WO2018092064A1

WO2018092064A1 - Combinations of mdm2 inhibitors and bcl-xl inhibitors

Info

Publication number: WO2018092064A1
Application number: PCT/IB2017/057177
Authority: WO
Inventors: Emilie Chapeau; Eric Durand; agnieszka GEMBARSKA; Michael Rugaard Jensen; Emeline MANDON
Original assignee: Novartis Ag
Priority date: 2016-11-18
Filing date: 2017-11-16
Publication date: 2018-05-24

Abstract

The present disclosure relates to a pharmaceutical combination comprising (a) an Mdm2 inhibitor and (b) a Bcl-xL inhibitor, particularly for use in the treatment of a cancer. This disclosure also relates to uses of such combination for preparation of a medicament for the treatment of a cancer; methods of treating a cancer in a subject in need thereof comprising administering to said subject a jointly therapeutically effective amount of said combination; pharmaceutical compositions comprising such combination and commercial packages thereto.

Description

COMBINATIONS OF MDM2 INHIBITORS AND BCL-XL INHIBITORS

FIELD OF THE DISCLOSURE

The present disclosure relates to a pharmaceutical combination comprising (a) an Mdm2 inhibitor and (b) Bcl-xL inhibitor, particularly for use in the treatment of a cancer. This disclosure also relates to uses of such combination for preparation of a medicament for the treatment of a cancer; methods of treating a cancer in a subject in need thereof comprising administering to said subject a jointly therapeutically effective amount of said combination; pharmaceutical compositions comprising such combination and commercial packages thereto.

BACKGROUND OF THE DISCLOSURE

The advent of targeted therapies for cancer has increased patient lifespan for various malignancies and helped to appreciate the complexity of tumors through the study of drug resistance mechanisms. The fact that clinical responses to targeted agents are generally incomplete and/or transient results from a multitude of factors that can be broadly put into two classes: toxicities that prevent optimal dosing of drugs and consequently limit target engagement (Brana and Siu 2012, Chapman, Solit et al. 2014), and the ability of cancers to adapt and maintain their proliferative potential against perturbations (Druker 2008, Chandarlapaty 2012, Doebele, Pilling et al. 2012, Duncan, Whittle et al. 2012, Katayama, Shaw et al. 2012, Lito, Rosen et al. 2013, Sullivan and Flaherty 2013, Solit and Rosen 2014). Combinations of drugs can address both these factors by improving overall efficacies and at the same time targeting tumor robustness and complexity to counter resistance (Robert, Karaszewska et al. 2015, Turner, Ro et al. 2015). It is not yet clear how many drugs are required and which processes need to be targeted in combination to overcome cancer. But it is almost certain that different pathways or drivers need to be inhibited, most likely requiring two or more drugs (Bozic, Reiter et al. 2013). This is supported by the successes of combining conventional chemotherapeutic agents to treat cancers (DeVita 1975), and combination therapies for infectious diseases such as HIV (Porter, Babiker et al. 2003), as well as by theoretic approaches showing how biological robustness can be challenged by increasing the order of perturbations (Lehar, Krueger et al. 2008).

In spite of numerous treatment options for patients with specific types of cancer, there remains a need for effective and safe combination therapies that can be administered for the effective long-term treatment of cancer.

SUMMARY OF THE DISCLOSURE It is an object of the present disclosure to provide for a medicament to improve treatment of a cancer, in particular to improve treatment of cancer through inhibition of cell growth (proliferation) and induction of apoptosis. It is an object of the present disclosure to find novel combination therapies, which selectively synergize in inhibiting proliferation and/or in inducing apoptosis.

Such inhibitors as Mdm2 inhibitors and Bcl-xL inhibitors, as a monotherapy, demonstrate anti-proliferative (cytostatic) and pro-apoptotic (cytotoxic) activities in vitro and in vivo pre-clinical assays. Surprisingly it has been found that a pharmaceutical combination comprising

(a) an MDM2 inhibitor selected from HDM201, i.e. (6S)-5-(5-Chloro-l-methyl-2- oxo-l,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l- (propan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof, and CGM097, i.e. (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6- methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans-cyclohexylmethyl]- amino} -phenyl)- l,4-dihydro-2H-isoquinolin-3 -one, or a pharmaceutically acceptable salt thereof; and

(b) Bcl-xL inhibitor selected from A-l 155463, A-1331852, WEHI-539 or a pharmaceutically acceptable salt thereof,

has a beneficial synergistic interaction, improved anti-cancer activity, improved antiproliferative effect, and improved pro-apoptotic effect. These combinations demonstrated a synergistic effect in cell growth inhibition and induction of cell death by apoptosis.

Further, it has been found that a combination of

(a) an MDM2 inhibitor selected from (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2- dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan- 2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof, and (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4- {methyl- [4- (4-methyl-3 -oxo-piperazin- 1 -yl)-trans-cyclohexylmethyl] -amino } -phenyl)- 1 ,4- dihydro-2H-isoquinolin-3-one, or a pharmaceutically acceptable salt thereof; and (b) Bcl-xL inhibitor selected from A-l 155463, i.e. 2-[8-(l,3-Benzothiazol-2- ylcarbamoyl)-3,4-dihydroisoquinolin-2(lH)-yl]-5-(3-[4-[3-(dimethylamino)prop-l- yn-l-yl]-2-fluorophenoxy]propyl)-l,3-thiazole-4-carboxylic acid, A-1331852, WEHI- 539 or a pharmaceutically acceptable salt thereof,

may advantageously comprise further inhibitors selected from MEK inhibitors (e.g. trametinib, 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid (2 -hydroxy ethoxy)-amide, (S)-5-fluoro-2-(2-fluoro-4- (methylthio)phenylamino)-N-(2-hydroxypropoxy)- 1 -methyl-6-oxo- 1 ,6-dihydropyridine- 3-carboxamide, PD0325901, PD-184352, RDEA119, XL518, AS-701255, AS-701173, AS703026, RDEA436, E6201, R04987655, RG7167, and RG7420 or a pharmaceutically acceptable salt thereof) EGFR inhibitors, PI3K inhibitors and BRAF inhibitors. In addition, CDK4/6 inhibitor or standard of care such as paclitaxel can be added to a combination of MDM2 inhibitor ("MDM2i") and trametinib, which can lead to further synergistic effect or strong induction of apoptosis. A combination of the MDM2 inhibitor with a Bcl-xL inibitor can be supplemented by a BRAF inhibitor (e.g. dabrafenib) and CMET inhibitor (e.g. PF-04217903) to form a quadruple combination. The latter combination was found to be weakly synergistic, but with strongly inducing apoptosis. In another aspect, the present disclosure relates to a pharmaceutical composition comprising the pharmaceutical combination of the disclosure and at least one

pharmaceutically acceptable carrier.

In one aspect, the present disclosure relates to the pharmaceutical combination or the pharmaceutical composition of the disclosure for use as a medicine.

In another aspect, the present disclosure relates to the pharmaceutical combination or the pharmaceutical composition of the disclosure for use in the treatment of cancer.

In another aspect, the disclosure provides the use of to the pharmaceutical combination of the disclosure for the preparation of a medicament for the treatment of a cancer.

In yet another aspect, the present disclosure relates to a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination of the present disclosure, or the pharmaceutical composition of the present disclosure.

Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination, as listed in the claims below.

DESCRIPTION OF THE DRAWINGS

Figl Tumoral insertional landscapes

Genetic landscape of PB insertions in the 327 RosaPB/+;ATP2/+;Arf-/- tumors sequenced. GOF: predicted

gain of function, LOF: predicted loss of function.

Fig2 PiggyBac insertional patterns in BRaf oncogene. Red bars represent insertions in same sense of gene, and blue bars in opposite sense as the gene.

Fig3 Characterization of response to HDM201 treatment of RosaPB/+; ATP2/+;Arf-/- allografted tumors

Activity of HDM201 in RosaPB/+; ATP2/+;Arf-/- allograft bearing mice. Model ID and tumor type are indicated on the figure. Yellow curves are vehicle treated, and red curves are from tumors of mice that were treated with HDM201 lOOmg/kg twice weekly.

Fig4 Response to HDM201 p53-Mdm2 inhibitor allografted mice

Kaplan-Meyer curve representing the survival of mice post treatment first dose. All 21 RosaPB/+;ATP2/+;Arf-/- tumor models are combined. The median survival is 0.71 weeks (n=106) for vehicle treated animals, and 2.48 (n=139) for the animals treated twice weekly (P<0.0001 Log-rank test).

Fig5 Comparative analysis of insertional patterns between vehicle-treated tumors and tumors that emerged from HDM201 -treated and resistant tumors. 87 genes were found significantly differentially inserted (red) in resistant versus untreated tumors (FDR=0.01). Fig6 Characterization of Bel -XL transposon-induced expression in HDM201 -resistant tumors

Left) PB insertional patterns in Bcl211 gene, from HDM201 -resistant tumors. Red bars represent insertions in same sense of gene, and blue bars in opposite sense as the gene. Insertional pattern in Bcl211 gene suggests a gain of function. Right) Western-blot analysis of tumors shows that Bcl-xL is the Bcl211 isoform expressed when PB insertion is found in resistant tumors (veh : vehicle-treated tumors, res: HDM201 -resistant tumors).

Fig7. Bcl-xL expressed in patient-derived xenograft models that became resistant to HDM201

Western-blot analysis showing BCL-XL protein expression in patient-derived tumor xenograft models (PDXs) resistant to HDM201 (res) or vehicle -treated (veh).

