CN116615195A - Combination therapy using BAX activators - Google Patents

Abstract

A pharmaceutical combination is provided comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound, an anti-apoptotic protein inhibiting compound. The present disclosure also provides a method of treating cancer in a subject by administering a B-cell lymphoma 2 associated protein X (BAX) activating compound in combination with an anti-apoptotic protein inhibiting compound (such as BCL-XL, BCL-2, BFL-1, BCL-w, or MCL-1 inhibiting compound).

Description

Combination therapy using BAX activators

Citation of related applications

The present application claims priority from U.S. provisional application serial No. 63/109,097, filed on month 11, 3, 2020, and U.S. provisional application serial No. 63/079,720, filed on month 9, 17, 2020, both of which are incorporated herein by reference in their entireties.

Statement of federal research

The present application was completed with government support under grant No. T32GM007491, awarded by the national institutes of health, and grant No. R01CA178394, F31CA236434, P30CA013330 and 1S10D01630, awarded by the national institutes of health NCI. The government has certain rights in this application.

Background

Apoptosis is a hallmark of cancer. Cancer cells prevent apoptosis to ensure their survival and growth and to develop resistance to current therapies. The intrinsic or mitochondrial pathway of apoptosis is regulated by BCL-2 family proteins, including pro-apoptotic or effector proteins (BAX, BAK and BOK), anti-apoptotic or survivin proteins (e.g., BCL-2, BCL-w, BFL-1, BCL-XL, MCL-1) and pro-apoptotic BH3 proteins classified as activators (e.g., BIM, BID) or sensitizers (e.g., BAD, HRK). Typically, cancer cells up-regulate anti-apoptotic BCL-2 family members to inhibit pro-apoptotic BCL-2 members BAX, BAK and BH 3-only proteins, thereby preventing apoptosis. More resistant cancers also down-regulate or inactivate pro-apoptotic BH3 proteins to inhibit apoptosis, rendering these tumors less sensitive to current treatments.

Pro-apoptotic BAX is an effector of mitochondrial apoptosis induced by most BH3 mimics and chemotherapeutic agents. Typically, under pro-apoptotic stimuli, BH 3-only proteins use their BH3 domain helices to trigger BAX activation, resulting in translocation and oligomerization of BAX at the Mitochondrial Outer Membrane (MOM). This results in the release of MOM permeabilization (MOMP) and pro-apoptosis (apotagenes), such as cytochrome c and Smack/Diablo, which activate the apoptotic caspase cascade. Elucidation of the BAX trigger site for helical binding of the BH3 domain to induce BAX activation enables the discovery of direct small molecule BAX activators that bind to (engage) the trigger site and mimic BH 3-only proteins, thereby inducing full conformational activation and apoptosis of BAX.

Direct activation of BAX using targeting small molecules can prevent down-regulation of the activator BH 3-only protein in cancer. However, there remains a need to develop additional and improved direct BAX activators, especially for apoptosis refractory tumors.

Disclosure of Invention

The present disclosure provides a pharmaceutical combination comprising: b-cell lymphoma 2 associated protein X (BAX) activating compounds; and anti-apoptotic protein inhibiting compounds such as B-cell lymphoma 2-oversized protein (BCL-XL) inhibiting compounds, B-cell lymphoma 2 (BCL-2) inhibiting compounds, B-cell lymphoma 2-like protein (BCL-w) inhibiting compounds, myeloid leukemia 1 (MCL-1) inhibiting compounds, BFL-1 inhibiting compounds, or BCL-B inhibiting compounds.

In a pharmaceutical combination, the BAX activating compound is a compound having the structure of BTSA1 or BTSA1.2 or a pharmaceutically acceptable salt thereof.

The present disclosure also provides a method of treating cancer in a subject in need thereof, the method comprising: obtaining a biological sample comprising cancer cells from a subject; detection of BAX immunoprecipitated from cancer cells: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: the level of MCL-1 complex, and/or detecting whether a cancer cell is anti-apoptotic BCL-XL, BCL-2, BCL-w, BFL-1, or MCL-1 dependent, or not triggering apoptosis; and administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound, and a B-cell lymphoma oversized protein (BCL-XL) inhibiting compound, or a B-cell lymphoma 2 (BCL-2) inhibiting compound, or a cell lymphoma 2-like (BCL-w) inhibiting compound, or a myeloid leukemia 1 (MCL-1) inhibiting compound, in an amount effective to treat the cancer.

The present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: obtaining a biological sample from a subject; measuring the expression level of at least one gene in the biological sample, wherein the gene comprises MUC13, EPS8L3, IGFBP7, or a combination thereof; and administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptotic protein inhibiting compound. The method may comprise comparing the expression level of the MUC13, EPS8L3 or IGFBP7 gene or a combination thereof in the biological sample to a standard expression level of any of these genes and administering an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptotic protein inhibiting compound if the expression level of the gene in the biological sample is higher than the standard expression level of the gene. MUC13, EPS8L3, IGFBP7 markers were identified using the BTSA 1.2/Navitocrax (Navitocrax) combination. Naviotok is considered a BCL-XL and BCL-2 inhibitor. In certain embodiments, the anti-apoptotic inhibiting compound is a B-cell lymphoma 2 inhibiting compound, or preferably is a B-cell lymphoma oversized protein (BCL-XL) inhibiting compound.

The above described and other features are exemplified by the following figures and detailed description.

Drawings

The following figures are exemplary embodiments.

Fig. 1A to 1K. Resistance to BAX activation and BCL-XL inhibition is regulated by BCL-XL upregulation and non-priming states. Fig. 1A: different sets of cancer cell lines (n=46) were treated with BTSA1.2 for 72 hours. Box plots correspond to tissue type mean cell viability IC ₅₀ mu.M, cell lines were classified as sensitive (IC ₅₀ <3. Mu.M) or resistance (IC ₅₀ >3 μm). FIG. 1B, correlation of sensitivity to BTSA1.2 with BAX and BCL-XL related protein levels using the pearson correlation method. Relevant protein levels were normalized to b-actin loading control and p-values were calculated using student t-test. Fig. 1C: BAX translocation after 4 hours of treatment with BTSA1.2 in BxPC-3 cells. Fig. 1D: BAX co-immunoprecipitation (co-IP) was performed after 4 hours of treatment with BTSA1.2 in BxPC-3 cells. Data represent n=3 independent experiments. Fig. 1E: different cancer cell collections (n=46) were treated with natatoric for 72 hours. Box plots correspond to tissue type mean cell viability IC ₅₀ mu.M, cell lines were classified as sensitive (IC ₅₀ <1.5. Mu.M) or resistance (IC ₅₀ >1.5 μm). Fig. 1F: sensitivity to navitock and BCL-XL and BAX using pearson correlation: correlation of BCL-XL related protein levels. Normalization of relevant protein levels to b-actin loading control P-values were calculated using student t-test. Fig. 1G: the heat map of% mitochondrial depolarization of 20 cancer cell lines indicates that these cancer cell lines are classified as different apoptosis blocks based on BH3 profiling (BH 3 profiling) methods. Fig. 1H to 1I: BH3 profiling predicts the apoptosis block associated with resistance to (H) BTSA1.2 and (I) naviatoxin. Fig. 1J: the Venn plot compares the resistance of cell lines to BTSA1.2 and naviatoxin as single agents. Fig. 1K: the schematic illustrates a therapeutic strategy for combination therapy of BAX activator (BTSA 1.2) and BCLXL inhibitor (navitock) to enhance apoptotic cell death. The data in fig. 1G and 1J are the average of three replicates of n=2 independent experiments.

FIGS. 1L-1Q. BTSA1.2, analogs of BTSA1, improved binding to BAX, cellular activity and targeted adapter activity (target engagement activity). FIGS. 1L-1O: IC for post-treatment lymphoma cell lines with BTSA1 or BTSA1.2 ₅₀ A curve. Fig. 1P: cell thermal shift assay (CETSA) of BAX melting curves in BxPC-3 cells treated with vehicle (DMSO) or 40. Mu.M BTSA1.2 for 15 min. The blots represent three independent experiments. Fig. 1Q: the data in fig. 1P were quantified by fluorescence intensity using LiCor Odyssey Clx and normalized to generate a melting curve. Data are mean ± SD of n=3 experiments.

Fig. 2A to 2H. BTSA1.2 and naviatoka cooperate to inhibit cell viability and induce apoptosis in resistant tumor cell lines. Fig. 2A: navioc and BTSA1.2 (1.25. Mu.M or 5. Mu.M) screening. Fig. 2B: with Navinotok and constant sensitization concentration of BTSA1.2 (loss of cell viability<20%) cell viability IC of the cancer cell line group (n=46) for 72 hours of combination treatment ₅₀ Bar graph of fold change (μm). The red bar graph corresponds to an IC ₅₀ Fold change>5x; the green bar graph corresponds to an IC ₅₀ Fold change 2-4x; and the gray bar corresponds to the IC ₅₀ Fold change<2x. Predicting that cells are sensitive to the combination (IC ₅₀ Fold change>5 x), with moderate sensitivity (IC) ₅₀ Fold change 2-4 x) or resistance (IC ₅₀ Fold change<2 x). Fig. 2C: the mutant status of TP53 and RAS in cancer cell lines is classified as sensitive or resistant to the combination. Fig. 2D: in the presence of different doses of BTSA1.2, in the group of resistant cancer cell lines (white bloodDisease = U937, colon = SW480, pancreas = BxPC-3, NSCLC = Calu-6), n = 3. Fig. 2E: briss synergy scoring heat maps for combination treatment of BTSA1.2 and naviatox in different cancer tissue types in b, n=3. Fig. 2F: caspase 3/7 activity, n=3, in different cancer cell lines treated with BTSA1.2 and naviatogram, alone or in combination, measured at 8 hours. Fig. 2G: WT and CRISPR/Cas9 BAX KO Calu-6 cell lines treated with Navigator alone in the presence of a fixed sensitization concentration of BTSA1.2 showed cell viability (loss of viability <10%). Comparison of BAX and BAK protein expression levels in the indicated cell lines, n=3. Fig. 2H: caspase 3/7 activity (loss of viability) in WT and CRISPR/Cas9BAX KO Calu-6 cell lines after 8 hours of treatment with Navinotor alone and in combination with a fixed sensitization concentration of BTSA1.2<10%), n=3. Statistical data were obtained using two-factor analysis of variance: * P, p<0.05；**，p<0.01；***，p<0.001；****，p<0.0001。

FIGS. 2I-2J. Fig. 2I: cell viability at 72 hours of WT and CRISPR/Cas9BAX KO Calu-6 cell lines treated with different doses of staurosporine. Fig. 2J: caspase 3/7 activity in WT and CRISPR/Cas9BAX CO Calu-6 cell lines were treated with different doses of staurosporine for 24 hours. Data are ± SD of four technical replicates of n=3 independent experiments.

Fig. 3A to 3J. The interaction of BAX with BCL-XL determines the sensitivity to the combination of BTSA1.2 and naviatogram. Fig. 3A: BH3 profiling predicts apoptosis retardation associated with the sensitivity of the combination of BTSA1.2 and naviatoka. FIGS. 3B-3C (FIG. 3B) immunoblot analysis of BAX Co-IP in NSCLC and colorectal cell sets (FIG. 3C). Fig. 3D: quantification of co-immunoprecipitated BAX with BCL-XL in the group of solid tumor cell lines grouped between sensitivity to the BTSA1.2 and the naviatogram combination (fig. 3B-C). Fig. 3E-3F: immunoblot analysis of BAX IP of NSCLC cancer cell line Calu-6 (fig. 3E) and colorectal cancer cell line SW480 (fig. 3F) after 4 hours of treatment with BTSA1.2 and naviatoka. Fig. 3G-3H: cleaved caspase-3 apoptosis markers detected by immunoblot analysis in (3G) NSCLC cancer cell line Calu6 and (fig. 3H) colorectal cancer cell line SW480 after 4 hours of treatment with BTSA1.2 and naviatoka. Fig. 3I: schematic of sensitive cells of the combination of BTSA1.2 and naviatoka. Data represent three independent experiments. Fig. 3J: apoptosis initiation using the activator BIM BH3 peptide was increased in sensitive cell lines following combination therapy, but not in resistant cells.

Fig. 4A to 4G. The combination of BTSA1.2 and navicular is well tolerated and does not potentiate the toxicity of the navicular drive in the hematopoietic system. Fig. 4A: schematic of a BTSA1.2 and naviatoxin combination toxicity study. Fig. 4B: body weight measurements on CD1-IGS mice were performed 0, 3, 7, 11 and 14 days after the first treatment with vehicle, 100mg/kg of Naviotor, 200mg/kg of BTSA1.2 or a combination. Fig. 4C-4F: counts of peripheral (C) erythrocytes, (D) leukocytes, (E) lymphocytes, and (F) platelets in CD1-IGS mice treated with vehicle, 100mg/kg of Navinotor, 200mg/kg of BTSA1.2, or a combination after 1 day and 7 days of treatment. The normal blood count range of CD-IGS male mice is indicated in gray. The data (fig. 4B-4F) represent mean ± SD (vehicle, BTSA1.2, and naviatoxin=5, combination n=6). Scale bar, 100 μm. Statistical data were obtained using student's t-test: * P <0.05; * P <0.01; * P <0.001; * P <0.0001. Fig. 4G: representative tissue sections of spleen, bone marrow, heart, liver, brain, lung and kidney of mice stained with hematoxylin and eosin (H & E) following vehicle, 100mg/kg of navitole, 200mg/kg of BTSA1.2 or combination therapy.

Fig. 5A to 5I. Combination treatment of BTSA1.2 and naviatoxin showed strong efficacy in resistant colorectal tumor xenografts. Fig. 5A: schematic of SW480 xenograft efficacy study. Fig. 5B: nu/Nu mice body weight at 0, 7 and last day were measured with vehicle, 100mg/kg of Naviotor, 200mg/kg of BTSA1.2 or combination treatment. Fig. 5C: tumor volume curves for vehicle, naftopsides, BTSA1.2 or combination cohort. Fig. 5D: tumor weight after completion of the study. (B-D) data represent mean ± SD (vehicle, BTSA1.2 and naviatoxin=5, combination n=6). Fig. 5E: schematic of SW480 pharmacodynamic xenograft study. Fig. 5F: examples of kinetics curves of mitochondrial potential in tumors treated with vehicle or combination under BH3-BIM peptide, puma2A, CCCP, or promethacin stimulation. Fig. 5G: tumor dynamic BH3 profile of mice treated with vehicle, or a combination of natalcok and BTSA 1.2. Bar graphs represent tumor cell mitochondrial depolarization of JC-1 detected after BH3-BIM derived peptide or DMSO treatment (vehicle n=2; combination n=3). Fig. 5H-5I, cleaved caspase-3 and cleaved PARP apoptosis marker in SW480 tumors were detected by immunoblot analysis, n=3. Relevant protein levels were normalized to b-actin loading control. Statistical data were obtained using one-way analysis of variance: * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

Fig. 6A to 6L. The predictive markers identify tumors that are sensitive to the combination treatment of BTSA1.2 and naviatoxin. Fig. 6A: schematic representation of tumor features predictive of clinical susceptibility by BH3 profiling and BAX co-IP. Fig. 6B: BH3 spectra of colorectal PDX. Heat maps represent the% of mitochondrial depolarization of isolated tumor cells detected by JC-1 following BH 3-derived peptide treatment, n=3. Fig. 6C: quantification of co-immunoprecipitated BAX with BCL-XL in colorectal PDX. Fig. 6D: cell viability of COLO-1PDX isolated cells after 24 hours of treatment with 1.25 μm of that valtolk, 10 μm of BTSA1.2 or combination, n=3. Fig. 6E: schematic of COLO-1PDX efficacy study. Fig. 6F: body weight measurements on NOD SCID mice at 0, 6 and last day were performed with vehicle, 50mg/kg of Navizodone, 200mg/kg of BTSA1.2 or combination treatment. Fig. 6G: tumor volume curves for vehicle, naftopsides, BTSA1.2 or combination cohort. The data in fig. 6F-6G represent single measurements (vehicle n=9, btsa1.2, navitock and combination n=12). Fig. 6H: survival of COLO-1PDX after 18 days of treatment with vehicle, 50mg/kg of Navinotor, 200mg/kg of BTSA1.2 or combination, n=8. Fig. 6I: dynamic BH3 profile of COLO-1 tumors in mice treated with vehicle, or a combination of natalck and BTSA 1.2. Bar graphs represent% mitochondrial depolarization of tumor cells detected by JC-1 after BH3-BIM, BH3-BID, or Puma 2A-derived peptide treatment, n=2. Fig. 6J: schematic of COLO-2PDX efficacy studies. Fig. 6K: tumor volume curves for vehicle, naftopsides, BTSA1.2 or combination cohort. Fig. 6L: the levels of BCL-XL protein were significantly increased in mice tumors treated with BTSA1.2 or Navinotor, while the MCL-1 level remained constant. The data represent individual measurements (vehicle n=5, btsa1.2, navitock and combination n=8). Statistical data were obtained using one-way analysis of variance: * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

Fig. 7A to 7E. Bioinformatics analysis predicts markers of sensitivity and resistance to the combination of BTSA1.2 and naviatoxin. Fig. 7A: volcanic diagrams show their ICs combined from navitock to BTSA1.2 and navitock alone ₅₀ The expression changes and significance levels of genes between the defined sensitive and resistant cell line groups were varied (corresponding to fig. 2B). The first 250 predicted susceptibility (red) and resistance (gray) markers are highlighted. Fig. 7B: in cell lines classified as sensitive or resistant to the combination, optimal markers (top hits) associated with sensitive and resistant to the combination were verified by RT-qPCR. Related gene expression was normalized using RPL 27. Fig. 7C: using the pearson correlation method, the BCL2L1 (corresponding to BCL-XL protein) related gene expression levels and MUC13 gene expression levels correlated in cell lines classified as sensitive or resistant to the combination (corresponding to fig. 2B). Fig. 7D: correlation of MUC13 expression with sensitivity to combination (corresponding to FIG. 2B). Fig. 7E: the expression data of MUC13 cancer patients used other non-redundant data in TCGA and cbioport. Statistical data were obtained using student's t-test: * P, p<0.05；**，p<0.01；***，p<0.001；****，p<0.0001。

Fig. 8A to 8B. BTSA1.2 is an improved analog of BTSA1, active in a diverse collection of human cancer cell lines. Fig. 8A: BTSA1 and BTSA1.2 structures. Fig. 8B: competitive fluorescence polarization assay with BTSA1 and BTSA1.2 competing for BIM BH3 binding to BAX. Data represent n=2 independent experiments.

