METHOD FOR SCREENING INHIBITORS TARGETING ANTI-APOPTOTIC SURVIVAL PATHWAYS

Abstract

A method of identifying inhibitors of the anti-apoptotic survival pathway in cancer cells is disclosed. The method comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentratioh for a predetermined period of time and determining cell viability aftet the exposure to the candidate inhibitor; (b) exposing two or more cell lines of specifically MCL-1 or BCL-2 or BCL-X.sub.1 addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor, and (c) indentifying the candidate inhibitor as a MCL-1 or BCL-2 or BCL-X.sub.1 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). The disclosed method provides a way to identify inhibitors which selectively inhibit specific members of the BCL-2 family (e.g., MCL-1) by screening two or more cell lines with addictions to different and specific members of the BCL-2 family of proteins.

Claims

1. A method of identifying MCL-1 or BCL-2 or BCL-X.sub.L inhibitors, the method comprising: (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing two or more cell lines independently addicted to MCL-1 or BCL-2 or BCL-X.sub.L to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a MCL-1 or BCL-2 or BCL-X.sub.L inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b).

2. The method of claim 1, further comprising the step of (d) identifying the candidate inhibitor as a selective inhibitor MCL-1 or BCL-2 or BCL-X.sub.L if the cell viability of one of the cell lines in step (b) is significantly higher than the cell viability of the other cell lines in step (b).

3. The method of claim 1, wherein step (b) comprises exposing the candidate inhibitor to MCL-1 addicted cells and BCL-2 addicted cells.

4. The method of claim 1, wherein step (b) comprises exposing the candidate inhibitor to MCL-1 addicted cells and BCL-X.sub.L addicted cells.

5. The method of claim 1, wherein step (b) comprises exposing the candidate inhibitor to MCL-1, BCL-2, and BCL-X.sub.L addicted cells.

6. The method of claim 1, whrein the candidate inhibitor is identified as a MCL-1 inhibitor if the cell viability in step (a) is significantly higher than said cell viability of the MCL-1 addicted cells in step (b).

7. The method of claim 1, wherein the candidate inhibitor is identified as a selective MCL-1 inhibitor if the cell viability of BCL-2 and/or BCL-X.sub.L addicted cells in step (b) is greater than the cell viability of MCL-1 addicted cells in step (b).

8. The method of claim 1, wherein one of the cell lines in step (b) comprise MCL-1 addicted cells, and wherein the MCL-1 addicted cells express higher levels of MCL-1 and BIM than the wild-type cells.

9. The method of claim 8, wherein the higher levels of MCL-1 and BIM are expressed from a MCL-1-IRES-BIM construct.

10. The method of claim 1, wherein the candidate inhibitor is a small molecule.

11. (canceled)

12. The method of claim 1, wherein the candidate inhibitor is a NOXA mimetic.

13. The method of claim 1, wherein the candidate inhibitor is a BAD mimetic.

14. The method of claim 1, wherein the wild-type cells and addicted cells are independently embryonic fibroblasts.

15. The method of claim 14, wherein the wild-type cells and addicted cells are independently mouse embryonic fibroblasts.

16. The method of claim 1, wherein the wild-type cells and addicted cells are independently human cells.

17. The method of claim 16, wherein the wild-type cells and the addicted cells are independently human cancer cells.

18. A kit comprising: wild type cells; two or more cell lines, wherein each cell line comprises MCL-1 or BCL-2 or BCL-X.sub.L addicted cells; and optionally instructions for performing the method of claim 1.

19. A method of treating a disease in a subject, the method comprising: administering to the subject a therapeutically effective amount of selective MCL-1 inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof.

20. The method of claim 19, wherein the disease is cancer.

21. The method of claim 19, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.

[0038] FIG. 1 shows the system for hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies.

[0039] FIGS. 2A-2B. FIG. 2A depicts selective inhibition of BCL-2/BCL-X.sub.L and MCL-1 by BAD mimetics and NOXA mimetics, respectively. FIG. 2B shows that BAD displaces BIM/PUMA from BCL-2 or BCL-X.sub.L whereas NOXA displaces BIM/PUMA from MCL-1 to activate BAX- and BAK-dependent apoptosis.

[0040] FIGS. 3A-3B. FIG. 3A shows H23 is addicted to MCL-1 for survival because knockdown of MCL-1 induces apoptosis in H23 but not A549 cells. Isogenic H23 cancer cell lines with selective addiction to MCL-1, BCL-2, or BCL-X.sub.L. FIG. 3B shows that selective addiction of engineered H23 cells was confirmed by treating these cell lines with inhibitors of BCL-2 (ABT-737 and ABT-199), BCL-X.sub.L (ABT-737), and MCL-1 (F9). As expected, the BCL-2-addicted H23 cells are sensitive to ABT-737 and ABT-199 but not inhibitors of MCL-1, the BCL-X.sub.L-addicted H23 cells are sensitive to ABT-737 but not ABT-199 or inhibitors of MCL-1, and parental H23 cells are only sensitive to inhibitors of MCL-1. The structure of compound F9 is shown.

[0041] FIG. 4 shows BCL-2 Family: 3 Subfamilies, including the anti-apoptosis (Anti-Death) subfamily of the BCL-2 family, which includes BCL-2, BCL-X.sub.L, MCL-1, A1 (BCL2A1), and BCL-W.

[0042] FIG. 5 shows Death Signals.

[0043] FIG. 6 shows A Two Class Model of BH3-Only Molecules.

[0044] FIG. 7 shows A BAD mimetic or ABT-737/263 displaces BIM/PUMA from BCL-2/BCL-XL to activate BAX/BAK and induce apoptosis.

[0045] FIG. 8 shows a BAD mimetic or ABT-737/263 is not able to displace BIM/PUMA from MCL-1 to Activate BAX/BAK.

[0046] FIG. 9 shows a NOXA mimetic displaces BIM/PUMA from MCL-1 to activate BAX/BAK and induce apoptosis.

