Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates in general to the field of treatments for metastatic cancers, and more particularly, to overcoming resistance to certain chemotherapeutic agents by inhibiting MCL-1.
• United States Patent Application Publication No. 20130217721 filed by Lo, et al, is directed to compositions and methods for detection and treatment of B-RAF inhibitor-resistant melanomas. Briefly, these applicants are said to teach the use of specific, targetable molecules mediating acquired resistance of B-RAF-mutant melanomas to a B-RAF inhibitor, thereby providing materials and methods for the treatment and detection of B-RAF inhibitor resistant cancers, such as melanoma.
• United States Patent Application Publication No. 20120148533 filed by Dhingra, et al, is directed to a combination therapy that combines propane- 1 -sulfonic acid ⁇ 3-[5-(4-chloro- phenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]-amide ⁇ or salts thereof, and an interferon for treating a patient suffering from a proliferative disorder or solid tumors, for example, colorectal cancer, melanoma, and thyroid cancer.
• the present invention relates to such a therapy wherein the interferon is peg interferon alfa-2a and the disorder is melanoma containing the V600E b-Raf mutation.
• the present invention includes methods for inhibiting cancer growth or extending the life span of a subject which is bearing cancer or reducing the size of a solid tumor in a subject or altering the adhesion characteristic of membrane of cancer cells or inducing apoptosis of cancer cells comprising contacting the cells or administering to the subject with an effective amount of an inhibitor of at least one of a MAPK pathway protein, a mutant MAPK pathway protein, or a MAPK pathway protein kinase and an Mcl-l inhibitor to the subject wherein the cancer is refractory to one or more MAPK pathway protein inhibitors.
• the MAPK pathway protein is a wild type or a mutated BRAF and the inhibitor of the BRAF is a small interfering RNA (siRNA) that inhibits expression of BRAF or reduces the overall BRAF activity in a cell.
• the inhibitor of Mcl-l is a small interfering RNA (siRNA) that inhibits expression of mcl-l .
• the tumor is insensitive to the inhibitor of the MAPK pathway protein, the mutant MAPK pathway protein, or the MAPK pathway protein kinase administered alone.
• the method further comprises pre-determining a level of expression of Mcl-l in the tumor of the subject.
• the Mcl-l inhibitors is selected from at least one of an antibody that blocks Mcl-l activity, omacetaxine mepesuccinate, 2-(R)-(l-Ethyl-2-hydroxyethylamino)-6- benzylamino-9-isopropylpurine (Seliciclib), a small interfering RNA (siRNA) and small hairpin RNA (shRNA) that inhibits expression of mcl-l, antibodies against mcl-l, antagonists of mcl-l isotype 1, agonists of mcl-l isotype 2, benzylisothiocyanate, phenethylisothiocyanate, diindolyl methane, curcumin, piperlongumine, Marinopyrrole A, Cucurbitacin B, Penfluridol, Perphenazine, Bcl-2 inhibitors, Bcl-2 siRNA/shRNA, Bcl-XL inhibitors
• the tumor is insensitive to the inhibitor of the MAPK pathway protein or the kinase administered alone.
• the method further comprises pre-determining a level of expression of Mcl-l in the tumor of the subject.
• the tumor that overexpresses Mcl-l is a primary tumor.
• the tumor that overexpresses Mcl-l is a tumor metastasis.
• the present invention includes a method for treating tumors or tumor metastases in a patient, comprising: identifying a patient that has become refractory to an inhibitor of a MAPK pathway protein or a kinase; obtaining a sample of the tumor; determining the level of expression of Mcl-l in the tumor cells; and administering to the patient simultaneously or sequentially a therapeutically effective amount of a combination of a MAPK pathway protein or kinase inhibitor and an Mcl-l inhibitor to treat the tumors or tumor metastases if the tumor overexpresses Mcl-l .
• the MAPK pathway protein is selected from at least one of BRAF, CRAF, MEK 1, MEK 2, ERK1, ERK2, NRAS, KRAS.
