of treatment. The method of the invention specifically provides for the treatment of cancer which comprises the administration of an inhibitor of the PI3K and/or mTOR pathway in combination with a c-MYC and/or NOTCH1 inhibitor.

In accordance therewith, there is specifically provided a combination comprising an amount of an inhibitor of the PI3K and/or mTOR pathway, and an amount of a c- MYC and/or NOTCH1 inhibitor, optionally in association with one or more

pharmaceutically acceptable carriers. Such combination can be a combination product or a kit of parts, prepared for concomitant, simultaneous, parallel or consecutive treatment.

Specific MYC and/or NOTCH 1 inhibitors that may be used in a combination treatment are, for instance, antibodies directed at NOTCH receptors or ligands (OMP- 21 M18, AV-232), inhibitors or NOTCH cleavage (e.g. ADAM/ TACE inhibitors, ILX, LY41 1575, LY450139, DAPT, dibenzazepine, MK0752 SL-301 , SL-302, RO4929097, tarenflurbil), inhibitors interfering with NOTCH binding to co-factors, such as SAHM peptides, and specifically including targeted therapies, including siRNA treatment, therapies employing affinity binders, such as immunotherapies employing antibodies or other receptors or ligands.

According to the invention there is further provided a novel use of a the repertoire of isogenic epithelial, mammalian cells, such as human epithelial cell lines, like breast cell lines, for determining one or more factors that influence the

susceptibility to an inhibitor of the PI3K and/or mTOR pathway.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 | Barcode screen set-up, detection and performance, (a) Isogenic cell lines infected with a lentiviral vector carrying a unique 24 base pair barcode sequence and specific genetic modification (e.g. cDNA or RNAi) are pooled, seeded in multi-well plates and subsequently treated with drug or DMSO control (left). The relative abundance of the barcodes in the population of cells is a proxy for the cellular fitness. In the example the cells with the "orange" barcode display a synthetic sick/lethal interactions with Drug X. After drug treatment the pooled isogenic cell lines are harvested, genomic DNA (gDNA) is isolated and barcodes are amplified (right). Labeled product is then hybridized to Luminex microspheres and the mixture is measured on a Luminex machine to determine the relative abundance for each of the 100 barcode sequences, (b) 5' biotinylated DNA oligos representing the linear amplification (LAMP) product were hybridized to xMAP microspheres at the indicated concentrations and fluorescent intensities were measured. Shown are the results for four independent barcode sequences, (c) Scatter plots of barcode signals derived from a pool of barcoded pLKO.2 vectors (left) or genomic DNA derived from HeLa cells infected with the same pool (right). Pearson correlation coefficients were calculated and shown are representative examples of three independent experiments, (d) One hundred pools with 99 barcoded plasmids (each missing a different barcode) were quantified using the Luminex assay. For each measurement a Z-score was calculated for the missing barcode and plotted.

Figure 2 | Negative selection in pooled cell populations, (a) MCF10A cells were infected with 96 distinct barcoded lentiviral vectors one of which lacking the puromycin resistance gene and treated with puromycin or left untreated (upper panels). Barcodes amplified from pooled cells subjected to puromycin selection for 3 days were analyzed and compared to unselected cells (lower panel). The experiment was performed in quadruplicate and standard deviations are indicated, (b) Barcoded cells expressing the inactive FANCD2-K561 R cDNA were mixed into a pool of barcoded cells expressing wild type FANCD2 and treated with MMC (15 ng/ml) for 5 days. Shown are the median signals for all barcodes of four independent drug treatments compared to DMSO control.

Figure 3 | Combinatorial breast cancer gene small compound screen, (a) Gene ontology annotation of the seventy breast cancer genes selected for the screen, (b) Overview of the primary targets of the drug library used in the screen, (c) Radial gene-drug interaction plot displaying the 7743 (89 isogenic cell lines x 87 drugs) pairwise drug-gene measurements. Distance from the center indicates significance and dot size is proportional to the magnitude of the drug vs. control effect. P-values for selected hits are indicated.

Figure 4 | Validation of selected screen hits, (a) Dose-response analysis of c- MYC, ICN1 and control MCF10A cells with the Aurora kinase inhibitor AT9283. Cells were treated with the indicated concentrations for 5 days and relative cell number was assessed. The experiment was repeated three times in triplicate and standard deviations are indicated, (b) The mean resistance scores (see Methods) for all ICN1 drug treatments were divided into PI3K and non-PI3K inhibitors and the mean for each group was calculated (p=0.02, one-sided Mann-Whitney test). MDM2 drug treatments served as a control. BEZ-235 was left out of the analysis, (c) Dose-response analysis of ICN1 and control MCF10A cells. Cells were treated with the indicated

concentrations BEZ-235 for 5 days and relative cell numbers were measured. The experiment was repeated four times in triplicate and standard deviations are indicated. (d) Bar graph showing relative viability of ICN1 or control MCF10A cells treated with PIK90 (250 ng/ml) for 5 days. Standard deviations of three replicates are indicated, (e) Crystal violet stained culture dishes of colony formation experiment. Cells were seeded at low density and treated with 30 ng/ml BEZ235 for 10 days. Figure 5 | NOTCH activation renders breast cancer cells resistant to

