NEW STRATEGY FOR TREATING PANCREATIC CANCER

Abstract

The present invention relates to the treatment of pancreatic cancer. In this study, the results of the inventors led them to highlight the non-explored but relevant pathway in the pancreatic cancer field, the Fatty Acid Oxidation (FAO) pathway. Interestingly, they found that the mitochondrial respiration of PDAC cells depends mostly on this pathway. Thus they hypothesized that inhibition of FAO could be an effective therapeutic strategy against PDAC. 10 Their data support the hypothesis that this metabolic pathway plays a crucial role in PDAC, as it has been reported in other types of cancer. Thus, the invention relates to an inhibitor of fatty acid oxidation (FAO) for use in the treatment of pancreatic cancer in a patient in need thereof.

Claims

1. A method of treating pancreatic cancer in a patient in need thereof, comprising, administering to the patient a therapeutically effective amount of an inhibitor of fatty acid oxidation (FAO).

2. The method of claim 1, wherein the inhibitor of FAO is administered simultaneously, separately or sequentially with a therapeutic compound used to treat pancreatic cancer.

3. The method according to claim 1 wherein the patient has a high OXPHOS profile.

4. The method according to according to claim 1, wherein the inhibitor of FAO is an inhibitor of CPT1, CACT, CPT2 or 3-KAT.

5. The method according to claim 4 wherein the inhibitor of FAO is an inhibitor of CPT1.

6. The method according to claim 1 wherein the inhibitor of CPT1 is Etomoxir, Perhexiline, Oxfenicine, Methyl palmoxirate, S-15176, Metoprolol, or amiodarone.

7. The method according to claim 2 wherein the therapeutic compound used to treat pancreatic cancer is gemcitabine, 5-fluorouracil (5-FU), Capecitabine, oxiplatin, cisplatin, irinotecan, or Nab-Paclitaxel, or a combination of folinic acid, 5-FU, irinotecan and oxaliplatin.

8. A therapeutic composition comprising an inhibitor of fatty acid oxidation (FAO) formulated for use in the treatment of pancreatic cancer in a patient in need thereof.

9. (canceled)

10. An in vitro method for predicting FAO inhibitor response of a patient in need thereof and treating the patient, comprising: i) determining, in a sample obtained from the patient, an expression level of a CPT1C isoform; ii) determining that the expression level determined at step i) is lower than a reference value, and iii) treating the patient determined to have an expression level that is lower than the reference value with an FAO inhibitor.

11. An in vitro method for monitoring FAO inhibitor treatment in a subject in need thereof and then treating the subject, comprising the steps of i) determining, in a sample obtained from said subject after treating the subject with the FAO inhibitor, an expression level of CPT1C isoform; ii) determining that the expression level determined at step i) is lower than a reference value, and iii) treating the subject with the FAO inhibitor.

12. The in vitro method according to claim 11, wherein the reference value is the expression level of CPT1C determined in samples obtained from the subject before FAO inhibitor treatment.

13. A method for treating pancreatic cancer in a patient in need thereof comprising i) determining, in a sample obtained from the patient, an expression level of a CPT1C isoform; and ii) administering a therapeutically effective amount of an FAO inhibitor to the patient determined to have an expression level of CPT1C lower than a reference value.

Description

FIGURES

[0226] FIG. 1. PDAC cells exhibit different sensitivity to pharmacological FAO inhibitors.

[0227] A. Relative cell viability of PDAC cells treated with Etomoxir (62.5 μM) for 72 h, compared to non-treated cells. The dotted line represents the mean of the treated cells (0.75). B. Relative cell viability of PDAC cells treated with Perhexiline (7 μM) for 72 h, compared to non-treated cells. The dotted line represents the mean of the treated cells (0.72).

[0228] C. Relative cell viability of PDAC cells treated with Trimetazidine (62.5 μM) for 72 h, compared to non-treated cells.

[0229] Data are presented as the mean of triplicates±SEM, and are representative of three independent experiments.

[0230] Each number below the bars corresponds to the number of the anonymized name of patients (for example 84 corresponds to patient PDAC084T as used in FIGS. 3 to 8). Viability is measured by Crystal violet assay.

[0231] FIG. 2. The OXPHOS rates correlate with sensitivity to FAO inhibition in PDAC cells.