• the Mcl- 1 inhibitors is selected from at least one of an antibody that blocks Mcl- 1 activity, omacetaxine mepesuccinate, 2-(R)-(l-Ethyl-2-hydroxyethylamino)-6-benzylamino-9- isopropylpurine (Seliciclib), a small interfering RNA (siRNA) and small hairpin RNA (shRNA) that inhibits expression Mcl-l, antibodies against mcl-l, antagonists of Mcl-l isotype 1, agonists of Mcl-l isotype 2, benzylisothiocyanate, phenethylisothiocyanate, diindolyl methane, curcumin, piperlongumine, Marinopyrrole A, Cucurbitacin B, Capsa
• the tumor that overexpresses Mcl-l is a primary tumor. In another aspect, the tumor that overexpresses Mcl-l is a tumor metastasis. In another aspect, the tumor is selected from colorectal cancer, thyroid cancer, melanoma, lung cancer or cancers treated the inhibitor of the MAPK protein or the kinase.
• the present invention includes a method for diagnosing and treating tumors or tumor metastases in a patient, comprising: identifying a patient that has become refractory to an inhibitor of a MAPK pathway protein or kinase; obtaining a sample of the tumor and isolating tumor cells therefrom; determining the level of expression of Mcl-l in the tumor cells; contacting the tumor cells with a therapeutically effective amount of a combination of an inhibitor of the MAPK pathway protein or kinase and Mcl-l inhibitor and determining the effect of the inhibitor of the MAPK pathway protein or kinase and Mcl-l inhibitor on tumor cell growth; and administering to the patient simultaneously or sequentially a therapeutically effective amount of a combination of the inhibitor MAPK pathway protein or kinase and Mcl-l inhibitor to treat the tumors or tumor metastases if the tumor overexpresses Mcl-l if the tumor cells are susceptible to the combination of the MAPK pathway protein or kinase inhibitor and Mcl-l inhibitor.
• Figures 1A to 1J shows that Vemurafenib treatment induces Mcl-l expression in melanoma cells.
• Figure 1A A375 cells, and in Figure IB SK-MEL-28 cells, were treated with various concentrations of vemurafenib for 72 hours. Following the treatment, the cells were stained with sulforhodamine B and the surviving cells were quantified spectrophotometrically. The study was performed at least three times independently, each time with eight replicates and the data is expressed as mean ⁇ S.D.
• Figure 1C A375 cells, and in Figure ID SK-MEL-28 cells, were treated with 0.1, 0.2 and 0.4 ⁇ vemurafenib for 72 hours.
• Figures 2A to 2D show Vemurafenib resistant melanoma cells exhibit Mcl-1 overexpression.
• Figure 2A shows that A375, A-375-R, A375X/R, SK-MEL-28 and SK-MEL-28-R cells were treated with various concentrations of vemurafenib for 72 hours following which the cell survival was analyzed by sulforhodamine B assay. The study was performed at least three times independently, each time with eight replicates and the data is expressed as mean ⁇ S.D.
• Figure 3C A375 and Figure 3D SK-MEL-28 cells untransfected or transfected with Mcl-1 plasmid were treated with 0.4 ⁇ vemurafenib for 72 hours. Following the treatment, the lysates were subjected to western blotting and analyzed for Mcl-1, cleaved caspase 3 and cleaved PARP. ⁇ Actin was used as a loading control. Each study was performed at least three times independently.
• Figure 3E A375 and A375-Mcl-1+/+ or Figure 3F, SK-MEL-28 and SK-MEL-28-Mcl-l+/+ were treated with 0.2 ⁇ and 0.4 ⁇ vemurafenib for 72 hours. The cell survival was evaluated by sulforhodamine B assay. The study was performed at least three times independently, each time with four replicates and the data is expressed as mean ⁇ S.D. *, p ⁇ 0.05 when compared with control.
• FIG. 5C Tumor lysates from 6 mice were subjected to western blotting and analyzed for Mcl-l, p-ERK, cleaved caspase 3 and cleaved PARP. ⁇ actin was used as a loading control.
• Figure 5D Formalin fixed paraffin embedded tumor sections were subjected to immunohistochemistry. Representative images of the tumor sections stained with Mcl-l, p-ERK and cleaved caspase 3.
• FIGS. 6A to 6D show silencing Mcl-l suppresses the growth of melanoma tumors resistance to vemurafenib.
• Figure 6B at day 30, the mice were sacrifice, tumors were extracted and weighed. The values are plotted as mean ⁇ S.D. *, p ⁇ 0.05 as compared to control.