Fig. 9A-9B. BTSA1.2 activity in different collections of human cancer cell lines. Fig. 9A: cell viability curves of different cell lines after 72 hours of BTSA1.2 treatment. Fig. 9B: cell viability IC arranged by sensitivity ₅₀ Bar chart of (. Mu.M), red IC ₅₀ <3. Mu.M; orange 3<IC ₅₀ <10. Mu.M; yellow IC ₅₀ >10. Mu.M. Data are mean ± SD of three technical replicates of n=2 independent experiments.

Fig. 10A to 10D. Correlation analysis of BCL-2 family protein expression levels and BTSA1.2 activity in solid tumor and hematological cancer cell lines. FIG. 10A) protein expression levels of key BCL-2 family members detected by Licor. Beta-actin served as a loading control. Fig. 10B-10D: sensitivity to BTSA1.2 and (fig. 10B) MCL-1, BCL-2 using pearson correlation; (FIG. 10C) BIM, BAK; and (fig. 10D) BAX: correlation of BCL-XL related protein levels. Related protein levels were first normalized to β -actin loading control and p-values were calculated using student t-test. Data represent n=2 independent experiments.

Fig. 11A to 11G. BCL-XL regulates BAX activation through BTSA1.2 resistance. Fig. 11A: quantification of translocation BAX after 4 hours of treatment with BTSA1.2 in BxPC-3 cell line (associated with figure 1C). Fig. 11B: BAX translocation after 4 hours of treatment with BTSA1.2 in SW40 cell line. Fig. 1C: BAX translocation 18 hours after treatment with BTSA1.2 in BxPC-3 cell line. Fig. 11D: after 4 hours of treatment with BTSA1.2 in BxPC-3 cell line, co-immunoprecipitated BAX was quantified with anti-apoptotic BCL-XL and MCL-1 (FIG. 1D). Fig. 11E: BAX co-IP of mitochondria and cytosol after treatment with 10 μm BTSA1.2 for 4 hours in BxPC-3 cell line. Fig. 11E: BAX co-IP of mitochondria and cytosol after treatment with 10 μm BTSA1.2 for 4 hours in BxPC-3 cell line. Data represent at least n=3 independent experiments. Immunoblot analysis of BAX Co-IP in the group of NSCLC (fig. 11F) and colorectal cells (fig. 11G). Data represent n=3 independent experiments.

Fig. 12A to 12C. Analysis of the activity of and correlation of the activity of naviatoxin in different sets of human cancer cell lines. Fig. 12A: cell viability curve of cell lines after 72 hours of treatment with natatorium. Fig. 12B: cell viability IC arranged by sensitivity ₅₀ Bar chart of (. Mu.M), red IC ₅₀ <1.5. Mu.M; orange 1.5<IC ₅₀ <10. Mu.M; yellow IC ₅₀ >10. Mu.M. Correlation of sensitivity to Navigator with MCL-1, BCL-2, BIM, BAK and BAX related protein levels using the Pearson correlation method. Fig. 12C: related protein levels were first normalized to β -actin loading control and p-values were calculated using student t-test. Three technical replicates for independent experiments with data of at least n=2Is + -SD of (C).

Fig. 13A to 13D. BH3 spectra of solid tumors and hematological cancer cell lines. Fig. 13A: % mitochondrial depolarization after BH 3-derived peptide-only treatment. Fig. 13B: BH3 profiling predicts apoptosis retardation associated with BTSA1.2 sensitivity. Fig. 13C: BH3 profiling predicts apoptosis retardation associated with that of naviatogram sensitivity. Fig. 13D: BH3 profiling predicts apoptosis blockade associated with BTSA1.2 and naviatogram resistance.

Fig. 14A to 14C. BTSA1.2 and naviatoxin combine to inhibit cell viability of resistant tumor cell lines. Fig. 14A: a hematologic cell line; fig. 14B: NSCLC, colorectal, melanoma, and ovarian cell lines; and fig. 14C: pancreatic, breast and HNCC cell lines. Viability curve of cell lines treated with natalcok in combination with a constant sensitization concentration (induced viability loss of < 20%) of BTSA 1.2. Data are ± SD of three technical replicates of at least n=2 independent experiments.

Fig. 15A to 15B. BTSA1.2 and naviatoka cooperate to inhibit cell viability of resistant tumor cell lines. Fig. 15A: dose-response curves for single agent, navitock, in the resistant cancer cell line group from various tissue types (colon=dld1, pancreas=mia PaCa-2, lymphoma=su-DHL-5) in the presence of different doses of BTSA 1.2. The effect on cell viability was measured by CellTiter-Glo 72 hours after treatment, n=3. Fig. 15B: bliss synergy scoring heatmap for combination treatment of BTSA1.2 and Navinotor in different cancer tissue types. Data are ± SD of three technical replicates of at least n=3 independent experiments.

Fig. 16A to 16G. Pharmacokinetic and maximum tolerated dose analysis of BTSA 1.2. Fig. 16A: after a dose of 3mg/kg of BTSA1.2 administered perioral (p.o.), the concentration of BTSA1.2 (ng/mL) in the plasma of the mice. Fig. 16B: after intravenous (i.v.) administration of 1mg/kg of BTSA1.2 mg/kg, the concentration of BTSA1.2 (ng/mL) in the plasma of mice was measured. Fig. 16C: BTSA1.2 is well tolerated in vivo: schematic of MTD and toxicity studies of BTSA 1.2. Increased concentrations of BTSA1.2 were orally administered daily to CD-IGS female and male mice for 5 days. Body weight and blood cell count were measured on the indicated dates. Mice were sacrificed 14 days after the first treatment and organs were collected for pathological analysis, n=6. Fig. 16D: body weight measurement of CD1-IGS mice following treatment with vehicle or BTSA 1.2. Fig. 16E: representative tissue sections of spleen, heart, liver, lung and kidney of mice stained with hematoxylin and eosin (H & E) following vehicle or BTSA1.2 treatment. Scale bar, 100 μm. Fig. 16F-16G: peripheral white blood cell count (FIG. 16F) and neutrophil count (FIG. 16G) in CD1-IGS mice treated with vehicle, 200mg/kg BTSA1 or 200mg/kg BTSA1.2 at day 0 and 2 post-treatment. The compounds are administered orally. The normal blood count range of CD-IGS male mice is indicated in gray. Data are ± SD of n=3 mice. Statistical data were obtained using student t-test (student t-test): * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

Fig. 17A to 17E. The combination therapy of BTSA1.2 and navicular is well tolerated, does not potentiate the toxicity of the navicular drive to the hematopoietic system, and triggers tumor apoptosis in vivo. Fig. 17A-17C: combination treatment of BTSA1.2 and naviatoka was well tolerated in vivo: counts of peripheral (17A) erythrocytes, (17B) platelets and (17C) leukocytes of CD1-IGS mice treated with vehicle, 100mg/kg of Navizodone, 200mg/kg of BTSA1.2 or combination at days 0, 1, 2, 7 and 12 post-treatment. The data in fig. 17A-17C represent mean ± SD (vehicle, BTSA1.2, and naviatoxin=5, combination n=6). Statistical data for these groups were obtained using one-way anova: * P <0.05; * P <0.01; * P <0.001; * P <0.0001. Fig. 17D-17E: dynamic BH3 profile of tumors in mice treated with vehicle, or a combination of natalcok and BTSA 1.2. Fig. 17D: examples of kinetic profiles of mitochondrial potential in tumors treated with vehicle or combination under stimulation with BH3-BID peptide, puma2A, CCCP or promethacin. Data represent mean ± SD of three replicates of n=2 independent experiments. Fig. 17E: bar graph shows mitochondrial depolarization of tumor cells detected by JC-1 after BH 3-BID-derived peptide or DMSO treatment, vehicle n=2; combination n=3. Statistical data for this group was obtained using one-way anova: * P <0.05; * P <0.01; * P <0.001; * P <0.0001.

Fig. 18A to 18F. Navik and BTSA1.2 combined sensitive predictive markers. Fig. 18A:BH3 spectra of two colorectal PDXs. Bar graphs represent the% of mitochondrial depolarization of isolated tumor cells detected by JC-1 following BH 3-derived peptide treatment. Data represent mean ± SD of triplicates of n=3 mice. Fig. 18B: immunoblot analysis of BAX Co-IP in colorectal PDX tumors. Fig. 18C: cell viability of COLO-1 and COLO-2PDX isolated cells after 24 hours of treatment with either nataker or BTSA1.2, n=3. Fig. 18D: cell viability of COLO-2PDX isolated cells after 24 hours of treatment with 10 μm of that valtolk, 10 μm of BTSA1.2 or combination, n=3. Fig. 18E: the heat map shows an IC based on a combination from that of novatok alone to that of BTSA1.2 and that of novatok ₅₀ The first 150 (selected by the adjusted p-value) differentially expressed genes of the resistant and sensitive cell lines were compared for changes. Fig. 18F: optimal genes (top hits) associated with sensitivity and resistance to combinations were verified by RT-qPCR in cell lines classified as sensitive or resistant to combinations (corresponding to fig. 2A). Related gene expression was normalized using RPL 27.

Fig. 19A and 19B. In resistant AML cell lines, cell viability was a function of the combination of BAX activator (BTSA 1) and BCL-2 inhibitor (Venetoclax). Fig. 19A shows the synergistic effect of BTSA1 and valnemulin combination in THP-1 cells. Fig. 19B shows the synergistic effect of BTSA1 and valnemulin combination in OCI-AML3 cells.

Fig. 20. In PDX, 10 primary patient AML samples were treated with single agents and in combination with valnemulin. In fig. 20, the percentage of transplantation (measured as a percentage change in hcd45+ cell number) is plotted as a function of time (weeks) for 10 AML tumor cell samples established from patient-derived xenografts (PDX). PDX mice were treated daily for 3 weeks with vehicle alone, ABT-199 (valnemulin), BTSA1 alone, or a combination of valnemulin and BTSA1. Although both the valnemulin and the BTSA 1/valnemulin combinations reduced the percentage of implantation, the post-treatment effect remained sustained only in animals administered the BTSA 1/valnemulin combination.

Fig. 21A and 21B. The combination of BTSA1 and valnemulin accelerates the induction and efficacy of apoptosis. Figure 21A shows increased apoptosis as measured by the caspase 3/7 activation assay of BTSA1, vinatorac and BTSA 1/vinatorac combinations. Figure 21B immunoblots show increased BAX activation by the BTSA 1/valnemtock combination.

Figure 22 shows the whole blood count (CBC) and percentages of several blood cell types in mice treated with vehicle, valnemulin, BAX activator (BTSA 1) and the valnemulin/BTSA 1 combination.

Fig. 23A and 23B. Leukemia cell (OCI-AML 3) viability treated with valnemulin alone, or a combination of BTSA1.2 and valnemulin. Fig. 23A: vitamin c or vitamin c+1.25 μm BTSA1.2, fig. 23B: vitamin c or vitamin c+2.5 μm BTSA1.2.

Fig. 24A and 24B. Viability of HL60 and ML2 leukemia cells treated with either natalcok alone or natalcok+1.25 μm BTSA 1.2. Fig. 24A: HL60 cells; fig. 24B: ML2 cells.

Fig. 25A and 25B. Viability of SU-DHL-4 and SU-DHL-5 lymphoma cells treated with either nadir alone or a combination of nadir plus 0.500 μm BTSA 1.2. Fig. 25A: SU-DHL-4 cells; fig. 25B: SU-DHL-5 cells.

Fig. 26A-26E. BTSA1.2 and BCL-XL selective inhibitor a1331852 cooperate to inhibit cell viability in tumor cell lines sensitive to the combination of naviatoxin/BTSA 1.2. Fig. 26A and 26B: dose-response curves for BCL-XL selective inhibitor a1331852 or BCL-2 selective inhibitor vinatoxin in the presence of different doses of BTSA1.2 in cancer cell lines sensitive (SW 480) or resistant (COLO-320) to the combination of that vinatoxin/BTSA 1.2. The effect on cell viability was measured by CellTiter-Glo 72 hours after treatment. Bliss synergy scoring heat maps for combination treatments. Data are mean ± SD of three technical replicates of n=3 independent experiments. Fig. 26C and 26D: dose-response curves for BCL-XL selective inhibitor a1331852 or BCL-2 selective inhibitor valnemtock in the presence of different doses of BTSA1.2 in OCI-AML3 or U937 hematology cell lines sensitive to the combination of natatock/BTSA 1.2. The effect on cell viability was measured by CellTiter-Glo 72 hours after treatment. Bliss synergy scoring heat maps for combination treatments. Data are mean ± SD of three technical replicates of n=3 independent experiments. Fig. 26E: comparison of BCL-2 family protein levels with sensitivity to BTSA1.2 and naviatoxin combinations: protein expression levels are correlated with the combination. Correlation of sensitivity to the combination of BTSA1.2 and Navigator with MCL-1, BCL-XL, BCL-2, BIM, BAK and BAX related protein levels using the pearson correlation method. Related protein levels were first normalized to b-actin loading control and p-values were calculated using student t-test. Data represent at least n=2 independent experiments.

Detailed Description

Disclosed herein are pharmaceutical combinations of a B-cell lymphoma 2 associated protein X (BAX) activating compound and a B-cell lymphoma ultra-large protein (BCL-XL) inhibiting compound, a B-cell lymphoma 2 (BCL-2) inhibiting compound, a myeloid leukemia 1 (MCL-1) inhibiting compound, a BCL-B inhibiting compound, or a BFL-1 inhibiting compound. (BFL-1 is a BCL-1 related protein that is first recognized in fetal liver.) also disclosed herein are methods of treating cancer in a subject comprising administering to the subject a BAX activating compound in combination with an anti-apoptotic protein inhibiting compound, such as a BCL-XL inhibiting compound, a B cell lymphoma 2 (BCL-2) inhibiting compound, or a myeloid leukemia 1 (MCL-1) inhibiting compound, in an amount effective to treat cancer in the subject. When an anti-apoptotic protein inhibiting compound (such as a BCL-XL, BCL-2, or MCL-1 inhibiting compound) is administered with a BAX activating compound, cancer cell death is increased compared to the BAX activating compound or anti-apoptotic protein inhibitor compound alone (such as a BCL-XL, BCL-2, or MCL-1 inhibitor compound). The present disclosure also relates to methods of treating a patient by first determining the sensitivity of a patient's cancer to a combination therapy of a BAX activating compound and an anti-apoptotic inhibiting compound, and treating the patient if the patient's cancer is determined to be sensitive to a therapy of a BAX activating/(BCL-XL, BCL-2, or MCL-1) inhibiting combination.

