Method for Predicting Therapeutic Response to Serine-Theronine Kinase Braf Inhibitor Drugs

Abstract

The present invention relates to an in vitro method which enables the prediction of therapeutic response to treatment with BRAF inhibitor drugs, such as vemurafenib, in cancer patients associated with oncogenic mutations in said kinase, such as BRAF positive melanoma. Said method is based on the measurement of cytoplasmic ERK1/2 levels through the detection and quantification of ERK1/2 phosphorylated in serine 301/284, respectively, in an isolated biological sample of the patient. The invention also provides a specific antibody against ERK1/2 phosphorylated in serine 301/284 and a kit comprising it, which are of use in the described method.

Claims

1-6. (canceled)

7. An in vitro method to predict the response to treatment with a serine-threonine kinase BRAF inhibitor in a patient, wherein said method comprises the following steps: a. Quantifying the levels of ERK1 phosphorylated in serine 301 and of ERK2 phosphorylated in serine 284 in an isolated biological sample of the patient, b. Comparing the levels quantified in step (a) with a reference value, wherein said reference value comes from the quantification of the levels of ERK1 phosphorylated in serine 301 and of ERK2 phosphorylated in serine 284 in an isolated biological sample of a patient who does not respond to treatment with a serine-threonine kinase BRAF inhibitor, and c. Assigning the patient from step (a) to the group of individuals who will respond adequately to treatment when the quantification value obtained in step (a) is significantly higher than the reference value, and wherein the quantification of the levels of ERK1 phosphorylated in serine 301 and of ERK2 phosphorylated in serine 284 is carried out by using a specific antibody against the peptide consisting of SEQ ID NO: 1, wherein the serine at position 9 of SEQ ID NO: 1 is chemically phosphorylated.

8. The method according to claim 7, wherein the serine-threonine kinase BRAF inhibitor is vemurafenib and/or dabrafenib.

9. The method according to claim 7, wherein the patient suffers from cancer.

10. The method according to claim 9, wherein the cancer is melanoma or non-small cell lung cancer.

11. The method according to claim 7, wherein the serine-threonine kinase BRAF inhibitor is vemurafenib and the patient suffers from melanoma.

12. The method according to claim 7, wherein the patient is human.

13. (canceled)

14. A specific antibody against the peptide consisting of SEQ ID NO: 1, wherein the serine at position 9 of SEQ ID NO: 1 is chemically phosphorylated.

15. A kit comprising the antibody according to claim 14.

16-21. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0083] FIG. 1. Specificity of the antibody of the invention (anti-phospho ser284/301 antibody). HEK294 cells were transfected with human (Hs) or zebrafish (Dr) ERK2, wherein the homologous residue of serine 284 is a proline, therefore, not phosphorylatable. It is observed that the antibody only recognises human ERK2, in cells under stimulation, in this case with EGF. An anti-FLAG antibody was used as a control.

[0084] FIG. 2. ERK1/2 phosphorylated in ser 301/284 are located exclusively in the cytoplasm. HeLa cells were stimulated with EGF for 5 min, and the subcellular localisation of phosphorylated endogenous ERK1/2 in the aforementioned residues was analysed by means of immunofluorescence and confocal microscopy. It is observed that ERK1/2 phosphorylated in said positions are completely excluded from the nucleus, marked with DAPI.

[0085] FIG. 3. Correlation between the levels of ERK1/2 phosphorylated in ser 301/284 and sensitivity to vemurafenib. From the BRAF positive melanoma cell line M249, a subline resistant to vemurafenib (vemR) was obtained and the levels of phospho-ser 284/301 were analysed in said subline in comparison with the parental line. It is observed that these levels are much higher in the parental line, sensitive to vemurafenib.

[0086] FIG. 4. Correlation between the levels of ERK1/2 phosphorylated in ser 301/284 and sensitivity to vemurafenib. The sensitivity to vemurafenib was evaluated in different lines of melanoma, analysing the reduction in the total activity of ERK1/2, measured by the decrease in the levels of canonical phosphorylation (p-ERK). It is observed that the most sensitive lines are 8505C and A375P. The levels of phospho-ser 284/301 were also analysed in said lines. It is observed that said levels, before treatment with vemurafenib, are much higher than those observed in the resistant lines MELJUSO and SKMEL2.