Fig8 Best combinations identified with the TP53-MDM2 inhibitor CGM097

A) 51 compounds were screened in combination with CGM097 (8) in 138 TP53 wild-type cell lines. The CGM097+ ABT-263 combination showed the 3rd best combination activity (green bar). Only the 25 best combinations are represented; bars are light gray when no statistical significance was found. A hit is defined as a combination with a synergy score above 2 and a maximum growth inhibition above 0.7 in individual cell lines over all assayed cell lines. B) Representative examples of CGM097 / ABT-263 combination responses. The % growth inhibition is represented in the visualization matrix. Cell line name is indicated at the top of each matrix. The number of replicates is indicated on the right side of each matrix.

Fig9 Syngergy between ABT-263 and HDM201 in p53 wild-type cell lines

Score plots for the combination of CGM097 and ABT-263 from an in vitro combination screen on 485 cancer cell lines treated with ranges of concentrations for CGM097 and ABT-263. Cell lines with no TP53 mutation are shown in blue (p53 wt), and cell lines with TP53 modification in green (p53 mt). Boost describes the maximal growth inhibition for any combination versus the highest single agent activity.

Figure 10 Bcl-xL modulates sensitivity of cell lines to TP53-MDM2 inhibition

A) Shift of sensitivity to HDM201 treatment in WM226.4 cell line transiently transfected with Bcl-xL, compared to cells transfected with a control plasmid. The curve is representative of two independent experiments of 3-day cell viability assay. The IC50 was of 6 folds higher in average upon Bcl-xL expression. The Western-blot shows expression of Bcl-xL after transient transfection in WM226.4 cells. B) Synergistic effect of combinations of HDM201 TP53-MDM2 inhibitor and A-l 155463 Bcl-xL inhibitor in two cell lines. SNG-M cells (upper panels) or LS-513 cells (lower panels) were treated for 3 days with a 7x7 dose matrix of HDM201 and A-l 155463. Percent inhibition is shown in the left panels, each field representing the average of three replicates. Right panels show the additional (or reduced) effect level in percent relative to drug self-combination based on the Loewe model.

Fig 11 Response and resistance to HDM201 in mice transplanted with piggyBac allografted tumors

A) Kaplan-Meyer curve representing the conditional survival of mice post treatment first dose. Two lymphoma and 4 medulloblastoma RosaPB/+;ATP2/+;Arf-/- tumor models are combined altogether. The median survival was 0.86 week (n=57) for vehicle treated animals, 2.71 weeks (n=39) for the animals treated twice weekly (P<0.0001 Log-rank test), and 3.14 weeks (n=60) for the animals treated daily (PO.0001 Log-rank test). B) The average of % body weight change was calculated for each mouse during the course of the treatment. Each dot represents one mouse. Unpaired t test demonstrate a significant difference (P=0.0018) in body weight change between continuous and intermittent regimens. The 40mg/kg HDM201 daily treatment was not well tolerated and 16 mice out of 60 (27%) had to be euthanized for body weight loss. The lOOmg/kg intermittent arm was better tolerated; one animal (2.6%) was euthanized due to body weight loss. C,D) Comparative analysis of insertional patterns between vehicle-treated tumors and tumors that emerged from HDM201 -treated resistant tumors. Red datapoints represent CIS genes found significantly inserted in resistant versus untreated tumors (FDR=0.01). C) Intermittent lOOmg/kg schedule and D) daily 40mg/kg schedule.

DETAILED DESCRIPTION OF THE DISCLOSURE

Introduction

The genes most commonly altered in human cancer, regardless of tumor type, are p53 tumor suppressor (1) and CDKN2A (INK4a/ARF) (2). The latter complex locus encodes two tumor suppressor proteins. One is pl6INK4a (3), an inhibitor of cyclin D- dependent kinases that regulates the ability of the retinoblastoma protein (Rb) to control cell cycle. The other is pl9ARF (4), a negative regulator of MDM2 function, that activates p53, thereby inducing cell cycle arrest or apoptosis. The INK4a/ARF locus is very frequently disrupted in human tumors, and consequently, these two tumor suppressor genes are disabled, through deletion, mutation or epigenetic silencing either in whole or in part (5). Subsequently, p53 tumor suppressor is degraded by MDM2. Compound specifically inhibiting the interaction between MDM2 and p53, thus preventing p53 degradation, have been discovered. Such agents induce p53 reactivation in tumors where p53 gene is wild-type (6-9). Although pharmacological effect of MDM2 inhibitors anticancer drugs was found beneficial, the tumors commonly relapse most likely because of the selection and growth of drug resistant cells (10, 11). Better understanding of the mechanisms of resistance would be beneficial to patient survival through identification of rational combinations and second line therapies.

In this study, we ought to investigate resistance arising upon treatment of mice with HDM201, a novel highly specific and potent p53/Mdm2 inhibitor (10, 12), structurally similar to CGM097 (6-8). To discover candidate cancer genes that function as key drivers of HDM201 resistance, we initiated a resistance screen in mice using transposon-based insertional mutagenesis. Tumor-prone Arf null mice (13) were crossed with mice carrying a constitutive piggyBac (PB) transposon mutagenesis system (14), constituted of the PB DNA transposon ATP2-S 1 and a PB transposase expressed from the Rosa26 locus(14). PB transposon system presents cut-and-paste properties without leaving undesired footprints, and has the ability to integrate randomly throughout the entire genome. We expected that the mice harboring the active PB transposon would not only acquire mutations that accelerate the rate of tumorigenesis in this Arf-/- sensitized model, but also acquire mutations in the process of progression to HDM201 -resistance. Because monitoring emerging resistance is technically challenging with spontaneous tumors, we decided to perform the screen after transplanting these tumors in flank of recipient mice and expanding these allografted tumors in larger cohorts of animals, as commonly performed for patient-derived tumor xenograft (PDX) models (10).

Transposon-based mutagenesis has been widely used to identify candidate cancer genes in various types of cancers (14-17). However, in only a few studies has this method been used successfully to characterize resistance mechanisms in vitro (18-20) or in mice (21). Our current results shed light on the diversity of resistance mechanisms encountered upon cancer therapy involving disruption of p53/Mdm2 interaction. Our screen also reinforced transposon-based mutagenesis as a powerful tool for the identification of novel resistance genes and mechanisms in genetically modified mouse models, and constitutes the first in vivo resistance screen for p53-Mdm2 inhibition. Our results may lead to better combination strategies in patients with p53 wild-type tumors relapsing while on treatment with Mdm2-p53 inhibitors.

In one aspect, the present disclosure relates to a pharmaceutical combination comprising

(a) an MDM2 inhibitor selected from (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2- dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan- 2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof, and (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4- {methyl- [4- (4-methyl-3 -oxo-piperazin- 1 -yl)-trans-cyclohexylmethyl] -amino } -phenyl)- 1 ,4- dihydro-2H-isoquinolin-3-one, or a pharmaceutically acceptable salt thereof; and

(b) Bcl-xL inhibitor selected from A-l 155463, A-1331852, WEHI-539, or a pharmaceutically acceptable salt thereof.

It has been determined that the combination could be used to efficiently treat cancer. In particularly, it has been determined that the combination could be used to efficiently treat cancer due to a synergistic effect in inhibition of cell proliferation and / or induction of apoptosis. Accordingly, the combinations of the present disclosure, in particular triple and further combination, may shift a "cytostatic" response to a

"cytotoxic" response, thus achieving cancer regression.

The terms "a" and "an" and "the" and similar references in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Where the plural form is used for compounds, patients, cancers and the like, this is taken to mean also a single compound, patient, or the like.

The term "synergistic effect" as used herein refers to action of two or three therapeutic agents such as, producing an effect, for example, slowing the progression of a proliferative disease, particularly cancer, or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of a drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

In particular, it has been demonstrated that combined inhibition of MDM2 and Bcl- xL in TP53 wild-type cell lines provides significant synergy compared to each single agents (see examples herein). Thus, the combinations of the present disclosure provide an effective therapy option capable of improving responses compared to each of the single agents and can lead to more durable responses in the clinic.

The term "MDM2 inhibitor" or "HDM2 inhibitor" or "Mdm2 inhibitor" as used herein, refer to any compound inhibiting the HDM2/p53 (Mdm2/p53) interaction association. HDM2 (Human homolog of murine double minute 2) is a negative regulator of p53. Mdm2 inhibitors are useful in pharmaceutical compositions for human or veterinary use where inhibition of Mdm2/p53 association is indicated, e.g., in the treatment of tumors and/or cancerous cell growth. In particular, Mdm2 inhibitors are useful in the treatment of human cancer, since the progression of these cancers may be at least partially dependent upon overriding the "gatekeeper" function of p53, for example the overexpression of Mdm2. According to the present disclosure, the Mdm2 inhibitor is a compound selected from the group consisting of

(6S)-5-(5-Chloro-l-methyl-2-oxo-l,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2- (2,4-dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol- 4(lH)-one, or a pharmaceutically acceptable salt thereof, and

(S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3- oxo-piperazin- 1 -yl)-trans-cyclohexylmethyl] -amino } -phenyl)- 1 ,4-dihydro-2H- isoquinolin-3-one, or a pharmaceutically acceptable salt thereof.