The antitumor activity of chemotherapeutic and targeted drugs is a result of their induction of cancer cell apoptosis. Cancer cells inhibit apoptosis by various mechanisms to promote survival and proliferation, and thus, treatment of cancer refractory to various treatments with a single therapeutic agent often results in moderate to weak antitumor activity due to ineffective induction of apoptosis. In particular, the release of the anti-apoptotic BCL-2 family interaction network ensures the regulation of apoptosis resistance by cancer, which is a significant challenge for current therapies. For purposes of this discussion, the "BCL-2 family" includes anti-apoptotic proteins BCL-XL, BCL-2, BCL-w, BFL-1, BCL-B and MCL-1. Cancer cells typically evade apoptosis by upregulating BCL-2 anti-apoptotic proteins. More resistant cancers also down-regulate or inactivate pro-apoptotic BH3 proteins to inhibit apoptosis.

Given the key role of anti-apoptotic BCL-2 proteins in cancer cell apoptosis resistance, and the interactions between BCL-2 family members, selective drugs have been designed to inhibit anti-apoptotic BCL-2 proteins, known as BH 3-mimetics. Selective inhibitors of these anti-apoptotic BCL-2 proteins (e.g., vitamin Nanetock (CAS registry number 1257044-40-8), nanetock (CAS registry number 923564-51-6), S63845 (CAS registry number 1799633-27-4), S64315 (also MIK665, CAS registry number 1799631-75-6) and AMG176 (Amgen, CAS registry number 1883727-34-1)), induce apoptosis primarily by releasing BH 3-only proteins (e.g., BIM and BID) from the anti-apoptotic BCL-2 protein to activate BAX and BAK sequentially. In preclinical and clinical studies, BH3 mimics show significant efficacy in tumors when cell viability is highly dependent on targeting anti-apoptotic BCL-2 family proteins. However, these molecules show limited single agent activity in many cancers, especially in solid tumors that rely on or up-regulate additional non-targeted anti-apoptotic BCL-2 family proteins to ensure survival. Thus, the full potential of BH3 mimetics to induce tumor apoptosis is to be determined by using rational and safe combination therapies to help overcome the resistance mechanisms to apoptosis and identify predictive biomarkers for accurate treatment.

Direct activation of BAX with targeting small molecules is likely to overcome the barrier to down-regulation of the activator BH 3-only protein in cancer. Based on the understanding that functional BAX, which contains an inactive cytoplasmic conformation in cancer cells, and that BAX is rarely mutated or not expressed, the inventors focused on developing specific BAX activators as a therapeutic strategy for cancer. As a result of these studies, it has been found that apoptosis resistance is mediated by an uninduced apoptotic state and over-expression of BCL-XL, limiting direct and indirect activation of pro-apoptotic BAX, in the scope of various hematological malignancies and solid tumors. The pharmacological combination of BAX activation and BCL-XL inhibition overcomes these survival mechanisms. In addition, functional assays and genomic markers have been identified to predict tumor susceptibility to combination therapy. The published findings facilitate an understanding of the mechanism of apoptosis resistance and demonstrate a novel therapeutic strategy for direct BAX activation and BCL-XL inhibition combinations as cancer therapies.

In various aspects, it has surprisingly been found that the combination of the orally bioavailable BAX activator BTSA1.2 and the clinical BCL-XL inhibitor, navitolg, exhibits synergistic efficacy in protecting healthy tissues while at the same time, in apoptosis resistant cancer cells, xenografts and patient-derived tumors.

As used herein, "BAX" refers to a BCL-2 related X protein. BAX is a mammalian protein, and in some aspects a human protein.

BAX activating compounds are compounds that activate cytosolic BAX and/or mitochondrial BAX. BAX activation plays a role in initiating apoptosis. The pharmaceutical composition comprises a BAX activating compound in an amount effective to activate BAX in the cell.

In some aspects, the BAX activating compound is BTSA1 or a pharmaceutically acceptable salt thereof.

In some aspects, the BAX activating compound is BTSA1.2 or a pharmaceutically acceptable salt thereof.

The BAX activating compound may be a compound of formula a, wherein formula a has the structure

Wherein the method comprises the steps of

A is N or CH;

b isWherein->Represents a point of attachment to the backbone;

x is CH or N;

y is O, S, NH, CO, CS or-ch=x;

R ¹ 、R ² 、R ⁴ and R is ⁵ H, F, cl, br, I, OH, SH, NO independently ₂ 、CF ₃ 、COOH、COOR ⁶ 、CHO、CN、NH ₂ 、SO ₄ H、SO ₂ NH ₂ 、NHNH ₂ 、ONH ₂ NHC＝(O)NNH ₂ 、NHC＝(O)NH ₂ 、NHC＝(O)H、NHC(O)-OH、NHOH、OCF ₃ 、OCHF ₂ 、NHR ⁶ 、NHCONH ₂ 、NHCONHR ⁶ 、NHCOR ⁶ 、OCR ⁶ 、COH、COR ⁶ 、CH ₂ R ⁶ 、CH ₂ R ⁶ 、CONH ₂ 、CON(R ⁶ R ⁷ )、CH＝N＝OR ⁶ 、CH＝NR ⁶ 、OR ⁶ 、SR ⁶ 、SOR ⁶ 、SO ₂ R ⁶ 、CH ₂ N(R ⁶ R ⁷ )、N(R ⁶ R ⁷ ) Or optionally substituted lower (C ₁ -C ₄ ) Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, wherein the optional substituents are F, CF ₃ 、Cl、Br、I、OH、SH、NO ₂ 、R ₆ 、COOH、COOR ⁶ 、CHO、CN、NH ₂ 、NHR ⁶ 、NHCONH ₂ 、NHCONHR ₆ 、NHCOR ⁶ 、NHSO ₂ R ⁶ 、OCR ⁶ 、COR ⁶ 、CH ₂ R ⁶ 、CON(R ⁶ R ⁷ )、CH＝N-OR ⁶ 、CH＝NR ⁶ 、OR ⁶ 、SR ⁶ 、SOR ⁶ 、SO ₂ R ⁶ 、COOR ⁶ 、CH ₂ N(R ⁶ R ⁷ ) Or N (R) ⁶ R ⁷ ) One or more of (a) and (b);or (b)

R ¹ And R is ² May form a cyclic, heterocyclic, aryl or heteroaryl ring, wherein the aryl or heteroaryl ring is optionally substituted with OH, CO ₂ H or SO ₂ NH ₂ Substitution;

R ³ and R is ¹⁰ H, F, CF independently ₃ 、Cl、Br、I、OH、SH、CF ₃ 、NO ₂ 、R ₆ 、COOH、COOR ⁶ 、CHO、CN、NH ₂ 、SO ₄ H、NHNH ₂ 、ONH ₂ 、NHC＝(O)NHNH ₂ 、NHC＝(O)NH ₂ 、NHC＝(O)H、NHC(O)-OH、NHOH、OCF ₃ 、OCHF ₂ 、NHR ⁶ 、NHCONH ₂ 、NHCONHR ₆ 、NHCOR ⁶ 、NHSO ₂ R ⁶ 、OR ⁶ 、SR ⁶ 、SOR ⁶ 、SO ₂ R ⁶ 、COOR ⁶ 、CH ₂ N(R ⁶ R ⁷ )、N(R ⁶ 、R ⁷ ) Lower (C) ₁ -C ₄ ) Alkyl, alkenyl or alkynyl;

R ⁶ and R is ⁷ H, C independently ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl, C ₃ -C ₆ Cycloalkyl, C ₁ -C ₆ Alkoxy, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Thioalkoxy or C ₁ -C ₆ A thiohaloalkoxy group; or a pharmaceutically acceptable salt thereof.

Another example of BAX activating compounds are those disclosed in U.S.2020/0093802, the entire contents of which are incorporated herein by reference. Any BAX activating compound disclosed in U.S.2020/0093802, or a combination thereof, may be included as a BAX activating compound in the combinations disclosed herein.

As used herein, "BCL-XL" refers to B-cell lymphoma oversized proteins. "BCL-2" is a B cell lymphoma 2 protein. "MCL-1" is a myeloid leukemia 1 protein. BCL-XL, BCL-2 and MCL-2 are mammalian proteins and in some aspects human proteins. BCL-XL, BCL-2 or MCL-1 proteins are anti-apoptotic proteins that play a role in inhibiting apoptosis by a number of different mechanisms, one of which is inhibition of BAX. In some aspects, a BCL-XL, BCL-2, or MCL-1 inhibiting compound is a compound that binds to an anti-apoptotic protein, such as a BCL-XL, BCL-2, or MCL-11 protein, thereby inhibiting its function in apoptosis. The BCL-XL, BCL-2 or MCL-1 inhibiting compound may be any compound capable of inhibiting the functions of BCL-XL, BCL-2 and MCL-1, in particular anti-apoptotic functions, in a cell. The pharmaceutical composition comprises an anti-apoptotic protein inhibiting compound (such as a BCL-XL, BCL-2 or MCL-1 inhibiting compound) in an amount effective to inhibit the anti-apoptotic activity of an anti-apoptotic protein (such as BCL-XL, BCL-2 or MCL-1) in the cell.

Non-limiting examples of BCL-XL inhibiting compounds include that of nadotok, a1331852, a1155463, pharmaceutically acceptable salts thereof, or combinations thereof. In some aspects, the BCL-XL inhibiting compound is naviatogram, also known as ABT-263 or 4- (4- { [2- (4-chlorophenyl) -5, 5-dimethyl-1-cyclohexen-1-yl ] methyl } -1-piperazinyl) -N- [ (4- { [ (2R) -4- (4-morpholinyl) -1- (phenylsulfanyl) -2-butanyl ] amino } -3- [ (trifluoromethyl) sulfonyl ] phenyl) sulfonyl) benzamide. BCL-XL inhibitors can be used alone or conjugated to antibodies capable of targeting specific cell types. The BCL-XL inhibitor can be linked to an E3 ligase ligand to form a BCL-XL PROTAC degradation agent, e.g., DT2216, which can lead to BCL-XL protein degradation. (Khan, s., et al, nature Medicine, (2019) 25:1938-1947.)

Other BCL-XL inhibitors useful in the combinations of the present disclosure include AZD0466 (a drug dendrimer (dendrimer) conjugate comprising the dual BCL2/XL inhibitor AZD-4320), AZD-4320 (Astra Zeneca), ABBV-155 (Abbvie) and APG-1252 (Ascentage Pharma), DT2216 (Dialectic Therapeutics).

As used herein, an anti-apoptotic protein is an anti-apoptotic protein of the BCL-2 protein family, examples of which include BCL-XL, BCL-2, BCL-w, BFL-1 or MCL-1. Anti-apoptotic protein inhibiting compounds include ABT-737 from Abbvie (CAS accession number 852808-04-9) that binds with high affinity (< 1 mol/L) to BCL-2, BCL-XL and BCL-w anti-apoptotic proteins; ABT-263 (navic) (CAS accession number 923564-51-6) from Abbvie, which binds with high affinity to BCL-2, BCL-XL and BCL-w anti-apoptotic proteins; ABT-199 (valnemotok) from Abbvie (CAS accession number 1257044-40-8), which is highly specific for BCL-2, is approved for the treatment of hematological cancers including Chronic Lymphocytic Lymphomas (CLL) (including recurrent and refractory CLL), small lymphomas (SLL), and Acute Myelogenous Leukemia (AML), and is also useful for the treatment of solid tumors; AMG-176 from Amgen (CAS registry number 1883727-34-1, an MCL-1 inhibitor) for the treatment of Multiple Myeloma (MM); AMG 397 (CAS registry number 2245848-05-7); AZD-4320 from Astra Zeneca (CAS registry number 1357576-48-7, a BCL-2 and BCL-XL inhibitor) for use in the treatment of lymphomas: AZD-0466 (a BCL-2 and BCL-XL inhibitor) from Astra Zeneca and Starcharm, a conjugate of AZD-4320 and Starcharm dendrimer, for use in the treatment of advanced solid tumours, lymphomas and multiple myeloma; VU661013 (CAS registry number 2131184-57-9, an MCL-1 inhibitor) from Vanderbilt and Boehringer Ingelheim; s65487 from Servier and Novartis (a BCL-2 inhibitor) for the treatment of acute myelogenous leukemia, multiple myeloma and non-hodgkin' S lymphoma; s64315 (MIK 665) (CAS accession No. 1799631-75-6, an MCL-1 inhibitor) from Servier and Novartis for the treatment of multiple myeloma, non-hodgkin lymphoma and multiple myeloma; and APG1252 (pellcitoclax) (CAS registry number 1619923-36-2, a BCL-2, BCL-XL, and BCL-w inhibitor) for the treatment of Small Cell Lung Cancer (SCLC) and other solid tumors.

The present disclosure also includes combinations of BAX activators and MCL-1 inhibitors, such as AMG-176 (Amgen), AZD5991 (Astra Zeneca), S64315 (MIK 665) and VU661013 (Vanderbilt University).

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As disclosed herein, a combination of a BAX activating compound and an anti-apoptotic protein inhibitor, such as BCL-XL, BCL-2, BCL-w, BFL-1 or MCL-1 inhibitor, induces apoptosis in cancer cells (solid and hematologic cancers). In some aspects, the combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound produces a synergistic therapeutic effect. In other words, the combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound (such as a BCL-XL, BCL-2, BCL-w, BFL-1 or MCL-1 inhibiting compound) results in an improved therapeutic effect compared to either compound alone. Preferably, the combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound allows for therapeutic effects at lower doses of the anti-apoptotic protein inhibiting compound than in the absence of the BAX activating compound.

In some aspects, the combination is a pharmaceutical combination. The "pharmaceutical combination" may be a single pharmaceutical composition containing the BAX activating compound and the anti-apoptotic protein inhibitor, or may be separate pharmaceutical compositions that independently contain the BAX activating compound or the anti-apoptotic protein inhibitor and are sold or packaged together.

BAX activating compounds and anti-apoptotic protein inhibiting compounds may be administered in the form of a composition comprising the compound and a pharmaceutically acceptable carrier. In particular, the compounds disclosed herein are administered in the form of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The compounds and compositions can be administered to a subject using any known route of administration. For example, it may be applied to a specific site, either systemically or locally. Routes of administration include, but are not limited to, oral, rectal, sublingual, buccal, intravenous, intramuscular, transdermal, dermal, subcutaneous, intrathecal, nasal, vaginal, or combinations thereof. In some aspects, the route of administration is oral.

The compounds and compositions are administered to a subject, particularly a subject suffering from cancer. The subject is a mammalian subject. For example, the mammalian subject may be a human, rodent, monkey, cat, dog, bovine (cow, beef cow, bull), sheep, monkey, or primate. In some aspects, the mammalian subject is a human.

Disclosed herein are methods of treating cancer in a subject comprising administering to the subject a combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound in an amount effective to treat cancer in the subject. In some aspects, the cancer is a hematologic cancer or a solid tumor.

The cancer may be breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, melanoma, malignant melanoma, ovarian cancer (ovarian cancer), brain or spinal cord cancer, primary brain epithelial cancer, medulloblastoma, neuroblastoma, glioma, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, stomach cancer, renal cancer, placental cancer, gastrointestinal cancer, renal cancer, cancer of the brain or spinal cord, glioblastoma, hepatoma, bladder cancer, gastric cancer, renal cancer, placental cancer, and cervical cancer Small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck epithelial cancer, breast epithelial cancer, endocrine cancer, eye cancer, genitourinary cancer, vulvar cancer, ovarian cancer (cancer), uterus or cervical cancer hematopoietic cancer, myeloma, leukemia, lymphoma, ovarian epithelial cancer, lung epithelial cancer, renal cell carcinoma, cervical epithelial cancer, testicular epithelial cancer, bladder epithelial cancer, pancreatic epithelial cancer, and the like gastric epithelial cancer, colonic epithelial cancer, prostate epithelial cancer, genitourinary epithelial cancer, thyroid epithelial cancer, esophageal epithelial cancer, myeloma, multiple myeloma, adrenal epithelial cancer, renal cell epithelial cancer, endometrial epithelial cancer, adrenal cortical epithelial cancer, malignant pancreatic insulinoma, malignant carcinoid epithelial cancer, chorionic epithelial cancer, mycosis mycotica, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, kaposi's sarcoma, polycythemia vera, primary thrombocythemia, hodgkin's disease, non-hodgkin's lymphoma, soft tissue carcinoma, soft tissue sarcomas, osteosarcomas, sarcomas, primary macroglobulinemia, central nervous system cancers, retinoblastomas, or combinations thereof. In some aspects, the cancer is colon cancer, rectal cancer, or colorectal cancer.

In certain embodiments, the cancer may be a hematologic cancer, such as non-hodgkin's lymphoma, multiple myeloma, acute myelogenous leukemia, small lymphocytic lymphoma, chronic lymphocytic lymphoma (including recurrent and/or refractory chronic lymphocytic lymphoma).

Also disclosed herein are methods of determining that a cancer is sensitive or resistant to a combination treatment with a BAX activating compound and a BCL-XL inhibiting compound. Advantageously, functional assays and genomic markers have been found that can be used to predict that a given cancer is sensitive or resistant to combination therapy. The detection includes quantitative reverse transcription PCR to determine the level of the gene messenger RNA in the biological sample.