[0232] Scatterplot of the basal Oxygen Consumption Rate (OCR) versus relative mean cell viability after 72 hours-treatment with Etomoxir (62.5 μM) or Perhexiline (7 μM) in PDAC cells shown in FIGS. 2A and 2B, respectively.

[0233] The OCR is significantly correlated with the relative cell viability for Perhexiline treatment, with a Pearson correlation coefficient (r) of −0.8186 (negative correlation).

[0234] No significant negative correlation is observed between Etomoxir treatment and relative cell viability (r=−0.5501).

[0235] Pearson correlation and two-tailed t-test were used to generate the correlation coefficient and associated P value.

[0236] FIG. 3. The FAO inhibitor Perhexiline in combination with Gemcitabine is synergistic specifically in high OXPHOS cells

[0237] Dose-response assays were performed in four selected PDAC cells: PDAC084T (FIG. 3A), PDAC012T (FIG. 3B), PDAC032T (FIG. 3C), and PDAC022T (FIG. 3D), according to the sensitivities to the FAO inhibitors. Cells were treated for 72 h with increasing doses of Gemcitabine, with Perhexiline (5 μM) or with the combination. Cell viability was measured by Crystal violet viability assay. Figures show the cell viability (%) of each treatment: Perhexiline (5 μM), Gemcitabine at one dose (1 nM), or the combination.

[0238] To determine the synergistic effect in the combination treatments, we calculated the predicted values by multiplying the cell viability in the Gemcitabine and Perhexiline groups. Then, when the observed value for the combination is less than the predicted value, we consider it as a synergistic effect. Data are presented as mean±SEM of triplicates. P values from Student's t-test.

[0239] FIG. 4. Impact of FAO inhibition in subcutaneous PDAC xenografts. Tumor growth progression in immunodeficient mice treated during one month with Gemcitabine (120 mg/kg IP twice a week), Perhexiline (5 mg/kg IP every other day), Gemcitabine plus Perhexiline (with the same indications), and vehicle.

[0240] A. Perhexiline not only enhances the antitumoral effect of Gemcitabine but also results in complete tumoral regression in a High OXPHOS PDAC xenograft (PDAC084T). B. The combination treatment shows a better efficiency than Gemcitabine alone in the intermediate responder xenograft (PDAC012T). C, D. No effect was seen upon combination treatment compared to Gemcitabine alone in the low responder xenografts (PDAC032T, PDAC022T). Data are presented as mean±SEM. ***P<0.001, **P<0.01, *P<0.05 from Two-way ANOVA test, compared to Gemcitabine treatment alone.

[0241] FIG. 5. The FAO inhibitor Perhexiline enhances the antitumor activity of chemotherapy by inducing apoptosis in vitro. Cell survival was measured using an AnnexinV/PI apoptosis assay and viable cells (AnnexinV.sup.−/PI.sup.− cells) were quantified. A. Quantification showing the cell survival (%) of four cell lines treated with Perhexiline (7 μM) for 72 h compared with the controls. Data are presented as mean±SEM of three independent experiments performed in duplicates. B. Quantification showing the viability of PDAC084T cells treated for 24 h with Gemcitabine (1 μm) Perhexiline (7 μm) or the combination. Data are presented as mean±SEM of one experiment performed in duplicates. P values from Student's t-test.

[0242] FIG. 6. The FAO inhibitor Perhexiline enhances the antitumor activity of chemotherapy by inducing apoptosis in vivo. Quantification of cleaved-Caspase 3 (A), Ki67 (B), and Masson's Trichrome staining (C), in tumor sections from PDAC084T xenografts treated with Gemcitabine, or the combination Perhexiline plus Gemcitabine.

[0243] FIG. 7. The FAO inhibitor Perhexiline enhances the antitumor activity of chemotherapy by inducing energetic stress in vitro. A. Mitochondrial respiration (Oxygen Consumption Rate, OCR) was measured on Seahorse oxygraph in PDAC084T cells after 6 h of Perhexiline, Gemcitabine, or combination treatment. B. The basal and maximal respiration, ATP production by mitochondria, and spare respiratory capacity were calculated.