[0047] FIG. 10 shows a cell-based screening strategy to identify MCL-1 inhibitors. MEFs expressing MCL-1-IRES-BIM are addicted to MCL-1 for survival whereas wild-type MEFs are not addicted to any single anti-apoptotic BCL-2 member for survival. According, MCL-1 inhibitors, such as NOXA mimetics, can displace BIM from MCL-1 to activate BAX- and BAK-dependent apoptosis in MEFs expressing MCL-1-IRES-BIM but not in wild-type MEFs. Accordingly, wild-type MEFs and MEFs expressing MCL-1-IRES-BIM are subjected to chemical screenings to identify chemicals that induce more apoptosis in MEFs expressing MCL-IRES-BIM than wild-type MEFs. The identified chemicals include MCL-1 inhibitors that disrupt the interaction between MCL-1 and BIM and regulators of MCL-1 expression or protein stability. The same screening strategy can be performed in the isogenic H23 cancer cell lines with selective addiction to MCL-1, BCL-2, or BCL-X.sub.L as shown in FIG. 3B.

[0048] FIG. 11 shows three cell lines (DMS53, SW1417, H82) that were identified as having differential addiction to BCL-2, BCL-XL, and MCL-1. The structure of inhibitor F9 is shown in FIG. 3B.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0049] New therapies based on inhibition of anti-apoptotic proteins are needed, and therefore new methods of screening potential inhibitors are crucially important. In one aspect, the present invention provides methods of engineering cells that mimic the primed cell death state of many cancers with a specific addiction to anti-apoptotic proteins for survival. The engineered cells can be used in methods for screening candidate inhibitors of the anti-apoptotic proteins, as described herein. In another aspect, the present invention provides cell lines and kits for practicing these methods. Once inhibitors of anti-apoptotic proteins are identified, these inhibitors may be used to treat diseases or disorders in a subject. Therefore, in yet another embodiment, the present invention provides methods for treating a disease in a subject using inhibitors of anti-apoptotic proteins. Certain embodiments of several aspects of the present invention follow.

Methods for Screening Inhibitors

[0050] In one aspect, the present invention provides methods of engineering cells that mimic the primed cell death state of many cancers with a specific addiction to anti-apoptotic proteins for survival. In certain embodiments, the anti-apoptotic protein is a member of the BCL-2 family. In certain embodiments, the anti-apoptotic protein is selected from the group consisting of BCL-2, BCL-X.sub.L, and MCL-1. In some embodiments, the method comprises expressing one or more anti-apoptotic BCL-2 member proteins in a cell. In certain embodiments, the BCL-2 member is selected from the group consisting of BCL-2, BCL-X.sub.L, and MCL-1. In certain embodiments, the BCL-2 member is BCL-2. In certain embodiments, the BCL-2 member is BCL-X.sub.L. In certain embodiments, the BCL-2 member is MCL-1. Any method known in the art can be used to express one or more anti-apoptotic BCL-2 member proteins in cells, including, but not limited to, transfection, plasmid-based expression, and viral vector expression. Expression of one or more proteins in the cells may be confirmed using any method known in the art, including, but not limited to, reporter gene assays, western blot, and ELISA (enzyme-linked immunosorbent assay).

[0051] In certain embodiments, the method comprises the steps of (a) expressing different combinations of anti-apoptotic BCL-2 members and activator BH3s in cells; and (b) converting the addiction of a cancer cell line to a specific anti-apoptotic BCL-2 member for survival to another anti-apoptotic BCL-2 member. In certain embodiments, the BCL-2 member expressed in the cell is selected from the group consisting of BCL-2, BCL-X.sub.L, MCL-1, and combinations thereof. In certain embodiments, the cells are fibroblasts. In certain embodiments, the cells are mouse embryonic fibroblasts. In certain embodiments, the activator BH3 is BIM, PUMA or BID. In certain embodiments, the method comprises the steps of (a) expressing different combinations of anti-apoptotic BCL-2 members and activator BH3s such as BIM, PUMA and BID in mouse embryonic fibroblasts; and (b) converting the addiction of a cancer cell line to a specific anti-apoptotic BCL-2 member for survival to another anti-apoptotic BCL-2 member. In certain embodiments, the method comprises the steps of (a) expressing different combinations of BCL-2, BCL-X.sub.L, and MCL-1, and activator BH3s such as BIM, PUMA and BID in mouse embryonic fibroblasts; and (b) converting the addiction of a cancer cell line to specific anti-apoptotic BCL-2 members (e.g., BCL-2 and/or BCL-X.sub.L) for survival to an addiction to MCL-1.

[0052] As described herein, once one or more BCL-2 members are expressed in the cell, the addiction of the cancer cell for survival to another anti-apoptotic BCL-2 member can be effected. For example, in certain embodiments, the mouse embryonic fibroblasts expressing MCL-1-IRES-BIM or MCL-1-IRES-PUMA are addicted to MCL-1 for survival, the mouse embryonic fibroblasts expressing BCL-2-IRES-BIM or BCL-2-IRES-PUMA are addicted to BCL-2 for survival, and the mouse embryonic fibroblasts expressing BCL-X.sub.L-IRES-BIM or BCL-X.sub.L-IRES-PUMA are addicted to BCL-X.sub.L for survival. In some instances, cells (e.g., H23, a K-RAS mutant lung cancer cell line) are dependent on MCL-1 for survival because knockdown of MCL-1 induces robust apoptosis, and its addiction to MCL-1 could be converted to BCL-2 or BCL-X.sub.L addiction by overexpressing BCL-2 or BCL-X.sub.L followed by knockdown of MCL-1. Likewise, in certain embodiments of the invention, addiction to BCL-2 and/or BCL-X.sub.L can be converted to MCL-1 addiction by overexpression of MCL-1 followed by knockdown of BCL-2 and/or BCL-X.sub.L.

[0053] In another aspect, the present invention provides methods of identifying inhibitors of anti-apoptotic survival pathways. In certain embodiments, the anti-apoptotic survival pathway involves overexpression of one or more member of the BCL-2 family (e.g., BCL-2, BCL-X.sub.L, MCL-1). In certain embodiments, the anti-apoptotic survival pathway involves overexpression of MCL-1. In certain embodiments, the anti-apoptotic survival pathway involves overexpression of BCL-2. In certain embodiments, the anti-apoptotic survival pathway involves overexpression of BCL-X.sub.L.