PI3K/mTORC1 inhibition, (a) Bar graph showing relative viability of ICN1 or control MCF10A cells treated with PP242 (3.0 uM) for 5 days. Shown is the mean of an experiment performed in triplicate and standard deviations, (b) Indicated MCF10A cells were treated for 5 days with 10 uM rapamycin. Shown is the mean of a representative experiment performed in triplicate (^* p<0.05). (c) BT549 or MCF7 cells were infected with a lentivirus expressing ICN1 and GFP and treated as indicated for 3 days after which the fraction of GFP positive cells was determined.

Figure 6 | ICN1 mediated induction of c-MYC uncouples PI3K-mTOR signaling from proliferation, (a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371 ) and total mTOR as a loading control, (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Dose response curve of c-MYC or control MCF10A cells with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. Four independent experiments were performed. Error bars indicate standard deviations, (d) Western blot analysis of c-MYC expression in control and ICN1 expressing cells. PDK1 served as a loading control, (e) Quantitative RT-PCR of c-MYC expression in wild type MCF10A (black), ICN1 cells (light grey) and ICN1 cells transfected with c-MYC siRNA (dark grey), (f) Cells as in (e) were treated with BEZ- 235 or vehicle for 3 days and relative cell number was determined (^*** p<0.001 ). Supplementary Figure 1 | The pLKO.2 vector. Cloning strategy, vector map and sequence.

Supplementary Figure 2 | PCR of barcodes shows limited PCR bias. PCR product of a pool of barcoded vectors (PCR, black bars) was used for a nested second PCR (re-PCR, blue bars) and fluorescent intensities were measured. Signals were normalized to the first PCR and the experiment was performed in triplicate. Error bars represent standard deviation.

Supplementary Figure 3 | FANCD2-K561 R cells display increased

sensitivity to Mitomycin C. Indicated cells were treated with MMC or left untreated for 5 days. Relative cell viability was measured using Cell titer Glo and shown are the means and standard deviations of a triplicate experiment.

Supplementary Figure 4 | Validation of cDNA and shRNA expressing MCF10A cells, (a, b) Protein or mRNA from infected and selected cells were analyzed by Western blot or qRT-PCR as indicated.

Supplementary Figure 5 | ICN3 expressing cells are not resistant to BEZ- 235. Dose response curve for ICN3 or control MCF10A cells treated with indicated concentrations of BEZ-235 for 5 days. Indicated are the standard deviations.

Supplementary Figure 6 | Phosphorylation of the mTORCI downstream target 4EBP1 is inhibited in MCF10A cells expressing ICN1. Western blot analysis of cells treated with BEZ-235 for 6 hours. AKT served as a loading control.

Supplementary Figure 7 | MCF10A-MYC cells show trend towards resistance to PI3K inhibitors in the screen. The mean resistance scores (see Methods) for all c-MYC drug treatments were divided into PI3K and non-PI3K inhibitors and the mean for each group was calculated. BEZ-235 was left out of the analysis (p= 0.08).

Supplementary Table 1 | General reference table, (worksheet 1) Overview of all cancer genes selected for the screen, including type of aberration, estimated frequency and references, (worksheet 2) cDNA expression vectors used in screen, (worksheet 3) Genes selected for siRNA knockdown, (worksheet 4) Used shRNA sequences, including (literature) reference (worksheet 5) Sequences of xMAP barcode sequences, (worksheet 6) Selected drugs for screen, (worksheet 7)

qRTPCR primers used in study. DETAILED DESCRIPTION OF THE INVENTION

The term c-MYC, also called MYC, as used herein refers to a gene (NCBI GenelD 4609 ) encoding a MYC protein, or else the MYC protein, which is known to bind to the DNA of other genes, commonly known as a transcription factor. In addition to its role as a classical transcription factor, MYC also functions to regulate global chromatin structure by regulating histone acetylation both in gene-rich regions and at sites far from any known gene.

The term shall include the proto-oncogene, and specifically the cancerous version of the gene, called an oncogene. A mutated version of MYC is found in many cancers which causes MYC to be persistently expressed. This leads to the unregulated expression of many genes some of which are involved in cell proliferation and results in the formation of cancer. A major effect of MYC is B cell proliferation.