[0087] FIG. 5. Sensitivity to vemurafenib of the lines of melanoma used in FIG. 4, evaluated by the concentration necessary to stop proliferation (GI50). It is observed that the most sensitive lines are 8505C and A375P, which show the highest levels of phospho-ser 284/301 in FIG. 4.

EXAMPLES

[0088] Next, the invention will be illustrated by means of assays carried out by the inventors that show the effectiveness of the method of the invention and of the antibody generated to be used in said method.

Example 1. Material and Methods

[0089] The cell lines used in the assays were:

[0090] HEK293T: Human kidney embryonic cells, T antigen positive.

[0091] HeLa: cervical cancer epithelial cells.

[0092] A375p: melanoma epithelial cells, having a BRAF mutation.

[0093] SKMEL2: melanoma epithelial cells, having a BRAF mutation.

[0094] Parental M249 and Vemurafenib-resistant M249: melanoma epithelial cells, having a BRAF mutation.

[0095] Mel JUSO: melanoma epithelial cells.

[0096] The cell lines were grown in DMEM culture medium supplemented with 10% Foetal Bovine Serum and antibiotics (Penicillin and Streptomycin).

Western Blot:

[0097] To obtain the total protein extracts, the cells were lysed with the suitable volume of lysis buffer (20 mM HEPES pH 7.5, 10 Mm EGTA, 40 mM β-glycerophosphate, 1% non-ionic detergent NP40, 2.5 mM MgCl.sub.2, 1 mM orthovanadate, 1 mM DTT and extemporaneously 10 μg/ml aprotinin and 10 μg/ml leupeptin). The cells were harvested and centrifuged at 12,000 rpm, for 10 minutes and at 4° C. The protein extracts were separated from the rest of the components of the cells and the protein concentration of each lysate was quantified. To determine the amount of protein, the Bradford method was used. Approximately 50 μg of protein were taken, to which 5× Laemmli buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 20 mM DTT and 0.005% bromophenol blue) was added. After boiling the samples for five minutes, they were subjected to vertical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of 12% acrylamide. The proteins were transferred to a nitrocellulose membrane setting the amperage at 400 mA for 1 hour. After the transfer had finished, the membranes were incubated for one hour, at room temperature and with stirring, in a solution of TBS-T with 4% BSA, to block unspecific sites. After that, the filters were incubated with the phosphospecific primary antibody of the invention (0.2-0.4 μg/ml diluted in 4% BSA in TBS-T) for one hour at room temperature or overnight at 4° C. while stirring. Two washes with TBS-T were performed for a total of fifteen minutes, after which the filters were incubated with the corresponding peroxidase-conjugated secondary antibody, diluted 1:5,000 or 1:10,000 in 0.4% BSA in TBS-T for one hour at room temperature. Two washes with TBS-T were performed again and the protein was detected by chemiluminescence using the ECL kit. An autoradiography of the filters was carried out with Konica films, which enabled the detection of a band where the primary antibody had specifically recognised the phosphorylated protein of interest. As an internal loading control, a specific antibody that recognises the total protein of interest was used.

Immunofluorescence

[0098] Cells were grown in DMEM 10% Foetal Bovine Serum to subconfluence on sterile 10 mm diameter glass cover slides. At the time of immunofluorescence, cells were washed with 1×PBS and fixed with a 4% solution of paraformaldehyde in 1×PBS, for 10 minutes at room temperature. The cells were permeabilised by incubating for 5 minutes with a 0.5% dilution of Triton X-100 in PBS, followed by three washes of 1×PBS of five minutes each. Next, cells were incubated with the phosphospecific primary antibody of the invention at a concentration of 1/100 for one hour in a humid chamber. After three washes with PBS, of five minutes each, the FITC fluorophore-conjugated secondary antibody was added and incubated for 45-50 minutes in a humid chamber and in the dark. After that time, two new washes were carried out, of five minutes each, con 1×PBS. Lastly, a drop of Prolong mounting medium with DAPI was added on a slide and the cover was then placed on top with the cells facing down. The cells were examined by means of confocal microscopy (Leica TCS SP8). The images were digitised and processed using the Fiji Image J program.