The MDM2 inhibitor can be (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2- dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan-2- yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof. The Mdm2 inhibitor (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2-dihydropyridin-3-yl)- 6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6- dihydropyrrolo[3,4-d]imidazol-4(lH)-one belongs to a novel class of imidazopyrrolidinone compounds, and shows potent inhibition of the MDM2/p53 interaction (this term including in particular Hdm2/p53 interaction). In particular, this compound acts as an inhibitor of MDM2 interaction with p53 by binding to MDM2. The MDM2 inhibitor (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2-dihydropyridin-3-yl)-6-(4- chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4- d]imidazol-4(lH)-one, which is the most preferred Mdm2i inhibitor according to the present disclosure, is a compound of formula I, and described in Example 102 of WO2013/111105, which is hereby incorporated by reference in its entirety:

The crystalline forms of (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2-dihydropyridin-3- yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)- l-(propan-2-yl)-5,6- dihydropyrrolo[3,4-d]imidazol-4(lH)-one are described as EX6, EX7 and EX8 in WO2013/111105. The disclosure encompasses succinic acid co-crystal of the (6S)-5-(5- Chloro-1 -methyl -2 -oxo-1, 2-dihydropyridin-3 -yl)-6-(4-chloropheny l)-2-(2,4- dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one compound. The compound can be also be in a form of an ethanol solvate.

The MDM2 inhibitor can also be (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6- methoxy-2-(4-{memyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans-cyclohexylmethyl]- amino} -phenyl)- l,4-dihydro-2H-isoquinolin-3 -one, or a pharmaceutically acceptable salt thereof. The Mdm2 inhibitor (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4- {methyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans-cyclohexylmethyl]-amino}-phenyl)- l,4-dihydro-2H-isoquinolin-3-one is a compound of formula II, and described in Example 106 of WO2011/076786, which is hereby incorporated by reference in its entirety:

(II).

In one embodiment, the pharmaceutically acceptable salt of (S)-l-(4-Chloro- phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans- cyclohexylmethyl] -amino} -phenyl)- l,4-dihydro-2H-isoquinolin-3 -one is bisulphate salt. Crystalline form of the bisulfate salt of (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6- methoxy-2-(4-{memyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans-cyclohexylmethyl]- amino}-phenyl)-l,4-dihydro-2H-isoquinolin-3-one is described in WO2012/066095.

The term "Bcl-xL inhibitor" or "BCL-XL inhibitor" or "Bcl-X_L inhibitor" is defined herein to refer to a compound which targets, decreases or inhibits the protein Bcl- XL of the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family which is composed of proteins such as Bcl-2, Bcl-X_L, Bcl-w, Mcl-1, Bfll/A-1, and/or Bcl-B. The term "Bcl-xL inhibitor" is preferably used herein to refer to those inhibitors which selectively inhibit Bcl-xL and do not inhibit e.g. Bcl-2.

In one embodiment, pharmaceutical combination of the present disclosure includes at least one Bcl-xL inhibitor (Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al), preferably that one Bcl-xL inhibitor is selected from A-l 155463, A- 1331852, WEHI-539, more preferably the BcL-xL inhibitor is A-l 155463.

According to the present disclosure the pharmaceutical combination may comprise the MDM2 inhibitor and the Bcl-xL inhibitor. According to the present disclosure the pharmaceutical combination comprising the MDM2 inhibitor and Bcl-xL inhibitor may further advantageously comprise a further inhibitor, which even further improves antitumor activity of the combination, e.g. a MEK inhibitor. Preferably, said MEK inhibitor is selected from the group consisting of trametinib, 6-(4-bromo-2-fluorophenylamino)-7- fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide, (S)-5- fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N-(2-hydroxypropoxy)- 1 -methyl-6-oxo- l,6-dihydropyridine-3-carboxamide, PD0325901, PD-184352, RDEA119, XL518, AS- 701255, AS-701173, AS703026, RDEA436, E6201, R04987655, RG7167, and RG7420 or a pharmaceutically acceptable salt thereof. The term "a MEK inhibitor" is defined herein to refer to a compound which targets, decreases or inhibits the kinase activity of MAP kinase, MEK. A target of a MEK inhibitor includes, but is not limited to, ERK. An indirect target of a MEK inhibitor includes, but is not limited to, cyclin D 1.

Pharmaceutical combinations of the present disclosure can include at least one

MEK inhibitor compound selected from the group consisting of trametinib, 6-(4-bromo-2- fluorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2- hydroxyethoxy)-amide, (S)-5-fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N-(2- hydroxypropoxy)- 1 -methyl-6-oxo- 1 ,6-dihydropyridine-3-carboxamide, PD0325901 , PD- 184352, RDEA119, XL518, AS-701255, AS-701173, AS703026, RDEA436, E6201, R04987655, RG7167, and RG7420, or a pharmaceutically acceptable salt thereof.

Preferably, the MEK inhibitor is trametenib (N-(3-{3-cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino] -6, 8-dimethyl-2,4,7-trioxo-3 ,4,6,7-tetrahydropyrido [4,3 -d]pyrimidin- l(2H)-yl}phenyl)acetamide, also referred to as JPT-74057 or GSK1120212). Trametinib (GSK1120212) is described in PCT Publication No. WO05/121142, which is hereby incorporated by reference in its entirety. The compound has been approved as Mekinist^®.

According to the present disclosure, another suitable MEK inhibitor for the combination of the present disclosure is a compound 6-(4-bromo-2-fluorophenylamino)- 7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide of formula (III)

The MEK inhibitor compound 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H- benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide is described in the PCT Application No. WO 03/077914, and methods for its preparation have been described, for example, in Example 18 therein.

Additional suitable MEK inhibitor for the combination of the present disclosure is compound (S)-5-fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N-(2-hydroxypropoxy)- l-methyl-6-oxo-l,6-dihydropyridine-3-carboxamide is a compound of formula (IV)

(IV)

The MEK inhibitor compound (S)-5-fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N- (2-hydroxypropoxy)-l-methyl-6-oxo-l,6-dihydropyridine-3-carboxamide is described in Example 25-BB of PCT Application No. WO2007/044084, and methods for its preparation have been described therein.

An especially preferred salt of 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3- methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide is a

hydrochloride or sulfate salt. Additional pharmaceutically acceptable salts of 6-(4-bromo- 2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2- hydroxyethoxy)-amide and (S)-5-fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N-(2- hydroxypropoxy)-l-methyl-6-oxo-l,6-dihydropyridine-3-carboxamide suitable for the present disclosure include the salts disclosed in PCT Application No. WO 03/077914 and PCT Application No. WO2007/044084, which are both hereby incorporated into the present application by reference.

Additional MEK inhibitors that may be used in the combination of the present disclosure include, but are not limited to, PD0325901 (Pfizer)(See PCT Publication No. WO02/06213), PD-184352 (Pfizer), RDEA119 (Ardea Biosciences), XL518 (Exelexis), AS-701255 (Merck Serono), AS-701173 (Merck Serono), AS703026 (Merck Serono), RDEA436 (Ardea Biosciences, E6201 (Eisai)( See Goto et al, Journal of Pharmacology and Experimental Therapeutics, 3331(2): 485-495 (2009)), R04987655 (Hoffmann-La Roche), RG7167, and/or RG7420.

Similarly, the pharmaceutical combinations of the present disclosure comprising (a) the MDM2 inhibitor and (b)(i) the Bcl-xL inhibitor, and/or (ii) the MEK inhibitor may further advantageously comprise an EGFR inhibitor.

The term "an EGFR inhibitor" is defined herein to refer to a compound which targets, decreases or inhibits the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) or bind to EGF or EGF related ligands.

The EGFR inhibitor compound used in the combination of the present disclosure is selected from the group consisting of erlotinib, gefitinib, lapatinib, canertinib, pelitinib, neratinib, (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2-enoyl)azepan-3 -yl)- 1 H- benzo[d]imidazol-2-yl)-2-methylisonicotinamide, panitumumab, matuzumab, pertuzumab, nimotuzumab, zalutumumab, icotinib, afatinib and cetuximab, and pharmaceutically acceptable salt thereof.

Preferably, the EGFR inhibitor is erlotinib, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the pharmaceutical combination comprises the MDM2 inhibitor selected from (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2-dihydropyridin-3-yl)-6-(4- chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4- d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof, and (S)-l-(4-Chloro- phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans- cyclohexylmethyl] -amino } -phenyl)- 1 ,4-dihydro-2H-isoquinolin-3 -one, or a

pharmaceutically acceptable salt thereof; the Bcl-xL inhibitor A-l 155463 or

pharmaceutically acceptable salt thereof; the MEK inhibitor trametinib, or

pharmaceutically acceptable salt thereof, and the EGFR inhibitor erlotinib, or a pharmaceutically acceptable salt thereof.

According to the present disclosure, the pharmaceutical combinations of the present disclosure comprising (a) the MDM2 inhibitor and (b)(i) the MEK inhibitor, and/or (ii) the Bcl-xL inhibitor may further advantageously comprise a PI3K inhibitor.

The term "a phosphatidylinositol 3-kinase inhibitor" or "a PI3K inhibitor" is defined herein to refer to a compound which targets, decreases or inhibits PI3 -kinase. PI3- kinase activity has been shown to increase in response to a number of hormonal and growth factor stimuli, including insulin, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, colony-stimulating factor, and hepatocyte growth factor, and has been implicated in processes related to cellular growth and transformation.