[0244] FIG. 8. CPT1C isoform as a key actor in the response to FAO targeting in PDAC. A. The high responder PDAC084T cells exhibit significantly lower mRNA levels of the CPT1A and CPT1C isoforms compared to the intermediate and low responder PDAC cells (PDAC012T and PDAC022T, respectively). The gene mRNA levels of CPT1 (A, B, C) and CPT2 isoforms were measured by RT-qPCR in three PDAC cells used in in vivo experiments. Data are presented as mean±SEM of three independent experiments performed in duplicates. P values from Student's t-test. B. The CPT1C mRNA levels correlate with response to FAO targeting with Perhexiline. The plot shows the mean of CPT1C mRNA levels in the previous Figure, compared with the relative cell viability with Perhexiline at 7 (see FIG. 1B).

[0245]

TABLE-US-00001 TABLE 1 Basal Oxygen Consumption Rates (OCR) in 10 primary PDAC cells from Patient-Derived Xenografts. The OCR (pmol/min/10 000 cells) is presented in decreasing order. Patient # OCR 84 102.29 27 60.96 12 46.13 21 44.54 03 44.4 82 31.73 01 24.5 22 23.51 32 23.27 85 15.52

EXAMPLE

[0246] Material & Methods

[0247] Cell Culture

[0248] We used 21 PDAC primary cells obtained from Patient-Derived Xenografts (PDX) from the PaCaOmics cohort [53], in which patients were included under the Paoli-Calmettes Institute clinical trial number 2011-A01439-32. Consent forms of informed patients were collected and registered in a central database. PDAC cells were obtained and maintained in serum-free ductal media (SFDM) as described [54], and incubated at 37° C. in a 5% CO2 incubator. Cell lines were monthly tested for Mycoplasma contamination and found to be negative.

[0249] Real-Time Metabolic Analysis (XF Mito Fuel Flex Test)

[0250] We assessed the mitochondrial capacities of the 21 PDAC primary cells to oxidize the three main energetic sources: Glucose, Glutamine, and Fatty Acids.

[0251] Measurements were performed using the Seahorse Bioscience XFe24 Extracellular Flux Analyzer (Agilent). Sixteen hours before the assay, cells at exponential growth were seeded into Seahorse 24-well plates and cultured at 37° C. with 5% CO2. The number of seeded cells was optimized to ensure 70-80% confluence the day of analysis.

[0252] The Seahorse XF Mito Fuel Flex Test Kit was used to determine the Dependency of cells to oxidize the three energetic fuels. Culture medium was replaced by OXPHOS assay medium and the plate was pre-incubated for 1 h at 37° C. in a non-CO2 incubator. Inhibitors of mitochondrial pyruvate carrier (UK5099 2 μM), glutaminase (BPTES 3 μM), and CPT1a (Etomoxir 4 μM) were used to inhibit glycolysis, glutaminolysis, and FAO pathways, respectively. The rate of oxidation of each fuel was determined by measuring the oxygen consumption rate (OCR) by mitochondria in the presence or absence of fuel pathway inhibitors according to the manufacturers' instructions.

[0253] Dose-Response Viability Experiments In Vitro (Chemograms)

[0254] We tested the sensitivity of PDAC cells to three different drugs targeting the FAO pathway: Etomoxir, Perhexiline, and Trimetazidine (all provided by Sigma-Aldrich, Saint-Quentin Fallavier, France), and to the standard chemotherapy Gemcitabine (Gemzar, Eli Lilly & Co).

[0255] Cells were seeded in 96-well plates (5,000 cells per well) and 24 h later, the medium was supplemented with increasing concentrations of the drugs in triplicates. For the combination treatment, we used Perhexiline at 5 μM with increasing doses of Gemcitabine. Cell viability was determined 72 h later by Crystal violet viability assay, which is independent from cell metabolism. Briefly, cells were fixed in Glutaraldehyde 1%, washed twice with PBS, stained with crystal violet 0.1% for 10 min, and then washed three times with PBS. Crystals were solubilized in SDS 1%, and absorbance was measured at 600 nm using an Epoch-Biotek spectrophotometer.

[0256] To determine the synergistic effect in the combination treatments, we calculated the predicted values by multiplying the cell viability in the Gemcitabine and Perhexiline groups. Then, when the observed value is less than the predicted value, we consider it as a synergistic effect.

