COMPOSITIONS FOR TREATING MELANOMA

Abstract

Inventors have shown that targeting DDR1 and DDR2 collagen receptors by Imatinib resensitizes melanoma tumors to BRAFV600E to targeted therapy and normalizes the fibrotic stromal reaction. These findings provide the rationale to combine Imatinib (or other DDR inhibitors) and MAPK-targeting agents to disrupt the influence of the matrix microenvironment in order to delay or prevent the emergence of therapy-resistant cells. They have shown that inhibition of DDR1 and DDR2 kinase activities by Imatinib suppressed the protection of melanoma cells against Vemurafenib (BRAFi) and Trametinib (MEKi) co-drugging and led to cell cycle arrest and cell death. Similar biochemical cell cycle and apoptotic events were promoted in presence of Nilotinib. They validated this anti-tumor activity of Imatinib combined with Vemurafenib in a pre-clinical xenograft model of melanoma and showed that targeting DDR1/2 signaling delays tumor relapse. Accordingly, the present invention relates to a method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of: i) an inhibitor of BRAF, ii) an inhibitor of MEK, and iii) an inhibitor of DDR1/2.

Claims

1. A method for treating melanoma in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of: i) an inhibitor of BRAF, ii) an inhibitor of MEK and iii) an inhibitor of DDR1/2.

2. The method according to claim 1, wherein the melanoma is resistant melanoma.

3. The method according to claim 1, wherein, i) the inhibitor of BRAF is vemurafenib; ii) the inhibitor of MEK is cobimetinib and iii) the inhibitor of DDR1/2 is imatinib.

4. The method according to claim 1, wherein, i) the inhibitor of BRAF, ii) the inhibitor of MEK and iii) the inhibitor of DDR1/2 are administered in combination with a classical treatment of melanoma.

5. The method according to claim 1, wherein, i) the inhibitor of BRAF, ii) the inhibitor of MEK and iii) the inhibitor of DDR1/2 are administered in combination with an anti-PD-1 antibody.

6. A pharmaceutical composition comprising i) an inhibitor of BRAF, ii) an inhibitor of MEK and iii) an inhibitor of DDR1/2.

7. (canceled)

8. (canceled)

Description

FIGURES

[0083] FIG. 1. (A) Fibroblast-derived 3D ECM confers drug-protective action to melanoma cells against anti-BRAFV600E therapies. Proliferation of 501Mel cells plated on plastic or on decellularized 3D cell-derived matrices generated from melanoma-associated fibroblasts (MAF) for the indicated times. After 2 days, cells were treated with vehicle (DMSO) or 2 μM Vemurafenib (BRAFi). Time-lapse analysis of fluorescently NucLight™-labeled cells using the IncuCyte ZOOM system. (B) Cell cycle distribution of 501Mel cells cultured on plastic or MAF-derived ECM for 48 h and treated for an additional 48 h with 2 μM vemurafenib (BRAFi). Cell cycle profiles were analyzed by flow cytometry of PI-stained cells. The percentage of cells in different phases of the cell cycle is indicated. (C) Inhibition of DDR1/2 by Imatinib overcomes ECM-mediated resistance to combined BRAF and MEK inhibition, and leads to apoptotic cell death. Upper panel, 1205Lu melanoma cells cultured on MAF-derived ECM for 48 h were treated for an additional 48 h with 2 μM Vemurafenib (BRAFi), 10 μM Imatinib (DDR1/2i) alone or a combination of both. Apoptosis was analysed by flow cytometry after labelling of cells with AnnexinV-FITC and propidium iodide (PI). The percentage of different forms of cell death based on Annexin-FITC and PI positivity is indicated. Lower panel, Western blot analysis of phosphorylated form of Rb and Caspase 3 cleavage in 1205Lu cells cultured on 3D-ECM and treated with Vemurafenib (BRAFi), Trametinib (MEKi), Imatinib (DDR1/2i) alone or in combination as indicated.