As disclosed herein, anti-apoptotic protein dependent or non-apoptosis inducing cancer cells are sensitive to combination therapies of BAX activating compounds and anti-apoptotic protein inhibiting compounds. Thus, methods comprising determining whether cancer cells in a subject are anti-apoptotic protein dependent or non-apoptotic triggering can be used to determine whether cancer in a subject with cancer will respond to treatment after administration of a combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound. BH3 profiling can be used to determine whether cancer cells are anti-apoptotic protein dependent or not triggering apoptosis. In some aspects, BH3 profiling comprises contacting a cancer cell with a BH3 domain peptide, measuring the amount of BH3 domain peptide-induced mitochondrial depolarization in the cancer cell, and comparing the amount of BH3 domain peptide-induced mitochondrial outer membrane permeabilization in the cancer cell to a population of control cells of the same type (i.e., non-cancer cells).

Surprisingly, it was also found that sensitivity of cancer cells to compound combination therapy with immunoprecipitation forms BAX: there is a correlation between the BCL-XL complexes. In particular, it has been found that cancer cells that are sensitive to a combination of compounds form higher levels of BAX than cell lines that are resistant to a combination of compounds: BCL-XL complex. Given the sensitivity of certain cancer cells to other anti-apoptotic protein inhibitors (such as BCL-2, bfl-2, and MCL-1), the sensitivity of cancer to specific BAX activator/anti-apoptotic protein inhibitor combination therapies is expected to be combined with immunoprecipitation detected BAX: there is a correlation between the formation of anti-apoptotic protein complexes. For example, BAX detected by immunoprecipitation: BCL-2 complex is understood to be predictive of the susceptibility of cancer to BAX activator/BCL-2 inhibitor combination therapy.

In some aspects, a method of determining the sensitivity or resistance of a cancer to treatment with an anti-cancer agent comprising a BAX activating compound in combination with an anti-apoptotic protein inhibiting compound, the method comprising obtaining a biological sample from a subject having cancer and detecting BAX immunoprecipitated from cancer cells: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: the level of MCL-1 complex, and/or detection of cancer cells is anti-apoptotic protein dependent or non-apoptosis inducing. Biological samples from a subject include cancer cells.

Detection that cancer cells are anti-apoptotic protein dependent or that do not trigger apoptosis includes BH3 profiling of cancer cells. BH3 profiling involves contacting cancer cells with a BH3 domain peptide, and measuring the amount of BH3 domain peptide-induced mitochondrial depolarization in the cancer cells, and comparing to a control cell population of the same type.

BAX: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: MCL-1 complexes are formed by co-immunoprecipitation of BAX and anti-apoptotic proteins from cancer cells and quantified (e.g., immunoblots and banding) using known methods to determine the expression of the relevant proteins. The method further comprises BAX in the cancer cell when compared to a normal cell of the same type: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: when the level of MCL-1 complex is increased, cancer cells are determined to be sensitive to anticancer agents.

A method of treating cancer in a subject in need thereof, comprising: obtaining a biological sample comprising cancer cells from a subject; detection of BAX immunoprecipitated from cancer cells: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: the level of MCL-1 complex, and/or detecting that the cancer cell is anti-apoptotic protein dependent or non-apoptotic; and administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptotic protein inhibiting compound (such as B-cell lymphoma oversized protein (BC 2-XL), BCL-2, BCL-w, BFL-1, or MCL-1 inhibiting compound) in an amount effective to treat the cancer. In some aspects, the method further comprises BAX in the cancer cell when compared to a normal cell of the same type: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: when the level of MCL-1 complex is increased, cancer cells are determined to be sensitive to anticancer agents.

Analysis of the expression of various genetic markers indicated that genes highly expressed in sensitive cancer cell lines include MUC13, EPS8L3 and IGFBP7, and genes highly expressed in resistant cancer cell lines include NR4A3, IRF4 and SLC7A3.

The MUC13 gene encodes the protein mucin-13, an epithelial and hematopoietic transmembrane mucin. The EPS8L3 gene encodes the epidermal growth factor receptor kinase substrate 8-like protein 3. The IGFBP7 gene encodes insulin-like growth factor binding protein 7. The NR4A3 gene encodes a member 3 protein of group A of nuclear receptor subfamily 4 (a transcriptional activator). The IRF4 gene encodes interferon regulatory factor 4 (a transcriptional activator). The SLC7A3 gene encodes a cationic amino acid transporter 3 that mediates uptake of arginine, lysine and ornithine in a sodium independent manner.

In some aspects, a method of determining the sensitivity or resistance of a cancer to treatment with an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound in combination with an anti-apoptotic protein inhibiting compound, such as B-cell lymphoma oversized protein (BCL-XL), BCL-2, BCL-w, BFL-1, or MCL-1 inhibiting compound, the method comprising obtaining a biological sample from a subject having the cancer; detecting the expression level of a gene in a biological sample, wherein the gene comprises MUC13, EPS8L3, IGFBP7, NR4A3, IRF4, SLC7A3, or a combination thereof; and determining the sensitivity or resistance of the cancer to an anticancer agent. In certain embodiments, the anti-apoptotic protein inhibiting compound is BCL-XL and the detected gene is MUC13, EPS8L3 or IGFBP7. When the expression level of the genes MUC13, EPS8L3, IGFBP7, or a combination thereof in a biological sample is increased compared to a control sample, it can be determined that the cancer cells are sensitive to a combination of a BAX activating compound and an anti-apoptotic protein inhibiting compound. When the expression level of the genes NR4A3, IRF4, SLC7A3 or a combination thereof in a biological sample is increased compared to a control sample, the combined resistance of cancer cells to the BAX activating compound and the anti-apoptotic protein inhibiting compound can be determined.

A method of treating cancer in a subject in need thereof, comprising: obtaining a biological sample from a subject; measuring the expression level of at least one gene in the biological sample, wherein the gene comprises MUC13, EPS8L3, IGFBP7, or a combination thereof; and administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptotic protein inhibiting compound in an amount effective to treat the cancer. The method further comprises determining that the expression level of the genes MUC13, EPS8L3, IGFBP7, or a combination thereof in the biological sample is increased compared to a control sample prior to administering the sample. In some aspects, the biological sample comprises cancer cells and the control sample comprises normal cells of the same type. The detection includes quantitative reverse transcription PCR to determine the level of the gene messenger RNA in the biological sample.

A "pharmaceutical composition" is a composition comprising an active agent and at least one other substance (such as an excipient). The excipient may be a carrier, filler, diluent, filler or other inactive or inert ingredient. The pharmaceutical composition optionally comprises one or more additional active agents. If specified, the pharmaceutical composition meets the GMP (good manufacturing practice) standard of the United states FDA for human or non-human drugs.

By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient or carrier, other ingredients, or combination of ingredients, alone or together, that provides a carrier or vehicle with which one or more compounds of the invention are formulated and/or administered, each of which is pharmaceutically acceptable as a whole. Also included are any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

By "pharmaceutically acceptable salts" is meant those salts which retain the biological effectiveness and properties of a given compound and are biologically or otherwise desirable. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. By way of example only, salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di (substituted alkyl) amines, tri (substituted alkyl) amines, alkenylamines, dienylamine, trialkenylamine, substituted alkenylamines, di (substituted alkenyl) amines, tri (substituted alkenyl) amines, cycloalkylamines, di (cycloalkyl) amines, tri (cycloalkyl) amines, substituted cycloalkylamines, di-substituted cycloalkylamines, tri-substituted cycloalkylamines, cycloalkenyl amines, di (cycloalkenyl) amines, tri (cycloalkenyl) amines, substituted cycloalkenyl amines, di-substituted cycloalkenyl amines, tri-substituted cycloalkenyl amines, aryl amines, diarylamines, triarylamines, tri-heteroaryl amines, heterocyclic amines, diheterocyclic amines, tri-heterocyclic amines, mixed diamines and tri-amines (wherein at least two substituents on the amines are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkenyl, aryl, heteroaryl, and the like). Also included are amines in which two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl.

Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (such as amines); a base or organic salt of an acidic residue (such as a carboxylic acid); etc. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts and quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like); from organic acids (such as acetic acid, propionic acidSuccinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid (pamoic), maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC- (CH) ₂ ) _n -COOH (wherein n is 0-4), etc.).

As used herein, "Treating" includes providing a compound disclosed herein as the sole active agent or sufficient, together with at least one additional active agent: (a) inhibiting cancer, i.e., arresting its development; (b) alleviating the disease, i.e., causing regression of the cancer.

An "effective amount" of an active ingredient or pharmaceutical composition/combination comprising an active ingredient is an amount effective to provide a therapeutic benefit when administered to a subject.

Throughout this disclosure and in the claims, the open transition phrase "comprising" includes the intermediate transition phrase "consisting essentially of and the closed transition phrase" consisting of or consisting of. The claims that use the "comprising" may be modified with intervening and closed transition phrases to specify a particular embodiment.

In some aspects, one or more additional therapeutic agents may be included in the pharmaceutical composition. Additional therapeutic agents include: for example, agents that induce apoptosis; polynucleotides (e.g., antisense, ribozyme, siRNA); polypeptides (e.g., enzymes and antibodies); a biological mimetic; an agent that binds to and inhibits an anti-apoptotic protein (e.g., an agent that inhibits an anti-apoptotic protein such as BCL-2, BCL-XL, BCL-w, BFL-1, or MCL-1 protein); an alkaloid; an alkylating agent; antitumor antibiotics; antimetabolites; a hormone; a platinum compound; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anticancer drugs, toxins, defensins, and the like), toxins, radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-. Alpha., etc.) and interleukins (e.g., IL-2, etc.); adoptive immunotherapeutic agent; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans retinoic acid, etc.); gene therapy agents (e.g., antisense therapeutic agents and nucleotides); tumor vaccine; an angiogenesis inhibitor; protein body inhibitors: NF- κβ modulators; an anti-CDK compound; and HDAC inhibitors. Agents that induce apoptosis include: for example, radiation (e.g., X-rays, gamma rays, UV); kinase inhibitors (e.g., epidermal Growth Factor Receptor (EGFR) kinase inhibitor, vascular Growth Factor Receptor (VGFR) kinase inhibitor, fibroblast Growth Factor Receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitor (e.g., GLEEVEC)); an antisense molecule; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN and AVASTIN); antiestrogens (e.g., raloxifene and tamoxifen); antiandrogens (e.g., flutamide, bicalutamide, finasteride, aminoglutethimide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory agents (e.g., phenylbutazone, DECADRON, prednisone, dexamethasone (dexamethasone), dexamethasone (dexamethasone intensol), DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxybenzone, PEDIAPRED, phenylbutazone, plaguenil, prednisolone, prednisone, pre, and TANDEARIL); and cancer chemotherapeutic agents (e.g., irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, tamotore, or tamol); a cell signaling molecule; ceramide and cytokines; and staurosporine.

The invention will be better understood from the following experimental details. However, those skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow.

Experimental details

A cell line. Cell lines were purchased from ATCC and DSMZ. The head and neck cancer cell lines HN30, HN31, UMSCC6, MDA686LN, and HN5 are provided by dr. Ovarian, NSCLC, colon, leukemia, lymphoma, bxPC-3 and ASPC1 cell lines were maintained in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 100U/ml penicillin/streptomycin, 2mM L-glutamine and 50. Mu.M mercaptoethanol. Mammary gland, melanoma, HCT116, MIA PaCa-2 and HEY cell lines were maintained in DMEM (Gibco) supplemented with 10% FBS, 100U ml-1 penicillin/streptomycin and 2mM l-glutamine. The head and neck cancer cell line was maintained in DMEM (Gibco) supplemented with 10% FBS, 1X vitamins, 1X sodium pyruvate, 1X non-essential amino acids, 100U ml-1 penicillin/streptomycin and 2mM L-glutamine. Capan-1 was maintained in Iscove's modified Dulbecco's medium (Gibco) supplemented with 10% FBS, 100U ml-1 penicillin/streptomycin and 2mM l-glutamine. Capan-2 was maintained in McCoy 5a medium (modified) (Gibco) supplemented with 10% FBS, 100U ml-1 penicillin/streptomycin and 2mM l-glutamine. OCI-AML3 was maintained in MEM alpha (Gibco) supplemented with 10% FBS, 100U ml-1 penicillin/streptomycin, 2mM l-glutamine and 50. Mu.M beta-mercaptoethanol.

A mouse

For toxicity studies, 6-8 week old D1-IGS male and female mice were purchased from Charles River. For xenograft studies and pharmacodynamic analysis experiments, 6-8 week old nude (nu/nu) mice and NOD SCID male mice were purchased from Charles River. All mice were maintained under standard and dietary conditions and had a body weight of greater than 20 grams.

Patient-derived xenograft samples. Human colorectal tumor xenografts were taken from the eduard o Vilar, university of texas, ambroxen cancer center, medical doctor. Samples were taken from two patients with metastatic colorectal cancer (see table 1 below). According to IRB approved protocols, patients provided written informed consent for patient-derived xenografts (PDX). Animal experiments using PDX were performed according to IACUC approved protocols.

A compound. Corresponding to the BH3 domain of BIMIs a binding peptide (Hydrocarbon-stabilized peptide), FITC-BIM SAHB _A2 ：FITC-βAla-EIWIAQELRS5IGDS5F`NAYYA-CONH ₂ Wherein S5 represents insertion of unnatural amino acids for olefin metathesis, synthesized by CPC Scientific Inc. to>95% purity purified and characterized as described previously.

The BTSA1 and BTSA1.2 compounds were synthesized at Albert Einstein College of Medicine. Synthesis of BTSA1 is as previously described in Reyna et al, cancer cell.2017Oct9;32 (4) 490-505.e10. Synthesis and analytical characterization of BTSA1.2 is described below. Chembridge and Molport provide other BAX activators with purity > 98%. Navy (purity 99.97%) from MedCheM Express was used for in vivo studies and Navy (purity 99.53%) from SelleckChem was used for in vitro studies. A-1331852 was purchased from Selleckchem (purity 99.8%), and Winetropox was purchased from Selleckchem (99.7%) and staurosporine (purity 99.61%). The following BH3 peptides in table 2 were purchased from Genscript with purity >95%.

Peptides have acetylation as an N-terminal modification and amidation as a C-terminal modification.

The compounds were reconstituted in 100% DMSO and diluted in aqueous buffer or cell culture medium for assay.

And (5) chemical synthesis. All chemicals and solvents were taken from commercial sources (Aldrich, acros, fisher) and used without further purification unless otherwise indicated. Using pure Solv ^TM The AL-258 solvent purification system yielded anhydrous solvents (tetrahydrofuran, toluene, methylene chloride, diethyl ether). Ethanol was dried over activated 4A molecular sieves. The microwave reaction was performed on Anton Paar Monowave 300. Use of disposable silica gel column (4, 12 and 24 g) at Teledyne ISCO CombiFlash R _f 200Chromatography was performed on i. Analytical Thin Layer Chromatography (TLC) was performed on an aluminium backed Silicicle silica gel plate (film thickness 250 μm, indicator F254). Using a dual wavelength (254 and 365 nm) UV lamp and/or with CAM (cerium ammonium molybdate) or KMnO ₄ The staining visualizes the compounds. NMR spectra were recorded on Bruker DRX 300 and DRX 600 spectrometers. ¹ H and ¹³ the C chemical shift (delta) is relative to tetramethylsilane (TMS, 0.00/0.00 ppm) as an internal standard or relative to the residual solvent (CD ₃ OD：3.31/49.00ppm；CDCl ₃ :7.26/77.16ppm; dmso-d6:2.50/39.52 ppm). Mass spectra were recorded on Shimadzu LCMS 2010EV (direct injection unless otherwise indicated). High resolution electrospray ionization mass spectrometry (ESI-MS) was obtained at the institute of eibert einstein medical college macromolecular analysis and proteomics laboratory or externally from intelek USA, inc (Whitehouse, NJ). Unless otherwise indicated, the purity of the synthesized compound was 3% as judged by 1H-NMR traces.

Synthesis of BTSA 1.2. The synthesis of BTSA1.2 is summarized in the reaction scheme shown below.

Synthesis of 3- (2-thiocarbamoylhydrazino) -3-propionic acid phenyl ester; compound S2

Hydrazine thiocarboxamide (2.00 g,22.0mmol,1.10 eq.) was dissolved in 5% aqueous HCl (64.2 mL,90mmol,4.50 eq.) and ethanol (30.0 mL). Ethyl 3-oxo-3-phenylpropionate (3.83 g,20.0mmol,1.00 eq.) was added with vigorous stirring. The resulting mixture was stirred vigorously overnight (> 12 h) at room temperature. The white precipitate that had formed was filtered and washed with a small amount of water. The resulting white fluffy solid (4.24 g) was washed with EtOH (15.0 mL), filtered and dried under high vacuum to give phenyl (Z) -3- (2-thiocarbamoylhydrazono) -3-propionate (S2; 3.54g,13.3mmol,67%, purity. Gtoreq.90% as measured by 1H-NMR).