The term NOTCH1 , also called NOTCH, as used herein refers to a gene (NCBI gene ID 4851 , UniProtKB/Swiss-Prot: NOTC1_HUMAN, P46531 ) encoding a NOTCH protein, or else the NOTCH protein. The gene encodes a member of the Notch family. Members of this Type 1 transmembrane protein family share structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like (EGF) repeats, and an intracellular domain consisting of multiple, different domain types. Notch family members play a role in a variety of developmental processes by controlling cell fate decisions. The Notch signaling network is an evolutionarily conserved intercellular signaling pathway which regulates interactions between physically adjacent cells. When activated by extracellular ligands this protein is cleaved by gamma secretase and the resulting intracellular domain of NOTCH activates gene expression. NOTCH functions as a receptor for membrane bound ligands, including JAGGED1 , JAGGED2, DELTA-likel , Delta-like3 and Delta-like4

The term "inhibitor of the phosphoinosite 3-kinase (PI3K) and/or mammalian target of rapamycin (mTOR) pathway" as used herein shall refer to an antagonist of the pathway that would interact with the corresponding genes or expression products to inhibit the activation of such genes or expression products and the process of signal transduction. The term specifically refers to kinase inhibitors or other inhibitors, which downmodulate the kinase enzymatic activity in a direct or indirect way, in particular inhibitors of PI3K and/or mTOR and/or Akt inhibitors. The term specifically includes PI3K kinase inhibitors, mTOR kinase inhibitors or PI3K/mTOR dual kinase inhibitors well-known in antitumor therapy. In particular such inhibitors are orally bioavailable small molecule targeting the phosphatidylinositol 3 kinase (PI3K) and/or mammalian target of rapamycin (mTOR) kinases in the PI3K/mTOR signaling pathway, with antineoplastic activity. Such antineoplastic activity may be tested in suitable functional and binding assays. PI3K/mTOR kinase inhibitors inhibits either PI3K kinase, mTOR kinase or both, which may result in tumor cell apoptosis and growth inhibition in susceptible tumor cell populations. Activation of the PI3K/mTOR pathway promotes cell growth, survival, and resistance to chemotherapy and radiotherapy.

The term "response" or "responsiveness", as used herein refers to a tumor response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth. The term shall also refer to an improved prognosis, e.g. reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause.

The term "sensitive" or "sensitivity" refers to the susceptibility of e.g. a tissue or cell population to treatment with a drug or drug composition compared to the reference standard, cell, or cell population. The reference may for example be the corresponding healthy tissue, cell, or cell population, for example be the corresponding non-tumor counterpart (wild type).

The term "resistant" or "resistance" as used herein shall refer to the reduced susceptibility of e.g. a tissue or cell population to the treatment a drug or drug composition compared to the above reference standard, cell, or cell population . The resistance in particular cells or tissues obtained from a patient, determines that said cells or tissue is likely to not to respond to the administration of a drug or composition. Thus, the clinician would consider using another drug or composition for the treatment of said patient.

Susceptibility to the treatment with a drug or drug composition is typically monitored as the response to the treatment with a drug or drug composition in terms of rate of survival and/or growth rate in the presence of said drug or composition. Thus, increased survival or maintained or increased growth compared to the reference indicates resistance to said drug or drug composition. It is to be understood that a cells may change in susceptibility depending on the status of the patient or stage of disease. Therefore, in specific cases, the susceptibility or possible resistance is determined from time to time.

The term "upregulation" with respect to MYC and/or NOTCH, as used herein refers to a differential, increased level of MYC and/or NOTCH, e.g. by a differential expression of the genes, an increased level of genes and gene products or an increased level of activity. When upregulated, the level of the gene(s) is significantly higher in a drug-resistant sample as compared to a reference level.

The term "level" as understood herein always refers to the number of copies, concentration or activity of the gene(s), gene products, expression products including encoded polypeptides or proteins, cleavage products and metabolites, specifically those resulting from an activation of the gene(s) or encoded proteins. In accordance therewith, the "NOTCH level" or "MYC level" is particularly understood to refer to the level of NOTCH pathway activity.

As used herein, the term "reference level" refers to a level of a substance which may be of interest for comparative purposes. In one embodiment, a reference level may be the expression level of a protein or nucleic acid expressed as an average of the level of the expression level of a protein or nucleic acid from samples taken from a control population of healthy or disease-free subjects. In another embodiment, the reference level may be the level in the same subject at a different time, e.g., before the present assay, such as the level determined prior to the subject developing the disease or prior to initiating therapy. In general, samples are normalized by a common factor. For example, body fluid samples are normalized by volume body fluid and cell- containing samples are normalized by protein content or cell count. For instance, the reference level may be the level of the same parameter measured in a control sample, e.g. a mean level for a drug-susceptible or drug-resistant sample, or may represent a cut-off or threshold level or fold change level for a sensitive or resistant designation. The upregulation is typically determined if the increase is at least 2 fold the standard deviation, preferably at least 3 fold, 4 fold or 5 fold, and possibly more than 10 fold. It is understood that the upregulation of the gene(s) may also be determined by measuring the level of effectors upstream or downstream the MYC and/or NOTCH gene(s) in the respective pathway, e.g. effectors upstream of MYC, including NOTCH. Thus, according to the invention it is possible to determine the MYC level indirectly by determinig other effectors in the pathway, such as NOTCH.