Phosphatidylinositol -3-kinase (PI3K) inhibitors suitable for the present disclosure are selected from the group consisting of 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl- 2,3-dihydro-imidazo[4,5-c]quinolin-l-yl)-phenyl]-propionitrile, or a pharmaceutically acceptable salt thereof, 5-(2,6-di-moφholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl- pyridin-2-ylamine, or a pharmaceutically acceptable salt thereof; and (S)-Pyrrolidine-l,2- dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-l,l-dimethyl-ethyl)-pyridin- 4-yl] -thiazol-2-yl} -amide), or a pharmaceutically acceptable salt thereof.

WO2006/122806 describes imidazoquinoline derivatives, which have been described to inhibit the activity of PI3K. The compound 2-methyl-2-[4-(3-methyl-2-oxo- 8-quinolin-3 -yl-2,3 -dihydro-imidazo [4,5 -c]quinolin- 1 -yl)-phenyl] -propionitrile has the chemical structure of formula (V)

The compound, its utility as a PI3K inhibitor and synthesis of 2-methyl-2-[4-(3- methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-l-yl)-phenyl]- propionitrile and its monotosylate salt are described in WO2006/122806, which is hereby incorporated by reference in its entirety hereto, for instance in Example 7 and Example 152-3 respectively. The compound 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3- dihydro-imidazo[4,5-c]quinolin-l-yl)-phenyl]-propionitrile may be present in the form of the free base or any pharmaceutically acceptable salt thereto. Preferably, 2-methyl-2-[4- (3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-l-yl)-phenyl]- propionitrile is in the form of its monotosylate salt.

WO07/084786 describes specific pyrimidine derivatives which have been found to inhibit the activity of PI3K. The compound 5-(2,6-di-mo holin-4-yl-pyrimidin-4-yl)-4- trifluoromethyl-pyridin-2-ylamine has the chemical structure of formula (VI)

The compound, its salts, its utility as a PI3K inhibitor and synthesis of the compound 5-(2,6-di-moφholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine are described in WO 2007/084786, which is hereby incorporated by reference in its entirety hereto, for instance in Example 10. The compound 5-(2,6-di-morpholin-4-yl- pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine may be present in the form of the free base or any pharmaceutically acceptable salt thereto. Preferably, 5-(2,6-di- moφholin-4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine is in the form of its hydrochloride salt.

WO2010/029082 describes specific 2-carboxamide cycloamino urea derivatives which have been found to be highly selective for the alpha isoform of PI3K and can be added to the combinations of the present disclosure. The compound (S)-Pyrrolidine-l,2- dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-l,l-dimethyl-ethyl)-pyridin- 4-yl]-thiazol-2-yl} -amide) has the chemical structure of formula (VII)

(VII).

The compound, its salts, its utility as an alpha-isoform selective PI3K inhibitor and synthesis of the compound (S)-Pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4- methyl-5-[2-(2,2,2-trifluoro-l,l-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide) are described in WO2010/029082, which is hereby incorporated by reference in its entirety, for instance in Example 15. The compound (S)-Pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-l, l-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl} -amide) may be present in the form of the free base or any pharmaceutically acceptable salt thereto. Preferably, (S)-Pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2- (2,2,2-trifluoro- 1,1 -dimethyl -ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide)is in the form of its free base.

Preferably, the PI3K inhibitor compound used in the combination of the present disclosure is (S)-Pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2- trifluoro-l,l-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide), or any pharmaceutically acceptable salt thereof.

In one embodiment, the pharmaceutical combination comprising the MDM2 inhibitor and the Bcl-xL inhibitor may further advantageously comprise the PI3K inhibitor. It has been surprisingly found that this triple combination synergistic inhibition (over the drug pairs in 2/5 cell models tested (Example 4, Table 9) and showed stronger apoptosis compared to the pair wise combinations (Example 4, Figure 14).

pharmaceutically acceptable salt thereof; the Bcl-xL inhibitor A-l 155463, or

pharmaceutically acceptable salt thereof, and the PI3K inhibitor (S)-Pyrrolidine-l,2- dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2 rifluoro-l,l-dimethyl-ethyl)-pyridin- 4-yl]-thiazol-2-yl} -amide), or any pharmaceutically acceptable salt thereof.

Furthermore, according to the present disclosure, the pharmaceutical combinations of the present disclosure comprising (a) the MDM2 inhibitor and (b) (i) the Bcl-xL inhibitor, and/or (ii) the MEK inhibitor may further advantageously comprise a BRAF inhibitor.

Furthermore, the pharmaceutical combination of the present disclosure may advantageously comprise (a) the MDM2 inhibitor, (b) the Bcl-xL inhibitor, (c) the MEK inhibitor, and (d) a BRAF inhibitor.

The term "a BRAF inhibitor" is defined herein to refer to a compound which targets, decreases or inhibits the activity of serine/threonine-protein kinase B-Raf.

The pharmaceutical combination according to any one of the preceding claims, wherein the BRAF inhibitor is selected from the group consisting of RAF265, dabrafenib (S)-methyl-l-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-l-isopropyl-lH- pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate, methyl N-[(2S)-l-({4-[3-(5- chloro-2-fluoro-3-methanesulfonamidophenyl)-l-(propan-2-yl)-lH-pyrazol-4- yl]pyrimidin-2-yl}amino)propan-2-yl]carbamate and vemurafenib, or a pharmaceutically acceptable salt thereof.

According to the present disclosure, the BRAF inhibitor is preferably dabrafenib, or a pharmaceutically acceptable salt thereof. In one embodiment, the BRAF inhibitor added to the combination is RAF265.

The combination of the present disclosure, particularly the combination of the MDM2 inhibitor and a MEK inhibitor (such as trametinib) can further comprise a CDK4/6 inhibitor. "Cyclin dependent kinase 4/6 (CDK4/6) inhibitor" as defined herein refers to a small molecule that interacts with a cyclin-CDK complex to block kinase activity. The Cyclin-dependent kinases (CDK) is a large family of protein kinases that regulate initiation, progression, and completion of the mammalian cell cycle. Preferably, the CDK4/6 inhibitor is 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-l-yl)pyridin-2- yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, or pharmaceutically acceptable salt thereof.

The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compound and which typically are not biologically or otherwise undesirable. The compound may be capable of forming acid addition salts by virtue of the presence of an amino group.

Unless otherwise specified, or clearly indicated by the text, reference to therapeutic agents useful in the pharmaceutical combination of the present disclosure includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds. The term "combination" or "pharmaceutical combination" is defined herein to refer to either a fixed combination in one dosage unit form, a non-fixed combination or a kit of parts for the combined administration where the therapeutic agents may be administered together, independently at the same time or separately within time intervals, which preferably allows that the combination partners show a cooperative, e.g. synergistic effect. Thus, the single compounds of the pharmaceutical combination of the present disclosure could be administered simultaneously or sequentially.

Furthermore, the pharmaceutical combination of the present disclosure may be in the form of a fixed combination or in the form of a non-fixed combination. The term "fixed combination" means that the therapeutic agents, e.g., the single compounds of the combination, are in the form of a single entity or dosage form.

The term "non-fixed combination" means that the therapeutic agents, e.g., the single compounds of the combination, are administered to a patient as separate entities or dosage forms either simultaneously or sequentially with no specific time limits, wherein preferably such administration provides therapeutically effective levels of the two therapeutic agents in the body of the subject, e.g., a mammal or human in need thereof.

The pharmaceutical combinations can further comprise at least one

pharmaceutically acceptable carrier. Thus, the present disclosure relates to a

pharmaceutical composition comprising the pharmaceutical combination of the present disclosure and at least one pharmaceutically acceptable carrier.

As used herein, the term "carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Generally, the term "pharmaceutical composition" is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to a subject, e.g., a mammal or human. The present pharmaceutical combinations can be formulated in a suitable pharmaceutical composition for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. If not indicated otherwise, these are prepared in a manner known per se, for example by means of various conventional mixing, comminution, direct compression, granulating, sugar-coating, dissolving, lyophilizing processes, or fabrication techniques readily apparent to those skilled in the art. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount may be reached by administration of a plurality of dosage units. The pharmaceutical composition may contain, from about 0.1 % to about 99.9%, preferably from about 1 % to about 60 %, of the therapeutic agent(s). One of ordinary skill in the art may select one or more of the aforementioned carriers with respect to the particular desired properties of the dosage form by routine experimentation and without any undue burden. The amount of each carriers used may vary within ranges conventional in the art. The following references disclose techniques and excipients used to formulate oral dosage forms. See The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2003). These optional additional conventional carriers may be incorporated into the oral dosage form either by incorporating the one or more conventional carriers into the initial mixture before or during granulation or by combining the one or more conventional carriers with granules comprising the combination of agents or individual agents of the combination of agents in the oral dosage form. In the latter embodiment, the combined mixture may be further blended, e.g., through a V-blender, and subsequently compressed or molded into a tablet, for example a monolithic tablet, encapsulated by a capsule, or filled into a sachet. Clearly, the pharmaceutical combinations of the present disclosure can be used to manufacture a medicine.

The present disclosure relates to such pharmaceutical combinations or

pharmaceutical compositions that are particularly useful as a medicine.

Specifically, the combinations or compositions of the present disclosure can be applied in the treatment of cancer.

The present disclosure also relates to use of pharmaceutical combinations or pharmaceutical compositions of the present disclosure for the preparation of a medicament for the treatment of a cancer, and to a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination according to the present disclosure, or the

pharmaceutical composition according to the present disclosure.

