WO2020089398A1

WO2020089398A1 - Combinatorial drug treatment for cancer

Info

Publication number: WO2020089398A1
Application number: PCT/EP2019/079843
Authority: WO
Inventors: Lilia Alberghina; Rosa Maria MORESCO; Silvia VALTORTA; Daniela GAGLIO
Original assignee: Universita' Degli Studi Di Milano Bicocca; Consiglio Nazionale Delle Ricerche
Priority date: 2018-10-31
Filing date: 2019-10-31
Publication date: 2020-05-07
Also published as: IT201800009974A1

Abstract

The present invention concerns the field of cancer, and in particular to oncogene-driven metabolic alterations which can simultaneously influence multiple metabolic pathways, thus generating unique cancer phenotypes. The present invention relates to a pharmaceutical kit for the treatment of cancer, particularly to the combination of therapeutic treatment of glycolysis/PI3K and of glutamine metabolism targets, using anti-cancer therapeutic agents showing enhanced anti-cancer effects.

Description

“COMBINATORIAL DRUG TREATMENT FOR CANCER”

FIELD OF THE INVENTION

The present invention concerns the field of cancer, and in particular to K-RAS oncogene-driven metabolic alterations which can simultaneously influence multiple metabolic pathways, thus generating unique cancer phenotypes.

The present invention relates to a pharmaceutical kit for the treatment of cancer, particularly to the combination of therapeutic treatment of glycolysis/PI3K and of glutamine metabolism targets, using anti-cancer therapeutic agents showing enhanced anti-cancer effects.

STATE OF THE ART

Lung cancer is the most common cancer worldwide followed by breast and colorectal cancer. Notoriously cancer is characterized by enhanced growth and altered metabolism. Once diagnosed, cancer is usually treated with one or combination of surgery, chemotherapy and radiotherapy.

Surgery generally is only effective for treating the earlier stages of cancer and in removing tumors located ad certain sites. However it cannot be used in treatment of tumors located in other areas inaccessible to surgeons, nor in the treatment of disseminated neoplastic condition. Therefore, to date chemotherapy is frequently considered as the first line of treatment for many types of cancers. Advances have been made by the identification of oncogenic mutations and the construction of drugs able to inhibit targets, such as EGFR and ALK/Ros1 . However, a significant number of cases is still in need of an appropriate therapy, either because patients do not respond to available drugs or they develop drug resistance often due to the mutation phenotype of cancer cells.

Oncogenic K-RAS mutations have been found in approximately 35% of lung adenocarcinomas and 45% of colorectal cancers and are associated with increased tumorigenicity and poor prognosis. Most of mutations K-Ras protein affect single amino acid substitutions in codon-12 (80%) or codon-13 (20%), which disrupt its GTPase activity in the active GTP-bound state, leading to hyperactive signaling by mitogen- activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K), hyper proliferation and altered metabolism. Moreover, direct targeting of K-Ras or key Ras effectors, including the Mapk pathway components Raf, Mek, and Erk, have produced unsuccessful results because of toxicities associated with their sustained inhibition and/or adaptive resistance mechanisms.

Recent targeted therapeutics studies are currently focusing on combinatorial treatments against either metabolic altered phenotype or downstream effectors of activated K-Ras, such as for example PI3K. Moreover, oncogene-driven metabolic alterations can simultaneously influence multiple metabolic pathways due to an intrinsic regulation of protein activity by metabolites, thus generating a unique cancer phenotype. Furthermore, the ability to activate a wide variety of alternative pathways provides dynamicity and robustness to cancer cells and it is crucial for environmental adaptation, as well as to confer acquired drug resistance.

Due to the severity and breadth of lung cancer disease, wherein the versatile metabolic phenotype of cancer cells induce uncontrolled proliferation and adaption to limited nutrient condition, there is a recognized need for the additional effective lung and/or oncogenic-driven K-RAS cancer therapies.

Object of the present invention is therefore the identification of combinatorial therapeutic treatment of cancer which induces tumor growth inhibition, involving metabolic profiling to provide tailor-made personalized cancer therapy.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical kit for use in the treatment of cancer. In particular the pharmaceutical kit of the present invention was seen surprisingly effective in the treatment of oncogenic-driven K-RAS tumors and/or tumors showing glycolytic and glutamine altered metabolism. Particularly, the invention provides the combination of compounds active on glycolysis/PI3K and glutamine metabolism targets using synergistic therapeutic agents, which show enhancement anti-cancer effect.