[0054] In certain embodiments, the method of identifying inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing BCL-2 member protein addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a BCL-2 family member inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b).

[0055] In certain embodiments, determining cell viability involves measuring apoptosis. In certain embodiments, determining cell viability involves measuring caspase activity. In certain embodiments, determining cell viability involves measuring cytochrome c release. In certain embodiments, determining cell viability involves measuring cell membrane permeability.

[0056] In certain embodiments, the method of identifying inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing MCL-1- or BCL-2-or BCL-X.sub.L-addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a MCL-1 or BCL-2 or BCL-X.sub.L inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the inhibitors identified by the inventive method are inhibitors of one or more members of the BCL-2 family (e.g., BCL-2, BCL-X.sub.L, MCL-1).

[0057] The methods of identifying inhibitors described herein can be used to identify inhibitors that selectively inhibit a specific BCL-2 family member by employing two or more cell lines independently addicted to different members of the BCL-2 family. In certain embodiments, the method of identifying inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing two or more cell lines independently addicted to MCL-1 or BCL-2 or BCL-X.sub.L to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a MCL-1 or BCL-2 or BCL-X.sub.L inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the inhibitor is identified as a selective inhibitor of a specific BCL-2 family protein if the cell viability of one cell line of specifically addicted BCL-2 cells is significantly higher than the other cell lines of specifically addicted BCL-2 cells employed in step (b).

[0058] In certain embodiments, two cell lines independently and specifically addicted to two different BCL-2 family proteins are employed in step (b). In certain embodiments, two cell lines independently addicted to MCL-1 or BCL-2 or BCL-X.sub.L are employed in step (b). In certain embodiments, one cell line in step (b) is specifically addicted to MCL-1, and the other cell line is specifically addicted to BCL-2. In certain embodiments, one cell line in step (b) is specifically addicted to MCL-1, and the other cell line is specifically addicted to BCL-X.sub.L. In certain embodiments, one cell line in step (b) is specifically addicted to MCL-1, and the other cell line is addicted to BCL-2 and BCL-X.sub.L. In certain embodiments, the candidate inhibitor is identified as a MCL-1 inhibitor if the BCL-2- and/or BCL-X.sub.L-addicted cells show significantly higher cell viability than the MCL-1-addicted cells in step (b).

[0059] In certain embodiments, three cell lines independently and specifically addicted to different BCL-2 family proteins are employed in step (b). In certain embodiments, three cell lines independently addicted to MCL-1, BCL-2, and BCL-X.sub.L are employed in step (b). In certain embodiments, the candidate inhibitor is identified as a MCL-1 inhibitor if the BCL-2- and or BCL-X.sub.L-addicted cells show significantly higher cell viability than the MCL-1-addicted cells in step (b). In certain embodiments, more than three cell lines independently and specifically addicted to different BCL-2 family proteins are employed in step (b). The BCL-2 family proteins may be selected from the group consisting of BAK (BAK1), BAX, parent BCL-2, A1 (BCL2A1), BCL-XL (BCL2L1), BCL-W (BCL2L2), BCL-B (BCL2L10), BCL-RAMBO (BCL2L13), BCL-G (BCL2L14), BOK, and MCL-1.

[0060] As described herein, a candidate inhibitor is identified as a BCL-2 member inhibitor if the cell viability in step (a) of the method is significantly higher than the cell viability in step (b) of the method. In certain embodiments, the cells in step (a) are wild-type cells, the cells in step (b) are MCL-1 addicted cells, and the candidate inhibitor is identified as a MCL-1 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the cells in step (a) are wild-type MEFs, the cells in step (b) are MCL-1 addicted MEFs and the candidate inhibitor is identified as a MCL-1 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b).

[0061] In certain embodiments, the cells in step (a) are wild-type cells, the cells in step (b) are BCL-2 addicted cells, and the candidate inhibitor is identified as a BCL-2 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In some embodiments, the cells in step (a) are wild-type MEFs, the cells in step (b) are BCL-2 addicted MEFs and the candidate inhibitor is identified as a BCL-2 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b).

[0062] In certain embodiments, the cells in step (a) are wild-type cells, the cells in step (b) are BCL-X.sub.L-addicted cells, and the candidate inhibitor is identified as a BCL-X.sub.Linhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the cells in step (a) are wild-type MEFs, the cells in step (b) are BCL-X.sub.L-addicted MEFs and the candidate inhibitor is identified as a BCL-X.sub.L inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b).

[0063] In certain embodiments, the method of screening inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing MCL-1 addicted cells and BCL-2 or BCL-X.sub.L addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a MCL-1 inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the method further comprises the step of (d) identifying the candidate inhibitor as a selective inhibitor of MCL-1 if the cell viability of the BCL-2- or BCL-X.sub.L-addicted cells is significantly higher than the cell viability of the cell viability of the MCL-1-addicted cells in step (b).

[0064] In certain embodiments, the method of screening inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing MCL-1 addicted cells and BCL-2 addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a BCL-2 family inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the method further comprises the step of (d) identifying the candidate inhibitor as a selective inhibitor of MCL-1 if the cell viability of the BCL-2 addicted cells is significantly higher than the cell viability of the cell viability of the MCL-1 addicted cells in step (b).

[0065] In certain embodiments, the method of screening inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing MCL-1-addicted cells and BCL-X.sub.L-addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a BCL-2 family inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the method further comprises the step of (d) identifying the candidate inhibitor as a selective inhibitor of MCL-1 if the cell viability of the BCL-X.sub.L-addicted cells is significantly higher than the cell viability of the cell viability of the MCL-1-addicted cells in step (b).