• FIG. 6C tumor lysates from 6 mice were subjected to western blotting and analyzed for Mcl-l, p-ERK, cleaved caspase 3 and cleaved PARP. ⁇ actin was used as a loading control.
• Figure 6D Formalin fixed paraffin embedded tumor sections were subjected to immunohistochemistry. Representative images of the tumor sections stained with Mcl-l, p-ERK and cleaved caspase 3.
• Figures 7A to 7D shows the Dabrafenib-Trametinib combination induces Mcl-l expression in melanoma cells.
• A375, SK-MEL-28, SK-MEL-5 and WM-239 were treated (Figure 7A) dabrafenib and trametinib and (Figure 7B) vemurafenib and trametinib for 72 hours.
• Mcl-l expression was analyzed by western blotting.
• Figure 7C shows the cytotoxicity of dabrafenib at 72 hours in wildtype and resistant A375, SK-MEL-28 and WM-239 was evaluated by SRB assay.
• FIG. 7D shows the expression of Mcl-l in A375, SK-MEL-28 and WM-239 wild type cells dabrafenib resistant cells was evaluated by western blotting, ⁇ actin was used as a loading control in all the western blots. Each study was performed at least three times independently.
• Figures 8A to 8D shows the Mcl-l overexpressing melanoma cells are resistant to combined BRAF inhibitor and MEK1/2 inhibitor treatment.
• Figure 8A (A375) and Figure 8B (SK-MEL- 28) untransfected or transfected with Mcl-l plasmid were treated with ⁇ dabrafenib or 2nM trametinib for 72 hours.
• Figure 8C A375 and Figure 8D SK-MEL 28 cells untransfected or transfected with Mcl- 1 plasmid were treated with 400nM vemurafenib or 2nM trametinib for 72 hours. The cell survival was evaluated by sulforhodamine B assay.
• the study was performed at least three times independently, each time with four replicates and the data is expressed as mean ⁇ S.D. *, p ⁇ 0.05 when compared with control. #, p ⁇ 0.05 when compared with the respective untransfected controls. Each study was performed at least three times independently.
• Figures 9A to 9D show that inhibiting Mcl-l suppresses the growth of melanoma tumors resistance to vemurafenib.
• Figure 9B at day 30, the mice were sacrifice, tumors were extracted and weighed. The values are plotted as mean ⁇ S.D.
• FIG. 9C SK-MEL-28 and Figure 9D shows Sk-MEL-2 cells were treated with 5, 7.5 and 10 ⁇ PL for 48 hours. Cells were lysed, subjected to western blotting and analyzed for Mcl-l and Cl.PARP. Actin was used as a loading control.
• Mcl-l Melanoma cells with stable overexpression of Mcl-l were completely resistant to the treatment with BRAF inhibitors. Melanoma cells resistant to BRAF inhibitors showed significantly higher expression of Mcl-l as compared to respective parent cell lines. Treatment of resistant cell lines with vemurafenib and Mcl-l inhibitor resulted in remarkable growth inhibition. Silencing of Mcl-l using siRNA completely sensitized resistant melanoma cells to cell growth suppression mediated by BRAF inhibitors. A375-R xenografts implanted in athymic nude mice did not respond to vemurafenib treatment but showed substantial tumor growth suppression when treated with a combination of vemurafenib with TW-37 or Mcl-l siRNA.
• Mcl-l Myeloid cell leukemia - 1
• Bcl-2 Myeloid cell leukemia - 1
• Mcl-l Myeloid cell leukemia - 1
• Bcl-2 Myeloid cell leukemia - 1
• Mcl-l is pro-survival member of Bcl-2, which is known to promote oncogenesis not through cell proliferation but by inhibition of apoptosis, hence leading to immortalization of malignant cells (12, 13). Its expression is regulated by transcription factors like STATs, cAMP response elements and NFKB (14).
• Mcl-l is frequently overexpressed in a variety of human cancers thereby providing protection to the tumor cells from apoptosis (15, 16).
• Mcl-l has been identified as an important target in majority of human cancers (17, 18) and several therapeutic strategies focusing on Mcl-l inhibition are currently under development (19-23).
• the present inventors establish herein that acquired resistance to vemurafenib in melanoma cells was due to overexpression of Mcl-l. Moreover, these results demonstrate that the combination of vemurafenib with Mcl-l targeted therapies were successful in overcoming acquired resistance in melanoma in vitro and in vivo.