[0084] FIG. 2. (A) Targeting DDR1 and DDR2 collagen receptors by Imatinib sensitizes melanoma tumors to BRAFV600E targeted therapy and normalizes the fibrotic stromal reaction. 1205Lu cells (1×10.sup.6) were injected sc into nude mice. Tumor volume was assessed with caliper and when tumors reached 100 mm3, mice were randomized into four groups and treated at the indicated date with vehicle or ip injections of Vemurafenib (BRAFi) (35 mg/kg), Imatinib mesylate (60 mg/kg), or Vemurafenib plus Imatinib for 30 days. Graphs show tumor growth following treatment by indicated drugs (mono or combo therapy). Data shown are mean±SEM of tumor volume (n=6; ****p<0.0001). Two-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analyses. Sections of 1205Lu xenografts were stained with picrosirius red and imaged under polarized light microscopy to ascertain collagen maturity and fiber thickness. Birefringence hue and amount of collagen fibers were quantified as a percent of total tissue area. (B) Kaplan-Meier survival curves of 1205Lu melanoma-bearing mice treated with Vehicle (n=5), Imatinib mesylate alone (n=5), Vemurafenib alone (n=10) and Vemurafenib+Imatinib mesylate (n=10). Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for Vemurafenib versus Vmurafenib+Imatinib mesylate: ***p<0.0001 and hazard ration (log rank): 0.2403 (95% Cl of ration, 0.08123 to 0.7106).

EXAMPLE

[0085] Material & Methods

[0086] Cell and Reagents

[0087] Melanoma cells and MAFs were cultured in Dulbecco's modified Eagle Medium (DMEM) plus 7% FBS (Hyclone). Culture reagents were purchased from Thermo Fisher Scientific. All other reagents were purchased from Sigma-Aldrich unless stated otherwise. PLX4032 (Vemurafenib), GSK1120212 (Trametinib), RO5126766, Imatinib mesylate, Nilotinib were from Selleckem. Collagen I rat tail was from Thermo Fisher Scientific.

[0088] RNAi Studies

[0089] Non-targeting control, DDR1 and DDR2 siRNA duplexes were designed by Invitrogen (Thermo Fisher Scientific). Transfection of siRNA was carried out using Lipofectamine RNAiMAX (Thermo Fisher Scientific), at a final concentration of 50 nM.

[0090] Cell-Derived Extracellular Matrix (ECM) Preparation and MM-DR Assay

[0091] Gelatin-coated tissue culture dishes were seeded with fibroblasts and cultured for 8 days in complete medium, supplemented with 50 μg/ml ascorbic acid every 48 h. Cell cultures were then washed with PBS and matrices were denuded following a 2 min treatment with pre-warmed extraction buffer (PBS 0.5% Triton X-100, 20 μM NH.sub.4OH). Matrices were then gently washed several times with PBS. For the drug-protection assay (MM-DR), melanoma cells were seeded onto the decellularized 3D matrices for 48 h at 37° C. in 5% CO2, and then cultivated in complete medium for another 48 h period in presence or not of the indicated amounts of targeted agents. Cells were detached and either fixed in 80% ethanol for cell cycle analysis or in RIPA buffer for immunoblot analysis.

[0092] Cell Proliferation Assay

[0093] For realtime analysis of cell growth using the IncuCyte™ Zoom imaging system (Essen Bioscience), cells stably expressing the nuclear-restricted non-perturbing fluorescent label (red IncuCyte® NucLight Reagent) were plated in triplicate in complete medium on 96-well plates (5×103 cells/well) coated or not with fibroblast-derived ECM. Phase contrast images were taken every hour over a 3-day period. Cell proliferation is quantified by counting the number of fluorescent nuclei over time to give cell growth rates. Growth curves were generated using the IncuCyte™ cell proliferation assay software.

[0094] Flow Cytometry Analysis

[0095] Cell cycle profiles were determined by flow cytometry analysis of propidium iodide (PI)-stained cells. Melanoma cells cultured onto cell-derived matrices and treated with or without 2 μM vemurafenib for 48 h were washed, fixed in 80% ethanol and incubated at −20° C. for 24 h. Cells were then stained for 20 min at 37° C. in buffer containing 40 μg/ml PI and 20 μg/ml ribonuclease A from bovine pancreas. Cell cycle profiles were collected on a FACS Canto. Cell death was evaluated by flow cytometry following staining with AnnexinV/PI (eBioscience). Melanoma cells cultured onto cell-derived matrices were double stained with AnnexinV-FITC and PI and analysed using a BD FACSCanto cytometer.