[0066] In certain embodiments, the method of screening inhibitors comprises the steps of (a) exposing cultured wild-type cells to a candidate inhibitor at a predetermined concentration for a predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; (b) exposing MCL-1-, BCL-X.sub.L-, and BCL-2-addicted cells to the candidate inhibitor at the predetermined concentration for the predetermined period of time and determining cell viability after the exposure to the candidate inhibitor; and (c) identifying the candidate inhibitor as a BCL-2 family inhibitor if the cell viability in step (a) is significantly higher than the cell viability in step (b). In certain embodiments, the method further comprises the step (d) identifying the candidate inhibitor as a selective inhibitor of MCL-1 if the cell viability of the BCL-X.sub.L addicted cells and BCL-2 addicted cells is significantly higher than the cell viability the MCL-1 addicted cells in step (b).

[0067] Cells used in the inventive methods (i.e., both wild-type cells and BCL-2 family member addicted cells) can be any type of cell. In certain embodiments, the wild-type cells are cancer cells. In certain embodiments, the wild-type cells are human cells. In certain embodiments, the addicted cells are cancer cells. In certain embodiments, the addicted cells are human cells. In certain embodiments, the wild-type cells are non-human animal cells. In certain embodiments, the addicted cells are non-human animal cells. In certain embodiments of the method, the wild-type cells are human cancer cells. In certain embodiments, the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are human cancer cells. In certain embodiments, the wild-type cells and the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are human cancer cells. In certain embodiments, the wild-type cells and the MCL-1-addicted cells are human cancer cells. In certain embodiments, the wild-type cells and the BCL-2-addicted cells are human cancer cells. In certain embodiments, the wild-type cells and the BCL-X.sub.L-addicted cells are human cancer cells. In certain embodiments, the wild-type cells are isogenic human cancer cells. In certain embodiments, the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are isogenic human cancer cells. In certain embodiments, the wild-type cells and the MCL-1 addicted cells are isogenic human cancer cells. In certain embodiments, the wild-type cells and the BCL-2 addicted cells are isogenic human cancer cells. In certain embodiments, the wild-type cells and the BCL-X.sub.L addicted cells are isogenic human cancer cells. In certain embodiments of the invention, the wild-type cells are embryonic fibroblasts. In certain embodiments, the MCL-1 or BCL-2 or BCL-X.sub.L addicted cells are embryonic fibroblasts. In certain embodiments, the wild-type cells and MCL-1 or BCL-2 or BCL-X.sub.L addicted cells are embryonic fibroblasts. In certain embodiments, the wild-type cells and MCL-1 addicted cells are embryonic fibroblasts. In certain embodiments, the wild-type cells and BCL-2 addicted cells are embryonic fibroblasts. In certain embodiments, the wild-type cells and BCL-X.sub.L addicted cells are embryonic fibroblasts. In certain embodiments, the embryonic fibroblasts are mouse embryonic fibroblasts (MEFs). In certain embodiments of the invention, the wild-type cells are mouse embryonic fibroblasts. In certain embodiments, the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are mouse embryonic fibroblasts. In certain embodiments, the wild-type cells and MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are mouse embryonic fibroblasts. In certain embodiments, the wild-type cells and MCL-1-addicted cells are mouse embryonic fibroblasts. In certain embodiments, the wild-type cells and BCL-2-addicted cells are mouse embryonic fibroblasts. In certain embodiments, the wild-type cells and BCL-X.sub.L-addicted cells are mouse embryonic fibroblasts. In certain embodiments, the wild-type and/or MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are K-RAS mutant cells. In certain embodiments, the wild-type cells are H23 parental cells. In certain embodiments, the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are H23 parental cells. In certain embodiments, the wild-type and the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells are H23 parental cells. In certain embodiments, BCL-X.sub.L-addicted cells are engineered H23 parental cells. Further examples of cell lines that can be used in the inventive method include, but are not limited to, A427 non-small cell lung cancer, H82 small cell lung cancer (SCLC), and DMS114 SCLC that are addicted to MCL-1, SK-LU-1 lung adenocarcinoma and SW1417 colorectal cancer cell lines that are addicted to BCL-X.sub.L, and DMS53 SCLC that is addicted to BCL-2.

[0068] The candidate inhibitor screened in the inventive method can be any molecular agent. In certain embodiments, the candidate inhibitor is selected from the group consisting of small molecules, proteins, peptides, polymers, and nucleic acids. In certain embodiments, the candidate inhibitor is a protein. In certain embodiments, the candidate inhibitor is a peptide. In certain embodiments, the candidate inhibitor is a polymer. In certain embodiments, the candidate inhibitor is a small molecule. In certain embodiments, the candidate inhibitor is a therapeutic small molecule. In certain embodiments, the candidate inhibitor is a small molecule drug. In certain embodiments, the candidate inhibitor is an organic small molecule. In certain embodiments, the candidate inhibitor is an inorganic molecule. In certain embodiments, the candidate inhibitor is an organometallic molecule. In certain embodiments, the candidate inhibitor is a NOXA mimetic. In some embodiments, the candidate inhibitor down-regulates MCL-1 mRNA or protein. In some embodiments, the candidate inhibitor is a BAD mimetic. In some embodiments, the candidate inhibitor down-regulates BCL-2 mRNA or protein. In some embodiments, the candidate inhibitor is a BAD mimetic. In some embodiments, the candidate inhibitor down-regulates BCL-X.sub.L mRNA or protein.

[0069] The candidate inhibitors may be screened via low-throughput screening (LTS) or high-throughput screening (HTS). In certain embodiments, the inventive method involves LTS of candidate inhibitors. In other embodiments, the method involves HTS of candidate inhibitors. In certain embodiments of the inventive method, cells expressing MCL-1-IRES-BIM or MCL-1-IRES-PUMA are addicted to MCL-1 for survival and can be utilized for HTS for MCL-1 inhibitors. In certain embodiments, the cells expressing BCL-2-IRES-BIM or BCL-2-IRES-PUMA are addicted to BCL-2 for survival and could be utilized for HTS for BCL-2 inhibitors. In certain embodiments, the cells expressing BCL-X.sub.L-IRES-BIM or BCL-X.sub.L-IRES-PUMA are addicted to BCL-X.sub.L for survival and could be utilized for HTS for BCL-X.sub.L inhibitors.