Immunohistochemistry

[0099] Tissue samples or cell pellets were fixed with 4% parapholmaldehyde and embedded in paraffin. The sections were mounted on positively charged slides (Genex-Brand®), recommended for immunohistochemistry. Dewaxing was achieved by passing the sections through xylene (10 min), and decreasing graduations of ethyl alcohol (100° 10 min, 96° 5 min, and 70° 5 min). Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide solution in methanol for 15 min, and incubation in distilled water for 10 min. Subsequently, the sections were incubated with a 1% BSA bovine serum albumin solution in TBST buffer for 30 min with the intention of blocking non-specific binding sites. Subsequently, sections were incubated with the phosphospecific antibody of the invention at a 1:100 dilution in PBS, overnight at 4° C., in a humid chamber. The development of the reaction was carried out by the DAKO chromogen DAB technique. Contrast staining was performed by immersing the sections in Mayer's hematoxylin for 1 min; they were then placed under a stream of running water for development. Mounting was done with aqueous mounting medium (VectaMount AQ, Vector Lab Ind). Observation of the preparations was made on a Nikon microscope. Photographs were taken with an Olympus C4000 digital camera.

Example 2. Results

[0100] To obtain an antibody that specifically recognises the phosphorylated ser 301/284 of ERK1/2, respectively, rabbits were immunised with the peptide consisting of SEQ ID NO: 1 previously mentioned, following the usual protocols routinely used for this purpose. The immunoreactivity of the resulting serum was analysed by means of western blot (FIG. 1) in HEK293 cells transfected with plasmids encoding human (Hs) or zebrafish (Dr) ERK2. In the latter, the residue corresponding to serine 284 is a proline (proline 293), so it is not phosphorylatable, and is used as a negative control. It was observed that after stimulation with EGF for 5 min, which induces phosphorylation of canonical TEY residues in both human and zebrafish ERK2s, phosphorylation of ser 284 is detected only in human ERK2.

[0101] Once the specificity of the antibody has been demonstrated, the cell sublocalisation of ERK1/2 phosphorylated in ser 301/284 was analysed. To that end, immunofluorescence was performed on HeLa cells, that were fasting (starved) or stimulated with EGF for 5 min (FIG. 2). It was observed, by means of confocal microscopy, that endogenous ERK1/2 phosphorylated in ser 301/284 are located exclusively in the cytoplasm, being completely excluded from the cell nucleus, stained by means of DAPI.

[0102] Subsequently, the correlation of the levels of ERK1/2 phosphorylated in ser 301/284 with sensitivity towards BRAF inhibitors in melanoma cells carrying BRAF mutations was analysed. For this purpose, the M249 cell line was used, from which a subline resistant to vemurafenib (vemR) was obtained. The levels of endogenous ERK1/2 phosphorylated in ser 301/284 were analysed by means of western blot (FIG. 3) comparing the resistant subline with the parental line, sensitive to vemurafenib. It was observed that the levels of phospho-ser 301/284 are much higher in the parental line, sensitive to vemurafenib, demonstrating a positive correlation between the levels of phospho-ser 301/284 and the response to the anti-tumour compound.

[0103] To verify the previous point, the correlation between the phosphorylation levels of ser 284/301 in ERK1/2 and the sensitivity to vemurafenib in different lines of melanoma was evaluated. It was observed that the reduction in the total activity of ERK1/2, measured by the decrease in the levels of canonical phosphorylation (p-ERK) by means of western blot, after the treatment of the different cell lines with vemurafenib, was correlated with the highest levels of phospho-ser 284/301. It is observed that said levels before treatment with vemurafenib are much higher in the most sensitive lines, 8505C and A375P, compared to those observed in the most resistant lines, MELJUSO and SKMEL2 (FIG. 4). This correlation is also observed when the concentration of vemurafenib necessary to stop the proliferation (GI50) of the different cell lines of melanoma is evaluated (FIG. 5). It is observed that the most sensitive lines to treatment with vemurafenib are 8505C and A375P, which show the highest levels of phospho-ser 284/301 in FIG. 4.