The term "treatment" as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject, increasing progression-free survival, overall survival, extending duration of response or delaying progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer. Within the meaning of the present disclosure, the term "treatment" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease in a patient, e.g., a mammal, particularly the patient is a human. The term "treatment" as used herein comprises an inhibition of the growth of a tumor incorporating a direct inhibition of a primary tumor growth and / or the systemic inhibition of metastatic cancer cells.

A "subject," "individual" or "patient" is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, simians, humans, farm animals, sport animals, and pets.

The term "a therapeutically effective amount" of a compound (e.g. chemical entity or biologic agent) of the present disclosure refers to an amount of the compound of the present disclosure that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one embodiment a therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg.

The optimal dosage of each combination partner for treatment of a cancer can be determined empirically for each individual using known methods and will depend upon a variety of factors, including, though not limited to, the degree of advancement of the disease; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art. The amount of each combination partner that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.

Frequency of dosage may vary depending on the compound used and the particular condition to be treated or prevented. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

A therapeutic amount or a dose of (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2- dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan-2- yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one may range between 100 and 1500 mg every three weeks, particularly between 100 and 800 mg every three weeks, or between 50 and 600 mg daily, when administered per os. A therapeutic amount or a dose of (6S)- 5 -(5 -Chloro- 1 -methyl -2 -oxo- 1 ,2-dihydropyridin-3 -yl)-6-(4-chlorophenyl)-2-(2,4- dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one can be 400 mg, more preferably is 300 mg for daily administration for the first 21 days of every 28 day cycle. Alternatively, a total therapeutic amount or a total dose of (6S)-5-(5- Chloro-1 -methyl -2 -oxo-1, 2-dihydropyridin-3 -yl)-6-(4-chloropheny l)-2-(2,4- dimethoxypyrimidin-5-yl)-l-(propan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one is 560 mg per cycle (40 mg qd 2 wks on / 2 wks off, or 80 mg qd 1 wk on / 3 wks off). Intravenous doses would need to be lowered accordingly.

A therapeutic amount or dose of (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy- 2-(4- {methyl-[4-(4-methyl-3 -oxo-piperazin- 1 -yl)-trans-cyclohexylmethyl] -amino} - phenyl)- l,4-dihydro-2H-isoquinolin-3-one is between 500 and 2000 mg, particularly between 500 and 1200 mg, when administered per os. In a preferred embodiment, a therapeutic amount or dose of (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4- {methyl-[4-(4-methyl-3-oxo-piperazin-l-yl)-trans-cyclohexylmethyl]-amino}-phenyl)- l,4-dihydro-2H-isoquinolin-3-one is 500 mg, more preferably 800 mg. Intravenous doses would need to be lowered accordingly.

The Bcl-xL inhibitor A-l 155463 has been dosed 5χ10^Λ-3 g/kg i.p. s.d. in preclinical studies in mice (PROUS integrity records). The dose escalation study in man will allow to identify the maximum tolerated dose, and will allow to define the recommended clinical dose for pivotal clinical studies.

The recommended dose of the MEK inhibitor trametinib is 2 mg daily. The management of adverse reactions may require dose reduction up to 1 mg daily.

The MEK inhibitor compound 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3- methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide may be administered to a suitable subject daily in single or divided doses at an effective dosage in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to a preferable dosage range of about 0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day.

The MEK inhibitor compound (S)-5-fluoro-2-(2-fluoro-4- (methylthio)phenylamino)-N-(2-hydroxypropoxy)- 1 -methyl-6-oxo- 1 ,6-dihydropyridine- 3-carboxamide may be administered daily to a suitable subject in single or divided doses at an effective dosage in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 mg/kg/day to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to a preferable dosage range of about 0.07 to 2.45 g/day, preferably about 0.05 to about 1.0 g/day.

An effective dose of the Bcl-2 inhibitor navitoclax may range from about 100 mg to about 500 mg daily. The dose may be reduced or a 150 mg 7-day lead-in dose employed. After the lead-in dose a 325 mg dose or up to 425 mg dose can be administered daily.

The recommended dose of the EGFR inhibitor erlotinib is 100 mg or 150 mg daily. The PI3K inhibitor compound (S)-pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4- methyl-5-[2-(2,2,2 rifluoro-l,l-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide) is generally administered orally at a dose in the range from about from 30 mg to 450 mg per day, for example 100 to 400 mg per day in a human adult. The daily dose can be administered on a qd or bid schedule. (S)-pyrrolidine-l,2-dicarboxylic acid 2-amide 1- ( {4-methyl-5-[2-(2,2,2-trifluoro- 1 , 1 -dimethyl -ethyl)-pyridin-4-yl]-thiazol-2-yl} -amide) may administered to a suitable subject daily in single or divided doses at an effective dosage in the range of about 0.05 to about 50 mg per kg body weight per day, preferably about 0.1-25 mg/kg/day, more preferably from about 0.5-10 mg/kg/day , in single or divided doses. For a 70 kg human, this would amount to a preferable dosage range of about 35-700 mg per day. More preferably, the dosage range is of about 35 - 400 mg per day.

The PI3K inhibitor compound 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3- dihydro-imidazo[4,5-c]quinolin-l-yl)-phenyl]-propionitrile is generally administered orally at a dose in the range from about 100 mg to 1200 mg, or about 200 mg to 1000 mg, or about 300 mg to 800 mg, or about 400 mg to 600 mg per day in a human adult. The daily dose can be administered on a qd or bid schedule.

The PI3K inhibitor compound 5-(2,6^ί^ο ηο1ίη-4^1^ΓΐΓ^ίη-4^1)-4- trifluoromethyl-pyridin-2-ylamine is generally administered orally at a dose in the range from about 30 mg to 300 mg, or about 60 mg to 120 mg, or about 100 mg per day in a human adult. The daily dose can be administered on a qd or bid schedule.

The recommended dose of the BRAF inhibitor dabrafenib is 150 mg orally twice daily as a single agent or in combination with trametinib 2 mg orally once daily.

It is understood that each therapeutic agent may be conveniently administered, for example, in one individual dosage unit or divided into multiple dosage units. It is further understood that that each therapeutic agent may be conveniently administered in doses once daily or doses up to four times a day.

The term "cancer" is used herein to mean a broad spectrum of tumors, in particular solid tumors. Examples of such tumors include, but are not limited to a benign or malignant tumor of the lung (including small cell lung cancer and non-small-cell lung cancer), bronchus, prostate, breast (including sporadic breast cancers and sufferers of Cowden disease), pancreas, gastrointestinal tract, colon, rectum, colon carcinoma, colorectal cancer, thyroid, liver, biliary tract, intrahepatic bile duct, hepatocellular, adrenal gland, stomach, gastric, glioma, glioblastoma, endometrial, kidney, renal pelvis, bladder, uterus, cervix, vagina, ovary, multiple myeloma, esophagus, neck or head, brain, oral cavity and pharynx, larynx, small intestine, a melanoma, villous colon adenoma, a sarcoma, a neoplasia, a neoplasia of epithelial character, a mammary carcinoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, polycythemia vera, essential thrombocythemia, a leukemia (including acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemia, and myeloid leukemia), a lymphoma (including non-Hodgkin lymphoma and Hodgkin's lymphoma), myelofibrosis with myeloid metaplasia, Waldenstroem disease, and Barret's adenocarcinoma.

Preferably, the cancer is colorectal cancer, melanoma, liposarcoma, glioblastoma, neuroblastoma, lymphoma or leukemia. In a preferred embodiment the cancer is colorectal cancer. The term "colorectal cancer", as used herein, refers to cancer in the colon or rectum, also known as colon cancer, rectal cancer or bowel cancer. In one embodiment, the present disclosure relates to metastatic colorectal cancer.

The combination is expected to achieve superior effects in functional p53 or p53 wild-type cancers. The TP53 gene is one of the most frequently mutated genes in human cancers. Thus, tumor suppressor p53 is functionally impaired by mutation or deletion in nearly 50% of human cancers. In the remaining human cancers, p53 retains wild-type status but its function is inhibited by its primary cellular inhibitor, the murine double minute 2 (Mdm2, MDM2; HDM2 (human homo log of murine double minute 2)). Mdm2 is a negative regulator of the p53 tumor suppressor. Mdm2 protein functions both as an E3 ubiquitin ligase, that leads to proteasomal degradation of p53, and an inhibitor of p53 transcriptional activation. Often Mdm2 is found amplified in p53 wild-type tumors. Because the interaction between Mdm2 and p53 is a primary mechanism for inhibition of the p53 function in cancers, which are retaining wild-type p53, the combination of the present disclosure comprising the MDM2 inhibitor is particularly useful for treatment of functional p53 or p53 wild-type cancers.

In addition, the efficacy of the combination is expected to be increased in cancer, which is characterized by one or more of KRAS mutation and/or BRAF mutation and/or MEK1 mutation and/or PIK3CA mutation and/or PIK3CA overexpression.

Patients with colorectal cancer harboring KRAS or BRAF mutations, which together make up 50%-60% of reported colorectal cancer cases (Fearon 2011), are generally associated with a poor prognosis (Arlington, Heinrich et al. 2012, Safaee Ardekani, Jafarnejad et al. 2012). The combinations of this disclosure are particularly useful for treatment of cancer, which comprises one or more of KRAS mutation or one or more of BRAF mutation.

Examples of BRAF mutations include, but not limited to V600E, R46 II, I462S,

G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, A727V. Most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions .V600E mutation has been detected in a variety of cancers, and is due to a substitution of thymine with adenine at nucleotide

1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to as V600E).