[0070] In certain embodiments, the MCL-1-addicted cells express higher levels of MCL-1 and BIM from a MCL-1-IRES-BIM construct. In certain embodiments, the MCL-1-addicted cells express higher levels of MCL-1 and PUMA from a MCL-1-IRES-PUMA construct. In some embodiments, the BCL-2-addicted cells express higher levels of BCL-2 and BIM from a BCL-2-IRES-BIM construct. In some embodiments, the BCL-2-addicted cells express higher levels of BCL-2 and PUMA from a BCL-2-IRES-PUMA construct. In some embodiments, the BCL-X.sub.L-addicted cells express higher levels of BCL-X.sub.L and BIM from a BCL-X.sub.L-IRES-BIM construct. In some embodiments, the BCL-X.sub.L-addicted cells express higher levels of BCL-X.sub.L and PUMA from a BCL-X.sub.L-IRES-PUMA construct.

[0071] As described herein, cell viability and/or extend of apoptosis can be measured by any method known in the art. Examples of methods for measuring cell viability and/or apoptosis include, but are not limited to, caspase activity assays, cytochrome c release assays, cell membrane permeability assays, fluorescent detection methods (e.g., live/dead cell viability assays), trypan blue assays, ATP tests, calcein AM assays, clonogenic assays, Evans blue assays, fluorescein diacetate hydrolysis/Propidium iodide staining, flow cytometry, formazan-based assays, green fluorescent protein assays, lactate dehydrogenase assays, methyl violet assays, Propidium iodide stain, Resazurin assays, and TUNEL assays.

[0072] In certain embodiments, the cell viability of wild-type cells is considered significantly higher than the cell viability of MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells if the cell viability of the wild-type cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the cell viability of the addicted cells. In certain embodiments, the cell viability of cells addicted to one or more specific BCL-2 members is significantly higher than the cell viability of cells addicted to other BCL-2 members if the cell viability of the first BCL-2 member-addited cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the cell viability of the second BCL-2 member-addicted cells. For example, the cell viability of BCL-2 and/or BCL-X.sub.L addicted cells is significantly higher than the cell viability of MCL-1 addicted cells if the cell viability of the BCL-2 and/or BCL-X.sub.L addicted cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the cell viability of the MCL-1 addicted cells. In certain embodiments, cell viability of one group of cells is significantly higher than cell viability of another group of cells if the cell viability of the first group of cells is more than 1-fold, not less than 2-fold, not less than 5-fold, not less than 10-fold, not less than 30-fold, not less than 100-fold, not less than 1,000-fold, or not less than 10,000-fold greater than the cell viability of the second group of cells.

[0073] In certain embodiments, the extent of apoptosis of MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells is considered significantly higher than the extent of apoptosis of wild-type cells if the extent of apoptosis of the MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than extent of apoptosis of the wild-type cells. In certain embodiments, the extent of apoptosis of cells addicted to one or more specific BCL-2 members is significantly higher than the cell viability of cells addicted to other BCL-2 members if extent of apoptosis of the second BCL-2 member-addited cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the extent of apoptosis of the second BCL-2 member-addicted cells. For example, the extent of apoptosis of MCL-1 addicted cells is significantly higher than the extent of apoptosis of the BCL-2 and/or BCL-X.sub.L addicted cells if the extent of apoptosis the MCL-1 addicted cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the extent of apoptosis the BCL-2 and/or BCL-X.sub.L addicted cells. In certain embodiments, extent of apoptosis of one group of cells is significantly higher than extent of apoptosis of another group of cells if the cell viability of the second group of cells is more than 1-fold, not less than 2-fold, not less than 5-fold, not less than 10-fold, not less than 30-fold, not less than 100-fold, not less than 1,000-fold, or not less than 10,000-fold greater than the extent of apoptosis of the first group of cells.

[0074] In certain embodiments, the cell viability of wild-type cells is considered significantly higher than the cell viability of MCL-1- or BCL-2- or BCL-X.sub.L-addicted cells if the normalized caspase activity of the addicted cells is greater than the normalized caspase activity of the wild-type cells. In certain embodiments, the cell viability of wild-type cells is considered significantly higher than the cell viability of MCL-1 or BCL-2 or BCL-X.sub.L addicted cells if the normalized caspase activity of the addicted cells is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the normalized caspase activity of the wild-type cells. In certain embodiments, the normalized caspase activity of the addicted cells is between 1% and 40% greater than the normalized caspase activity of the wild-type cells. In certain embodiments, the normalized caspase activity of the addicted cells is at least 40% greater than the normalized caspase activity of the wild-type cells Likewise, in certain embodiments, the cell viability of a specific BCL-2 member addicted cell is considered significantly higher than the cell viability of another BCL-2 member addicted cell if the normalized caspase activity of one cell line is at least 1%, 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the other cell line. In certain embodiments, the normalized caspase activity is between 1% and 40% greater than the normalized caspase activity of the wild-type cells. In certain embodiments, the normalized caspase activity is at least 40% greater.

Cell Lines and Kits

[0075] The present invention provides cell lines for carrying out the methods described herein. The cell lines may comprise any of the cells described herein, including wild-type and BCL-2 family protein addicted cells. In certain embodiments, the cell lines comprise any of the wild-type cells described herein. In certain embodiments, the cell lines comprise any of the BCL-2 family addicted cells described herein. In certain embodiments, the cell lines comprise BCL-2, MCL-1 or BCL-X.sub.L addicted cells, or any combination thereof. In certain embodiments, the cell lines comprise MCL-1 addicted cells.

[0076] As described herein, cells used in the inventive methods (i.e., both wild-type cells and BCL-2 family member addicted cells) can be any type of cell. Examples of wild-type cells and BCL-2 member addicted cells include, but are not limited to, human cells (e.g., human cancer cells, isogenic human cancer cells) and non-human animal cells (e.g., embryonic fibroblasts, including, but not limited to, mouse embryonic fibroblasts). In certain embodiments, the cells include H23 parental cells. In certain embodiments, the cells include H23 parental cells engineered to express one or more members of the BCL-2 family (e.g., BCL-2, BCL-X.sub.L, MCL-1)

[0077] Also provided herein are kits comprising cell lines for carrying out the inventive methods. The cell lines can be any of the cell lines described herein, which can comprise any of the cells described as being useful in the inventive methods. In certain embodiments, the kit comprises cell lines comprising wild-type cells and cell lines comprising BCL-2 member protein (e.g., BCL-2, MCL-1, BCL-X.sub.L) addicted cells. In certain embodiments, the kit comprises cell lines comprising wild type cells and cell lines comprising MCL-1 addicted cells. Any of the kits described herein may further comprise instructions for performing or executing the methods described herein. Any of the kits may further comprise one or more candidate inhibitors for screening using the inventive method. In certain embodiments, the kit further comprises control compounds. In certain embodiments, the kit further comprises buffers useful for practicing the inventive method described herein. In certain embodiments, the kit comprises instructions for practicing the inventive method described herein.