To facilitate the quantitative assessment of functional drug-gene interactions in human cells, we developed a method to multiplex cellular fitness measurements of up to one hundred isogenic cell lines using molecular barcodes. This method assists the systematic assessment of the impact of cancer aberrations on proliferation in response to a collection of drugs. In a specific query, a 70 x 87 drug-gene interaction matrix in breast cancer cells, the interrogation of over six thousand drug-gene pairs was possible. Besides several previously identified drug-gene interactions, we surprisingly found a novel mechanism of resistance to PI3K inhibitors, which are currently in clinical trials (Workman, P.et al. Cancer research 70, 2146-2157 (2010)).

Using an unbiased drug-gene functional interaction screen we identified that MYC expression levels determine the response of breast cancer cells to PI3K pathway inhibitors, including mTOR inhibitors (eg rapamycin, BEZ235). Cells that express high levels of MYC, either due to exogenous expression of MYC or endogenous activation (eg NOTCH 1 acitvation), display marked resistance to these compounds. Based on these findings, it is concluded that mechanisms resulting in upregulation of MYC activity and/or protein levels would render breast cancer cells resistant to PI3K inhibitors. This finding has important clinical implications as patients with high MYC levels are unlikely to benefit from treatment with PI3K pathway inhibitors. The resistance to PI3K inhibitors is not limited to one cell line and could also be found in the MCF7 cell line, indicating that MYC activation in estrogen positive breast cancers also confers resistance to PI3K inhibitors. Furthermore, all PI3K inhibtors tested showed the same result.

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. In a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, the systematic analysis of thousands of gene-drug interactions was possible. Applying this approach to breast cancer revealed various synthetic lethal interactions and drug resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to PI3K inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC overrode the dependency of cells on the PI3K/mTOR pathway for

proliferation. These data reveal a novel mechanism of resistance to PI3K inhibitors and this has direct clinical implications.

It has particularly proven that such a combined synthetic lethality and drug resistance screen rcould reveal that NOTCH 1 activation is a mechanism of resistance for PI3K inhibitors in solid tumors or cancer, specifically breast cancer, which is confirmed by respective predictions and treatment effects.

Using a panel of cell lines from various solid tumor cancers it can be shown that the MYC amplification status is a predictor to sensitivity to PI3K and/or mTOR inhibitors. The invention thus refers to solid tumor cancers in general, in particular epithelial cancers.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purpose of illustration only, and are not intended to limit the scope if the invention.

EXAMPLES

Example 1

A platform for combinatorial cellular fitness screens in human cells

The first step in building a platform to multiplex large numbers of combinations of genetic and chemical perturbations was to develop a sensitive and quantitative method to allow the identification of populations of cells carrying specific genetic modifications within a complex mixture. Molecular barcodes are short non-transcribed stretches of DNA which when integrated into the genomic DNA of a cell line introduce a molecular beacon that can be selectively quantified by PCR. In a mixed population of cells, each containing a unique barcode, the relative number of cells containing a particular vector can therefore be determined by quantification of the barcodes. By pairing genetic modifications of cells (e.g. the expression of an oncogene or

knockdown of a tumor suppressor) with these barcodes, the cellular fitness upon drug treatment can be followed in a multiplexed fashion. Thus, we first generated one hundred lentiviral vectors carrying unique molecular barcodes flanked by common primer sites for efficient delivery into human cells (Supplementary Fig. 1 ).

To identify the effect of individual genetic changes on cell growth (i.e. fitness) in response to a specific drug, and bypass the difficulty of comparing heterogeneous cell lines with their multitudes of genetic changes, we used an isogenic cell line approach.

Individual genetic modifications were introduced into cells with the same genetic background using overexpression and RNA interference (RNAi). To systematically analyze the effects of a drug library on this heterogeneous population of cells, each unique barcode was then paired with one genetic modification, so that the cellular fitness upon drug treatment could be followed in a multiplexed fashion (Fig. 1 a).

To quantify the barcodes we used hybridization based Luminex xMAP technology which uses a set of fluorescent microspheres that are analyzed by flow cytometry. An advantage of this methodology over massive parallel sequencing is that it is fast and the cost per sample is independent of the size of the experiment, making the method highly flexible and affordable. Briefly, barcodes were amplified from genomic DNA by PCR, fluorescently labeled and hybridized to microspheres that are coupled to the antisense barcode sequence. Subsequent analysis of the beads then reveals the relative abundance of each barcode (Fig. 1 a).

We subjected the screening platform to specific tests to determine its reliability and power for identifying drug-gene interactions. The typical dynamic range and linearity of the barcode detection extended over two orders of magnitude and the relative signals were maintained upon re-amplification, indicating limited PCR bias (Fig. 1 b and Supplementary Fig. 2) Furthermore, the method was highly robust as illustrated by the high correlation coefficients of both technical and biological replicates (r² >0.98, Fig. 1 c).