• Chemicals. Vemurafenib, dabrafenib, trametinib and TW-37 were purchased from Selleck Chemicals (Houston, YX, USA). All the antibodies and Mcl-1 siRNA were procured from Cell Signaling Technology Inc. (Danvers, MA, USA). Plasmid overexpressing Mcl-1 was acquired from Addgene (Cambridge, MA, USA).
• A375 was a kind gift from Dr. Tyler Wakenda (Rochester University, NY), which was originally purchased from ATCC (Manassas, VA, USA).
• SK-MEL-28 and WM-239 cells were purchased from ATCC.
• SK-MEL-5 was a kind gift from Dr. Randy Burd. The authenticity of these cell lines was confirmed by STR analysis at Texas Tech University Health Sciences Center core facility (Lubbock, TX, USA). All the cell lines were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum.
• EMEM Eagle's Minimum Essential Medium
• Vemurafenib resistant clones of A375 and SK-MEL-28 were generated by continuous exposure of cells to escalating concentrations of vemurafenib over a period of one year.
• the cells were treated with vemurafenib for 72 hours after which fresh media was added to the cells.
• the cells were allowed to recover for 24 hours after which they were again exposed to vemurafenib as shown in Table 1.
• cells were exposed weekly to two treatments of vemurafenib for 72 hours each with a 24 hour recovery period between the treatments.
• the initial concentration of vemurafenib used was 0.2 ⁇ which was eventually increased to as high as ⁇ .
• dabrafenib resistant cells were cultured by incubating the cells with increasing concentrations of the drug for a period of 3 months as described above. The fold resistant was intermittently evaluated by cytotoxicity analysis.
• Vemurafenib resistant cell lines were referred to as SK-MEL-28-R or A375-R and dabrafenib resistant cell lines were referred to as A375-DR, SK-MEL-28-DR or WM-239-DR.
• Another type of resistant cell line generated was A375 xenograft-resistant (A375 X/R) cells.
• A375 cells were injected subcutaneously in nude mice.
• mice When palpable tumors were formed, the mice were orally treated with 30 mg/kg vemurafenib twice a day. After a month, the tumors the resisted vemurafenib treatments were extracted and cultured in vitro. These cells were called as A375 X/R cells.
• SK-MEL-28, A375, WM-239, SK-MEL-28-R, A375-R, A375-DR, SK- MEL-28-DR and WM-239-DR cells were treated with various concentrations of vemurafenib or dabrafenib and cytotoxicity analysis was performed by SRB assay as previously described by the present inventors (24, 25).
• TW-37 treatment SK-MEL-28, A375, SK-MEL-28-R, A375-R and A375-X/R were plated in a six- well plate at a density of 0.3 x 10 6 cells/well and left overnight to attach. Next day, cells were treated with 500nM TW-37 for one hour followed by treatment with 0.4 ⁇ vemurafenib for 72 hours. Cells were collected and processed for SRB assay or western blotting.
• Mcl-l overexpression A375 or SK-MEL-28 cells were transiently or stably transfected with plasmid overexpressing Mcl-l by nucleofection kit (Lonza, Basel, Switzerland) according to manufacturer's protocol and previously described by the inventors (26). Briefly, 2 x 10 6 cells were suspended in a reaction mixture from the kit specific to the cell line (Kit V for A375 and Kit R for SK-MEL-28 cells). The cells were transferred to the cuvettes and nucleofected using the nucleofector instrument (Amaxa, Cologne, Germany). To achieve stable overexpression, after transfection, cells were exposed to puromycin with an initial concentration of 1 ⁇ g/ml, which was gradually increased to 5 ⁇ g/ml.
• the resistant colonies were picked by colony picking cylinder and were cultured in presence of puromycin (5 ⁇ g/ml). Stable overexpression of Mcl-l was intermittently tested by western blotting. A375 or SK-MEL-28 cells with stable overexpression of Mcl-l were denoted as A375-Mcl-1+/+ or SK-MEL-28-Mcl-l+/+ respectively.
• Mcl- 1 silencing Silencing of Mcl- 1 was achieved according to the protocol described by the inventors (27). Briefly, 0.3 x 10 6 cells were plated in OPTI-MEM without antibiotics and transfected with either Mcl-l siRNA or scrambled siRNA. Complexes were prepared by incubating lOmM siRNA with 8 ⁇ 1 siPORT transfection reagent in 200 ⁇ 1 OPTI-MEM media without serum or antibiotic for 20 minutes. These complexes were then added to the cells. Six hours after transfection, complexes were replaced with fresh medium.