MEK1 mutation may be, for example, MEK1 S72G mutation. Examples of PIK3CA mutation and/or PIK3CA overexpression include, but not limited to , amplification of the alpha isoform of PI3K, somatic mutation of PIK3CA, germline mutations or somatic mutations of PTEN, mutations and translocation of p85ot that serve to up-regulate the p85-pl 10 complex, or amplification or overexpression of the beta isoform of PI3K.

The pharmaceutical combination of the present disclosure is particularly useful for the treatment of a cancer, particularly colorectal cancer, wherein the cancer is resistant to a treatment with an EGFR inhibitor, or is developing a resistance to a treatment with an EGFR inhibitor, or is under high risk of developing a resistance to a treatment with an EGFR inhibitor, particularly wherein the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib and afatinib.

The pharmaceutical combination of the present disclosure is also suitable for the treatment of poor prognosis patients, especially such poor prognosis patients having a cancer, particularly colorectal cancer, which becomes resistant to treatment employing an EGFR inhibitor, e.g. a cancer of such patients who initially had responded to treatment with an EGFR inhibitor and then relapsed. In a further example, said patient has not received treatment employing a FGFR inhibitor. This cancer may have acquired resistance during prior treatment with one or more EGFR inhibitors. For example, the EGFR targeted therapy may comprise treatment with gefitinib, erlotinib, lapatinib, XL- 647, HKI-272 (Neratinib), BIBW2992 (Afatinib), EKB-569 (Pelitinib), AV-412, canertinib, PF00299804, BMS 690514, HM781-36b, WZ4002, AP-26113, cetuximab, panitumumab, matuzumab, trastuzumab, pertuzumab, or a pharmaceutically acceptable salt thereof. In particular, the EGFR targeted therapy may comprise treatment with gefitinib, erlotinib, and afatinib. The mechanisms of acquired resistance include, but are not limited to, developing a second mutation in the EGFR gene itself, e.g. T790M, EGFR amplification; and / or FGFR deregulation, FGFR mutation, FGFR ligand mutation, FGFR amplification, or FGFR ligand amplification.

The pharmaceutical combinations as described herein are particularly useful for use in patients which have a resistance to mdm2 inhibitors. The resistance may be caused by regrowth of mdm2 inhibitor resistant cells or by genetic predisposition.

The pharmaceutical combinations as described herein may further comprise the use of a TPO receptor agonist to overcome cytopenias, such as thrombocytopenia and/or neutropenia. A preferred TPO receptor agonist is eltrombopag.

The present invention provides combinations of drug substances as described herein or any pharmaceutically acceptable salt thereof for use in the treatment of the indications as described herein. Alternatively, the present invention provides methods for the treatment of the indications as described herein in human patients in need of such treatment which comprises administering an effective amount of the combinations of drug substances as described herein or any pharmaceutically acceptable salt thereof.

As a further alternative the present invention provides the use of the combinations of the drug substances as described herein or any pharmaceutically acceptable salt thereof for the manufacture/preparation of medicaments for the treatment of the indications as described herein.

As a further alternative the present invention provides medicaments for the treatment of the indications as described herein comprising the combinations of drug substances as described herein or any pharmaceutically acceptable salt thereof.

The following Examples illustrates the disclosure described above, but is not, however, intended to limit the scope of the disclosure in any way. Other test models known as such to the person skilled in the pertinent art can also determine the beneficial effects of the claimed disclosure.

EXAMPLES

Results

piggyBac-induced spontaneous tumors in the Arf-/- background and derived allograft models and their transposon insertional mutagenesis landscapes

Before identification of resistance mechanisms to p53-Mdm2 inhibition, we generated a collection of tumors sensitive to such inhibitors. We crossed mice to combine genetic components of the constitutive PB system bearing ATP2-S 1 and Rosa26-transposase PBase (14) with an Arf null allele, deficient for the p53 regulator and tumor suppressor pl9Arf (13, 22). To further utilize these Arf-/- PB tumor models in efficacy experiments, the tumors were serially transplanted as for human patient-derived xenograft. To this end, fragments of spontaneous RosaPB/+;ATP2/+;Arf-/- tumors were implanted

subcutaneously in athymic Nude mice flanks. Once an allograft tumor grew, it was further expanded in 20 to 60 immunodeficient mice. To identify genes recurrently targeted by transposon with good statistical power, we sequenced the transposon insertion sites of 327 tumors obtained from RosaPB/+;ATP2/+;Arf-/- mice. Using a gene-centric common insertion sites (gCIS) calling method, that identifies density of transposon insertions within the coding regions of all RefSeq genes higher than predicted by chance (24, 25), we identified 2444 CIS that were found in at least 2 tumors (Figl). Consistent with previous Sleeping Beauty mutagenesis in the Arf-/- background (23), Braf was the most frequent transposon insertion found in 90.8% of tumors, indicating it may constitute a major cooperating pathway with Arf loss of function. Indeed, insertions at Braf could not be found in PB tumors with no Arf deletion. The Braf gene was targeted between exons 8 and 12 in a directional manner (Fig2), presumably leading to the expression of a specific constitutively active truncated protein as previously described (23, 27, 28). Similar human BRAF gene truncations or fusions were previously reported in human brain, pancreatic, and prostate tumors (27, 29-34).

Response and resistance to p53-Mdm2 inhibition in piggyBac allografts

Because of Arf deletion, we expected our RosaPB/+; ATP2/+;Arf-/- tumors to respond to HDM201. To assess the tumor sensitivity to HDM201, we treated 21 allograft models at the passage 1 level (Fig3). After random enrollment, 139 mice were treated twice weekly with lOOmg/kg of HDM201 and 106 mice were treated with vehicle. Overall, we observed a significant response across the 21 models (Fig4). After HDM201 treatment, 6 out of 139 mice (4.3%), did not relapse within 60 days post last dose. However, 5 out of 21 models displayed poor response to HDM201 : a B cell lymphoma, a choriocarcinoma, two MPNST, and a liposarcoma (Fig3). Overall, despite a good preclinical response rate to HDM201 for RosaPB/+; ATP2/+;Arf-/- implanted tumors, most tumors eventually become resistant (Fig3).

Identification of mechanisms of resistance to p53-Mdm2 inhibition by insertional mutagenesis

To define genetic causes of resistance to p53-MDM2 inhibition, genomic DNA from resistant and vehicle-treated tumors was subjected to splinkerette PCR and deep sequencing to define genetic landscapes based on gCIS. A differential integration analysis identified PB target genes that were significantly enriched in resistant tumors (Fig5). 87 genes were identified suggesting a diversity and/or heterogeneity of the resistance mechanism. Gene ontology analysis revealed that only the p53 pathway was found significantly enriched. The PB bidirectional pattern predicted a Trp53 loss of function. Bcl211 gene was found as the second major enriched target in HDM201 resistant tumors, with a gain of function insertional pattern that did not allow distinguishing between expressions of Bcl-xL or Bcl-xS transcripts (Fig6). However, immunoblotting experiment demonstrated that Bcl-xL protein, but not Bcl-xS, was expressed in resistant tumors with transposon insertion in the Bcl211 promoter (Fig6). Bcl-xL protein interaction with p53 is known to antagonize the antiapoptotic effect in p53 mitochondrial apoptotic pathway (43, 44), and overexpressing Bcl-xL may therefore allow inhibition of p53 apoptotic pathway.

Because Bcl-xL is a known druggable targets, we next investigated if similar resistance mechanisms could be found on breast and lung PDX models resistant to the Hdm2/p53 inhibitors HDM201 (10) (Fig 7). Bcl-xL overexpression was detected in 5 resistant human tumors. Bcl-xL and MDM2 inhibitors act synergistically in p53 wild-type tumor models

Bcl-xL can be chemically inhibited by dual Bcl2/Bcl-xL inhibitor like ABT-263 (49) or Bcl-xL selective inhibitor like A-1155463 (50). To understand if dual inhibition of Bcl-xL and MDM2 could be beneficial in a broad manner, we evaluated the synergistic effects of 69 compounds with CGM097 in an in vitro viability screen on 485 cancer cell lines(lO). ABT-263 was found the third best combination partner with CGM097 in the 138 of these cell lines that were wild-type for p53 (Fig8). Exposing the 485 cancer cell lines to a dose matrix of ABT-263 and CGM097 revealed significant synergy in 35 out of the 138 p53 wild-type cell lines, and no significant synergy in p53 mutant cell lines (Fig9). Overall, these data suggested that a fraction of patients with p53 wild-type tumors may generally benefit from a dual treatment with Bcl2/Bcl-xL inhibitor and Mdm2 inhibitor, consistent with previous observations in leukemia that combination treatment of MDM2 inhibitor and ABT-263 could achieve longer-term tumor regression (41). To confirm that the combination effects were mediated by Bcl-xL and not Bcl2 inhibition, we performed dose-matrix combination experiments with HDM201 and the Bcl-xL selective inhibitor A-1155463 (50) in two of the cell lines (SNG-M and LS-513) that responded well to CGM097 and ABT-263 combination. As expected, strong synergy between HDM201 and A-1155463 was observed in both lines (Fig 10). In addition, in vitro viability assay revealed that induced overexpression of Bcl-xL in a metastatic melanoma p53 wild-type cell line WM266.4 led to a marked reduced sensitivity to HDM201 (FiglO). The GI50 was 6 fold higher when Bcl-xL was overexpressed.