The invention therefore relates to a pharmaceutical kit for use in the treatment of cancer, said kit comprising a parenteral or oral dosage unit containing an inhibitor of glutaminase and a parenteral or oral dosage unit containing an inhibitor of PI3K or of glycolysis, and an instructions leaflet for use thereof.

As will be further described in the detailed description of the invention, the pharmaceutical kit of the present invention has the advantages of being specific for K- RAS-driven mutation lung cancer, but has also been advantageously used for other solid tumors showing glycolytic and glutamine altered metabolism, for example an epithelial derived cancer such as: melanoma, breast cancer, pancreatic cancer and ovarian cancer.

The invention further relates to a composition for use in the treatment of cancer comprising an inhibitor of glutaminase and an inhibitor of PI3K or of glycolysis, and pharmaceutically acceptable excipients.

The combination therapy, comprising an inhibitor of glutaminase and an inhibitor of PI3K/glycolysis, is particularly advantageous because K-RAS oncogenic-driven mutation tumors still in need of an appropriate therapy since either patients do not respond to available drugs or they develop drug resistance due to multiple metabolic pathways activation, in which both glucose and glutamine are essential nutrients able to provide dynamicity and robustness to cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Examples given for illustrative and non limiting purposes, and from the annexed Figures 1 -8, wherein:

Figure 1 : shows the schematic representation of the key actors of the TCA cycle in cancer metabolic rewiring (left) which generates enhanced tumor growth, and the same when inhibited according to the present invention (right) wherein the tumor growth is reduced.

Glucose and glutamine are required for enhanced cancer cell proliferation and their unique metabolic network configuration can affect both cancer phenotype and therapy response.

Figure 2: shows A) relative metabolite abundances involved in glycolysis and TCA cycle in A549 (si) and HCT1 16 (■) cells measured by mass spectrometry. B) Untargeted metabolic profiling of A549 lung and HCT 1 1 6 colon-rectal cancer cell lines. Hierarchical clustering heatmaps show significantly (p < 0.05) different intracellular metabolites by LC-MS and GC-MS (left panel). C) Mitochondrial respiration reflected by OCR levels was detected in A549 (■) and HCT1 16 (■) cancer cells under basal conditions or following the addition of oligomycin (O, 1 mM), the uncoupler FCCP (F, 0.5 pM) or the electron transport inhibitor Rotenone (R, 2 pM).(n=5).

Figure 3: A) Comparison of NVP-BKM120, CB-839, Idelalisib (-ID- FDA approved 50pM), NVP-BKM120 + CB-839 and ID+ CB-839 effectiveness treatments to inhibit cancer growth of A549 lung adenocarcinoma and HCT 1 1 6 colon-rectal cancer cells. Figure 4: PI3K/ALDO (NVP-BKM120) inhibitor and glutaminase inhibitor (CB839) cooperate to inhibit growth of A549 lung adenocarcinoma and HCT1 1 6 colon-rectal cancer cells. A) Aldolase activity and B) glutamate production of A549 lung adenocarcinoma and HCT1 1 6 colon-rectal cancer cells treated respectively with 1 pM NVP-BKM120 (A) and 50nM CB839 for 48h (B). C) A549 lung adenocarcinoma and HCT1 1 6 colon-rectal cancer cell lines were incubated with PI3K/ALDO inhibitor (NVP- BKM120 ), glutaminase inhibitor (CB839 D) or NVP-BKM120 + CB-839 (o) and CTR collected and counted at indicated time points. D) Morphological analysis of A549 lung adenocarcinoma and HCT 1 1 6 colon-rectal cancer cells treated with NVP-BKM120 + CB-839.