• mice were injected subcutaneously with 3.5 x 10 6 A375-R cells. When the tumor volume reached to 150 mm 3 , mice were randomly segregated into several groups with 6-7 mice in each group. Vemurafenib, formulated as MBP was suspended at a desired concentration in a vehicle containing 2% Klucel LF and adjusted to pH 4 with HC1, was given at a dose of 25 mg/kg twice a day through oral gavage.
• TW-37 in PBS/ethanol/Tween 80 was administered intraperitonially at a dose of 30 mg/kg thrice a week.
• animals were sacrificed and tumors were excised and fixed in formalin for immunohistochemistry or flash frozen in liquid nitrogen for western blotting analysis as described previously by the inventors (31).
• Vemurafenib treatment induces Mcl-1 expression in melanoma cells.
• the inventors initially performed a cell viability assay to determine the concentration dependent effects of vemurafenib. For this, the inventors used A375 and SK-MEL-28 melanoma cells, both of which harbor BRAF mutation at V600E.
• the IC5 0 of vemurafenib in A375 and SK-MEL-28 cells at 72 hours were 0.1 ⁇ and 0.075 ⁇ respectively (Fig 1A-B).
• A375 and SK-MEL-28 cells were treated with 0.1, 0.2 and 0.4 ⁇ vemurafenib for 72 hours (Fig 1C-D).
• the inventors separated the live and the dead cells after treatment and compared the levels of Mcl- 1 by western blotting. These results showed that there was a diminished expression of Mcl-l in the dead floating cells (Fig. 1E-F). In contrast, the cells that survived upon vemurafenib treatment had significant upregulation of Mcl-l as compared to control cells indicating that expression of Mcl-l perhaps protected the cells from the cytotoxic effects of vemurafenib (Fig. 1E-F).
• Mcl- 1 inhibitor enhances the growth suppressive effects of vemurafenib.
• TW-37 a Mcl-l inhibitor, enhances vemurafenib mediated growth suppression.
• Vemurafenib (0.4 ⁇ ) treatment reduced the cell viability of A375 and SK-MEL-28 by 52% and 55% respectively (Fig. 1G-H).
• TW-37 alone decreased the viability of A375 and SK-MEL-28 cells by 60% and 58% respectively (Fig. 1G- H).
• the combination of vemurafenib and TW-37 treatment reduced the cell survival by 85% and 84%, which was significantly higher than any of the single treatments (Fig. 1G-H).
• Vemurafenib resistant melanoma cells exhibit Mcl-l overexpression.
• the levels of Mcl-l in the cells with vemurafenib resistance was determined.
• the inventors generated A375-R, A375-X/R and SK-MEL-28-R vemurafenib resistant cell lines.
• the resistance in these cell lines was developed by treating the cells with increasing concentrations of vemurafenib over a period of one year according to the data shown in Tables 1 and 2.
• Table 1 shows that the Resistance to vemurafenib in A375 cells was developed by treating the cells with escalating concentrations of vemurafenib. Cells were exposed to two treatments of vemurafenib in a week for 72 hours each. Cytotoxicity of these cells was evaluated intermittently by sulforhodamine B assay.
• Table 2 shows that the resistance to vemurafenib in SK-MEL-28 cells was developed by treating the cells with escalating concentrations of vemurafenib. Cells were exposed to two treatments of vemurafenib in a week for 72 hours each. Cytotoxicity of these cells was evaluated intermittently by sulforhodamine B assay.
• the IC5 0 of vemurafenib in A375R and A375 X/R was 3 ⁇ and 2.15 ⁇ , respectively, and that in SK-MEL-28R was 3.3 ⁇ as compared to the IC 50 of ⁇ . ⁇ ⁇ and 0.075 ⁇ in A375 and SK- MEL-28 parent cell lines (Fig 2 A).
• the inventors achieved 30-40 fold resistance to vemurafenib in these cell lines.
• the resistant cells were absolutely not affected at the concentrations that suppressed more than 60% growth of the sensitive cell lines (Fig 1G-H and 2C).
• western blot results showed a massive increase in Mcl-l expression in vemurafenib resistant cell lines (Fig. 2B).