Methods for the Treatment of Disorders

[0078] In another aspect, the present invention provides methods for treating diseases or disorders with a BCL-2 family inhibitor. In certain embodiments, the present invention provides methods for treating diseases or disorders with an agent identified by the inventive screening method. In certain embodiments, the disease or disorder is associated with defective apoptosis (e.g., cancer, arthritis, inflammation, lymphoproliferative conditions, inflammatory diseases, and autoimmune diseases). In certain embodiments, the disease is an inflammatory disease, an autoimmune disease, a proliferative disease. In certain embodiments, the disease is a neoplasm or tumor. In certain embodiments, the disease is associated with angiogenesis. In certain embodiments, the disease is cancer In certain embodiments, the method comprises the step of administering to a subject in need thereof an effective amount of an MCL-1 or BCL-2 or BCL-X.sub.L inhibitor, or a pharmaceutical composition thereof. In certain embodiments, the method for treating cancer in a subject comprises administering to a subject in need thereof an effective amount of a MCL-1 inhibitor, or a salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the method for treating cancer in a subject comprises administering to a subject in need thereof an effective amount of a selective MCL-1 inhibitor, or a salt thereof, or a pharmaceutical composition thereof, wherein a selective MCL-1 inhibitor is an inhibitor that targets MCL-1 and not other members of the BCL-2 family (e.g., BCL-2, BCL-X.sub.L).

[0079] In other embodiments, the MCL-1 inhibitor is administered in combination one or more additional agents. In certain embodiments, the additional agent is a therapeutic agent. In certain embodiments, the additional agent is an anti-cancer agent, wherein anti-cancer agentis as defined herein. In some embodiments, the second agent is a BCL-2 or BCL-X.sub.L inhibitor, or a pharmaceutical composition thereof. In some embodiments, the second agent is ABT-737 or ABT-263, or a pharmaceutical composition thereof.

[0080] In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

[0081] Pharmaceutical compositions described herein may comprise one or more MCL-1, BCL-2, or BCL-X.sub.L inhibitors, and optionally a pharmaceutically acceptable excipient. Pharmaceutical compositions described herein may further comprise one or more additional therapeutic agents. Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

[0082] Certain modes for carrying out the invention are presented in terms of exemplary embodiments, discussed herein. However, the application is not limited to the described embodiments and a person skilled in the art will appreciate that many other embodiments of the application are possible without deviating from the basic concept of the application, and that any such work around will also fall under scope of this application. It is envisioned that other styles and configurations of the present application can be easily incorporated into the teachings of the present application, and only one particular configuration shall be shown and described for purposes of clarity and disclosure and not by way of limitation of scope.

EXAMPLES

[0083] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples provided in this application are offered to illustrate the methods, cell lines, and kits provided herein and are not to be construed in any way as limiting their scope.

Development and Empolyment of a Cell-Based Screening Strategy for MCL-1 Inhibitors

[0084] Cellular dependency on BCL-2, BCL-X.sub.L or MCL-1 for survival is governed by the relative abundance among these proteins (FIG. 1). Intricate interplays among the BCL-2 subfamilies govern cellular survival/death, and also provide a molecular blueprint concerning the clinical application of BH3-mimetics in killing cancer cells. Using MEFs that express different combinations of anti-apoptotic BCL-2 members and activator BH3s, a system has been built that can distinguish between the inhibition of BCL-2/BCL-X.sub.L and MCL-1 by BAD/BAD mimetics and MOXA/NOXA mimetics, respectively (FIGS. 2A and 2B). Because NOXA can only displace BIM or PUMA from MCL-1, but not from BCL-2/BCL-X.sub.L to activate BAX/BAK, NOXA selectively induces apoptosis in MCL-1-IRES-BIM or MCL-1-IRES-PUMA but not BCL-X.sub.L-IRES-BIM, BCL-X.sub.L-IRES-PUMA, BCL-2-IRES-BIM, or BCL-2-IRES-PUMA MEFs (FIGS. 2A and 2B).

[0085] In contrast, BAD induces apoptosis in BCL-X.sub.L-IRES-BIM, BCL-X.sub.L-IRES-PUMA, BCL-2-IRES-BIM or BCL-2-IRES-PUMA cells but not MCL-1-IRES-BIM or MCL-1-IRES-PUMA cells because BAD binds to BCL-X.sub.L and BCL-2 but not MCL-1 (FIGS. 2A and 2B). Importantly, this system mimics the primed cell death state of many cancers with abundant pre-assembled complexes of BCL-X.sub.L/BIM, BCL-X.sub.L/PUMA, BCL-2/BIM, BCL-2/PUMA, MCL-1/BIM or MCL-1/PUMA. Conversely, wild-type cells are less sensitive to BAD or NOXA due to the lack of pre-assembled cell death priming complexes (FIG. 2B). Based on these data, NOXA-mimetics will trigger more apoptosis in MCL-1-IRES-BIM than wild-type cells. In addition, compounds that downregulate MCL-1 mRNA and/or protein or induce endogenous NOXA, BIM, or PUMA will trigger similar death patterns.

[0086] Low-throughput screens using the NCI DTP (Developmental Therapeutics Program) Diversity Set 1,900 compounds and the ChemBridge DiverSet A (10,000 compounds) are used to identify compounds that display more than 20% growth-inhibitory effect in MCL-1-IRES-BIM than wild-type MEFs. Compounds are identified that trigger more apoptosis in MCL-1-IRES-BIM or MCL-1-IRES-PUMA than BCL-X.sub.L-IRES-BIM, BCL-X.sub.L-IRES-PUMA, BCL-2-IRES-BIM, BCL-2-IRES-PUMA or wild-type cells.