[0096] Immunoblot Analysis and Antibodies

[0097] Melanoma cells cultured onto cell-derived matrices were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Whole cell lysates were subjected to SDS-PAGE and immunoblot analysis. The following antibodies were used at working dilution of 1:1,000, unless otherwise stated. Anti ERK1/2, HSP90, HSP60 and DDR1 antibodies were from Santa Cruz Biotechnology. Anti DDR2, phospho-DDR1 (Tyr792), phospho-MEK1/2 (Ser217/221), phospho-ERK1/2 (T202/Y204), phospho-Rb (Ser807/811), p27KIP1, Survivin, BAX, BIM and Caspase 3 antibodies were from Cell Signaling Technology. Anti phospho-DDR2 (Tyr740) antibodies were from Bio-Techne. Peroxydase-conjugated anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology. Peroxydase-conjugated anti-goat antibody was from Dako. Secondary antibodies were used at 1:3,000 dilution. Proteins were detected by enhanced chemiluminescence (ECL) Amersham Western Blotting Detection Reagents (GE Healthcare Life Sciences).

[0098] Cell Line-Derived Xenograft (CDX) Tumor Models

[0099] Mouse experiments were carried out in accordance with the Institutional Animal Care and the local ethical committee. 1×10.sup.6 1205 Lu melanoma cells were subcutaneously implanted into both flanks of 6 week old female athymic nude nu/nu mice (Envigo). Tumor was measured by caliper and the volume was calculated using the formula: V=tumor width×tumor length 2×0.5. When tumor reached 100 mm.sup.3, mice were randomly grouped into control and test groups. Vemurafenib (35 mg/kg) and Imatinib mesylate were delivered (alone or in combination) intraperitoneally three times per week. Mice in control group were treated with vehicle. Mice were treated for 14 days and followed for up to 40 days or until tumors reached a pre-defined volume (1000 mm.sup.3). After animal sacrifice tumors were dissected, fixed in formalin and embedded in paraffin.

[0100] Histopathological Evaluation of Collagen Fibers

[0101] Paraffin-embedded xenograft tissues were cut into 10-μm sections and stained with Picrosirius red using standard protocols. Picrosirius red stained sections were analyzed under Polarizing Microscope to analyze the nature of collagen (thickness, organization, hue, density, and birefringence).

[0102] Results

[0103] To investigate the contribution of ECM components and of collagen signaling through DDR, in MM-DR, inventors generated 3D-ECM from MAFs and tested their effectiveness to protect BRAFV600E-mutated melanoma cells (501Mel or 1205Lu cells) against the anti-proliferative effects of inhibition of the BRAF oncogenic pathway. Cell-derived matrices were generated, denuded of cells and their architecture, topology and molecular composition were analyzed using proteomic and microscopic approaches. In their experimental conditions, MAFs produce and assemble in vitro a rich and complex ECM composed of Collagen and Fibronectin fibers that recapitulates many biochemical and biophysical properties of in vivo ECM (data not shown). 501Mel cells stably expressing a fluorescent nuclear label were cultured on matrices derived from MAF population isolated from melanoma metastatic biopsies, and treated or not with vemurafenib (BRAFi). Cell proliferation was monitored using live cell time-lapse imaging and quantified by counting the number of fluorescent nuclei. Cell growth inhibition induced by BRAFi was totally abrogated when cells are cultured on cell-derived ECM (FIG. 1A). Cell cycle analysis showed that experimentally produced ECM prevented the G0/G1 cell cycle arrest induced by the targeted therapy in contrast to standard cell culture conditions on plastic (FIG. 1B). Thus, ECM transmits signals that prevent the anti-proliferative action of BRAF inhibition. At the molecular level, matrix-mediated therapeutic escape from BRAF inhibition was associated in both 501Mel and 1205Lu cells with sustained levels of the proliferation marker CyclinD1, low levels of the cell cycle inhibitor p27KIP1 together with maintained phosphorylation of ERK1/2 in presence of the drug. Similar results were obtained with ECM generated from another MAF population (data not shown). Because clinical management of BRAF mutated melanomas currently involves combinatorial therapy integrating BRAF and MEK inhibitors, they next tested whether cell-derived ECM could be effective in protecting drug-sensitive melanoma cells from BRAF and MEK co-inhibition. Matrices generated by MAF also significantly prevented the effect of dual inhibition on 501Mel cells observed on plastic conditions, evidenced by maintaining phosphorylation of the proliferation marker Rb, low levels of p27KIP1 and expression level of the anti-apoptotic protein Survivin (data not shown). Upregulation of expression levels of the pro-apoptotic protein BIM observed in response to the drugs was also prevented after exposure of cells to cell-derived ECM. Note that expression levels of BAX, another pro-apoptotic protein, remained unchanged in the different experimental settings analyzed (data not shown).