• the fold increase in Mcl-l expression in A375-R and A375-X/R was 6.2 and 4.83 respectively, and that in SK-MEL-28-R was 10.1 1 as compared to respective sensitive cells (Fig. 2B).
• Mcl-l inhibitor overcomes vemurafenib resistance in melanoma cells.
• Vemurafenib at a concentration of 0.4 ⁇ , exhibited negligible effect on the survival of A375-R cells (Fig. 2C).
• the survival of A375-R cells when treated with TW-37 was decreased by 25% (Fig. 2C).
• the survival of A375-R cells was decreased drastically by 80% (Fig. 2C).
• SK-MEL- 28-R cells vemurafenib showed minimal effect on cell survival but when combined with TW- 37, the cell survival was drastically suppressed by about 80% (Fig. 2C).
• TW-37 treatment alone reduced the growth of SK-MEL-28-R cells by 25% only (Fig. 2C).
• vemurafenib when combined with TW-37 can be attributed to the inhibition of Mcl-l making the cells sensitive to vemurafenib. Similar results were observed in A375-X/R cells (Fig. 2C). The western blot results showed substantial expression of Mcl-l in control and interestingly vemurafenib treatment further increased the expression of Mcl-l in all the resistant cell lines and almost no cleavage of caspase 3 in three cell lines (Fig. 2D). However, vemurafenib in combination with TW-37, which significantly inhibited Mcl-l, substantially increased the cleavage of caspase 3 and PARP indicating apoptosis (Fig. 2D).
• vemurafenib treatment did not have any effect on p-ERK but when combined with TW-37, there was a significant down-regulation of p-ERK (Fig. 2D). These results clearly indicated the role of Mcl- 1 in inducing resistance to vemurafenib in malignant melanoma cells.
• Mcl-l overexpression reduce the sensitivity of melanoma cells to vemurafenib.
• Mcl-l in vemurafenib resistance To further characterize the role of Mcl-l in vemurafenib resistance, the inventors overexpressed Mcl-l in A375 and SK-MEL-28 cells by transfecting the cells with Mcl-l overexpressing plasmid. The survival of A375 cells was reduced by about 55% and 75% and SK-MEL-28 cells by 57% and 67% when treated with 0.2 ⁇ and 0.4 ⁇ vemurafenib respectively (Fig. 3A-B). However, upon Mcl-l overexpression, the effect of vemurafenib was significantly reduced in both the cell lines (Fig. 3A-B).
• A375-Mcl-1+/+ and SK-MEL-28-Mcl-l+/+ cell lines exhibiting stable overexpression of Mcl-l were evaluated. As expected, these cells behaved very similar to the resistant cells when treated with vemurafenib.
• the survival of A375-Mcl-1+/+ cells was reduced by only 20% and 25% when treated with 0.2 ⁇ and 0.4 ⁇ vemurafenib respectively (Fig. 3E).
• Mcl-l was silenced using siR A. Silencing of Mcl-l in all the cell lines was almost 100% (Fig. 4B and 4D). The inventors first tested the effect of Mcl-l silencing in A375-Mcl-1+/+ and SK- MEL-28-Mcl-l+/+ cells, since their response to vemurafenib was analogous to the resistant cell lines (A375-R and SK-MEL-28-R).
• vemurafenib treatment had very minimal effect on A375-Mcl-1+/+ cells and was totally ineffective in SK-MEL-28-Mcl-l+/+ cells (Fig. 4A).
• vemurafenib upon silencing of Mcl-lby siRNA, vemurafenib suppressed the survival of A375-Mcl-1+/+ cells by 73% and that of SK-MEL-28-Mcl-l+/+ cells by 70% (Fig. 4A).
• Mcl-l was silenced and the cells were treated with vemurafenib, apoptosis was induced as depicted by significant cleavage of caspase 3 and PARP (Fig. 4B).
• the inventors determined the effect of vemurafenib in A375-R, A375-X/R and SK-MEL- 28-R cells after silencing Mcl-l as these cells expressed very high levels of Mcl-l .
• About 80- 90% Mcl-l silencing was achieved in all the resistant cell lines.
• Mcl-l silencing there was a significant reduction in cell survival in all the resistant cell lines when treated with vemurafenib.