[0257] In Vivo Experiments

[0258] We assessed the FAO inhibition impact in vivo using the drug Perhexiline in combination with Gemcitabine. We performed subcutaneous xenografts in immunodeficient mice using four different primary PDAC cells. Recipient mice were 6-week-old female Swiss nude mice Crl:Nu(lco)-Foxn1nu purchased from Charles River, France. To obtain the xenografts, subcutaneous tumors from initial mouse donors were removed and finely minced with a scalpel. Then, 150 mg of tumor's pieces were mixed with 50 μl of Matrigel and implanted with a trocar (10 Gauge) in the subcutaneous space of anesthetized mice. Tumor volume was measured twice per week using a digital caliper and using the formula V=lenght×(width)/2. When tumor volume reached 200 mm.sup.3, mice were randomly assigned in a treatment scheme:

[0259] a) Gemcitabine: 120 mg/kg twice a week

[0260] b) Perhexiline: 5 mg/kg every other day

[0261] c) Combination treatment: Gemcitabine plus Perhexiline with the same described indications

[0262] d) Vehicle: PBS in the case of Gemcitabine treatment controls and 3% DMSO in PBS for combination treatment controls.

[0263] Mice were treated by intraperitoneal injection during one month, and mice in which tumor volume reached 1.5 cm.sup.3 during this period were ethically sacrificed and tumors removed.

[0264] All mice were kept under specific pathogen-free conditions and according to the current European regulation; the experimental protocol was approved by the Institutional Animal Care and Use Committee (#16711).

[0265] Flow Cytometry

[0266] Cells were seeded in 6-well plates in duplicates (150 000 cells per well) and the day after, the corresponding treatment was administered. We treated four PDAC cells with 7 μM of Perhexiline for 72 h (FIG. 5A), and in combination with 1 μM of Gemcitabine for 24 h for the PDAC084T cells. After the treatment period, cells were detached with Accutase, and resuspended in Annexin V-binding buffer (Biolegend). Cell death was determined using the Annexin V and Propidium Iodide (PI) staining (Biolegend), for apoptosis and necrosis, respectively. Ten thousand events per sample were acquired in a MAC SQuant-VYB (Miltenyi Biotec) and data analysis was done with the FlowJo software.

[0267] Ex Vivo Analysis (Immunohistochemistry and Histochemistry)

[0268] Tumor-bearing mice were sacrificed under treatment (middle-point of one-month treatment), and tumors fixed in 4% Paraformaldehyde, dehydrated and embedded in paraffin. Serial 4 μm sections were cut and stained with the Masson's Trichrome staining for collagen fibers detection, cleaved-Caspase 3 antibody (apoptosis), and ki67 (proliferation). Slides were scanned and images were captured using Calopix digital software. Quantification of stained areas were done with Fiji ImageJ.

[0269] Real-Time Metabolic Analysis (XF Cell Mito Stress Test)

[0270] We tested the impact of Gemcitabine, Gemcitabine, and the combination treatment (6 hours) on the mitochondrial respiration in PDAC084T cells. Measurements were performed using the Seahorse Bioscience XFe24 Extracellular Flux Analyzer (Agilent). Sixteen hours before the assay, cells at exponential growth were seeded into Seahorse 24-well plates and cultured at 37° C. with 5% CO.sub.2. Six hours before the assay, the media was replaced with vehicle DMSO (0.01%), Gemcitabine (1 μM), Perhexiline (10 μM), or the combination of the two drugs. After 6 h treatment, the media was replaced with OXPHOS assay medium (DMEM without phenol red [Sigma-Aldrich reference D5030], 143 mM NaCl, 2 mM glutamine, 1 mM sodium pyruvate and 10 mM glucose, pH 7.4) and the plate was pre-incubated for 1 h at 37° C. in a non-CO.sub.2 incubator. OCR was measured under basal conditions, and then after sequential injections of Oligomycin at 1 μM, carbonyl cyanide-p-trifluoromethox-yphenyl-hydrazon (FCCP, the concentration was optimized for each cell line), and 0.5 μM of Rotenone plus Antimycin A. Oligomycin is a respiratory Complex V inhibitor that allows to calculate ATP production by mitochondria, and FCCP is an uncoupling agent allowing the determination of the maximal respiration and the spare capacity. Finally, Rotenonte/Antimycin A are Complex I and III inhibitors, respectively that are injected to stop mitochondrial respiration enabling the calculation of the background (i.e., non-mitochondrial respiration driven by processes outside the mitochondria).