1H-NMR (600 MHz, dmso-d 6): delta 10.62 (s, 1H), 8.38 (s, 1H), 8.03 (s, 1H), 7.88-7.85 (m, 2H), 7.39-7.37 (m, 3H), 4.10-4.06 (m, 4H), 1.16 (t, J=7.1 Hz, 3H). 13C-NMR (151 MHz, dmso-d 6): delta 179.2,168.3,142.3,136.8,129.2,128.3,126.5,60.8,33.2,14.0). The crude product of compound S2 was used directly in the next step without further purification.

Synthesis of 5-phenyl-2- (4-phenylthiazol-2-yl) -1, 2-dihydro-3H-pyrazol-3-one; compound S3

In a 60mL centrifuge tube with a stirring bar and a lid, phenyl 3- (2-thiocarbamoylidene) -3-propionate (S2; 1.06g,4.00mmol,1.00 eq.) and 2-bromo-1-phenethyl-1-one (1.04 g,5.20mmol,1.30 eq.) are suspended in ethanol (21 mL). The mixture was stirred at room temperature. Almost immediately, all the starting material dissolved, and after about 1 minute, a thick white precipitate formed. After 60 minutes, sodium acetate (492 mg,6.00mmol,1.50 eq.) was added and the mixture was stirred overnight at room temperature>12h) A. The invention relates to a method for producing a fibre-reinforced plastic composite 20.0mL of water was added and the mixture was filtered under reduced pressure. And a small amount of water is added to rinse the residual raw materials in the reaction vessel. The resulting residue was washed with more water (total volume of collected aqueous phase: 60.0 mL). The crude product obtained was an off-white solid, dried under high vacuum. In Isco CombiFlash (0.0.fwdarw.60.0%CH ₂ Cl ₂ In hexane), the obtained 5-phenyl-2- (4-phenylthiazol-2-yl) -1, 2-dihydro-3H-pyrazol-3-one (S3) was a colorless solid (850 mg,2.66mmol, 71%).

TLC：R _f 0.75(50％CH ₂ Cl ₂ In hexane). ¹ H-NMR(600MHz,dmso-d ₆ +2dr py-d ₅ ):δ8.03(d,J＝7.3Hz,2H),7.89(d,J＝7.3Hz,2H),7.86(s,1H),7.51–7.45(m,5H),7.37–7.34(m,1H),6.08(s,1H). ¹³ C-NMR(151MHz,dmso-d ₆ +2dr py-d ₆ ) Delta 158.5,155.9,153.0,150.1,133.9,130.4,129.7,128.8,128.7,128.0,126.1,126.0,109.8,88.2.ESI-MS M/z (relative to internal standard) is (320.1 ([ M+H)] ⁺ ,100). For C ₁₈ H ₁₄ N ₃ HRMS of OS (m+h): calculated values: 320.0852; measurement value: 320.0853. (if NMR is in pure dmso-d) ₆ Is performed in the middle ofMixtures of tautomers were observed. The addition of pyridine shifts the equilibrium completely to one side, allowing a different set of signals to be detected. )

Synthesis of 4- (2- (4, 5-dimethylthiazol-2-yl) hydrazono) -5-phenyl-2- (4-phenyl-thiazol-2-yl) -2, 4-dihydro-3H-pyrazol-3-one (S5; BTSA 1.2); compound S5 (BTSA 1.2)

A solution of sodium nitrite (64.8 mg,0.939mmol,1.00 eq.) in water (0.47 mL) was added dropwise via pipette to a suspension of 4, 5-dimethylthiazol-2-amine (120 mg,0.939 mmol) in hydrochloric acid (semi-concentrated, 0.75mL,12.4mmol,13.2 eq.) in an open vessel in a wet ice/NaCl cooling bath (temperature maintained at +.5deg.C). The diazonium salt formed was a dark yellow solution. After about 10 minutes, the starting material was completely dissolved and the solution was stirred at-5 ℃ for a further 15 minutes. Simultaneously, 5-phenyl-2- (4-phenylthiazol-2-yl) -2, 4-dihydro-3H-pyrazol-3-one (S3; 300mg,0.939mmol,1.00 eq.) was dissolved in aqueous sodium hydroxide (2.50M; 0.94mL,2.35mmol,2.50 eq.) and ethanol (0.94 mL). After a few minutes a clear solution forms and is stirred at room temperature for a further 10 minutes. In these cases, water/ethanol (1:1) was added in small increments until a solution was formed. The solution of the anionic species formed above is then added dropwise to the diazonium salt. The deep red precipitate formed almost instantaneously. After complete addition, the mixture was warmed to room temperature and stirred for an additional 20 minutes. Reaction aliquots (directly diluted with small amounts of methanol or subjected to H ₂ O/EtOAc micro-treatment) showed consumption of thiazole amine, formation of new product, some more polar colored byproducts, and remaining compound S3.

The mixture was diluted with water (1.0 mL), filtered through a filter paper into a buchner funnel, washed with a small amount of water (about 3.0 mL) and then dried in the air stream of the filtration apparatus. The pre-dried material was transferred to a flask and additionally dried under high vacuum. In Isco CombiFlash (0.1@5.0% MeOH in CH ₂ Cl ₂ In) to obtain 4- (2- (4, 5-dimethylthiazol-2-yl) hydrazono) -5-phenyl-2- (4-phenyl-thiazol-2-yl) -2, 4-dihydro-3H-pyrazin-3-one (BTSA1.2S5; 261mg,0.569mmol, 61%) as bright red solid.

This synthesis scheme is highly sensitive to mixing/cooling problems that are complicated by precipitation of the product and reprotonated intermediates during the reaction. In some cases, considerable amounts of impurities may be formed due to the decomposition of the diazonium reagent. In these cases, additional chromatography and/or recrystallization of the product (most commonly from dioxane) is required. As a result, the yield may be lower as judged by TLC, although the conversion is generally acceptable.

TLC: rf 0.90 (5% MeOH in CH2Cl 2). 1H-NMR (600 MHz, dmso-d 6): delta 8.14 (d, J=7.1 Hz, 2H), 7.99 (dd, J=8.1, 1.0Hz, 2H), 7.80 (s, 1H), 7.54-7.45 (m, 5H), 7.35 (t, J=7.3 Hz, 1H), 2.25 (s, 3H), 2.18 (s, 3H). 13C-NMR (151 MHz, dmso-d 6): delta 177.7,154.7,152.5,149.7,149.2,134.9,134.2,130.9,129.7,129.6,128.7,128.4,128.0,127.9,125.9,119.1,108.8,11.7,11.4.ESI-MS m/z (relative intensity): (459.1 [ M+H ] ]+,100). For C ₂₂ H ₁₃ N ₆ O ₃ S ₂ HRMS of (m+h): calculated values: 459.1056; measurement value: 459.1051.

and (5) measuring cell viability. Cancer cells (1-2 x 10) ³ Cells/well) were inoculated in 384 Kong Baiban and incubated in FBS-free medium with serial dilutions of BAX activator compounds including BTSA1.2, naviatox, a-1331852, vinatoxin, staurosporine or vehicle (1% dmso) for 2 hours before replacement with 10% FBS to a final volume of 25 μl. Cell viability was determined at 72 hours by addition of CellTiter-Glo detection reagent according to the manufacturer's protocol (Promega) and luminescence was measured using an F200 PRO microplate reader (TECAN). For the combination experiments of navitock and BTSA1.2, cells were seeded as described above and co-treated with Navitoclaz and BTSA1.2 at the indicated doses. For the Navickers andBTSA1.2 combinations, cells were seeded as described above for the a-1331852 and BTSA1.2 combination and the vitamin c and BTSA1.2 combination experiments and co-treated with naviteclaz or a-1331852 or vitamin c and BTSA1.2 at the indicated doses. The high throughput drug screening was excluded and the viability assay was performed in at least triplicate, and the data normalized to 1% vehicle treated control wells. IC (integrated circuit) ₅₀ The values were determined by nonlinear regression analysis using Prism software (Graphpad). Dilution of compounds was performed from 10mM stock using a TECAN D300e digital dispenser. Bioinformatics.201615 as previously described in Di Veroli et al; 32 (18) BLISS calculations were performed using the combineflit procedure described in 2866-8.

Genomic changes in the panel of human cancer cell lines tested are shown in table 3 below.

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Production of recombinant BAX proteins. Human, recombinant and unlabeled BAX were expressed in Escherichia coli (Escherichia coli) and purified (Uchime, o.et al j. Biol. Chem,291,89-102 (2016), as previously reported). BAX wild type was purified by size exclusion chromatography in buffer containing 20mM HEPES ph7.2, 150mM KCl, 1mM DTT. Superdex 75/300 GL and 200 10/300GL (GE Healthcare) columns were used.

Fluorescence polarization binding assay. Fluorescence Polarization Assays (FPAs) as previously described in gavanithis, et al, nat Chem biol.2012jul;8 (7) 639-4. First, direct binding isotherms were generated by incubating FITC-BIM SAHBA2 (50 nM) with serial dilutions of full length BAX and fluorescence polarization was measured on an F200 PRO microplate reader (TECAN) at 20 minutes. Subsequently, in a competition assay, serial dilutions of small molecules or acetylated BIM SAHBA2 (Ac-BIM SAHB) are combined with FITC-BIM SAHBA2 (50 nM), followed by addition of recombinant protein at EC75 concentration as determined by direct binding assay (BAX: 500 nM) 。EC ₅₀ And IC ₅₀ Values were calculated by nonlinear regression analysis of competitive binding curves using Graphpad Prism software. Ki uses the following formula ki=ic 50/(1+ ([ L ])]Kd) slave IC ₅₀ Calculated, wherein [ L ]]Is the concentration of FITC-BIM SAHBA2, and Kd is the binding of FITC-BIM SAHBA2 to BAX.

Immunoblotting. Protein lysates were obtained by cell lysis in 1% NP-40 buffer (50 mM Tris-HCl, 150mM NaCl, 1mM EDTA, 10% glycerol, 1% NP-40, pH 7.50). Protein samples were separated by electrophoresis on 4-12% NuPage (Life Technologies) gel, transferred to a mobilon-FL PVDF membrane (Millipore) and immunoblotted. For visualization of proteins with Odyssey infrared imaging system (LI-COR Biosciences), membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences). Primary antibody was used at 1: the 1,000 dilution was incubated overnight at 4 ℃. After washing, 1:10,000 and 1: the membranes were incubated with either IRdye800 conjugated goat anti-rabbit IgG or IRdye800 conjugated goat anti-mouse IgG secondary antibody (LI-COR Biosciences) at 20,000 dilutions. Proteins were detected using an Odyssey infrared imaging system. Antibodies were used to detect the following proteins on the membrane: BCL-XL (Cell Signaling Cat.2762), MCL-1 (Cell Signaling Cat.4572), BAX (Cell Signaling Cat.2772), BCL-2 (BD.Cat.610539), BAK (Millipore Cat.06-536), BIM (Cell Signaling Cat.2933S), cleaved caspase-3 (Cell Signaling Cat.9664S), cleaved PARP (Cell Signaling Cat.5625S), COX-IV (Cell Signaling Cat.4850S), beta-actin (Sigma Cat.A 1978), beta-tubulin (Cell Signaling Cat.2146S).

Whole cell immunoprecipitation and immunoblotting. Protein lysates were obtained by cell lysis in 0.2% NP-40 buffer (50 mM Tris-HCl, 150mM NaCl, 1mM EDTA, 10% glycerol, 0.2% NP-40, pH 7.50). Immunoprecipitation was performed in 600mL of 400. Mu.g protein, which was pre-clarified by centrifugation and then exposed to 12. Mu.L (50% slurry) of protein A/G beads (Santa Cruz) for 30 min at 4 ℃. The clarified extract was incubated overnight with 2. Mu.L of anti-BAX antibody (Cell Signaling Cat.2772). The samples were then exposed to 20. Mu.L (50% slurry) of protein A/G beads (Santa Cruz) for 2 hours at 4deg.C, then centrifuged and washed three times with 0.2% NP-40 buffer and boiled in loading buffer (Life Technologies). Protein samples were separated by electrophoresis on 4-12% NuPage (Life Technologies) gel, transferred to a mobilon-FL PVDF membrane (Millipore) and immunoblotted. For visualization of proteins with Odyssey infrared imaging system (LI-COR Biosciences), membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences). Primary antibody was used at 1: the 1,000 dilution was incubated overnight at 4 ℃. After washing, 1:10,000 and 1: the membranes were incubated with either IRdye800 conjugated goat anti-rabbit IgG or IRdye800 conjugated goat anti-mouse IgG secondary antibody (LI-COR Biosciences) at 20,000 dilutions. Antibodies were used to detect the following proteins on the membrane: BCL-XL (Cell Signaling Cat.2762), MCL-1 (Cell Signaling Cat.4572), BAX (Cell Signaling Cat.2772), BCL-2 (BD.Cat.610539), beta-actin (Sigma, cat.A 1978).

Cell thermal displacement assay (CETSA). BxPC3 cells were seeded in 10-cm dishes to about 85% confluency. The medium was removed and replaced with FBS-free medium and cells were treated with 40 μm BTSA1.2 for 15 min at 37 ℃. The medium was removed, the cells were washed once with PBS and harvested with a cell scraper. The cells were resuspended in PBS to 10X 10 ⁶ cells/mL and 50 μl was transferred into PCR tubes. The cells were then heated in a Biorad C1000 touch thermocycler using temperature gradients (50, 52.1, 55.4, 59.4, 64.9, 69.2, 72.1 and 74 ℃) for 3 minutes. All cells were lysed by three freeze-thaw cycles using liquid nitrogen. The sample was then incubated at 2X 10 at 4℃ ⁴ Centrifuge at Xg for 15 min. Supernatants were collected, resolved by SDS-PAGE, and analyzed by immunoblotting with N-terminal BAX antibody (Cell Signaling, 2772S). The results were quantified by densitometry analysis using Image Studio software and normalized to 25 ℃ (100%) and blotting background (0%).

Cellular BAX translocation assay. Cells were inoculated in FBS-free medium and incubated with serial dilutions of BTSA1.2 or vehicle (1% DMSO). After 2 hours, FBS was supplemented to a final concentration of 10%. After 4 hours of treatment, cells were lysed in 100 μl of digitonin buffer [20mM Hepes (pH 7.2), 10mM KCl, 5mM MgCl2, 1mM EDTA, 1mM EGTA, 250mM sucrose, 0.025% digitonin (from 5% w/v stock) and complete protease inhibitor cocktail (complete protease inhibitors cocktail) (Thermo-Fisher) ] and incubated on ice for 10 minutes. The supernatant was isolated by centrifugation at 15,000Xg for 10 minutes and the mitochondrial particles were dissolved in 1% Triton X-100/PBS for 1 hour at 4deg.C. The particles were dissolved, spun at 15,000Xrpm for 10 minutes, and 50ng of protein was mixed with 25. Mu.L of LDS/DTT loading buffer. The equivalent fraction e of the corresponding supernatant samples was mixed with 25. Mu.L of LDS/DTT loading buffer. Mitochondrial supernatants and particle fractions (pellet fractions) were then separated with 4-12% NuPage (Life Technologies) gel and analyzed by immunoblotting with anti-BAX antibody (2772S,Cell Signaling), BCL-XL (Cell Signaling Cat.2762), MCL-1 (Cell Signaling Cat.4572). COX-IV (Cell Signaling Cat.4850S) and beta-tubulin (Cell Signaling Cat.2146S) were used for loading controls of mitochondrial and supernatant fractions, respectively.