Figure 5: Metabolic profiling of A549 lung adenocarcinoma and HCT1 1 6 colon-rectal cancer cells under PI3K/ALDO (NVP-BKM120) inhibitor and glutaminase inhibitor (CB839). A and B) Untargeted metabolic analysis of A549 lung adenocarcinoma and HCT1 1 6 colon-rectal cancer cell lines. Hierarchical clustering heatmaps show significantly (p < 0.05) different intracellular metabolites in the four experimental conditions by LC-MS and GC-MS. Enriched metabolic pathways were ranked according to their FDR values calculated by the MetPa method implemented in MetaboAnalyst 2.0 software. The most significant pathways combined treatment compared to CTR (Figure 4A and 4B) were represented by both the bigger/red dots and by those dots with higher log p value. The pathway impact is calculated as the sum of the importance measures of the matched metabolites normalized by the sum of the importance measures of all metabolites in each pathway. Figure 6: shows evaluation of A549 K-Ras^G12S lung adenocarcinoma tumors after treatment. A) Changes in tumor size were measured by caliper in mice and when tumors reached a volume of 130-150mm³, mice were treated for 15 days with vehicle (CTR) or a combination of BKM120 (50 mg/kg in NMP/PEG300 (10/90, v/v) o.g. daily) plus CB-839 (200 mg/kg dissolved in 25% (w/v) hydroxypropyl-b-cyclodextrin in 10 mmol/L citrate (pH 2.0) o.g. twice daily) (Treat) left panel. -Middle panel- [¹⁸F]FDG uptake in A549 K-Ras^G12S tumors exposed to the combinatorial treatment compared to CTR. -Right panel- Lactate labeling evaluated using [U-¹³C6]Glc infused in A549 K- Ras^G12S xenograft mice exposed to the combinatorial treatment compared to CTR and analyzed by GC-MS. B) Post mortem analysis of tumor volume (left panel) and weight (middle panel) and evaluation of hepatotoxic effect of drugs by assessing aspartate transaminase (GOT) and alanine transaminase (GPT) (right panel). C) Untargeted metabolic analysis of A549 lung adenocarcinoma tumors. Hierarchical clustering heatmap show significantly (p < 0.05) different intracellular metabolites. Enriched metabolic pathways were ranked according to their FDR values calculated by the MetPa method implemented in MetaboAnalyst 2.0 software. The most significant pathways combined treatment compared to CTR were represented by both the bigger/red dots and by those dots with higher log p value. The pathway impact is calculated as the sum of the importance measures of the matched metabolites normalized by the sum of the importance measures of all metabolites in each pathway. D) Kaplan-Meier survival curves of A549 K-Ras^G12S tumor bearing mice raised from 14 to 21 days (p < 0.0001 ).

Figure 7: shows evaluation of HCT1 16 K-Ras^G13D colon-rectal tumors after treatment. A) Changes in tumor size were measured by caliper in mice and when tumors reached a volume of 130-150mm³, mice were treated for 15 days with vehicle (CTR) or a combination of BKM120 (50 mg/kg in NMP/PEG300 (10/90, v/v) o.g. daily) plus CB- 839 (200 mg/kg dissolved in 25% (w/v) hydroxypropyl-b-cyclodextrin in 10 mmol/L citrate (pH 2.0) o.g. twice daily) (Treat) Figure 4A left panel. - Figure 4A Middle panel- [¹⁸F]FDG uptake in HCT1 1 6 K-Ras^G13D tumors exposed to the combinatorial treatment compared to CTR. - Figure 4A Right panel- Lactate labeling evaluated using [U- ¹³C6]GIC infused in HCT1 1 6 K-Ras^G13D xenograft mice exposed to the combinatorial treatment compared to CTR and analyzed by GC-MS. B) Post mortem analysis of tumor volume (left panel) and weight (middle panel) and evaluation of hepatotoxic effect of drugs by assessing aspartate transaminase (GOT) and alanine transaminase (GPT)(right panel). C) Untargeted metabolic analysis of HCT 1 1 6 lung adenocarcinoma tumors. Hierarchical clustering heatmap show significantly (p < 0.05) different intracellular metabolites. Enriched metabolic pathways were ranked according to their FDR values calculated by the MetPa method implemented in MetaboAnalyst 2.0 software. The most significant pathways combined treatment compared to CTR were represented by both the bigger/red dots and by those dots with higher log p value. The pathway impact is calculated as the sum of the importance measures of the matched metabolites normalized by the sum of the importance measures of all metabolites in each pathway. D) Kaplan-Meier survival curves of HCT1 1 6 K-Ras^G13D tumor bearing mice raised from 14 to 21 days (p < 0.0001 ).

Figure 8: shows the combined treatment effect in several cancer cell lines. PI3K/ALDO (NVP-BKM120) inhibitor and glutaminase inhibitor (CB839) cooperate to inhibit growth of metastatic melanoma cancer cell lines (Colo 794 and A375), triple negative breast cancer cell (MDA-MB231 ) and ovarian cancer cell lines (OC-31 6 and IGROV-1 ).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a combination therapy for the treatment of cancer, particularly to combination of therapeutic treatment of glycolysis/PI3K and glutamine metabolism targets using synergistic therapeutic agents, which show enhancement anti-cancer effect.

Cancer is a complex disease in which genetic alterations meeting altered metabolic pathways affect therapy response of patients. Cancer metabolic rewiring represents one of the most robust hallmark of tumors, because metabolites per se determine an intrinsic regulation able to generate a unique phenotype.

Cancer cells use glucose and glutamine to sustain the enhanced and unrestricted growth of cancer cells.