[0271] Gene Set Enrichment and RT-qPCR Analysis

[0272] We analyzed the RNA-sequencing data (RNA-seq) from five PDAC cells (#84, #82, #27, #12, #21) of the high and intermediate responder group to FAO targeting with Perhexiline; and three PDAC cells (#03, #32, #85) from the low responder group. Then, we used the KEGG database to determine the significantly upregulated or downregulated pathways between the two groups taking as reference the high/intermediate responder group.

[0273] Secondly, we measured the mRNA levels of the CPT1 (A, B, and C) and CPT2 isoforms by RT-qPCR in the PDAC cells used in the in vivo experiments (#84, #12, #22, #32). Briefly, cells were seed in 10 cm.sup.2 petri dishes (one million cells per dish), and the day after, cells were subjected to treatments: vehicle DMSO (0.01%), Perhexiline 5 μM, Gemcitabine 1 μM, and the combination. 24 hours later, cells were detached and RNA was extracted using the RNeasy Mini kit (Qiagen) according to manufacturer's instruction. Next, RNA samples were subjected to reverse-transcription (Takara), and quantitative real-time PCR was performed in duplicates.

[0274] Results

[0275] Mitochondrial Respiration of Primary PDAC Cells Shows Dependency Towards Fatty Acids

[0276] Besides glucose, fatty acids and glutamine are main nutrients that feed the TCA cycle for cellular respiration producing ATP. Therefore, we addressed the dependency of mitochondrial respiration towards these three energetic fuels in 21 primary PDAC cells obtained from Patient-Derived Xenografts (PDX of the PaCaOmics biobank). Inhibitors of mitochondrial pyruvate carrier (UK5099), glutaminase (BPTES), and CPT1a (Etomoxir) were used to inhibit glycolysis, glutaminolysis, and FAO pathways, respectively. The cells' mitochondrial dependency on each of these fuel sources is determined by measuring the decrease in oxygen consumption rate (OCR) after addition of the specific inhibitor, followed by inhibition of the two alternative pathways.

[0277] Our results show that mitochondrial respiration depends mainly on fatty acids in the 21 primary PDAC cells (data not shown). Moreover, the percentage of dependency towards fatty acids was significantly higher than on glucose and glutamine. Indeed, 12 of the 21 cells exhibited a high dependence for this fuel (≥60%) and the other 9 a moderate dependency degree. Regarding glucose, the majority of PDAC cells showed a moderate reliance on this nutrient for respiration. Interestingly, a very low dependency on glutamine (average of 8%) was observed in all the PDAC cells. Therefore, in sharp contrast with several studies, our result takes the focus away from the glycolysis and glutaminolysis pathways, and brings to light the importance of FAO in PDAC. In conclusion, this finding places the FAO pathway as a novel vulnerability in PDAC.

[0278] PDAC Cells Exhibit Different Sensitivities to Pharmacological FAO Inhibitors and OXPHOS Rates Correlate with Response to FAO Targeting

[0279] The dependency of mitochondrial respiration on fatty acids in all the PDAC cells suggest that the deprivation of this nutrient could result in energetic stress promoting cancer cell elimination. Therefore, we evaluated the effect of FAO inhibition in vitro. We performed dose-response viability experiments treating PDAC cells for 72 h with three different drugs: Etomoxir, Perhexiline and Trimetazidine. Etomoxir and Perhexiline are inhibitors of the Carnitine Palmitoyl-Transferase 1 (CPT1), and Trimetazidine targets the last step of beta-oxidation.

[0280] Our results show that PDAC cells exhibit different sensitivity to the FAO inhibitors Etomoxir and Perhexiline (FIG. 1). FIGS. 1A and 1B show the relative cell viability of PDAC cells upon treatment with Etomoxir (62.5 μM) and Perhexiline (7 μM) compared with non-treated cells. These findings led us to pinpoint PDAC cells that respond efficiently to Etomoxir and Perhexiline, i.e. showing relative cell viability below the mean (0.75 for Etomoxir and 0.72 for Perhexiline) that we consider as “High responders”. Another group of cells show moderate response with values close to the mean, and finally some cells show low response (“Low responders”) with relative cell viability above the mean. Interestingly, no effect in any cell was seen upon Trimetazidine treatment compared with the non-treated cells (FIG. 1C).