Immunoprecipitation of digitonin fractionated supernatant and mitochondrial extracts. Cells (10X 10) ⁶ Cells/well) were inoculated in 100mm dishes and incubated with serial dilutions of BTSA1.2 or vehicle (0.2% DMSO) in FBS-free medium with a final volume of 5 mL. After 2 hours, FBS was supplemented to a final concentration of 10%. After 4 hours of treatment, cells were incubated in 100. Mu.L digitonin buffer [20mM Hepes (pH 7.2), 10mM KCl, 5mM MgCl2, 1mM EDTA, 1mM EGTA, 250mM sucrose, 0.025% digitonin (from 5% w/v stock) and complete protease inhibitor cocktail (Roche Applied Science)]And incubated on ice for 10 minutes. The supernatant was isolated by centrifugation at 15,000Xg for 10 min and the mitochondrial particles were dissolved in NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150mM NaCl, 5mM MgCl) ₂ 1Mm EGTA, 10% glycerol, 0.2% NP-40). Immunoprecipitation was performed from supernatant and mitochondrial particle fractions in 600 μl of 500 μg protein. Briefly, the fractions were pre-clarified by centrifugation after 1 hour of exposure with 12. Mu.L (50% slurry) protein A/G beads (Santa Cruz) at 4 ℃. The clarified extract was incubated overnight with 1 μl of anti-BAX antibody (Cell Signaling cat.2772). The sample is then exposed at 4 DEG C Protein A/G beads (Santa Cruz) were washed three times (3,000G for 1 min) with NP-40 lysis buffer after 3 hours at 20. Mu.L (50% slurry) and boiled in loading buffer (Life Technologies) for 15 min. Protein samples were separated by electrophoresis on a 4-12% NuPage (Life Technology) gel, transferred to a mobilon-FL PVDF membrane (Millipore), and immunoblotted. For visualization of proteins with Odyssey infrared imaging system (LI-COR Biosciences), the membranes were blocked in PBS containing 5% milk powder (5% dry milk). Primary antibody was used at 1: the 1,000 dilution was incubated overnight at 4 ℃. After washing, 1: the membranes were incubated with irdyne 800 conjugated goat anti-rabbit IgG or irdyne 800 conjugated goat anti-mouse IgG secondary antibody (LI-COR Biosciences) at 5,000 dilutions. Antibodies were used to detect the following proteins on the membrane: BCL-XL (Cell Signaling Cat.2764S), MCL-1 (Cell Signaling Cat.4572), BAX (Cell Signaling Cat.2772), beta-tubulin (Cell Signaling Cat.86298S) and COX IV (Cell Signaling Cat.11967S).

Immunoblot protein quantification and pearson correlation. Densitometry of protein bands was obtained using an LI-COR Odyssey scanner. The immunoassay tools using Image Studio software were used for quantification and analysis. The relevant expression levels were quantified based on the protein expression of the respective loading control COX-IV, β -actin or β -tubulin. Comparison of cell viability IC for single agent and combination of nadir and BTSA1.2 values ₅₀ Protein quantification of different members of BCL-2 family proteins pearson correlation was determined using Prism software (Graphpad).

BH3 spectra. Cancer cell lines were compared by BH3 profiling under basal conditions. BIM BH3, BID BH3, BMF-y, PUMA, BAD, HRK-y and NOXA peptide (final concentration 10. Mu.M); puma2A peptide (final concentration 20 μm); propofol (final concentration 25. Mu.M); CCCP (final concentration 10 μm) was added to JC1-MEB staining solution (150 mM mannitol, 10mM HEPES-KOH, 50mM KCl, 0.02mM EGTA, 0.02mM EDTA, 0.1% BSA, 5mM succinate, pH 7.5) in black 384 well plates. Single cell suspensions were prepared in JC-1-MEB buffer, as previously described in Montero et al, cell.20150eb; 160 (5) 977-89. The cells were kept at room temperature for 10 minutes to allow permeabilization of the cellsAnd dye balancing. Cells were added to 384 well plates (1.0X10) ⁴ Cell/well to 2.0X10 ⁴ Cells/well) fluorescence was measured at 30 ℃ using an M1000 microplate reader (TECAN) at 590nm emission 545nm excitation, once every 15 minutes for 3 hours. The percentage of depolarization was calculated by AUC normalized to solvent-only control DMSO (0% depolarization) and positive control CCCP (100% depolarization), as previously described in Ryan et al methods.2013jun;61 (2) 156-64.

Caspase 3/7 activation assay. Cancer cells were treated with BTSA1.2, BTSA1, naviatoxin, vinatoxin, or staurosporine at the indicated concentrations following single or combination doses described in the previous cell viability assays. Caspase-3/7 activation was measured for BTSA1.2 and Navinotoxin at 8 hours and for staurosporine at 24 hours by adding caspase-Glo 3/7 chemiluminescent reagent according to manufacturer's protocol (Promega). Luminescence was detected by an F200 PRO microplate reader (TECAN). The assays were performed in at least triplicate.

Pharmacokinetic analysis. ICR (CD-1) male mice were fasted for at least 3 hours and water was available ad libitum prior to the study. The animal is placed in a controlled environment, target conditions: the temperature is 18-29 ℃ and the relative humidity is 30-70%. The temperature and relative humidity were monitored daily. An electronic timed illumination system was used to provide a 12 hour light/12 hour dark cycle. 3 mice were administered with BTSA1.2 in 1% DMSO, 30% PEG-400, 65% D5W (5% dextrose in water) and 4% Tween-80 at each indicated time point by oral gavage (3 mg/Kg) or intravenous injection (1 mg/Kg). Mice were sacrificed, plasma samples were collected at 0 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and analyzed for BTSA1.2 levels using LC-MS/MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.3.6.3. Experiments were performed in SIMM-SERVER in combination with biopharmaceutical laboratories.

Maximum Tolerated Dose (MTD) and in vivo toxicity studies. Female and male 6-8 week old CD1-IGS mice (Charles River) were divided into six groups (n=6 per group) and treated with vehicle, 200mg/kg BTSA1, 50mg/kg, 100mg/kg, 200mg/kg or 300mg/kg BTSA1.2 by daily oral gavage for 5 days. Mice were monitored daily and body weight was monitored on the indicated date. 14 days after the first treatment, mice were euthanized and necropsied (histology and comparative pathology institute, albert einstein medical institute) and tissues (e.g., spleen, liver, kidney, lung, heart) were harvested for fixation in 10% buffered formalin (Fisher Scientific) for pathology analysis. Paraffin-embedded sections (5 mm) were stained with H & E. Peripheral blood of CD1-IGS mice was obtained by facial venipuncture and collected in EDTA coated tubes (BD cat.365973). Blood counts were measured on a forgate veterinary blood analyzer (Oxford Science inc.). Necropsy studies were performed on 200mg/kg BTSA1 and 300mg/kg BTSA1.2 mice, confirming that they died from renal failure after 3 days of treatment. Histological and autopsy were assessed by a histology and comparative pathologist certified veterinary pathologist.

In vivo toxicity study of the combination of BTSA1.2 and naviatoxin. CD1-IGS male mice of 6-8 weeks of age were purchased from Charles River. Mice were divided into four groups (vehicle, BTSA1.2 and naviatox: n=5, combination: n=6) and treated by oral gavage daily with vehicle, 200mg/kg BTSA1.2, 100mg/kg Navitoclaz or combination of BTSA1.2 and naviatox for 7 days. Mice in the combination group were first administered 100mg/kg of Naviotor and 6-8 hours later administered 200mg/kg of BTSA1.2. Mice were monitored daily; body weight and peripheral blood counts were monitored on the indicated days. 14 days after the first treatment, mice were euthanized and necropsied (histology and comparative pathology institute, albert einstein medical college) and tissues (e.g., spleen, liver, kidney, lung, heart, bone marrow, brain) were harvested for fixation in 10% buffered formalin (Fisher Scientific) for pathology analysis. Paraffin-embedded sections (5 mm) were stained with H & E. Peripheral blood of CD1-IGS mice was obtained by facial venipuncture and collected in EDTA coated tubes (BD cat.365973). Blood counts were measured on a forgate veterinary blood analyzer (Oxford Science inc.).

Tumor xenograft study. Nu/nu nude mice of 6-8 weeks of age were purchased from Charles River. Will be about 2.5x10 ⁶ Individual SW480 cells were suspended in cold PBSAnd subcutaneously injected into the right flank of the mouse. Mice were divided into four groups (efficacy study: vehicle, BTSA1.2 and navitolg: n=5, combination: n=6; pharmacodynamic study: all groups: n=3) and treated by daily oral gavage with vehicle, 200mg/kg BTSA1.2, 100mg/kg Navitoclaz or combination of BTSA1.2 and navitolg. Mice in the combination group were first administered 100mg/kg of Navinotor and 6-8 hours later administered 200mg/kg of BTSA1.2. For curative effect, the curative effect research is that the tumor reaches 200mm ³ Is started at the time of volume of (2). Tumor volumes were monitored every 3 days by caliper measurements until the experiment stopped when the tumors reached an ethically unacceptable size for vehicle, BTSA1.2 or navico treated mice, and for mice administered the combination, the mice were euthanized the following day after euthanization of single agent or vehicle treated mice. Mice were monitored for body weight during treatment. For pharmacodynamics research, the tumor reaches 400mm ³ The treatment was started 3 days after daily treatment, mice were euthanized and tumors were collected for analysis.

Patient-derived xenograft study. NOD SCID male mice of 6-8 weeks of age were purchased from Charles River. Will be about 1.0x10 ⁶ Individual COLO-1 or COLO-2 cells were suspended at 1:1DMEM: matrigel, and subcutaneously injected into the right flank of mice. PDX characterization: mice were divided into two groups, COLO-1 and COLO-2 (n=3). In the case of tumor reaching 1,000mm ³ Tumors were collected after the volume of (2). COLO-1 efficacy study: mice were divided into four groups (vehicle, BTSA1.2, navitolk and combination: n=4) and treated by daily oral gavage with vehicle, 200mg/kg BTSA1.2, 50mg/kg navitolclaz or BTSA1.2 and navitolk combination. Mice in the combination group were first administered 50mg/kg of Navigator and 6-8 hours later administered 200mg/kg of BTSA1.2. In the case of tumor reaching 200mm ³ After the volume of (2) the treatment is started. Tumor volumes were monitored every 3-4 days by caliper measurement until tumors reached an ethically unacceptable size or the experiment was stopped after 18 days of daily treatment (first arrival). Mice were monitored for body weight during treatment.

Ex vivo BHS profile. By BH3 spectroscopy under basal conditionsSW480 xenograft vehicle and combination treated tumors were analyzed for COLO-1 and COLO-2PDX tumors. Single cells were isolated from the tumor by mechanically passing them through a 70 μm screen filter with cold PBS. SW480 xenograft tumor: BIM BH3 and BID Bh3, peptide (final concentration 10-0.5. Mu.M); puma2A peptide (final concentration 10 μm); propofol (final concentration 25. Mu.M); CCCP (final concentration 10 μm) was added to JC1-MEB staining solution (150 mM mannitol, 10mM HEPES-KOH, 50mM KCl, 1mM EGTA, 1mM EDTA, 0.1% BSA, 5mM succinate, pH 7.5) in a black 384 well plate. PDX tumor: BIM BH3 and BID Bh3, peptide (final concentration 25-1. Mu.M); PUMA, BMF-y, BAD and HRK (final concentration 100-10 μm); MS1 and FS1 (final concentration 25-10. Mu.M); and PUMA2A peptide (final concentration 100-25 μm); propofol (final concentration 25. Mu.M); CCCP (final concentration 10 μm) was added to JC1-MEB staining solution in 384 well plates. As described previously, at 1: single cell suspensions were prepared in 1JC-1-MEB buffer and kept at room temperature for 10 minutes to allow cell permeabilization and dye equilibration. Cells were added to 384 well plates (2.0X10) ⁴ Cells/well) fluorescence was measured at 30 ℃ using an M1000 microplate reader (TECAN) at 590nm emission 545nm excitation, once every 15 minutes for a total of 2 hours. The percentage of depolarization was calculated by normalizing to AUC of negative control Puma2A (0% depolarization) and positive control CCCP (100% depolarization), as described above. Mitochondrial membrane potential was calculated by normalizing AUC values to AUC of negative control solvent (1% dmso only).

Ex vivo cell viability. Single cells were isolated from COLO-1 and COLO-2PDX tumors by mechanically passing them through a 70. Mu.M screen filter with cold PBS. The isolated cells (10-20X 10 ³ Cells/well) were inoculated in 384 Kong Baiban and incubated with vehicle (1% dmso) or serial dilutions of BTSA1.2, navicular, or with navicular and BTSA1.2 (at the indicated dose) for 2 hours in FBS-free medium, then replaced with 10% FBS to a final volume of 25 μl. Cell viability was determined at 24 hours by addition of CellTiter-Glo detection reagent according to the manufacturer's protocol (Promega) and luminescence was measured using an F200 PRO microplate reader (TECAN). Vitality determination at leastDuplicate replicates were performed and data normalized to 1% vehicle treated control wells. IC (integrated circuit) ₅₀ The values were determined by nonlinear regression analysis using Prism software (Graphpad). Compounds were diluted from 10mM stock using a TECAN D300e digital dispenser (Digital Dispenser).

Bioinformatics analysis. We tested the individual drug candidates BTSA1.2 and naviatoxin, as well as the combination, on a total of 46 cancer cell lines. Cancer cell lines were defined as synergistic or non-synergistic for the combination by fold change from the IC50 of the navicular to that of the combination of navicular and BTSA 1.2. Two groups are defined: (a) synergistic group, IC50 fold change > =4; (B) non-synergistic group, IC50 fold change <2.RNA-Seq raw count data was taken from the Cancer Cell Line Encyclopedia (CCLE) database of BROAD institute (https:// ports. Broadenstitute. Org/cc/data). A total of 23 cell lines (8 non-synergistic and 15 synergistic) had RNA-Seq data from CCLE. Differential expression analysis was then performed using the DESeq2 package in comparing R in non-synergistic and synergistic groups based on the original RNA-Seq data. The first 150 differentially expressed gene heatmaps were generated using the pheeatmap package in R, comparing non-synergistic and synergistic cell line groups. Based on the adjusted p-values, literature searches for bioinformatics analysis optimal genes (top hits) were performed for genes previously associated with apoptosis, cancer treatment resistance, BCL-2 family, or poor prognosis of cancer. After literature evaluation, the first 8 genes selected were further verified by RT-q-PCR.

RNA preparation and real-time PCR. RNA was isolated from cells in culture using the e.z.n.a total RNA kit from Omega according to the manufacturer's instructions. The quality and quantity of RNA was determined spectrophotometrically using a NanoDrop 8000 spectrophotometer from Thermo Scientific. For quantitative reverse transcription PCR (RT-qPCR), RNA was reverse transcribed using a high capacity cDNA reverse transcription kit from Applied Biosystems according to the manufacturer's instructions. PCR was performed on the ViiA 7 real-time PCR system from Applied Biosystems using PowerUp SYBRGreen premix (PowerUp SYBRGreen Master Mix) from Applied Biosystems according to manufacturer's instructions. Cycling conditions included uracil-DNA glycosidase (UDG) activation at 50℃for 2 minutes followed by Dual-LockTaq DNA polymerase activation at 95℃for 2 minutes, followed by 40 amplification cycles consisting of denaturation at 95℃for 15 seconds, annealing at 60℃for 15 seconds and extension at 72℃for 1 minute. The specificity of the amplified DNA was confirmed by melting curves at the end of each RT-QPcr run. No template control (containing all reaction components except the cDNA samples) was used to identify PCR contaminants, as these samples should not return CT values. The gene expression results were normalized to the transcript amount of ribosomal protein RPL 27. Primers for PCR were designed using the on-line NCBI Primer-BLAST tool. Each RT-qPCR was performed at least three times. The following primers (Table 4) were purchased from Eurofins Genomics.

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Cancer patient gene expression. Survival of cancer patients and gene expression data for genes were obtained from a set of non-redundant studies planned for cbioport.

Quantification and statistical analysis. The plots and statistical tests were generated in GraphPad Prism 8.0. Data are expressed as mean ± SEM unless indicated otherwise. Statistical comparisons between the two groups were performed using unpaired single-tailed student t-test, or ANOVA with Tukey multiple comparison test. The P values are shown on the graph: * p <0.05, < p <0.01, < p <0.001, < p <0.0001. Additional statistical details are provided in the legend and method details.

Results

BAX activation in resistant cancer cells regulated by BCL-XL and apoptosis initiation

To improve the biological activity and in vivo properties of the small molecule BAX activator BTSA1 we performed further pharmacochemical optimisation. A new oral bioavailable analog, BTSA1.2, was produced that had two methyl groups on the thiazolyl group of BTSA1 to increase van der waals forces in contact with BAX trigger sites (based on previously determined BTSA1 binding postures) (table 5). Furthermore, the two methyl groups are installed to avoid potential formation of the reactive and toxic metabolite aminothiazole from BTSA1 in vivo metabolism. BTSA1.2 has a phenyl group as BTSA1 bound to a pyrazolone group, which provides significantly increased binding to BAX and apoptotic activity compared to BAM7 and compound 3 lacking the phenyl group (table 5). BTSA1.2 has a thiazole hydrazone moiety because compound 3 showed enhanced binding of thiazole hydrazone compared to ethoxyphenyl hydrazone of BAM7 (table 5). The carboxylic acid was attached to the phenylhydrazone of compound 5 and the phenylthiazole of compound 6 provided fewer active compounds than BTSA1.2 and BTSA1, indicating that the hydrophobic group was better tolerated by both rings (table 5).

Table 5: structure-activity relationship of BAX activators based on binding and cellular activity in vitro.