Because the quantification method is hybridization-based, we needed to exclude any cross-hybridization of barcode sequences as this could obscure the detection of individual barcodes. For this purpose we assembled one hundred pools of barcoded vectors in which a single vector was omitted and performed barcode measurements on PCR amplified material. In all cases the absence of the correct barcode was confirmed, indicating limited cross hybridization under these conditions (Fig. 1 d). Next, we determined if the method was able to detect differences in cellular fitness in a complex mixture of barcoded cells. We used drug hypersensitivity as a benchmark as it is technically more challenging to detect the absence of a cell within a population than the increase in proliferation occurring in drug resistance. Cells were infected with one of 95 barcoded vectors carrying a puromycin resistance gene or a barcoded vector lacking this cassette (#96). As expected, treatment with puromycin only killed the cells without the resistance gene, leaving all others unaffected (Fig. 2a, upper panel). In addition, when all cells were pooled and subsequently treated with puromycin, a strong and highly significant depletion of the barcode associated with the puromycin-less vector was detectable whereas all other barcodes remained

unchanged (Fig. 2a, lower panel). Thus, the approach was sensitive enough to detect the loss of one individual cell population within a complex mixture.

As an additional proof-of-principle experiment, we measured the known hypersensitivity of Fanconi Anemia complementation group D2 (FANCD2) patient cells for the DNA cross-linking agent Mitomycin C (MMC) in the multiplexed assay. A patient-derived cell line (PD20) stably transduced with a vector expressing wild type FANCD2 or an inactive point mutant (K561 R) were infected with barcoded lentiviruses, pooled and subsequently exposed to MMC. As predicted, the barcode derived from the cells expressing the inactive mutant protein was significantly depleted from the population, which could be clearly detected with our screening approach, thus confirming the MMC hypersensitivity of FANCD2 mutant cells (Fig. 2b, Supplementary Fig. 3).

Together, these experiments show that the screening platform provides a semiquantitative method to determine cellular fitness in a multiplexed format.

Example 2

A combined synthetic lethal and drug resistance screen in breast cancer

We applied our screening platform to interrogate drug-gene interactions in breast cancer cells. We first established an isogenic cell line model based on the non- tumorigenic human breast epithelial cell line MCF10A. The cell line was selected because it has a relatively normal karyotype and is thought to represent a multi-lineage progenitor as it has both basal and luminal transcriptional characteristics. Furthermore, the cells are responsive to most signaling pathways present in normal breast epithelial cells. A previously reported deletion of the INK4A locus and some other chromosomal aberrations could be confirmed by high-density SNP array.

We selected breast cancer-relevant genetic aberrations using an extensive literature and database search, excluding genes only loosely linked to tumorigenesis. This yielded a list of seventy genes that have been clearly linked to breast cancer, including HER2, BRCA1/2, c-MYC, NOTCH 1 and PTEN, which were selected for the drug-gene interaction screen (Fig 3a and Supplementary Table 1 ). To mimic the aberrations of these genes in cancer we manipulated their expression using cDNA overexpression or RNAi, and unique barcodes were introduced by lentiviral

transduction yielding a total of 89 isogenic cell lines (Supplementary Table 1 ). All cDNAs and the majority of knockdowns were confirmed using immunoblot and qRT- PCR and for a number of stable cell lines a marked morphological change was observed indicative of oncogenic transformation (Supplementary Fig. 4).

After pooling all barcoded cells they were screened against a custom compound library, which was selected to maximize the chance of identifying a drug-gene interaction that could be useful in the clinic. The library mainly consisted of clinically relevant kinase inhibitors and several tool compounds, together comprising 87 small molecules (Fig. 3b and Supplementary Table 1 ). The library was used at various concentrations and the screen yielded over 30 thousand data points (Fig. 3c).

Data analysis revealed various gene-drug interactions including synthetic lethal interactions between three components of the NOTCH signaling pathway (i.e. JAG1 , NOTCH 1 and c-MYC) and the Aurora kinase drugs AT9283 and SNS-314 (Fig. 3c). Dose-response analysis of cells expressing the intracellular active domain of NOTCH 1 (ICN1 ) or c-MYC confirmed the exquisite sensitivity to Aurora kinase inhibition (Fig. 4a). C-MYC and NOTCH 1 have recently been shown to display a synthetic lethal interaction with Aurora B kinase in retinal epithelial cells, corroborating our findings and further validating the approach. Furthermore, the observation that multiple components of a single pathway cluster with two drugs targeting the same gene product illustrates how large-scale drug-gene screens in human cells could be used to elucidate drug action and gene function, and is reminiscent of the powerful genetic screens previously limited to yeast. Example 3

NOTCH1 activation confers resistance to PI3K inhibition

Importantly, our screen revealed several novel drug-gene interactions. The most significant resistance hit in the screen was the intracellular active domain of NOTCH 1 (ICN1 ), conferring resistance to the dual PI3K/mTOR inhibitor BEZ-235 (Fig. 3c).

Given the clinical relevance of both PI3K inhibitors and NOTCH1 in breast cancer, and no reported connection between the two, we decided to study this observation further.