• Mcl-l suppresses the growth of melanoma tumors resistant to vemurafenib. Although it was very evident from the in vitro results that inhibition of Mcl-l completely reversed the acquired resistance of melanoma cells to vemurafenib, it was important to translate these observations in vivo. As such, A375-R cells, which are completely resistant to vemurafenib, were injected subcutaneously in athymic nude mice. The tumors were allowed to grow till the tumor volume reached 150 mm 3 . The tumor bearing mice were then randomly divided into four groups so that each group had seven mice.
• vemurafenib a combination of vemurafenib with TW-37, which is a Mcl-l inhibitor, was tested.
• the mice received 30mg/kg TW-37 by intraperitonial injection thrice a week and the tumors were also measured thrice a week.
• the average tumor volume of the control group was 1613.5 ⁇ 231.9 mm 3 while that of vemurafenib treated group was 1688 ⁇ 156.19 mm 3 (Fig. 5A).
• the tumors did not respond to vemurafenib treatment at all.
• the tumor volume of the mice that were treated with TW-37 alone was 870 ⁇ 187.8 mm 3 demonstrating a 48% and 46% reduction in tumor growths as compared to the tumor volumes of the mice from control as well as vemurafenib treated group, respectively (Fig. 5A).
• the mice that were treated with a combination of vemurafenib and TW-37 had tumor volumes that were drastically lower than all the three groups (Fig. 5 A).
• the average tumor volumes of these mice at the end of the study were 215.3 ⁇ 51.6 mm 3 , showing an impressive reduction in tumor growth by more than 85% as compared to control or vemurafenib group (Fig. 5A).
• mice from all the groups were sacrificed and the tumors were removed and weighed.
• Fig. 5B there was no difference in the tumor weight of control and vemurafenib treated mice.
• the weight of the tumor in TW-37 treated group was reduced by 47% as compared to control and 48% as compared to vemurafenib treated group (Fig. 5B).
• the tumor weight in the mice treated with TW-37 and vemurafenib was drastically reduced by more than 85%, consistent with tumor volume data.
• Mcl-l silencing suppresses the growth of melanoma tumors resistant to vemurafenib.
• the average tumor volume of the mice that were treated with Mcl-l siRNA alone at the end of the study was 875 ⁇ 134.3 mm 3 , which was significantly lower than that of control and vemurafenib treated mice (Fig. 6A).
• the average tumor volume of the mice treated with scrambled siRNA was 1553 ⁇ 650 mm 3 , showing no significant difference between the average volumes of the tumor of the mice treated with scrambled siRNA, vemurafenib or control mice (Fig. 6A).
• the average volume of the tumors in the mice that were treated with vemurafenib and Mcl-l siRNA was 292 ⁇ 48.12 mm 3 , which once again showed a remarkable suppression of tumor growth by more than 80%, as compared to control or vemurafenib treated group (Fig. 6A).
• the weight of the tumor in Mcl-l siRNA treated group was reduced by 40% as compared to control and 43% as compared to vemurafenib treated group (Fig. 6B).
• the tumor weight in the mice treated with Mcl- 1 siRNA and vemurafenib was drastically reduced by more than 85%, consistent with tumor volume data (Fig. 6B).
• the tumors were examined by western blotting as well as immunohistochemistry.
• the control tumors showed marked expression of Mcl-l (Fig. 5 C-D and Fig. 6 C-D).
• the tumors from vemurafenib treated group had significantly higher expression of Mcl-l than the tumors from control group (Fig. 5 C-D and Fig. 6 C-D).
• the tumors that were treated with either Mcl-l inhibitor (Fig. 5C-D) or Mcl-l siRNA (Fig. 6C-D) had diminished expression of Mcl-l .
• the tumors that were treated with a combination of vemurafenib with either Mcl-l inhibitor or Mcl-l siRNA had significantly lower expression of Mcl-l as compared to the tumors from control or vemurafenib treated group (Fig. 5 C-D and Fig. 6 C-D).
• the inventors also examined the expression of cleaved Caspase 3, cleaved PARP and p-ERK in these tumors. Tumors from control and vemurafenib group showed minimal cleavage of caspase 3 as well as PARP (Fig. 5C-D and Fig. 6 C-D).
• Dabrafenib-Trametinib combination induces Mcl-1 expression in melanoma cells.