Compared to BTSA1, BTSA1.2 showed increased binding to BAX and more potent cellular activity in a panel of lymphoma cell lines (table 5, fig. 1L-1O). Furthermore, a significant increase in the melting temperature of cellular BAX, as determined using the cell thermal transfer assay (CETSA), provided evidence that BTSA1.2 directly engaged with cellular BAX (fig. 1P-1Q). Pharmacokinetic analysis of BTSA1.2 shows that oral administration has good properties such as substantial half-life in mouse plasma (T _1/2 About 14 hours), good oral bioavailability (% F-50%) and significant plasma exposure (AUC-100 μΜhr) (fig. 16A-16B-1S and table 6). In vivo, comparison of BTSA1.2 and BTSA1 administered orally at a daily dose of 200mg/Kg also demonstrated better tolerability of BTSA1.2 administered orally, as mice survived at the daily dose of 5 without significant toxic effects. In contrast, mice taking BTSA1 died from renal failure after three daily doses, and on the next day, the mice exhibited increased leukocyte and neutrophil levels (fig. 16F-16G). Thus, BTSA1.2 (a reasonable BTSA1 analog) has improved binding to BAX, cytotoxicity, and in vivo Has better tolerance.

Table 6: pharmacokinetic parameters of BTS1.2 after oral and intravenous administration in mice.

Direct and indirect BAX activation in apoptosis-resistant solid tumors is regulated by BCL-XL and apoptosis initiation. The ability of BTSA1.2 to promote cytotoxicity in different groups of solid and hematological tumor cell lines (n=46) was evaluated. The group includes non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, melanoma, ovarian cancer, leukemia and lymphoma cell lines that contain genomic alterations common to cancer, including mutations in TP53, RAS, BRAF and/or PIK3 CA. BTSA1.2 treatment in leukemia and lymphoma cell lines (average IC ₅₀ <3. Mu.M) showed a higher than in most solid tumor cell lines (average IC ₅₀ >10 μm) was significantly better cytotoxicity (fig. 1A). Thus, similar to other BH3 mimics such as Venezutolk and S63845, BTSA1.2 shows better efficacy as a single agent in hematological malignancies (Souers et al, nat Med.2013Feb;19 (2): 202-8; kotschy et al, nature.2016Oct19;538 (7626): 477-82;Tron et al,Nat Commun.2018Dec;9 (1): 5341). However, to fully explore the potential of BTSA1.2, the use of rational and safe combination therapies can help overcome resistance to direct BAX activation.

It is speculated that anti-apoptotic BCL-2 proteins may promote resistance to BAX activator treatment. Quantitative protein expression profiles of key BCL-2 family proteins were performed in different cancer cell line groups and showed a strong correlation between BTSA1.2 cytotoxicity and BAX expression levels (fig. 1B, fig. 9A-9D). Interestingly, among the key BCL-2 family proteins, only anti-apoptotic BCL-XL). Interestingly, among the key BCL-2 family proteins, only anti-apoptotic BCL-XL levels correlated with lower potency of BTSA1.2, suggesting that BCL-XL is a key protein that promotes resistance to BAX activator treatment (fig. 1B). To evaluate the effect of BCL-XL on BTSA1.2 treatment, we examined whether BAX was activated in the solid tumor cell lines BxPC-3 and SW80, which are less sensitive to BTSA1.2 in the cell line group. In BxPC-2 cells and SW480 cells, translocation of cytosolic BAX to mitochondria was achieved at 10. Mu.M of BTSA1.2 at 4 hours (FIGS. 1C-1D, FIGS. 11A, 11B). However, lower concentrations of BTSA1.2 (2.5-5 μm) also promoted translocation of cytosolic BAX to mitochondria in BxPC-3 cells at a time point after 18 hours (supplementary fig. 11C). Interestingly, co-immunoprecipitation of BAX in SW480 and BxPC-3 cells showed that BAX was formed without BTSA1.2 treatment: BCL-XL complex and only BAX is increased after BTSA1.2 treatment: BCL-XL complex without increasing BAX: MCL-1 complex. (fig. 1D and 11D, 11E) furthermore, BAX was detected in several solid tumor cell lines without BTSA1.2 treatment: complexes of BCL-XL (supplementary fig. 11F, 11G). Taken together, these data indicate that BRSA1.2 induces BAX activation and translocation, but that BAX-mediated apoptosis may be hindered by BCL-XL interactions with BAX.

BCL-XL is considered a resistance factor to direct BAX activation in our cancer cell line group, we examined whether the clinical BCL-XL/BCL-2 inhibitor, navitock, has cytotoxicity promoting effects on the same cancer cell line group. Notably, inhibition of BCL-2 by that which is not explained by inhibition of BCL-2 by that which is responsible for the decrease in cell viability, as only a small fraction of solid tumor cell lines have detectable levels of BCL-2 protein (fig. 9A). Interestingly, several solid tumor cell lines resistant to BTSA1.2 treatment were found to also have a low BCL-XL inhibition response to that of navitock, which was insufficient to reduce cell viability of these several cell lines (average IC ₅₀ >10 μm) (fig. 1E, fig. 10A-10B). Analysis of the expression of key BCL-2 family proteins indicated that higher MCL-1 and BCL-XL levels correlated with resistance to the natatorium treatment (fig. 1F, fig. 9A, 10C). On the other hand, BAX: the BCL-XL ratio correlated with that of the tavern sensitivity, indicating that cells with higher BAX expression were generally more sensitive to BCL-XL inhibition (fig. 1F, fig. 10C). Taken together, these data indicate that BCL-XL inhibition as a single agent is insufficient to promote apoptosis in these resistant cancer cell lines, and underscores the close relationship with BAX activation in these solid tumor cancer cell lines.

To further determine the survival mechanism of cancer cell lines to avoid apoptosis, we performed BH3 profiling (an alternative method of determining the survival mechanism of cancer cell lines to avoid apoptosis) (fig. 1G and 13A). BH3 profiling shows that cell lines are dependent on: 1) "non-triggered" apoptosis; depolarization does not occur after treatment with sensitizer BH3 peptide (e.g., BAD, HRK, NOXA), but only after addition of activator BH3 peptide alone (e.g., BIM, BID, PUMA), consistent with BAX/BAK not being activated under basal conditions, or 2) depends on one or more anti-apoptotic BCL-2 proteins to survive; depolarization occurs after the addition of the specific sensitizer BH 3-only peptide, but not just after the addition of the activator BH3 peptide (fig. 1G, fig. 13A-C). Interestingly, most BTSA1.2 resistant and naviatoka resistant cell lines are classified into two major anti-apoptotic survival mechanisms: anti-apoptotic BCL-XL dependence or "non-apoptosis-triggering" (fig. 1H-I, fig. 13A-C). In fact, BH3 profiling data does not support that survival of most solid tumor cell lines is dependent on BCL-XL, as similar depolarization of HRK and BAD peptides is observed in only a few cell lines. BH3 spectral data does not exclude the case where BAX activated by either the BTSA1.2 or BIM BH3 peptide can still be controlled by the availability of BCL-XL to neutralize activated BAX. Thus, since some anti-apoptotic BCL-XL dependent cell lines are resistant to BCL-XL inhibition by naviatoxin (fig. 1D, 1H), this suggests that another pro-survival mechanism, such as "non-triggering" apoptosis, may also play a role in the apoptosis resistance of these cell lines. Indeed, by detecting the apoptosis-triggering status of solid tumors and hematological malignancies with the activator BIM BH3 peptide, we determined that solid tumors classified as BCL-XL-dependent were less triggered than hematological malignancies (fig. 1J). Furthermore, considering the cell lines that were resistant to both BTSA1.2 and that of the single treatment of navatoka, we found that most of these cell lines did not trigger apoptosis (fig. 1K).

Taken together, these data indicate that targeting one survival mechanism is insufficient to induce effective apoptosis in resistant solid tumor cell lines. Thus, we reasonably believe that dual treatment of BTSA1.2 and naviatoxin can overcome both survival mechanisms by enhancing the initiation of apoptosis through direct BAX activation, and inhibiting anti-apoptotic blockade to promote apoptosis (fig. 1K).

BTSA1.2 and naviatoxin cooperate to induce apoptosis in resistant tumor cell lines

We screened in our cancer cell line panel to compare the cytotoxic activity of navitecalx with that of navitock combined with a fixed sensitization concentration of BTSA1.2 (loss of cell viability<20%). Combination therapy of naftopsides with fixed sublethal doses of BTSA1.2 increases cytotoxicity in many cancer cell lines, including resistant solid tumors such as pancreatic and colorectal cancers, regardless of common genetic alterations (e.g., TP53, RAS) (fig. 2A-2C, fig. 12A). IC based on cell viability after drug treatment ₅₀ Fold Change (FC) of cell lines were classified as sensitive to combination (IC ₅₀ Fold change>5 x), have moderate sensitivity to combination (IC) ₅₀ Fold change 2-4 x) or resistance to combination (IC) ₅₀ Fold change <2 x) (fig. 2A-2C, fig. 12A). Cancer cell lines sensitive to the combination are predicted to have a synergistic effect after dual treatment. In fact, in cell lines from different tumor types, cell viability was synergistically reduced throughout the different concentrations following the dual treatment (fig. 2D-2E, fig. 12B-12C). Consistent with the synergistic efficacy for apoptosis induction, a significant increase in caspase 3/7 activation was observed following double treatment compared to the activity of the single agent (fig. 2F). Importantly, the synergistic effect of loss of viability and induction of apoptosis is considered BAX dependent, as Calu-6 cells sensitive to the combination of BTSA1.2 and navitolk (BNc) develop resistance to the combination in the absence of BAX expression by these cells. (FIGS. 2G-2H) however, calu-6 BAX KO cells were still sensitive to the general apoptosis inducer staurosporine by BAK-mediated apoptosis (FIGS. 2I, 2J). Thus, BNc appears to be a promising therapeutic strategy because these compounds synergistically promote apoptosis in solid tumors and hematological malignancies, regardless of the mutational background.

To further elucidate the contribution of BCL-XL or BCL-2 inhibition, we studied the combination of the selective BCL-XL inhibitor a-1331852 and the selective BCL-2 inhibitor vinatoxin with BAX activator BTSA1.2, since that vinatoxin is able to inhibit both proteins in cells. We assessed a panel of cell lines from solid tumors, such as SW480, COLO230 and hematological malignancies OCI-AML3, U937, in which the expression of BCL-XL or BCL-2 or both proteins could be detected (fig. 10A). In SW480 and COLO230 cell lines, no BCL-2 protein expression and good BCL-XL expression were detected by immunoblotting, valnemulin was not effective at sub-molar concentrations, and no synergy of the combination of valnemulin and BTSA1.2 was observed (FIGS. 26A, 26B). In contrast, BCL-XL specific inhibitor a-1331852 was potent in SW480 cell line and showed strong synergy with BTSA1.2 in SW480 (fig. 26A). A-1331852 may be ineffective in COLO230 due to MCL-1 (FIG. 10A), but it demonstrates synergy with BTSA1.2 (FIG. 26B). Furthermore, valnemtock was potent as a single agent in BCL-2 expressing OCI-AML3 and U937 cells and showed synergy when combined with BTSA1.2 (FIGS. 26C, 26D). More specifically, valnemulin was more potent in OCI-AML3 cells than in U937 cells and synergized with BTSA1.2, likely because OCI-AML3 was more dependent on BCL-2 protein and had higher BCL-2 protein levels (fig. 10A). A-1331852 was moderately effective as a single agent in OCI-AML3 and U937 cells, but it also showed a synergistic effect in combination with BTSA1.2 (FIGS. 26C, 26D). These data support that BCL-XL is an anti-apoptotic protein that controls BAX in most resistant solid tumor cell lines, as BCL-2 or BCL-2 inhibition is not detected in these cell lines to a limited extent.

The interaction of BAX with BCL-XL determines sensitivity to the combination of BTSA1.2 and Navinotog

To determine determinants of susceptibility to BNC, we observed the expression and interaction of BCL-2 family proteins. The combination of BTSA1.2 and Navelotor detected the protein expression level of the BCL-2 family member. BH3 spectra showed that cell lines sensitive to this combination were classified as either anti-apoptotic BCL-XL dependent or non-apoptosis-inducing (fig. 3A). Interestingly, resistant cell lines to single agent treatment with either that of BAX or BTSA1.2 employed these survival mechanisms (fig. 1H, 1I), suggesting that dual targeting of BAX and BCL-XL could overcome both survival mechanisms in previously categorized resistant cells to single agent treatment.

Next, we assessed how the BCL-2 family is regulated in BNc-sensitive cells. When we detected the protein expression level of BCL-2 family members, only BAX: BCL-XL levels correlate slightly with sensitivity to this combination (fig. 26E). This finding suggests that interactions between BCL-2 family members, rather than protein levels, may regulate sensitivity to the combination. To dissect this, we immunoprecipitated BAX and queried its binding to BCL-XL and MCL-1 anti-apoptotic proteins in several colorectal and non-small cell lung cancer cell lines (fig. 3B, 3C and 26E). In general, cell lines sensitive to this combination form higher levels of BAX than cell lines resistant to it: BCL-XL complex (fig. 3D). The BAX complex formed is consistent with this finding (BCL-XL plays a key role in the apoptosis resistance of these cancer cell lines) as immunoprecipitated BAX has more interactions with BCL-XL (fig. 1B, fig. 3-3C). Next, we treated colorectal cancer and non-small cell lung cancer cell lines that were sensitive to this combination. Treatment with naftopidic destroyed BAX: BCL-XL complex and after co-treatment with BTSA1.2, the additional complex was destroyed, alleviating BCL-XL inhibition of active BAX (fig. 3F, 3G). Importantly, only after treatment with the BTSA1.2 combination, the additional complex was destroyed and apoptosis induction was observed by caspase-3 activation (fig. 3E-3H). Consistently, the initiation of apoptosis of the activator BIM BH3 peptide increased in sensitive cell lines but not in resistant cells following combination therapy (fig. 3J). Thus, our data are consistent with the interaction of BCL-XL with BAX as a determinant of the synergistic apoptotic efficacy of the BTSA 1.2/navitock combination (BNc) (fig. 3I).

The combination of BTSA1.2 and naviatoxin is well tolerated in vivo

Next, the therapeutic potential with the combination of BTSA1.2 and naviatoxin was evaluated in vivo, targeting both survival mechanisms simultaneously. Pharmacokinetic analysis of BTSA1.2 by oral administration showed good properties such as substantial half-life in mouse plasma (T1/2-15 hours), good oral bioavailability (% F-50%) and significant plasma exposure (AUC-100 μΜhr) and peak concentration (Cmax-8 μΜ) (fig. 16A-16B). We performed a Maximum Tolerated Dose (MTD) study according to the standard MTD protocol, which included daily doses of BTSA1.2 at concentrations ranging from 50mg/kg/po to 300mg/kg/po for 5 days and monitored for 14 days. (FIG. 16C). MTD studies showed that orally administered BTSA1.2 was safe, well tolerated, at up to 200mg/kg without dose limiting toxicity (DLT at 300 mg/kg), with BTSA1.2 treated mice showing constant body weight and organs examined between normal histological limits (fig. 16C-16E). Thus, BTSA1.2 is a BAX activator that can be safely administered orally and has desirable pharmacokinetics to address therapeutic effects.

We then used the MTD of each of BTSA1.2 and navitock (200 mg/kg/po for BTSA1.2 and 100mg/kg/po for navitecalx) for toxicity studies on their combinations, as previously Tse et al, cancer res.20080may1; 68 (9) 3421-8 (FIG. 4A). After a single treatment with natatorium, lymphocyte, leukocyte and platelet counts reached a level below normal counts when body weight, erythrocyte count and organs examined were between normal parameters (fig. 4B-4G), as in previous Tse et al, cancer res.20080may1; 68 3421-8 and Whitecross et al blood.2009Feb 26;113 (9) 1982-91. Following BTSA1.2 treatment, the body weight and blood count measured were at normal levels, but a decrease in white blood cell and lymphocyte count was observed following repeated doses. BTSA1.2 was well tolerated with the co-administration of naviatox and no additional toxicity was observed in body weight, organ and blood count compared to single agent treatment (fig. 4B-4G). In addition, the mice treated with nadog/BTSA 1.2 appeared healthy at the time of treatment and daily monitoring, and reached normal blood cell count after the end of treatment (fig. 17A-17C). Compared to previous BH3 mimetics and combinations thereof, e.g., combinations of BCL-2 inhibitor and MCL-1 inhibitor (which exhibit additional toxicity compared to single agents), there is advantageously no toxicity in BTSA1.2 treated mice alone and in combination with naviatoxin. Overall, the data indicate that the combination of BTSA1.2 and naviatoxin is well tolerated and safe for in vivo use.