BCL-XL expression confers resistance to HDM201 treatment specifically with intermittent high dose scheduling

To understand whether the biology behind resistance differs with the schedule of HDM201 administration, we now run a similar resistance screen using a daily

HDM201dosing at 40mg/kg, instead of lOOmg/kg twice a week which we name intermittent. We considered 6 out of the 16 piggyBac;Arf-/- transplanted tumor models responsive to HDM201 (2 lymphoma and 4 medulloblastoma), and now decided to treat transplanted tumors of same passage with 40mg/kg HDM201 daily. After multiple dosing, similar response rates were seen across these 6 models for both dosing regimens (Figl 1A). The continuous treatment was not well tolerated in mice: body weight loss was detrimental and 16 mice out of 60 had to be terminated. On the contrary, the intermittent treatment was well tolerated and only one animal was euthanized due to body weight loss (Fig 1 IB).

We then identified insertional events linked to the development of resistance to TP53- MDM2 inhibition, genomic DNA from resistant and vehicle -treated tumors. A total of 7 and 10 genes were targeted by PB in tumors that relapsed from intermittent scheduling (Figl 1C) or daily scheduling (Figl ID) respectively. Consistently with previous findings, genes directly regulating TRP53 (Trp53, Mdm4, Trp63) were highly represented in treated relapsing tumors versus vehicle treated. Interestingly, Bcl211, encoding Bcl-xl isoform, was enriched only in the intermittent scheduling, thus highlighting Bcl-XL expression as a unique mechanism of resistance in intermittent high dose scheduling. These data suggests that different apoptotic induction between the two dose regimens likely play a role in the way the resistance takes place. Because BCL-xL can be targeted therapeutically (3, 4), our results provide evidence that Bel -XL inhibition may be beneficial for relapsing patients that were treated with HDM201 intermittent scheduling.

Discussion

We performed a screen to identify genes involved in the resistance to p53/Mdm2 inhibition. To this end, we utilized allografted tumors from Arf-/-; ATP2/+; Rosa-PB/+ tumor models sensitive to HDM201. CIS genes were found significantly targeted in resistant tumors and likely able to confer resistance to HDM201, in a differential analysis comparing vehicle- or HDM201 -treated samples from engrafted animals. The most significant transposon-altered genes included genes directly regulating the p53 pathway like Trp53 (50%), Mdm4(31.9%), ΔΝ isoform of Trp63(18.1%) and ΔΝ isoform of Trp73(16%) genes. Interestingly, the Bcl211 gene was the second most significant CIS gene enriched in HDM201 resistant tumors, after Trp53. Activating transposon insertions in these resistant samples led to enhanced Bcl-xL protein expression. To our knowledge, no therapy is available to counteract p53 mutations, ΔΝρ63 and ΔΝρ73. Only MDM4(56) and Bcl-xL (49, 50) can currently be targeted therapeutically. Therefore, upfront combination therapies may provide a more promising therapeutic strategy where complete killing of cancers may be attained. For instance, our study identified MEK inhibitor as the best combination partner for p53/MDM2 inhibition in p53 wild-type cell lines (FigS9A), consistent with possible cooperation of p53 inhibition through Arf deletion and MAPK pathway activation via BRaf truncation. Such MEK inhibition and p53/MDM2 inhibition combination was also identified as efficacious in long term colony formation assays and enhanced apoptosis induction. Our in vitro assays demonstrated that Bcl-xL inhibition sensitized cells to HDM201 inhibition. Therefore, triple combination of MEK, p53/MDM2, and Bcl-xL inhibition may provide further added benefits to prevent cancer recurrence or prolong partial remission, provided adverse effects can be managed.

Material and methods

Experimental animals

All animal studies were conducted in accordance to procedures covered by permit number BS-2604 and BS-1763 issued by the Kantonales Veterinaramt Basel-Stadt and strictly adhered to the Eidgenossisches Tierschutzgesetz and the Eidgenossische

Tierschutzverordnung. All animals were allowed to adapt for 7 days and housed in a pathogen-controlled environment (5 mice/Type III cage) with ad libitum access to food and water and were identified with transponders. Mice were housed in a specific pathogen-free facility with a 12-h light/12-h dark cycle. Conditional survival is defined by maximum tumor size estimation at 1.5cm diameter or when mice showed suffering or symptoms of morbidity/moribundity, or more than 15% body weight loss. The following genetic components were combined by crossing mice in order to obtain experimental animals from which derived the tumor fragments : heterozygous for RosaPB et ATP2-S 1, and homozygous for Arf deficient allele. ATP2-S 1 : CALB/FVB-TgTn(pb/sb- ATP2)SlBrd mouse line carries 15 transposon copies inserted in chromosome 17. ATP2- S 1 piggyBac transposon contains a unidirectional MSCV promoter and gene traps (splice acceptors and Poly A) acting in both orientations (5). The PB transposase knockin-mice, RosaPB: CALB/SV129-Gt(ROSA)26Liutml(ipb)Ww, carried the piggyBac transposase in RosaPB locus (5). The Arf-/- mouse line was FVB-Cdkn2atmlNesh (6, 7).

Generation of allograft tumor models

Approximately l-2mm3 tissue fragments were implanted subcutaneously with 50% Matrigel (#354234 Corning) into the flank region of athymic nude (nu/nu) female (Charles River, Germany) mice using a trocar. Engrafted tumor models were then passaged once. Tumor material on flank was collected in PBS and kept on wet ice for engraftment within 12 h after resection, or slow frozen. Necrotic and supporting tissues were carefully removed using a surgical blade. Immunodeficient models were used to prevent the tumor graft rejection since the experimental mice (RosaPB/+;ATP2/+;Arf-/-) were in a mixed genetic background.

Animal treatments

Treatments were initiated when the subcutaneous tumors were at least 200mm3. Efficacy studies, tumor response and relapse were reported with the measures of tumor volumes from the treatment start. HDM201 was administrated at lOOmg/kg, in 0.5%

methylcellulose and 0,1% Tween 80, orally twice a week, with alternation of intervals of 3 days and one of 4 days. Vehicles were generated according to respective formulations.

Splinkerette PCR for the amplification of transposon integration sites.

We adapted the protocol previously described (8). Briefly, genomic DNA was isolated, sheared to fragment length of 200-600bp on a Covaris sonicator. After end-repair and A- tailing, purified DNA fragments were ligated to a Splinkerette adaptor (obtained after annealing of 5'-gttcccatggtactactcatataatacgactcactataggtgacagcgagcgct-3' and 5'- /5Phos/gcgctcgctgtcacctatagtgagtcgtattataatttttttttcaaaaaaa-3'). Transposon-containing fragments were enriched with 18 cycles of transposon-specific PCR for both the 5' and 3' transposon ends in separate libraries (5'- gatatacagaccgataaaacacatgcgtca-3' for 3' arm of PB; 5'-gacggattcgcgctatttagaaagagag-3' for 5' arm of PB ; and common Splinkerette primer 5'-gttcccatggtactactcata-3'). Barcoding of individual samples and completion of Illumina adaptor sequences were achieved with an additional 12 cycles of transposon- specific PCR and a custom array of 96 unique bar-coding primers. For the 3' arm, we used 5'-XXXXXXXXacgcatgattatctttaacgtacgtcac-3', for the 5' arm, we used 5'- XXXXXXXXatgcgtcaattttacgcagactatc-3', and for the splinkerette side, we used 5'- ACTGAATCtaatacgactcactatagg-3 ' primers. The Xs represent the bar code made of 8 nucleotides. After size selection via magnetic bead purification (Beckman Ampure XP), libraries were pooled in two separate 5' and 3' pools and sequenced on the HiSeq Desktop Sequencer (Illumina). Mapping of insertion sequences to the mouse genome and identification of common integration sites.

We aligned integration reads to the mm 10 version of the mouse reference genome using bowtie2. We performed stringent query sequence filtering: we required paired reads to start with the exact splinkerette primer sequence on the one side, and the exact transposon primer sequence on the other side. We filtered the aligned reads for PCR duplicates by removing reads with the same start and end positions. The PB transposon requires a TTAA motif to insert itself in the genome. Therefore, we also filtered out the reads that did not align to a TTAA motif in the mouse genome. Finally, we required reads from the 3 ' and 5 ' arms of PB to align exactly 4bp (one TTAA) apart. The result of this stringent alignment procedure is a list of counts per sample and genomic position. We call such counts div counts (for Diversity). Div counts are then normalized to account for unequal library sizes.

In order to identify genes that are commonly integrated, we adapted the gCIS strategy first described for SB (10). Shortly, we defined a gene associated region as the gene transcription unit extended upstream by lOkb of promoter sequence. For each gene associated region, we counted the number of insertions (normalized div counts) and the number of TTAA motifs that fell inside and outside the region. We then performed a Fisher exact test on the resulting 2x2 contingency table. The gCIS method allows identifying genes in aggregates by adding normalized div counts for a pool of samples, but also for a single sample, enabling the analysis of rare/hard to obtain indications. Indeed, thanks to the stringent alignment procedure and the high number of PB integration sites identified in single samples, the gCIS method was verified to recover known cancer genes even in single sample analyses.

Differential integration analysis

Differential integration analysis was performed to identify integration sites likely to confer resistance to HDM201. We use the Bioconductor R package edgeR (11, 12) to perform the differential integration analysis. In order to account for the fact that tumors were passaged from 6 primary tumors, we used an experimental design in which we block for primary tumor. The div counts were normalized using edgeR's TMM normalization method.