[0281] Strikingly, no correlation was observed between response to pharmacological FAO inhibition and fatty acids dependency. By contrast, we found some link with basal mitochondrial respiration (basal oxygen consumption rate [OCR] shown in Table 1). Indeed, we observed a significant negative correlation between the OCR and the relative cell viability with Perhexiline (FIG. 2). This means that the High/moderate responders to Perhexiline correspond to the cells with High OCR (e.g. the highest OCR cell #84 is the most sensitive to Perhexiline), whereas the Low responders to Perhexiline are the ones with Low OCR activity. These observations were similar with the Etomoxir treatment, even if there is no statistical correlation. Altogether, our observations highlight the different sensitivities of PDAC cells to FAO inhibitors, and suggest that the basal OXPHOS status could predict the response to FAO targeting by Perhexiline.

[0282] The FAO Inhibitor Perhexiline in Combination with Gemcitabine is Synergistic Specifically in High Responder Cells

[0283] Furthermore, we wondered whether treating cells with a FAO inhibitor in combination with Gemcitabine could increase the efficacy of Gemcitabine. Based on our in vitro outcomes, we chose to use the potent FAO inhibitor Perhexiline with a low concentration (5 μM) that no impacts cell viability, or combined with increasing concentrations of Gemcitabine. For this, we treated four different PDAC cells with different sensitivities to Perhexiline (high, intermediate, and low responders). Interestingly, combining Perhexiline with Gemcitabine specifically sensitizes the High and intermediate responders cells PDAC084T and PDAC012T, showing a strong synergistic effect (FIG. 3A-B), which is not the case for the low responder cells PDAC022T and PDAC032T (FIG. 3C-D).

[0284] FAO Inhibition with Perhexiline in Combination with Gemcitabine Induces Complete Tumor Regression in a High Responder PDAC Xenograft

[0285] We then addressed the question of the impact of FAO inhibition on chemotherapeutic response in vivo. We performed subcutaneous xenografts in immunodeficient mice using the same four different primary PDAC cells treated with the combination in vitro. We selected the PDAC cells regarding the in vitro sensitivity to Perhexiline and the combination with Gemcitabine (FIGS. 1B and 3), and the basal OCR rate (Table 1). Accordingly, we worked with a high responder cell with high OXPHOS rate (PDAC084T), a moderate responder with intermediate OXPHOS rate (PDAC012T), and low responder cells with low OXPHOS rate (PDAC022T and PDAC032T). Tumor-bearing mice were treated during one month using Gemcitabine alone or in combination with Perhexiline.

[0286] FIG. 4 shows the progressive tumor growth in the four PDAC xenografts, upon treatment with Gemcitabine, Perhexiline, or the combination Gemcitabine+Perhexiline. Overall, we observed that Perhexiline per se doesn't have any impact on tumor growth compared with vehicle-injected mice. In both high and intermediate OXPHOS contexts (FIG. 4A-C), treatment with Gemcitabine alone is arresting the tumor growth, suggesting a cytostatic effect. Excitingly, in the High responder xenograft (PDAC084T), the co-administration of Perhexiline and Gemcitabine significantly potentiates the efficacy of Gemcitabine, and more importantly, induces a complete tumoral regression after one-month treatment (FIG. 4A). In the intermediate responder xenograft (PDAC012T), the mice treated with the combination showed a higher tumor growth inhibition compared with the chemotherapy alone (FIG. 4B). Finally, tumors from the low responder xenograft PDAC032T show a high sensitivity to Gemcitabine alone which induces tumor regression. No impact was observed with the combination treatment compared with Gemcitabine alone, in both low responder models PDAC032T and PDAC022T (FIG. 4C, D). Altogether, we demonstrate that targeting FAO using Perhexiline enhances the antitumoral activity of Gemcitabine in two of our pre-clinical models. Moreover, combining Perhexiline with Gemcitabine results in a complete tumor regression in the high responder PDAC xenograft. This means that applying this therapeutic strategy could notably improve the prognosis of PDAC in a subset of patients.

[0287] The FAO Inhibitor Perhexiline Enhances the Antitumor Activity of Chemotherapy by Inducing Apoptosis and Energetic Stress in Pancreatic Cancer

[0288] Based on the above outcomes, we further investigated the mechanism of cooperation between Perhexiline and Gemcitabine underlying the complete tumor regression in the high responder PDAC xenograft (PDAC084T). For that, we performed cell death assays demonstrating that Perhexiline is an apoptotic inductor, and we clearly illustrate the different responses in the four PDAC cells in terms of cell death (FIG. 5A). Moreover, the combination of Perhexiline plus Gemcitabine in PDAC084T cells results in an increase in cell death by apoptosis with the consequent decrease in cell viability (FIG. 5B).