Recently it has demonstrated that cancer cells are able to switch metabolism from glucose to glutamine and vice versa or increase glucose consumption as a result of a change in the in vivo microenvironment and/or when exposed to mono-treatment to avoid the drug effect. We observed a versatile role of glucose and glutamine in lung cancer A549 K-RasG12S cells and colon cancer HCT1 16 K-RasG13D cells essential to maintain aggressive cell proliferation (Figure 1 and 2).

The invention relates to a pharmaceutical kit for use in the treatment of cancer, said kit comprising a parenteral or oral dosage unit containing an inhibitor of glutaminase and a parenteral or oral dosage unit containing an inhibitor of PI3K or of glycolysis, and an instructions leaflet for use thereof.

The specific combination of glycolysis ad glutamine metabolism inhibitors surprisingly cooperate in vivo to induce tumor growth inhibition. It was seen that the treatment with only the glycolysis or the glutamine metabolism inhibitors did not allow to obtain the results obtained by the combination.

In a further aspect, the present invention relates to a pharmaceutical kit, wherein said inhibitor of glutaminase is CB 839.

CB-839 is a potent, selective, reversible and orally bioavailable inhibitor of human glutaminase of formula C26H24F3N7O3S and CAS No. : 1439399-58-2. The enzyme glutaminase, which converts glutamine to glutamate, has been identified as a critical choke point in the utilization of glutamine by cancer cells.

In a still further aspect, the present invention relates to a pharmaceutical kit, wherein said inhibitor of PI3K is BKM120.

BKM120 -or NVP-BKM120- (common name buparlisib) shows a strong synergistic inhibiting effect on the proliferation of various cancer cell types.

The aldolase inhibitor BKM120 (or NVP-BKM120) was chosen due to the fact that it is a 2-morpholino pyrimidine derivative pan-PI3K inhibitor, which inhibits all four class I PI3K isoforms and has CAS No. : 944396-07-0, on the contrary to the registered Idelalisib (Zydelig - Gilead Sciences, Inc. C22FI18FN70), a RI3Kd isoform inhibitor with more than 30-fold selectivity over other PI3Kisoforms, but which does not inhibit glycolysis and with significant lower effectiveness compared to NVP-BKM120 (Figure 3). In a preferred aspect, the pharmaceutical kit of the invention comprises a parenteral or oral dosage unit containing CB 839 and a parenteral or oral dosage unit containing BKM120 (Figure 3)

In a further aspect, the present invention relates to a pharmaceutical kit, wherein said cancer patient has a mutation in one or more of the genes chosen from the group consisting of k-RAS, PI3K or Aldolase.

In a further aspect, the present invention relates to a pharmaceutical kit, wherein said cancer patient is a patient having lung adenocarcinoma (LU-AD), colon adenocarcinoma (CO-AD), ovarian cancer, breast cancer, pancreatic cancer or melanoma (Figure 8).

In a further aspect, the present invention relates to a pharmaceutical kit, wherein said cancer therapy is a combined therapy with BKM120 and CB839 and wherein said cancer patient is a patient having lung adenocarcinoma (LU-AD) or colon adenocarcinoma (CO-AD).

In a further aspect, the present invention relates to a pharmaceutical kit, wherein :

- said parenteral or oral dosage unit containing CB 839 is in an amount in the range from 5 nM to 250 nM, for a patient administration in the range from 600 mg to 800 mg twice a day; and

- said parenteral or oral dosage unit containing BKM120 is in an amount in the range from 0.1 mM to 25 mM, for a patient administration per day in the range from 50 mg/day to 100 mg/day, preferably the patient administration is of 80 mg/day.

Throughout this document, the term "parenteral administration" encompasses all modes of administration, requiring injection, implantation or topical administration, except for the oral/intestinal route. Suitable examples of parenteral administration include intramuscular, intravenous, subcutaneous, intravaginal, transdermal and intranasal administration.

In a further aspect, the pharmaceutical kit further comprises a chemotherapeutic drug, wherein said chemotherapeutic drug is preferably bevacizumab for colon adenocarcinoma or pemetrexed for lung adenocarcinoma.

In a further aspect the invention relates to a composition for use in the treatment of cancer, wherein said inhibitor of glutaminase is CB 839.

In a still further aspect, said inhibitor of PI3K is BKM120.

In a preferred aspect the composition comprises CB 839 and BKM120.