Although our initial analysis revealed that ICN1 only showed a significant interaction with BEZ-235, we reasoned ICN1 cells might also be resistant to some of the other PI3K inhibitors used in the screen. Indeed, when all remaining PI3K inhibitors were analyzed as a group, the interaction with ICN1 was also significant, indicating that the resistance could be extended to other PI3K inhibitors (Fig. 4b). Consistent with this, we found that resistance to BEZ-235 and PIK90, a selective PI3K inhibitor, could be confirmed in dose-response experiments (Fig. 4c and Fig. 4d).

To extend this observation, we used a long-term colony formation assay, which also showed pronounced resistance of ICN1 -expressing cells to PI3K inhibition (Fig. 4e). The intracellular fragment of NOTCH3 did not show resistance to BEZ-235, consistent with previous observations that NOTCH3 acts as a repressor of NOTCH1 - mediated transcription (Supplementary Fig. 5).

To begin to uncover the mechanism whereby activation of NOTCH1 in cells confers resistance to PI3K inhibitors we analyzed one of the main downstream effector pathways of PI3K: the serine-threonine kinase mTOR, which resides in the two distinct protein complexes mTORCI and mTORC2. We found that ICN1 expressing cells were also less sensitive to PP242, an mTOR kinase inhibitor, and rapamycin, which is a selective mTORCI inhibitor (Fig 5a and Fig 5b). This indicates that activation of NOTCH 1 can bypass the cellular requirement for this growth pathway and that consistent with previous reports, in these cells PI3K inhibitors mainly exert their effect by acting on mTORCI .

Next, we investigated if the ICN1 -mediated resistance could also be observed in human breast cancer cell lines. Both the adenocarcinoma-like cell line MCF7 and the ductal carcinoma-like cell line BT549 showed marked resistance to BEZ-235 treatment upon expression of ICN1 . These results suggests that this effect of NOTCH 1 in uncoupling proliferation from the PI3K/mTOR pathway is a general phenomenon across breast cancer cell lines, and may be relevant for human breast cancer (Fig. 5c).

Example 4

ICN1 uncouples mTORCI signaling from proliferation by inducing c-MYC transcription

Ribosomal S6 Kinase (S6K) and the eukaryotic translation initiation factor 4E- binding protein 1 (4EBP1 ) belong to the main effector molecules of mTORCI and their phosphorylation stimulates protein translation. Interestingly, S6K and 4EBP1

phosphorylation was equally inhibited in ICN1 expressing cells as in control cells (Fig. 6a and Supplementary Fig. 6). This suggests that ICN1 uncouples mTORCI signaling from proliferation by a downstream mechanism.

Upon closer inspection of the screening data we found that c-MYC also displayed a moderate but significant resistance to BEZ-235 and resistance to other PI3K inhibitors (Fig. 6b and Supplementary Fig. 7, p=0.03). As c-MYC is a known direct NOTCH target gene in breast cancer this suggested a model whereby ICN1 renders cells resistant to PI3K/mTOR inhibition by activating c-MYC transcription. To test this we first analyzed ICN1 cells, and found an induction of c-MYC mRNA and protein (Fig. 6d and Fig 6e), consistent with the possibility that this could be the downstream mechanism of ICN1 -mediated resistance. To investigate this directly, we inhibited c-MYC expression by RNAi (Fig 6d). Knockdown of c-MYC to control levels completely reversed the resistance to BEZ-235 (Fig 6e). Thus, we suggest that

NOTCH pathway activation uncouples PI3K-mTORC1 signaling from proliferation by induction of c-MYC.

DISCUSSION

We identified a novel mechanism of resistance to PI3K inhibitors in breast cancer cell lines by activating NOTCH signaling and induction of c-MYC. NOTCH activation occurs in a significant subset of breast cancers and is associated with tumor progression and poor prognosis and MYC amplification is a relative frequent event. PI3K and mTOR targeting drugs have received much attention as the pathway is frequently hijacked in a variety of malignancies, including breast cancer. As tumors invariably acquire resistance to single agent treatments, the ability to anticipate drug resistance has enormous clinical and economic value. However mechanisms of resistance in human tumors to PI3K inhibitors have not yet been reported.

We could show that resistance occurs by the transcriptional activation of c-MYC and that this seems to uncouple mTORCI regulation of translation from proliferation. The stimulation of translation by c-MYC through the induction of eukaryotic initiation factor 4F (elF4) family members is a known mechanism whereby c-MYC drives protein translation and is implicated in c-MYC-driven tumorigenesis. This mechanism of how NOTCH 1 activation could induce resistance to PI3K inhibitors is an attractive model but remains to be confirmed. Together, these observations position NOTCH and MYC activation as mechanisms of resistance to PI3K inhibitors with direct clinical

implications.