Western blot analysis

Proteins were extracted from tumor powder using cold Giordano buffer containing phosphatase inhibitors 100X (Sigma P-0044, and P-5726) and proteases inhibitors 100X (Sigma, P-8340). Protein concentration was determined following Qubit® protein determination kit's protocol. 50ug of protein extract were separated by SDS-PAGE (CriterionTM XT Precast Gel, 4-12% Bis-Tris, BIO-RAD, #345-0124, blotting buffer XT MOPS, BIO-RAD, #161-0788) and transferred onto PVDF membranes (Immobilon-P, MILLIPORE, #IPVH00010) using a wet transfer system (Trans-Blot® Transfer Cell, BIO-RAD, #1703930). Membranes were probed with antibodies (diluted in 5% skim milk powder in PBS/T20 + 0,05% Sodium Azide) against Vinculin (V91131, Sigma) or Bcl-xL (Cell Signaling, 54H6) overnight. Secondary antibody was HRP -conjugated anti-mouse IgG antibody (7076, cell signaling) or HRP-conjugated anti-rabbit IgG antibody (7074, cell signaling) and blots were revealed with ECL substrate (WesternBright ECL, Advansta #K-12045-D20) on the Fusion FX7 imager.

Cell line combination synergy testing

The in vitro combination screen was performed on cancer cell lines and data calculations were previously described (10). Here we focused the data analysis on combinations with CGM097, an MDM2 inhibitor structurally similar to HDM201. In total, 485 cancer cell lines were treated with ranges of concentrations for CGM097 and for 25 other compounds. We integrated the information of p53 mutation status and differentiated cell lines with no p53 mutation from cell lines with p53 alteration. The synergistic effect of combinations of HDM201 and A- 1155463 was assessed using methods previously described (58).

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Claims

A pharmaceutical combination comprising

(b) Bcl-xL inhibitor selected from the group consisting of A-l 155463, A-1331852, WEHI-539, or a pharmaceutically acceptable salt thereof.

The pharmaceutical combination according to claim 1 comprising

(a) an MDM2 inhibitor selected from (6S)-5-(5-Chloro-l-methyl-2-oxo-l,2- dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-l-(propan- 2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(lH)-one, or a pharmaceutically acceptable salt thereof, and (S)-l-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4- {methyl- [4- (4-methyl-3 -oxo-piperazin- 1 -yl)-trans-cyclohexylmethyl] -amino } -phenyl)- 1 ,4- dihydro-2H-isoquinolin-3-one, or a pharmaceutically acceptable salt thereof;

(b) Bcl-xL inhibitor selected from the group consisting of A-l 155463, A-1331852, WEHI-539, or a pharmaceutically acceptable salt thereof and

(c) a MEK inhibitor.

The pharmaceutical combination according to claim lor claim 2, wherein the Bcl-xL inhibitor is A-l 155463, or a pharmaceutically acceptable salt thereof.

The pharmaceutical combination comprising according to claim 1, claim 2 or claim 3, wherein the MEK inhibitor is trametinib, or a pharmaceutically acceptable salt thereof.

The pharmaceutical combination according to any one of claims 1 to 4, wherein the combination further comprises an EGFR inhibitor.

The pharmaceutical combination according to claim 7, wherein the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, lapatinib, canertinib, pelitinib, neratinib, (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2-enoyl)azepan-3 - yl)- lH-benzo[d]imidazol-2-yl)-2-methylisonicotinamide, panitumumab, matuzumab, pertuzumab, nimotuzumab, zalutumumab, icotinib, afatinib and cetuximab, and pharmaceutically acceptable salt thereof.

The pharmaceutical combination according to claim 5 or claim 6, wherein the EGFR inhibitor is erlotinib, or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical combination according to any one of claims 1 to 6, wherein the combination further comprises a PI3K inhibitor.

The pharmaceutical combination according to claim 8, wherein the PI3K inhibitor is selected from the group consisting of 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl- 2,3 -dihydro-imidazo [4,5 -c]quinolin- 1 -yl)-phenyl] -propionitrile, 5 -(2,6-di-morpholin- 4-yl-pyrimidin-4-yl)-4-trifluoromethyl-pyridin-2-ylamine, and (S)-Pyrrolidine- 1 ,2- dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-l,l-dimethyl-ethyl)- pyridin-4-yl]-thiazol-2-yl}-amide), or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical combination according to claim 8 or claim 9, wherein the PI3K inhibitor is an alpha-isoform specific phosphatidylinositol-3 -kinase (PI3K) inhibitor (S)-Pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-l, l- dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl} -amide), or any pharmaceutically acceptable salt thereof.

11. The pharmaceutical combination according to any one of claims 1 to 10, wherein the combination further comprises a BRAF inhibitor.

12. The pharmaceutical combination according to claim 10, wherein the BRAF inhibitor is selected from the group consisting of RAF265, dabrafenib, (S)-methyl-l-(4-(3-(5- chloro-2-fluoro-3-(methylsulfonamido)phenyl)- 1 -isopropyl- lH-pyrazol-4- yl)pyrimidin-2-ylamino)propan-2-ylcarbamate, methyl N-[(2S)-l-({4-[3-(5-chloro-2- fluoro-3-methanesulfonamidophenyl)-l-(propan-2-yl)-lH-pyrazol-4-yl]pyrimidin-2- yl}amino)propan-2-yl]carbamate and vemurafenib, or a pharmaceutically acceptable salt thereof.

13. The pharmaceutical combination according to claim 13 or claim 14, wherein the

BRAF inhibitor is dabrafenib, or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein the combination further comprises a CD4/6 inhibitor. 15. The pharmaceutical combination according to claim 14, wherein the CD4/6 inhibitor is 7-cyclopentyl-N,N -dimethyl -2-((5-(piperazin- l-yl)pyridin-2 -yl)amino)-7H- pyrrolo[2,3-d]pyrimidine-6-carboxamide, or pharmaceutically acceptable salt thereof.

16. The pharmaceutical combination according to any one of claims 1 to 15, wherein the combination further comprises paclitaxel.

17. The pharmaceutical combination according to any one of claims 1 to 16, wherein the combination further comprises a cMET inhibitor.

18. The pharmaceutical combination according to claim 19, wherein the cMET inhibitor is PF-04217903.

19. The pharmaceutical combination according to any one of the preceding claims for simultaneous or sequential use.

20. The pharmaceutical combination according to any one of the preceding claims in the form of a fixed combination.

21. The pharmaceutical combination according to any one of claims 1 to 21 in the form of a non-fixed combination.

22. A pharmaceutical composition comprising the pharmaceutical combination according to any one of the claims 1 to 21 and at least one pharmaceutically acceptable carrier.

23. The pharmaceutical combination according to any one of claims 1 to 23 or the

pharmaceutical composition according to claim 19 for use as a medicine.

24. The pharmaceutical combination according to any one of claims 1 to 21, or the

pharmaceutical composition according to claim 19 for use in the treatment of a cancer.

25. Use of a pharmaceutical combination according to any one of claims 1 to 21 for the preparation of a medicament for the treatment of a cancer.

26. A method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination according to any one of claims 1 to 21, or the pharmaceutical composition according to claim 22.

27. The pharmaceutical combination for use according to claim 24, or the use of a pharmaceutical combination according to claim 25, or the method according to claim

26, wherein the cancer is a solid tumor.

28. The pharmaceutical combination for use according to claim 24, or the use of a pharmaceutical combination according to claim 25, or the method according to claim 26, wherein the cancer is selected from the group consisting of a benign or malignant tumor of the lung (including small cell lung cancer and non-small-cell lung cancer), bronchus, prostate, breast (including sporadic breast cancers and sufferers of Cowden disease), pancreas, gastrointestinal tract, colon, rectum, colon carcinoma, colorectal cancer, thyroid, liver, biliary tract, intrahepatic bile duct, hepatocellular, adrenal gland, stomach, gastric, glioma, glioblastoma, endometrial, kidney, renal pelvis, bladder, uterus, cervix, vagina, ovary, multiple myeloma, esophagus, neck or head, brain, oral cavity and pharynx, larynx, small intestine, a melanoma, villous colon adenoma, a sarcoma, a neoplasia, a neoplasia of epithelial character, a mammary carcinoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, polycythemia vera, essential thrombocythemia, a leukemia (including acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemia, and myeloid leukemia), a lymphoma (including non-Hodgkin lymphoma and Hodgkin's lymphoma), myelofibrosis with myeloid metaplasia, Waldenstroem disease, and Barret's adenocarcinoma.

29. The pharmaceutical combination for use according to claim 24, or the use of a pharmaceutical combination according to claim 25, or the method according to claim 26, wherein the cancer is a colorectal cancer, liposarcoma, glioblastoma, neuroblastoma, lymphoma, leukemia or melanoma.

30. The pharmaceutical combination for use according to any one of claims 24, the use of a pharmaceutical combination according to any one of claims 25, or the method according to any one of claims 26, wherein the cancer comprises functional p53 or wild-type TP53.

31. The pharmaceutical combination for use according to any one of claims 24, the use of a pharmaceutical combination according to any one of claims 25, or the method according to any one of claims 26, for use in patients which have a resistance to mdm2 inhibitors.

32. The pharmaceutical combination for use according to any one of claims 24, the use of a pharmaceutical combination according to any one of claims 25, or the method according to any one of claims 26, further comprising the use of a TPO receptor agonist, preferably said TPO receptor agonist is eltrombopag.