Detailed analysis of metabolism showed that glucose and glutamine are required for enhanced cancer cell proliferation and their unique metabolic network configuration can affect both cancer phenotype and therapy response (Figure 1 ). Particularly lung cancer A549 K-RasG12S cells showed catabolize high amounts of glucose leading to lactate production on the contrary of HCT1 1 6 K-RasG13D colon-rectal cancer cells that along this pathway also include TCA cycle and amino acids biosynthesis, generating more vascularized tumors (Figure 2). Moreover, both cancer cell lines showed glutamine as the major source of TCA cycle, in order to sustain anabolic processes (Figure 2).

This detailed metabolic signature, in which both glucose and glutamine are essential nutrients able to provide dynamicity and robustness to cancer cells, identified a combined therapeutic treatment of glycolysis/PI3K (Elvire Pons-Tostivint, 2017; Fleudel et al., 2017, Flu et al., 201 6), drug selected pan-class I PI3K inhibitor NVP-BKM120, and glutamine metabolism, drug selected CB839, targets (Figure 4A, B, C and D). Combinatorial treatments with BKM120 and CB839 showed a remarkable synergic effect both on cancer cell proliferation and on cancer metabolism of both human cancer cell lines (Figure 4C and D; 5A and B).

Surprisingly, the synergic antitumor effect of BKM120 plus CB839 combined treatment was also seen in in vivo A549 K-RasG12S lung cancer cells and HCT1 16 K-RasG13D colon cancer cells xenografts (Figure 6 and 7). We observed that significant tumor reduction and prolonged therapeutic responses in mice correlated with reduced 18F- FDG and uptake seen in both A549 K-RasG12S lung and HCT1 1 6 K-RasG13D colon xenografts (Figure 6A and 7A). Noteworthy, the combination of BKM120 and CB839 metabolic inhibitors in A549 K-RasG12S and HCT1 1 6 K-RasG13D xenografts mice induced severe energetic stress and metabolic crisis, which resulted in significant tumor growth regression (Figure 6B and 7B) without toxicities association (Figure 6B and 7B right panel).

In addition, combinatorial treatments with NVP-BKM120 and CB839 metabolic drugs blocking both metabolic routes was essential to prevent the tumor to adapt alternative metabolic pathways and therefore, these drugs could be used in the first-line treatment of patients with aggressive tumors correlated with poor prognosis and high adaptive resistance mechanisms as oncogenic K-Ras tumors.

Furthermore, in wfro and in vivo detailed metabolic signature of A549-K-RasG12S and FICT1 1 6-K-RasG13D was seen to be an effective combinatorial metabolic therapeutic strategy. Specifically, we demonstrated that: 1 ) oncogenic K-Ras in our biological models drove similar metabolic alterations stimulating both glucose and glutamine metabolism; 2) detailed metabolic profiling identified both PI3K inhibitor (BKM120) and glutaminase inhibitor (CB839) as effective tumor growth inhibitors and alternative pathway activation, such as amino sugar and proline metabolism to avoid drugs treatments effect.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

Example 1.

Glucose and Glutamine addiction in K-Ras human lung and colon cancer cells

To study an effective therapeutic strategy for lung and colon tumors able to tear down the versatile metabolic phenotype due to the environmental context (Davidson et al., 201 6; Gaglio et al., 201 6), we selected two biological cellular models carrying K-Ras, one of the most common mutations with poor prognosis. Specifically, we used the K- RasG1 2S-driven mutation lung cancer cells A549 and the K-RasG13D driven mutation colorectal cancer (CRC) cell line HCT1 1 6. To perform a detailed cellular typing, we performed basic metabolic analyses showing a similar metabolic phenotype behavior in A549 and HCT 1 16 cancer cell lines. Although oncogenic human K-Ras cancer cells showed a similar basic metabolic phenotype, further deepened analysis of the relative abundance of intracellular metabolites involved in glycolysis and TCA cycle metabolism suggested a preferential glucose oxidation via lactate in A549 lung cancer cells, as compared to HCT1 1 6 colon cancer cells (Figure 2). These results were confirmed by targeted metabolomic analysis using uniformly labeled glucose ([U-13C6]Glc) and glutamine ([U-13C5]Gln) via gas and liquid chromatography/mass spectrometry (GC/MS and LC/MS) (Data not shown) and by oxygen consumption rate (OCR) showing higher levels of basal oxidative phosphorylation (OXPHOS, indicated by OCR) in HCT1 1 6 as compared to A549 lung cancer cells (Figure 2C). Thus, although oncogenic K-Ras seems to drive as a general rule the nutrient-dependent phenotype, A549 lung cancer cell and H CT 1 1 6 colon cancer cells used nutrients in a slightly different way. In fact, A549 lung K-Ras cancer cells showed a perfect decoupling of nutrients in which glucose was converted via lactate and glutamine to maintain TCA cycle metabolite pool and sustain anabolic processes (Figure 2); on the other hand, HCT1 1 6 colon K-Ras cancer cells, which notoriously generate more vascularized tumors, used glucose and glutamine in a complementary and mutually reinforcing manner to sustain enhanced growth (Figure 2).