Identification of MCL-1-Addicted Cancer Cell Lines and Assessment of Small Molecule Inhibitors of MCL-1 Discovered in Pilot Screens

[0087] H23, a K-RAS mutant lung cancer cell line, requires MCL-1 for survival due to its high MCL-1 and low BCL-2/BCL-X.sub.L expression (FIG. 3A). In contrast, knockdown of MCL-1 in A549, another K-RAS mutant lung cancer cell line, induces minimal apoptosis. Nevertheless, knockdown of MCL-1 renders A549 cells susceptible to ABT-737-induced apoptosis because concurrent inhibition of BCL-2, BCL-X.sub.L and MCL-1 is required to activate BAX/BAK in A549 cells.

[0088] Candidate compounds that specifically antagonize MCL-1 will trigger apoptosis in H23 cells as a single agent and synergize with ABT-737 to trigger apoptosis in A549 cells. Of note, the synergistic effect is absent in Bax.sup./Bak.sup./ cells, confirming the activation of BAX/BAK. Since both H23 and A549 cell lines display similar EC50 for paclitaxel, the selective sensitivity of H23 to these compounds is not simply due to a death-prone phenotype of H23 cells.

Characterization of Small Molecule Inhibitors of MCL-1 Identified in Pilot Screens

[0089] There are three potential mechanisms by which compounds can inhibit the pro-survival function of MCL-1. They can (1) directly bind and inhibit the hydrophobic binding groove of MCL-1 as NOXA mimetics; (2) downregulate MCL-1 through transcriptional/translational/post-translational mechanisms; or (3) induce NOXA, BIM or PUMA. In some embodiments, compounds reduce MCL-1 protein by effectively inducing apoptosis in MCL-1-addicted H23 cancer cells. In particular embodiments, a compound will bind to the hydrophobic dimerization pocket of MCL-1. In other embodiments, a compound does not bind to MCL-1 and may reduce MCL-1 protein stability by either inhibiting the deubiquitinases of MCL-1 or activating the E3 ligases of MCL-1.

Establishment of and Performance of Cell-Based High-Throughput Screening to Identify Inhibitors of the MCL-1-Dependent Survival Pathway for Cancer Therapy

[0090] A. Establishment of Isogenic Cancer Cell Lines that are Selectively Cancer Cell Lines that are Selectively Addicted to BCL-2, BCL-X.sub.L or MCL-1 for Mechanistic Studies and High-Throughput Screening for MCL-1 Inhibitors

[0091] Pilot screens using engineered MEFs mimicking primed cell death state of cancers have led to the identification of mechanism-specific compounds. Hence, BCL-2, BCL-X.sub.L or MCL-1 singularly addicted isogenic cancer cell lines are generated to further validate the specificity of hit compounds against MCL-1 and for additional high-throughput screening (HTS).

[0092] K-RAS mutant H23 cell lines have been converted from MCL-1 addiction to BCL-2 or BCL-X.sub.L addiction by overexpressing BCL-2 or BCL-X.sub.L followed by knockdown of MCL-1. The selective addiction of engineered H23 cells was confirmed by treating these cell lines with inhibitors of BCL-2 (ABT-737 and ABT-199), BCL-X.sub.L (ABT-737), and MCL-1 (F9). As expected, the BCL-2-addicted H23 cells are sensitive to ABT-737 and ABT-199 but not inhibitors of MCL-1, the BCL-X.sub.L-addicted H23 cells are sensitive to ABT-737 but not ABT-199 or inhibitors of MCL-1, and parental H23 cells are only sensitive to inhibitors of MCL-1 (FIG. 3B).

[0093] To extend the study to different cancer types harboring distinct driver mutations, the Broad Novartis Cancer Cell Line Encyclopedia was mined to identify cancer cell lines that highly express one anti-apoptotic BCL-2 member and confirm their respective dependency using RNA interference technology. Thus far, A427 non-small cell lung cancer, H82 small cell lung cancer (SCLC), and DMS114 SCLC that are addicted to MCL-1, SK-LU-1 lung adenocarcinoma and SW1417 colorectal cancer cell lines that are addicted to BCL-X.sub.L, and DMS53 SCLC that is addicted to BCL-2 have been identified. The dependency of SK-LU-1 and SW1417 cell lines to BCL-X.sub.L is converted to MCL-1 addiction by overexpression MCL-1 followed by knockdown of BCL-X.sub.L. FIG. 11 shows three cell lines (DMS53, SW1417, H82) that were identified as having differential addiction to BCL-2, BCL-XL, and MCL-1. These cell lines can be employed to determine the specificity of candidate compounds against MCL-1 versus BCL-2 /BCL-X.sub.L. More importantly, the MCL-1-addicted, BCL-2 or BCL-X.sub.L-addicted isogenic cancer cell lines will be utilized for HTS proposed below.

B. Establishment of a Cell-Based High-Throughput Screening Platform to Identify Inhibitors of the MCL-1-Dependent Survival Pathway with Defined Mechanisms of Action

[0094] Pilot screens demonstrated that the proposed assay platform is able to identify mechanism-specific compounds with cellular activity. Herein, low-throughput Alamar Blue assays are adapted to high-throughput Caspase-Glo assays for higher sensitivity and specificity. CellTiter-Glo assays can be alternatives to Caspace-Glo assays for determining cell viability. Parallel screening is performed on wild-type and MCL-1-IRES-BIM expressing MEFs to identify chemicals that selectively induce apoptosis in MCL-1-IRES-BIM but not wild-type cells, which is the same approach as for the low-throughput screens. Parallel HTS is performed using MCL-1- and BCL-X.sub.L-addicted isogenic H23 cancer cells. The BCL-X.sub.L-addicted are chosen over the BCL-2-addicted cell lines as a control based on the fact that both MCL-1 and BCL-X.sub.L bind BAK with high affinity whereas BCL-2 preferentially interacts with BAX. Moreover, a promising selective BCL-2 inhibitor ABT-199 is currently in clinical trials. In contrast, no clinically applicable BCL-X.sub.L-specific inhibitor is available. These screens may also identify BCL-X.sub.L specific inhibitors.