[0289] More importantly, we investigated the mechanism of cooperation between Perhexiline and Gemcitabine in vivo. Using the same mouse models of PDAC xenografts, we excised the tumors from mice under treatment (middlepoint of one-month treatment) in the PDAC084T xenograft. Next, we performed immunohistochemistry analysis for apoptosis (cleaved-Caspase 3) and proliferation (ki67) markers, as well as histochemistry with the Masson's Trichrome staining (to detect fibrosis). Similarly to the cellular investigation, we observe that the PDAC084T tumors treated with the combination therapy show a higher staining for cleaved-Caspase 3 in comparison with Gemcitabine alone (FIG. 6A), suggesting that Perhexiline increases the Gemcitabine-induced apoptosis in vivo, having as a result a complete tumor regression. In contrast, no difference for ki67 (proliferation) is observed between Gemcitabine or combination treatment groups (FIG. 6B). However, the tumors from mice treated with the combination, show larger fibrotic areas compared with the other mice groups (FIG. 6C).

[0290] Finally, to continue deciphering the mechanism of cooperation between Perhexiline and Gemcitabine in PDAC084T, we determined the impact of Gemcitabine, Perhexiline, and the combination, on the mitochondrial respiration (Seahorse metabolic analyzer). FIG. 7A clearly illustrates that the FAO inhibitor Perhexiline is able to decrease mitochondrial respiration in about 50%. More importantly, the combination of Gemcitabine and Perhexiline drastically decreases basal and maximal mitochondrial respiration, ATP production, and spare respiratory capacity, leading cells to an energetic crisis (FIG. 7B).

[0291] CPT1C Isoform as a Key Actor in the Response to FAO Targeting in PDAC

[0292] To decipher the key molecular actors related to the response to FAO targeting combined with chemotherapy, we performed a transcriptomic analysis (RNA sequencing and RT-qPCR). For the first analysis, we analyzed the RNA-sequencing data (RNA-seq) from five PDAC cells belonging to the high and intermediate responder group, and 3 PDAC cells from the low responder group. In this data, we used the KEGG database to determine the significantly upregulated or downregulated pathways between the two groups. Interestingly, our results point to the CPT1C isoform, which is significantly downregulated in the high responder group. Then, for the second analysis, we measured the mRNA levels of the CPT1 (A, B, and C) and CPT2 isoforms by RT-qPCR in the PDAC cells used in the in vivo experiments. Consistently, we found that the mRNA level of the CPT1C isoform in the PDAC084T cells is significantly lower in comparison with the other cells and that the CPT1C mRNA levels correlate with the response to Perhexiline treatment (FIG. 8A-B).

[0293] Taking together this data, our molecular analysis points to the CPT1C enzyme as a key actor in the mechanism of cooperation between Perhexiline and Gemcitabine to induce complete pancreatic cancer regression in the PDAC084T xenograft. Currently, we are working on deciphering how the different treatments impact on the CPT1C gene expression, and more importantly, we are working on the manipulation of the CPT1C mRNA levels in PDAC084T. Our hypothesis proposes that the low mRNA level of CPT1C in PDAC084T is responsible for the complete tumor regression observed, and that by increasing these levels, the combination therapy will lose its efficacy.

CONCLUSION

[0294] In conclusion, the work of the inventors demonstrates that Fatty Acid Oxidation (FAO) inhibitors could be used for the treatment of pancreatic cancer (Pancreatic Ductal AdenoCarcinoma). CPT1, a key enzyme in the FAO pathway, can be pharmacologically targeted by drugs like Etomoxir and Perhexiline. Moreover, in this work, the inventors used Perhexiline and showed enhancement of the antitumoral effect of chemotherapy (Gemcitabine), resulting in a complete tumoral regression in preclinical assays for a subset of patients (“high responder”, namely “high OXPHOS”). Thus, such combinations could also be used in High OXPHOS patients. Further, this work provides the mechanism of cooperation between Perhexiline and Gemcitabine, and points to the CPT1C isoform as a potential biomarker that predict the response of patients to FAO targeting.

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