Taken together, these results suggested that oncogenic K-Ras mutations greatly alter glucose and glutamine fate to support aggressive proliferation, but mutations affecting different codons and the presence of a mutation (FI1047R) in the catalytic domain of PIK3CA gene in HCT 1 1 6 cells could lead a slight singular aptitude to use the same nutrients.

Example 2.

In Vitro combined metabolic inhibitors reduce enhanced cancer cell growth

The complexity of finding effective agents to inhibit K-Ras could be hampered by the ability of tumor cells to carry out a metabolic switching from glucose to glutamine and vice versa in accordance with the environmental context (Davidson et al., 2016). Therefore, based on: 1 ) limitations provided by previous published data suggesting that direct targeting K-Ras alone does not provide successful results for treatment (Kilgoz et al., 201 6); 2) results described above; 3) considering that Ras-PI3K signaling is involved in nutrients uptake (Lien et al., 201 6) and aldolase activity regulation (Hu et al., 201 6), we examined the impact of NVP-BKM120 (PI3K inhibitor) and CB839 (glutaminase inhibitor), in our cancer cell lines models (Figure 4). Following dose- response curves for BKM120 and CB839 to select appropriate concentrations, we examined their effect on aldolase activity and glutamate production in our models (Figure 4A and 4B). Consistently, BKM120 inhibitor induced a significant reduction of aldolase activity in all cellular models after treatment for 48h (A549, 34.0% and HCT 1 1 6, 36.8% (Figure 4A). Likewise, CB839 treatment for 48h produced a significant decrease of glutamate levels (A549, 71 .0% and HCT 1 1 6, 60.3% (Figure 4B). To further test the efficacy of treatments, we performed prolonged proliferation curves under either BKM120 or CB839 in single treatments, or in combination, BKM120 plus CB839 (Figure 4C and D). Specifically, A549 lung cancer cells showed a larger decrease of cell number when grown under prolonged single treatment with CB839 than under BKM120 (Figure 4C, left panel). Instead, we observed an increased reduction of proliferation in HCT1 1 6 grown under prolonged single treatment with BKM120, rather than with CB839, which showed not significant changes as compared to CTR (Figure 4C, right panel). Moreover, a dramatic reduction of cell proliferation was observed in all cellular models under combinatorial BKM120 plus CB839 treatment, as compared to CTR (Figure 4C and 4D), proposing a synergic effect of the combined treatment strategy to reduce cell viability. Furthermore, the amazing effect of BKM120 plus CB839 on cell proliferation was even more remarkable in untargeted metabolic profiling of A549 performed by metabolomics-mass-spec analysis displaying a whole metabolites reduction, as compared to single treatments (BKM120 or CB839) and CTR, as determined by ANOVA comparison of the four groups. The merge statistical analysis of GC-MS and LC-MS datasets in A549 revealed similar metabolic signatures between CB839 alone and combinatorial BKM120 plus CB839, as opposed to CTR and BKM120 alone (Figure 5A, left upper panel). In particular, the significant higher level of metabolites (such as G6P, F6P, G1 P, 6P-GL, R5P, X5P, N-A-Gln-1 P and N-A-Gln-6P, Figure 5A, left panel), involved in first step of glycolysis, pentose phosphate pathway and amino sugar metabolism (Figure 5A, left lower panel), suggested an attempt to activate alternative glucose-dependent pathways in stressed A549 under combined treatment, as compared to CTR. Stress was further reinforced due to decreased levels of Gin, Glu, 5-Oxo and All-Cys involved in glutathione metabolism (Figure 5A, left panel). Consistent with the drop of aggressive proliferation and the results described above, we observed a significant decrease of metabolites involved in nucleotides metabolism and in the TCA cycle (Figure 5A). A less relevant effect of combined treatment was observed in HCT 1 1 6 cancer cells metabolic profiling (Figure 5B). Similar to A549, the merge statistical analysis of GC-MS and LC-MS datasets showed a significant increase of metabolites involved in glycolysis and decreased levels of metabolites involved in TCA cycle metabolism under combined treatment and CB839 alone compared to BKM120 single treatment and CTR (Figure 5B). In addition, we observed that FICT 1 1 6 treated with combinatorial drugs showed a remarkable increase of metabolites, such as: Succ-S-A (Ala, Asp and Glu metabolism) able to generate succinate which could enter the TCA cycle, 3-M-Pyr involved in tryptophan metabolism, Acac involved in keton bodies metabolism and able to promote Mek-Erk signaling (Kang et al., 2015), and 3-S-Ala involved in taurine and hypotaurine metabolism (Figure 5B, upper and lower panels). The activation of the latter unusual metabolic pathway (taurine and hypotaurine metabolism) with antioxidant action (Fluang et al., 2016) could probably encourage to protect FHCT1 1 6 cancer cells from significant increased level of ROS generated by combined pharmacological treatment.