• BRAF inhibitor (dabrafenib) and MEKl/2 inhibitor (trametinib) for the treatment of late phase malignant melanoma has been approved by the FDA.
• the inventors determined whether the role of Mcl-1 in drug resistance is only specific to vemurafenib or applicable to other BRAF inhibitors as well.
• the inventors treated the cells with BRAF inhibitors (dabrafenib and vemurafenib) and MEK 1/2 inhibitor (trametinib) alone as well as in combination.
• Dabrafenib-trametinib resistant melanoma cells exhibit overexpression of Mcl-1.
• dabrafenib and trametinib resistant A375, SK-MEL-28 and WM-239 cells were generated that are designated: A375-DR, SK-MEL- 28-DR and WM-239-DR.
• the IC 50 of dabrafenib in A375, SK-MEL-28 and WM-239 was 5nM, 2nM and 6nM respectively, and that of A375-DR, SK-MEL-28-DR and WM-239-DR was greater than 100 nM (Fig. 7C). It was found that dabrafenib resistant melanoma cells also showed massive upregulation of Mcl-1 as compared to the respective wild type cells (Fig. 7D).
• Mcl-1 overexpressing melanoma cells are resistant to combined BRAF inhibitor and MEKl/2 inhibitor treatment.
• Mcl-1 overexpression did not only reduced the efficacy of either BRAF inhibitor or MEKl/2 inhibitor alone but also their combination (Fig. 8 A-D).
• Mcl-1 overexpression did not only reduced the efficacy of either BRAF inhibitor or MEKl/2 inhibitor alone but also their combination (Fig. 8 A-D).
• dabrafenib, trametinib or the combination reduced the survival of A375 and SK-MEL-28 cells by 60-70% (Fig. 8A-B).
• the reduction in cell survival with any of the treatments was at the most 25% (Fig. 8A-B).
• Mcl-1 overexpression completely blocked the effect of dabrafenib (Fig. 8B). Similar results were observed when Mcl-1 overexpressing melanoma cells were treated with a combination of vemurafenib and trametinib (Fig. 8C-D).
• Figures 9A to 9D show that inhibiting Mcl-1 suppresses the growth of melanoma tumors resistance to vemurafenib.
• Figure 9B at day 30, the mice were sacrifice, tumors were extracted and weighed. The values are plotted as mean ⁇ S.D.
• FIG. 9C SK-MEL-28 and Figure 9D shows Sk-MEL-2 cells were treated with 5, 7.5 and 10 ⁇ PL for 48 hours. Cells were lysed, subjected to western blotting and analyzed for Mcl-1 and Cl.PARP. Actin was used as a loading control.
• Mcl-1 Oncogenic Braf V600E in melanoma cells(35). Since BRAF inhibitors were developed to specifically target Braf V600E , theoretically, vemurafenib or dabrafenib should have suppressed the expression of Mcl-1, which was not observed by the present inventors. In fact, the opposite was observed. Not wanting to be bound by theory, it is possible (but not a limitation of the present invention) that the activation of some other pathways post treatment leading to upregulation of Mcl-1. Expression of Mcl-1 is promoted by various transcription factors like STATs, cAMP response elements and NFKB (14).
• Targeting upstream molecules for therapy often fails as the intermediary molecules often get mutated or influenced by cross talk with other pathways resulting in the ineffectiveness of therapy (38-40).
• the present inventors show herein using both in vitro and in vivo studies prove and establish Mcl-1 as the major culprit in inducing resistance to BRAF inhibitors in melanoma. Wild type A375 and SK-MEL-28 cells completely lost sensitivity to BRAF inhibitors alone or in combination with MEK1/2 inhibitors upon Mcl-1 overexpression and transformed them into resistant cells.
• the words “comprising” (and any form of comprising, such as “comprise” and “comprises"), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
• “comprising” may be replaced with “consisting essentially of or “consisting of.
• the phrase “consisting essentially of requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
• the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
• A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
• A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
• expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
• BB BB
• AAA AAA
• AB BBC
• AAABCCCCCC CBBAAA
• CABABB CABABB
• words of approximation such as, without limitation, "about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
• the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
• a numerical value herein that is modified by a word of approximation such as "about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
• Vaux DL Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988;335:440-2.
• Mcl-l is a potential therapeutic target in multiple types of cancer. Cell Mol Life Sci. 2009;66: 1326-36.

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