[0104] These findings demonstrate that melanoma cell lines were protected by the experimentally derived MAF matrix against the growth and survival inhibitory effects induced by MAPK targeting drugs compared with standard growth conditions on plastic. These results thus indicate a critical role for the ECM in creating a permissive niche for resistance to targeted therapies in melanoma.

[0105] The functional contribution of DDR1 and DDR2 signaling pathway to matrix-mediated resistance to BRAF inhibition was next explored. Immunoblot analysis of 1205Lu melanoma cells stimulated with collagen I revealed a slow but persistent increase in DDR1 and DDR2 tyrosine kinase activities, as measured by DDR1-Tyr792 and DDR2 Tyr740 autophosphorylation (data not shown). To address the contribution of the two receptors for collagens in MM-DR, a siRNA approach was used to target DDR1, DDR2 or both in 1205Lu cells. Immunoblot analysis showed specific protein reduction after siRNA transfection in single target and target combination treatments in 1205Lu cells cultured on MAF-derived ECM (data not shown). Compared to the single knockdown, the simultaneous knockdown of DDR1 and DDR2 overcame MM-DR to BRAF targeted therapy as revealed by decreased levels of phospho-Rb and Survivin. As DDRs are tyrosine kinase receptors and druggable targets, inventors used Imatinib to inhibit DDR kinase activities in the drug-protection assay. Imatinib is a tyrosine kinase inhibitor (TKI) developed as a BCR-ABL kinase inhibitor but later shown to inhibit DDR1 and DDR2 kinase activities highly efficiently. Imatinib belongs to therapeutic molecules used in the clinic for the treatment of Chronic Myeloid Leukemia (CML). Nilotinib, another BCR-ABL inhibitor has been developed later and approved for the treatment of patients with resistance to Imatinib. They first confirmed that Imatinib efficiently abrogated type I collagen-mediated DDR1 and DDR2 tyrosine phosphorylation in melanoma cells (data not shown). Inhibition of DDR1 and DDR2 kinase activities by Imatinib suppressed the protection of melanoma cells from Vemurafenib (BRAFi) and Trametinib (MEKi) co-drugging and led to reduced Rb phosphorylation and cleavage of Caspase 3, a sign of apoptotic cell death (FIG. 1C). Induction of apoptosis caused by Imatinib in presence of Vemurafenib was further confirmed on 1205Lu melanoma cells cultured on 3D-ECM by cytometry analysis (FIG. 1C). Similar biochemical cell cycle and apoptotic events were promoted in presence of Nilotinib (data not shown).

[0106] This indicates that DDR1 and DDR2 determine BRAF mutant melanoma cells responsiveness to targeted therapies and that the drug-protective action of DDRs is dependent of their enzymatic activities.

[0107] Finally, the anti-tumor activity of Imatinib combined with Vemurafenib was validated in a pre-clinical xenograft model of melanoma. BRAF-mutated melanoma cells 1205Lu were subcutaneously xenografted into nude mice (CDX model), which were exposed to Vemurafenib, Imatinib or Vemurafenib plus Imatinib. As expected, BRAF targeting induced a rapid inhibition of tumor growth, whereas Imatinib did not display any significant anti-melanoma effect. However, after 12 days, tumors treated with BRAFi alone had resumed growth, whereas combination with Imatinib markedly delayed the development of resistance and led to a significant reduction of tumor growth. Histochemical analysis of Collagen content and organization showed that Vemurafenib treatment triggered a profound remodeling of the melanoma ECM stroma, with marked increase of Collagen fibers thickness that was suppressed by Imatinib (FIG. 2A). This data suggests that treatment with Imatinib counteracts the adverse effect of targeted agents on aberrant Collagen deposition, a process potentially contributing to drug resistance and relapse. Moreover, the Vemurafenib/Imatinib combination significantly increased the survival of mice that were treated for 14 days (FIG. 2B) without apparent weight loss or signs of organ toxicity (data not shown).

[0108] From these observations, inventors conclude that targeting DDR1 and DDR2 collagen receptors by Imatinib sensitizes melanoma tumors to BRAFV600E targeted therapy, prevents tumor relapse seen with BRAFi alone, and normalizes the fibrotic stromal reaction. These findings provide the rationale to combine Imatinib (or other DDR inhibitors) and MAPK-targeting agents to disrupt the influence of the matrix microenvironment in order to delay or prevent the emergence of therapy-resistant cells.

REFERENCES

[0109] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.