Example 3.

BKM120 and CB839 combinatorial treatments inhibit in vivo K-RasG12S and lung and

K-RasG13D colon tumors To finally demonstrate the efficacy of the BKM120 plus CB839 combined therapy in K- Ras tumor growth inhibition, we evaluated drug treatments response in a mouse model of A549 K-RasG12S lung cancer cells and HCT1 16 K-RasG13D colon cancer cells xenografts (Figure 6 and 7). In accordance with published data, we administered mice xenografts with BKM120 (50 mg/kg) (Alagesan et al., 2015; Alikhani et al., 2013) and CB839 (200 mg/kg) (Davidson et al., 201 6; Gross et al., 2014) in combination. When tumors reached a volume of 130-150mm3, mice were treated for 15 days with vehicle (CTR) or a combination of BKM120 plus CB839 (T reat). Mice were monitored for tumor growth using caliper and glucose metabolism by [¹⁸F]FDG-PET scans performed before receiving the therapy (pre-treatment scans), during the therapy (7 days) and again following 14 days of treatment with either CTR or combined therapy (post treatment scans) (Figure 6 and 7). A remarkable tumor growth inhibition was observed during the entire therapeutic window, as determined by significant decreased tumor volume (Figure 6A and 6B) and confirmed by significant lower levels of post mortem tumor weight measured in A549 K-RasG12S tumors treated with BKM120 plus CB839 compared to CTR. These results were further confirmed by FDG-PET scan revealing significant decreased level of 18F-FDG uptake in A549 K-RasG12S tumors exposed to the combinatorial treatment compared to CTR (Figure 6A, middle panel). In addition, considering the valuable role of LDFI (lactate dehydrogenase) as a marker in patients with cancer, [U-¹³C6]Glc was infused in A549 K-Ras^G12S xenograft mice to evaluate lactate labeling. Consistent with ¹⁸F-FDG uptake, we found decreased lactate labeling in A549 K-Ras^G12S tumors treated with BKM120 plus CB839, as compared to vehicle (Figure 6A, right panel). Furthermore, post mortem untargeted metabolic profiling identified a significant decreased relative abundance of metabolites involved in amino acids metabolism (Lys metabolism, Tyr metabolism, Ala-Asp-Glu metabolism, Pyr metabolism), except for Ala and Cys, that showed higher levels in A549 K-RasG12S Treat tumors, compared to vehicle (Figure 6D). Moreover, applying Volcano Plot algorithm with a cutoff of P < 0.005 and fold change (FC) > 2, we identified four remarkable prognostic biomarkers (Figure 6D, right lower panel). In fact, significantly decreased levels of Lac and Ala reflect both the metabolic stroke-down and tumor growth inhibition of A549 K-RasG12S Treat tumors, in contrast to the increased levels of Cys and Ala that reflect a redox status stress induced by treatment and the attempt to activate a mechanism of glucose metabolic reprogramming (Figure 6D, right lower panel). To further substantiate the value of the therapy, we tested the hepatotoxicity of drugs by assessing aspartate transaminase (GOT) and alanine transaminase (GPT) activity, notoriously used as indicators of healthy and diseased states in patients. In addition to lack of significant weight changes between Treat and CTR, we did not find significant differences in transaminases activity between A549 K-RasG12S Treat and CTR (Figure 6C).