We established a screening platform to systematically search for synthetic lethal interactions and mechanisms of drug resistance in cancer cells. The ability to pair tumor genotype with cancer treatment is receiving increasing attention as rising cost of cancer treatment is placing a burden on the health care system. The multiplexed assay allowed the interrogation of thousands of gene-drug combinations with the potential to identify clinically relevant interactions that could lead to new patient-stratified medicine. The method is highly flexible, can be used with cDNA overexpression, RNAi or any cellular perturbation of interest and is applicable to all cells transducible with lentiviral vectors. Furthermore, the NOTCH pathway interaction with Aurora kinase inhibitors provides an example of how "guilt by association" can shed light on the mechanism of action of drugs or function of cancer genes. In summary, the ability to efficiently measure large numbers of drug-gene interactions in human cells has the potential to provide insight into various aspects of chemical biology.

METHODS

Cell culture, antibodies and compounds. MCF10A cells (ATCC) were cultured in DMEM/F12 supplemented with 5% horse serum, penicillin/streptomycin, insulin (10 ug/ml), cholera toxin (100 ng/ml), EGF (20 ng/ml) and hydrocortisone (500ng/ml). All other cells were grown in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. PDK1 antibody (E-3), anti-GFP and anti p53 (DO-1 ) were purchased from Santa Cruz Biotechnology. Anti beta-actin and anti-c-Myc antibody were obtained from Sigma-Aldrich. All other antibodies were acquired from Cell Signaling. Compounds were obtained from SynThesis Medchem (China) except for rapamycin, Mitomycin C and PP242 (Sigma).

Barcoded vectors and generation of isogenic cell lines

The stuffer fragment in the lentiviral vector pLKO.1 (Moffat, J. et al. Cell 124,

1283-1298 (2006)) was replaced with a short linker sequence and barcodes (Flexmap barcode tags, Supplementary Table 1 ) flanked by primer sites and inserted 5' of the U6 promoter. This vector (pLKO.2, see Supplementary Fig. 1 ) was then used to introduce stable DNA barcodes into cells by lentiviral transduction. Cloning oligos into pLKO.2 using the Agel and EcoRI restriction sites generated short hairpin RNA expressing vectors. An overview of all vectors used in this study is provided in Supplementary Table 1 .

MCF10A isogenic cell lines overexpressing cDNAs or shRNAs were produced by lenti- or retroviral transduction and selection. Stable isogenic lines were cultured for approximately four weeks prior to the screen and barcoded by a second infection, when applicable. Prior to siRNA SMARTPool (Dharmacon) transfections (siLentfect, Biorad) MCF10A were infected with barcoded lentivirus.

Screen set-up and Luminex assay

For each compound a 4-point dose-response curve was determined in MCF10A cells using the Celltiter Glo assay (Promega). From this data, concentrations were selected for the screen. All barcoded cell lines were pooled, counted and seeded in multiwell plates in quadruplicate. Compound or DMSO was added 16h after seeding using a liquid handling robot (Cybio). Medium was refreshed every second day and cells were cultured for a total of 9 days (split once) after which genomic DNA was isolated and barcodes were amplified. Genomic DNA extraction was performed with a liquid handler (Cybio) using the Genfind v2.0 kit (Agencourt). In brief, medium was removed and cells were washed twice with PBS. After lysis (1 % SDS, 10mM EDTA, 10mM NaCI 10mM Tris-HCI pH 8.0,), 100μΙ_ raw lysate was transferred into 96- deepwell plates and 60μΙ_ Agencourt binding buffer was added. Beads were washed six times with 70% ethanol and purified genomic DNA was eluted in dh O. Barcodes were amplified in a 2-step protocol by PCR (Fwd 5'-CGATTAGTGAACGGATCTC - Sequence ID No. 1 , Rev 5'-GAAGGTGAGAACAGGAGC - Sequence ID No. 2) and linear amplification was performed with a 5' biotinylated primer (5' Biotin- TGAG G ATAGCAG AG AAG G - Sequence ID No. 3). The single stranded product was hybridized to pre-coupled Luminex xMAP beads (as described by Stegmaier, K. et al. PLoS Med 4, e122 (2007)) for 1 .5h at 40°C in 384 well plates and streptavidin coupled phycoerythrin (SAPE, Invitrogen) was added for 30' at 40°C. Finally, beads were washed once and samples were measured in a Flexmap 3D plate reader (Luminex) at 40°C.

Luminex data analysis

Raw bead signal intensities were reduced to a single value for each barcode and well (i.e. drug treatment) by computing a median from the four replicates. Each well was then normalized by a factor determined by making the sum of bead signal medians equal and the normalized data was log-transformed. A first robust linear regression of the form data=well+barcode+residual completed the normalization and its residuals were interpreted as the well+barcode effect. These effects were submitted to a second robust linear regression effect=drug^*barcode+residual to deconvolute the drug-barcode interactions. The P-values of the regression coefficients were used to rank the interactions whereas the regression coefficients themselves estimated the magnitude of the interaction.

Resistance scores were calculated by first excluding all normalized data points in the sensitivity direction. Next, the fold change (drug vs. all) was calculated for each drug treatment of ICN1 , c-MYC and MDM2 as a control gene. The mean resistance scores were then calculated by taking all PI3K inhibitor data (minus BEZ-235) or all remaining drugs.