[0095] A library of over 300,000 diverse compounds, and two sets of cell lines, were screened. The first set includes wild-type MEFs and MEFs stably expressing MCL-1-IRES-BIM, which has been used in the pilot screens for the discovery of promising leads. The second set includes H23 parental cell line and the engineered BCL-X.sub.L-addicted H23 cell line. The HTS is performed in 384-well plates and the viability of cells is determined by caspase activity. Accordingly, Caspase-Glo 3/7 assays (Promega) are used to quantify effector caspase activation, which is more specific for apoptosis and at the same time provides a wide dynamic range.

[0096] The HTS assays are optimized by determining the cell seeding density, pre-treatment seeding time, compound treatment time, and DMSO tolerance (vehicle). Compounds are screened at 10 M concentration (0.2% DMSO). The relative caspase activity is expressed as the ratio of the luminescence signal of a compound-treated well minus the luminescence signal of a negative control well (0.2% DMSO) to the luminescence signal of a positive control well (staurosporine) minus the luminesce signal of a negative control. The hit criteria will be based on the relative activity of the sample compound versus intraplate positive (F9 at EC80 concentration) and negative (DMSO only) controls. If CellTiter-Glo assays are employed, the effect of compounds on viability will be expressed as percentage growth inhibition compared to positive (staurosporine) and negative controls (0.2% DMSO) using the following equation: % inhibition=((negative control averageread value of a compound-treated well)/(negative control averagepositive control average))100. A compound will be considered a hit if it induces2-fold growth inhibition in MCL1-IRES-BIM MEFs than WT MEFs. A statistically significant cutoff based on the z-score will be applied and the selected primary hits will be picked and tested in full 11-point concentration response experiments and in parallel secondary assays and analyzed in HPLC-MS for purity and structural integrity.

[0097] The hits identified in both MEFs and H23 cells represent the most specific inhibitors of the MCL-1-dependent survival pathway. Furthermore, BCL-X.sub.L-specific inhibitors may be identified that induce more apoptosis in the BCL-X.sub.L-addicted H23 cells than parental H23 cells.

C. Performance of Secondary Screening to Validate Apoptosis Induction through the Inhibition of MCL-1-Dependent Survival Pathway

[0098] The specificity of hit compounds in MCL-1 inhibition is confirmed by comparing their death-inducing effect in cell lines with selective addiction to MCL-1, BCL-X.sub.L or BCL-2 using annexin-V assays. Cytochrome c translocation, a hallmark of mitochondrial outer membrane permeabilization, is also assessed. Lastly, it is confirmed that these compounds do not have any effect on Mcl-1 KO MEFs.

[0099] It has been confirmed that hit compounds induce apoptosis in MCL-1-IRES-BIM and MCL-1-IRES-PUMA MEFs but not in wild-type, BCL-X.sub.L-IRES-BIM, BCL-X.sub.L-IRES-PUMA, BCL-2-IRES-BIM or BCL-2-IRES-PUMA MEFs. Cell viability is quantified by FACS analysis following annexin-V staining. Hit compounds that induce more apoptosis in MCL-1-addicted MEFs are further assessed for their ability in triggering cytochrome c translocation by immunofluorescence. The same assays are employed to confirm that hit compounds induce apoptosis in MCL-1-addicted but not BCL-2- or BCL-X.sub.L-addicted cancer cells. Along the same lines, it is determined whether the hit compounds synergize with ABT-737 or ABT-263 to induce apoptosis in A549 or wild-type MEFs that are not addicted to a single anti-apoptotic BCL-2 member for survival.

[0100] Finally, it is confirmed that the synergistic effect of hit compounds with ABT-737 or ABT-263 is dependent on MCL-1 using Mcl-1 KO MEFs and on BAX/BAK-dependent apoptosis using Bax.sup./Bak.sup./ MEFs. The activity of hit compounds is further assessed by determining their EC50 in killing H23 cells.

[0101] Additional MCL-1 inhibitors with defined mechanisms of action are identified, which include chemicals that downregulate MCL-1 mRNA, target MCL-1 for degradation or induce endogenous BH3-only proteins. Mcl-1 KO MEFs are instrumental in differentiating NOXA mimetics from chemicals that target MCL-1 for degradation.

D. Characterization of Identified Small Molecule Inhibitors of the MCL-1-Dependent Cancer Cell Survival Pathway

[0102] If hit compounds are NOXA mimetics, they will bind to the hydrophobic dimerization pocket of MCL-1 to displace BIM. First, hit compounds will disrupt the co-immunoprecipitation of MCL-1 and BIM in cells. Second, potential interactions between candidate compounds and MCL-1 are determined using surface plasmon resonance (SPR) assays. The equilibrium dissociation constant (K.sub.D, binding constant) is calculated from the association (k.sub.a, on rate) and dissociation rates (k.sub.d, off rate). An inhibitor of MCL-1 or BIM BH3 peptides will serve as positive controls. Recombinant MCL-1 proteins carrying mutations in the hydrophobic dimerization groove (W261A/G262A/R263A) that disrupt the heterodimerization between MCL-1 and BIM are also be included for comparison. A ProteOn XPR36 instrument (Bio-Rad) is used in these assays.

[0103] Third, the ability of compounds in disrupting the binding of FITC-labeled BIM BH3 peptides from recombinant MCL-1 protein is directly assessed using fluorescence polarization assays (FPA) to determine dissociation constants (K.sub.1). Recombinant BCL-X.sub.L and BCL-2 proteins are included for comparison in both SPR and FPA.

[0104] Bona fide NOXA mimetics are identified that display specific interaction with MCL-1 but not BCL-2 or BCL-X.sub.L. Noteworthy, the binding affinity of NOXA mimetics to truncated MCL-1 protein in vitro may not reflect their interaction in cells.

Equivalents and Scope

[0105] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0106] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0107] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0108] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.