Similar to A549 K-RasG12S Treat, HCT1 1 6 K-RasG13D colon tumors showed significant regression of tumor volume during the therapeutic window (Figure 7A, left panel) and post mortem tumor growth (Figure 7B), as well as significant decreased levels of 18F-FDG uptake (Figure 7A, middle panel) and [U-¹³C6]Glc lactate labeling under combinatorial treatment, as compared to vehicle. Nevertheless, as we found a significant effect of the combinatorial treatments in HCT 1 1 6 K-Ras^G13D xenograft mice, we confirmed in vivo the lower efficacy of BKM120 plus CB839 therapy in HCT1 16 K- Ras^G13D tumors, as compared to A549 K-Ras^G12S (Figure 6 and 7). The similar effect has been confirmed by post mortem untargeted metabolic profiling revealing a significant decreased relative abundance of metabolites involved in amino acids and nucleotides metabolism (Figure 7D, left and right panels) observed in T reat FICT 1 1 6 K- Ras^G13D tumors as compared to CTR. It was also interesting to note that the inhibitory effect on nucleotides was confirmed by Volcano Plot algorithm with a cutoff of P < 0.005 and fold change (FC) > 2 that identified significant decreased levels of metabolites, such as IMP, UMP, Asp. In addition, by Volcano Plot we observed significant decreased levels of ATP and ADP metabolites in Treat HCT1 16 K-Ras^G13D tumors, thus confirming the energetic status stress induced by the combinatorial treatment (Figure 7). Finally, we also tested hepatotoxicity of drugs by GOT and GPT activity and we observed a slight hepatic stress, but not toxic effect in Treat HOT 1 1 6 K-RasG13D tumors as compared to CTR (Figure 1C).

Moreover, groups of A549 K-Ras^G12S and HCT1 1 6 K-Ras^G13D tumors bearing mice were treated with the combination of BKM120 and CB839 or vehicle to monitor survival. The combination of the two drugs significantly increased survival of both tumor models (Figure 6D and 7D): in detail median survival of HCT1 16 K-Ras^G13D tumor bearing mice raised from 14 to 21 days (p < 0.0001 ) and that of A549 K-Ras^G12S tumor bearing mice from 44.5 to 62 days (p < 0.05). This data confirmed the lower efficacy of the combined treatment on HCT 116 K-Ras^G13D tumor than on A549 K-Ras^G12S and consequently the capability of HCT1 16 K-Ras^G13D tumor to use other sources of energy (Figure 6D and 7D).

Finally, to further tested the efficacy of combined treatments we tested in various kinds of cancer cell lines: Colo794 and A375 melanoma metastatic cancer cells, MDA-MB231 breast cancer cell, OC-316 and IGROV-1 ovarian cancer cells, observing a significant cell proliferation reduction of cancer cells grown under combined treatments as compared to CTR (Figure 8).

From the above description and the above-noted examples, the advantage attained by the compositions and kits described and obtained according to the present invention are apparent.

The present invention therefore resolves the above-lamented problem of drug resistant cancer due to oncogene-driven metabolic alterations with reference to the mentioned prior art, offering at the same time numerous other advantages, including allowing the development of diagnostic methods capable of predicting the therapeutic response so to refine not only the diagnostics but above all direct the best therapeutic choice.

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Claims

1 . A pharmaceutical kit for use in the treatment of cancer, said kit comprising a parenteral or oral dosage unit containing an inhibitor of glutaminase and a parenteral or oral dosage unit containing an inhibitor of PI3K, and an instructions leaflet for use thereof;

- wherein said inhibitor of glutaminase is CB 839, and

- wherein said inhibitor of PI3K is BKM120.

2. The pharmaceutical kit according to claim 1 , comprising a parenteral or oral dosage unit containing CB 839 and a parenteral or oral dosage unit containing BKM120.

3. The pharmaceutical kit according to anyone of claims 1 or 2, wherein said cancer patient has a mutation in one or more of the genes chosen from the group consisting of k-RAS, PI3K or Aldolase.

4. The pharmaceutical kit according to anyone of claims 1 to 3, wherein said cancer patient is a patient having lung adenocarcinoma (LU-AD), colon adenocarcinoma (CO- AD), ovarian cancer, breast cancer, pancreatic cancer or melanoma.

5. The pharmaceutical kit according to anyone of claims 1 to 4, wherein said cancer therapy is a combined therapy with BKM120 and CB839 and wherein said cancer patient is a patient having lung adenocarcinoma (LU-AD) or colon adenocarcinoma (CO-AD).

6. The pharmaceutical kit according to anyone of claims 1 to 5, wherein said parenteral or oral dosage unit containing CB 839 is in an amount in the range from 5 nM to 250 nM and said parenteral or oral dosage unit containing BKM120 is in an amount in the range from 0.1 mM to 25 mM.

7. The pharmaceutical kit according to anyone of claims 1 to 6, further comprises a chemotherapeutic drug, wherein said chemotherapeutic drug is preferably bevacizumab for colon adenocarcinoma or pemetrexed for lung adenocarcinoma.

8. A composition for use in the treatment of cancer comprising an inhibitor of glutaminase and an inhibitor of PI3K, and pharmaceutically acceptable excipients,

- wherein said inhibitor of glutaminase is CB 839; and

- wherein said inhibitor of PI3K is BKM120.