Quantitative real-time PCR

RNA was isolated from sub-confluent cells using Trizol (Invitrogen). After purification and DNAse treatment (Turbo-DNA free, Ambion) reverse transcription was performed using random hexamer primers and RevertAid reverse transcriptase (Fermentas). Quantitative real-time PCR was carried out using the iTaq SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. Measurements were performed in triplicate and related to GAPDH as a reference gene. All primer sequences are listed in Supplementary Table 1 . GFP competition assay

MCF7 and BT549 cells were infected with vectors carrying the cDNAs for ICN1 and GFP (EF-hlCN1-CMV-GFP) or an empty control vector. After infection, cells were pooled and distributed among multiple 6-well plates for BEZ-235 or DMSO treatment. On day 3 of drug incubation, medium was removed and replaced with fresh medium without drug. The next day cells were trypsinized and analyzed using an automated microscope (Leica DMI6000B). Ten randomly chosen fields were imaged for each cell line-drug combination and cells were quantified using CellProfiler (The Broad Institute)

Claims

1 . A method for determining the responsiveness of a mammalian tumor cell to treatment with an inhibitor of the phosphoinosite 3-kinase (PI3K) and/or mammalian target of rapamycin (mTOR) pathway, said method comprising determining the c-MYC and/or NOTCH 1 level in said tumor cell, wherein the c-MYC and/or NOTCH 1 upregulation is indicative for the resistance of said tumor cell to the treatment.

2. In vitro method for the identification of a resistance to treatment of solid tumor cancer with an inhibitor of the PI3K and/or mTOR pathway in a patient suffering from said cancer, said method comprising the following steps:

(a) obtaining a sample from a patient suffering from a solid tumor cancer, and

(b) evaluating in said sample the level of c-MYC and/or NOTCH1 expression, whereby the c-MYC and/or NOTCH 1 upregulation is indicative for the resistance of said patient to the inhibitor.

3. The method of any one of claims 1 or 2, wherein said inhibitor is a direct or indirect inhibitor of PI3K and/or Akt and/or mTOR.

4. The method of claim 3, wherein said inhibitor is a kinase inhibitor.

5. The method of claim 3 or 4, wherein said inhibitor is an mTOR and/or Akt and/or PIK3 inhibitor, such as BEZ235, PIK90, GDC-0941 , RAD001 (Everolimus), rapamycin (Sirolimus), CCI-779 (Temsirolimus), TORIN or KRX-0401 (Perifosine, Perifosineare).

6. The method of any one of claims 1 to 5, wherein said level is determined by measuring the quantity and/or activity of the genes or gene expression products.

7. The method of any one of claims 1 to 6, wherein said level is indirectly determined by analysing surrogate markers indicative for the level of c-MYC and/or NOTCH 1 and/or activation products of c-MYC and/or NOTCH 1 .

8. The method of any one of claims 1 to 7, wherein the level of a NOTCH 1 ligand, such as JAGGED ligands, including JAGGED1 , JAGGED2, DELTA-likel , Delta-like3 and Delta-like4 is determined.

9. The method of any one of claims 1 to 8, wherein said level is determined by a nucleic acid analysis selected from the group consisting of methods which employ amplification methods, among them nucleic acid amplification methods, RT-PCR, microarrays, nuclear localization analysis, hybridization and NOTCH1 mutation analysis.

10. The method of any one of claims 1 to 9, wherein said level is determined by a protein analysis selected from the group consisting of immunoassays, such as ELISA, EIA, RIA, western blot, protein arrays, immunocytochemistry or

immunohistochemistry methods.

1 1 . The method of any one of claim 1 to 10, wherein said cell or sample is obtained from a tumor of said patient.

12. An in vitro method of predicting the efficacy of a treatment of solid tumor cancer with an inhibitor of the PI3K and/or mTOR pathway, for a patient suffering from said cancer, comprising the steps of

a) determining in a cell or tissue sample obtained from said patient the level of c-MYC and/or NOTCH 1 , and

b) comparing the activity or level of said marker gene(s) determined in a) with a reference level, wherein the extent of an increase is indicative for the reduced predicted efficacy of the treatment.

13. An in vitro method of monitoring the efficacy of a treatment of solid tumor cancer with an inhibitor of the PI3K and/or mTOR pathway, in a patient suffering from said cancer comprising the steps

a) determining in a cell or tissue sample obtained from said patient the level of c-MYC and/or NOTCH 1 , and

b) comparing the activity or level of said marker gene(s) determined in a) with a reference level, wherein the extent of an increase is indicative for the reduced efficacy.

14. The method of claim 13, wherein said treatment of cancer comprises the administration of an inhibitor of the PI3K and/or mTOR pathway in combination with a c-MYC and/or NOTCH 1 inhibitor.

15. A combination comprising an amount of an inhibitor of the PI3K and/or mTOR pathway, and an amount of a c-MYC and/or NOTCH1 inhibitor.