Combination of BTSA1.2 and naviatoxin is effective in resistant colorectal xenografts

BCL-XL plays a key role in colorectal neoplasia and treatment resistance. However, since preclinical studies indicate that BCL-XL inhibition alone is not sufficient to effectively induce apoptosis, BH3 mimics are not used in clinical trials of colorectal tumors. Here, we found that bncs are able to promote apoptosis of colorectal tumors in vitro. To assess the therapeutic efficacy of the combination of BTSA1.2 and navitock in vivo, we chose to assess colorectal SW480 cells in a xenograft mouse model, as SW480 cells were resistant to BTSA1.2 or Navitoclaz treatment.

After xenograft establishment, mice were randomized into four groups for treatment with vehicle, BTSA1.2, naviatox and combination. When the tumor reaches 200mm ³ With MTD doses, daily oral administration started the treatment (fig. 5A). Although BTSA1.2 or navidoc as single agents had no significant efficacy in reducing tumor growth, oral co-administration of BTSA1.2 and navidoclaz significantly inhibited tumor growth compared to vehicle or single agent treatment, demonstrating the synergistic activity of the two drugs in vivo (fig. 5B-D). Importantly, body weight remained constant during the in vivo study, and mice appeared healthy after treatment with the compound (fig. 5B).

To further demonstrate the synergistic efficacy of the two pro-apoptotic drugs in vivo, we reached 400mm in tumors ³ In agreement with tumor growth data, we determined that tumors treated with only the combination of BTSA1.2 and navitolg exhibited significantly elevated apoptosis markers (fig. 5F-I) compared to vehicle or single agent treatment, and that in summary, our data indicated that the combination of BTSA1.2 and navitolg was synergistically effective and well tolerated in vivo.

Functional marker identification of colorectal tumors in combination-sensitive patients

The data indicate that cell lines classified as sensitive to the combination of BTSA1.2 and naviatoke (BNc) are characterized by BH3 profiling as anti-apoptotic BCL-XL dependent or "non-primed" and form increased levels of BAX compared to cell lines resistant to the combination: BCL-XL complex (fig. 3B-3E). Since these functional assays differentiated cancer cell lines sensitive and resistant to BNc, we assessed whether these functional assays could also be used to predict the efficacy of BNc in patient-derived xenograft (PDX) samples (fig. 6A). Two colorectal patient-derived xenograft (PDX) samples (FIG. 6A), COLO-1 and COLO-2 were analyzed. Analysis of PDX samples by quantitative co-immunoprecipitation of BAX with BCL-XL revealed that both PDX had similar levels of BAX: BCL-XL complex. However, the BH3 spectrum of the PDX sample designates COLO-1 as BCL-XL dependent, while COLO-2 is characterized as "non-triggering" apoptosis (FIGS. 6B-C, 18A-18B). Since BNC is effective in BCL-XL dependent and non-apoptotic inducing cancer cells, COLO-1 and COLO-2PDX are predicted to be sensitive to the combination of BTSA1.2 and Navinotok. Indeed, consistent with enhanced pro-apoptotic activity, ex vivo treatment of PDX showed an increase in BTSA1.2 and naviatoxin combination (BNc) induced loss of viability in both PDX samples when compared to single agent (fig. 6D, fig. 18C-14D).

Next, the therapeutic efficacy of the combination of BTSA1.2 and Navinotor was evaluated in vivo using the mouse PDX model from COLO-1 tumors. After establishment of the PDX model, mice were randomly divided into four groups, treated with vehicle, BTSA1.2, navitolg and combination, and when tumor reached-200 mm ³ To begin treatment (figure 6E). The compound was administered orally once a day using MTD of BTSA1.2, and this time the less toxic dose of that tavern (half of MTD) was tested as a single agent and combination therapy. Treatment was continued for up to 18 days or until the tumor size reached an ethically unacceptable level, and then mice were monitored to assess survival (fig. 6E). The combination of BTSA1.2 with a less toxic dose of Navitoclaz significantly inhibited tumor growth and achieved tumor regression over vehicle, BTSA1.2, or navidoc treatments, demonstrating the synergistic activity of the two drugs in vivo (fig. 6F-6G). Notably, some PDXs were shown to be responsive to BTSA 1.2-only treatment, as their tumor growth was affectedTo inhibition.

Consistent with tumor growth data, the combination of BTSA1.2 and naviatoxin also significantly improved survival after termination of treatment compared to vehicle or single agent treatment (fig. 6H). Furthermore, PDX tumors treated with the drug combination had increased mitochondrial depolarization, increased initiation of apoptosis compared to vehicle-treated PDX, confirming the pro-apoptotic efficacy of the combination (fig. 6I). Notably, body weight remained constant during the in vivo study, and mice appeared healthy after treatment with the compound (fig. 6F). Interestingly, the levels of BCL-XL protein were significantly increased in mice tumors treated with single agents BTSA1.2 or naftoplix, while MCL-1 levels remained constant (fig. 6J). This analysis further supported in vitro data indicating that BCL-XL upregulation confers resistance to single agent BTSA1.2 or naviatoxin therapy (fig. 1, 3C, 3D).

Furthermore, we evaluated COLO-2PDX in vivo to determine the use of BAX: the BCL-XL complex was characterized as whether the non-apoptosis-inducing PDX samples were indeed sensitive to the BTSA1.2 and naviatoxin combination in vivo (fig. 6B-6C, fig. 18C-18D). Similar to the COLO-1PDX study, the combination of BTSA1.2 with less toxic doses of Navitoclaz significantly inhibited COLO-2 tumor growth and achieved tumor regression over vehicle, BTSA1.2 or Navitock treatments, demonstrating the synergistic activity of the two drugs in vivo (FIGS. 6J-6K).

In summary, we were able to predict sensitivity to the combination of BTSA1.2 and Navinotok based on BH3 profiling and co-immunoprecipitation of BAX with BCL-XL. Our data indicate that BAX: BCL-XL complexes, "BCL-XL dependent" and "non-apoptosis-inducing" can be used as sensitive markers for such combination therapies. Importantly, these studies demonstrate the therapeutic efficacy of this combination in colorectal PDX models, even with low doses of navitropine that are less toxic to platelet counts.

Genomic markers predict sensitivity or resistance to the combination of BTSA1.2 and naviatogram

Genomic biomarkers that identify sensitivity or resistance to a combination of drugs can provide information that aids in patient selection and further biological research. BTSA1.2 and naviatoxin combinations were evaluated in a diverse set of solid tumors and hematological malignancies (fig. 2A, 2B) and significant therapeutic efficacy of the combinations in vivo was achieved in specific colorectal tumors (fig. 5C, 6J, 6K), we were interested in identifying genomic markers that could predict tumor sensitivity and resistance to drug combinations. We used genomic information, and in particular disclosed gene expression assays that could be used for several cell lines that were sensitive and resistant to drug combinations (FIG. 2B). Bioinformatics analysis determined significant differences in gene expression between the sensitive and resistant groups and identified-250 genes (hits) with high fold-change and statistical significance (fig. 7A, 18E). We examined the potential association of the most differentially expressed genes with apoptosis, resistance to current treatment, and/or poor prognosis of cancer using literature and patient database searches, and selected several genes for further validation. Genes MUC13, EPS8L3 and IGFBP7, which are highly expressed in sensitive cell lines, were predicted to be potential markers sensitive to the combination of BTSA1.2 and naviatoka. On the other hand, genes such as NR4A3, IRF4 and SLC7A3 were highly expressed in resistant cell lines, suggesting that these genes may be used as markers for resistance to this combination (fig. 7A, fig. 18E).

To confirm the relevance of these specific genes, we selected cell lines that were sensitive and resistant to the BTSA 1.2/naviatogram combination and confirmed the higher expression in sensitive or resistant cell lines by RT-qPCR (fig. 7B). Further analysis showed that the expression level of the cell surface receptor gene MUC13 showed a significant correlation with the BCL-XL gene expression level (fig. 7C). Thus, higher expression of the MUC13 marker correlates with up-regulation of BCL-XL, which also determines sensitivity to the BTSA 1.2/Navelocin combination. Notably, analysis of patient tumor data indicated that colorectal and other solid tumors (such as pancreatic and gastric cancers) had higher levels of MUC13, suggesting that tumor patients with high MUC13 levels were likely to be the most benefited patients from treatment with the combination of BTSA1.2 and naviatoka (fig. 7E). Also, uveal melanoma, thyroid and thymoma were probably the most resistant to combination treatment (fig. 7D). In summary, we were able to identify genomic markers for the combination of BTSA1.2 and naviatoxin based on whole genome bioinformatics, which could be used as sensitivity or resistance markers for this combination therapy.

Discussion of the invention

Many studies have established a key role for BCL-2 family proteins in regulating apoptosis in tumor development, maintenance, and resistance to targeted therapies and chemotherapies. In general, upregulation of the major anti-apoptotic members BCL-2, BCL-w, BFL-1, BCL-XL and MCl-1 blocks pro-apoptotic members and apoptosis. Potent and selective inhibitors of these proteins have been developed and are known as BH3 mimetics. These drugs have been shown to be active against a variety of hematological malignancies. Indeed, valnemulin is a selective BCL-2 inhibitor, the first drug approved for use in patients with in part chronic lymphocytic leukemia or acute myelogenous leukemia. Despite this success, many studies have shown that solid tumors are largely refractory when these drugs are used as single agents. For these more resistant tumors, multiple anti-apoptotic BCL-2 proteins are upregulated and/or the pro-apoptotic BH3 proteins required to activate BAX and BAK to induce apoptosis are kept inhibited.

The development of small molecules that directly activate BAX to induce apoptosis represents a tremendous progression in our ability to promote apoptosis in cancer cells. BAX activators can drive cancer cell apoptosis or enhance initiation of apoptosis and are independent of the availability of BH 3-only protein activators. Here we describe BTSA1.2, a small molecule BAX activator modified from BTSA1 previously described, with BTSA1.2 having improved potency, oral bioavailability and good in vivo tolerability. BTSA1.2 showed significant activity in leukemia and lymphoma cell lines for various solid tumors and hematological malignancies, but similar to other BH3 mimics, BTSA1.2 had reduced efficacy in the solid tumor cell lines tested.

Our studies indicate that higher BAX protein levels are associated with an increase in BTSA1.2 pro-apoptotic activity. BTSA1.2 activity in leukemia cells was consistent with the demonstrated significant efficacy of BTSA1 as a single agent treatment in the human AML model. This is consistent with the mechanism of BAX activation, as increased BAX protein levels can lead to increased activation of BAX by small molecule BAX activators and thus increased Mitochondrial Outer Membrane (MOMP) permeabilization and apoptosis induction. Furthermore, the data also indicate that BCL-XL is the primary regulator of BAX activation response. BCL-XL and BCL-2 have a higher affinity for BAX than MCL-1. Thus, in most solid tumor cell lines, as demonstrated by the absence of BCL-2 expression, BCL-XL is the major anti-apoptotic protein that sequesters activated BAX. When both BCL-XL and BCL-2 proteins are expressed at similar levels, as demonstrated primarily in hematological malignancies, then both proteins can modulate BAX activation. Our nadir tests on the same different cell lines showed that the activity of nadir is mainly dependent on the level of BAX and that targeting BCL-XL alone is insufficient to promote apoptosis in several cell lines. In addition, BH3 spectra underscores that most cells resistant to BTSA1.2 or naviatogram show a dependence on BCL-XL, or they do not trigger apoptosis. Thus, our study of the mechanism of apoptosis resistance in a range of malignancies reveals two survival mechanisms that limit direct and indirect BAX activation and apoptosis induction.

The combination of BTSA1.2 and naviatoka shows synergistic activity in different solid tumors and hematological cell lines, which are usually dependent on BCL-XL inhibition or they do not trigger apoptosis. Notably, this synergistic activity is not affected by common oncogenic mutations (such as TP53 or KRAS), which generally limit chemotherapeutic agents and targeted therapeutic efficacy in cancer. Thus, the combination of BTSA1.2 and naviatoxin can be more widely applied to various tumors.

By profiling the mechanisms of combined BAX activation and BCL-XL inhibition in sensitive cell lines, we found that BAX was formed in untreated conditions in cell lines sensitive to the combination: BCL-XL complex. Activation of cytosolic BAX by BTSA1.2 may promote additional BAX: the BCL-XL complex makes these cells more susceptible to anti-apoptotic inhibition by either nadotor or BCL-XL selective inhibitors. On the other hand, a selective inhibitor of either nado or BCL-XL can use, directly or indirectly, a unhindered BH 3-only protein to destroy BAX: BCL-XL complex. In these cases, the apoptotic activity of BCL-XL inhibition will depend on the level of activated BAX bound to BCL-XL and the level of BH 3-only protein bound to BCL-XL that can be unblocked to activate BAX. Thus, the combined activity of BAX activators and BCL-XL inhibitors provides an effective strategy to induce apoptosis, enabling increased MOMP and apoptosis induction by simultaneously increasing activated BAX levels and inhibiting chelation of activated BAX by BCL-XL.

The combination of BTSA1.2 and naviatoxin also showed a synergistic therapeutic effect in colorectal tumors while also being significantly tolerated in vivo. Indeed, this therapeutic strategy can be very promising for colorectal neoplasms given the previously convincing evidence that high expression levels of BCL-XL play a key role in colorectal neoplasia and therapeutic resistance. Despite these evidence, the use of naftopsides in colorectal tumors, including our work here, suggests that BCL-XL inhibition is inadequate as a single agent treatment and does not drive apoptosis effectively. Previous studies have shown that naftopsides is effectively synergistic with targeted therapies such as EGFR inhibitors in non-small lung cancer, KRAS mutated cancers and MEK inhibitors in BRAF mutated melanomas. These studies indicate that targeting the oncogenic driving pathway results in increased BH 3-only proteins (e.g., upregulation of BIM by MEK inhibitors), which enhances the initiation and efficacy of the tavertock-mediated BCL-XL inhibition. Active clinical trials are currently testing the efficacy of these combinations on patients (NCT 03222609, NCT02079740, NCT 01989585). However, these combination strategies rely on mutations in specific kinases to be effective. Since in our study we found that the mutated background of cancer cells did not affect the synergy between navitock and BTSA1.2, this supports the potential for this combination strategy to be effective against a variety of tumors. In addition, the effect of that on thrombocytopenia is hindered by that of that, and combination therapy in clinical trials requires an effective therapeutic window. Thus, it is notable that our combination studies in vivo demonstrate the synergistic therapeutic efficacy of reduced dose of natatorium, as well as the overall safety of tissue and blood counts. In addition, efforts are underway to develop clinical compounds targeting BCL-XL that have minimal platelet toxicity, and these compounds can alternatively be used to enhance the pro-apoptotic activity of BTSA 1.2.

Our work is based on BAX of cancer cells: the BCL-XL complex and BH3 spectra, functional assays and markers were determined to predict sensitivity to simultaneous BAX activation and BCL-XL inhibition. Diagnostic assays for identifying BAX-containing protein complexes and the development of BH3 spectra from solid tumor biopsies should be established next. While this has yet to be determined, our data on genomic analysis and identification of susceptibility or resistance genes to drug combinations provides information useful for biomarker screening. Our analysis identified that high levels of MUC13 are markers of sensitivity to the combination of BTSA1.2 and naviatoxin, indicating that this combination treatment is beneficial for cancer patients with high levels of MUC 13. Interestingly, MUC13 has been proposed as a poor marker of colorectal tumor prognosis, supporting our discovery of combined targeting of BAX and BCL-XL in resistant colorectal tumors. Furthermore, from a mechanistic point of view, our bioinformatic analysis provides stimulatory data for future studies to analyze the impact and relationship of markers such as MUC13 on modulation of expression and interactions in BCL-2 protein families and apoptosis induction.

Taken together, the data herein facilitate an understanding of the mechanism of cell death in cancer cells and demonstrate a novel therapeutic strategy that reasonably targets pro-apoptotic BAX and anti-apoptotic BCL-XL to overcome the mechanism of apoptosis resistance in a range of tumors. Our findings provide preclinical proof of concept for a new combination of BAX activator BTSA1.2 and navitolg, which may provide a broad therapeutic effect on tumors.

The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of: any suitable material, step, or composition disclosed herein. Additionally or alternatively, the compositions, methods, and articles of manufacture may be formulated to be free or substantially free of any material (or substance), step, or component otherwise not necessary to achieve the function or purpose of the compositions, methods, and articles of manufacture.

All ranges disclosed herein include endpoints, and endpoints are independently combinable (e.g., ranges of "up to 25wt.%, or, more specifically, 5wt.% to 20wt.%," inclusive of the endpoints and all intermediate values of the ranges of "5wt.% to 25wt.%," etc.). "combination" includes blends, mixtures, alloys, reaction products, and the like. The terms "first," "second," and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The terms "a" and "an" and "the" do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. "or" means "and/or" unless explicitly stated otherwise. Reference throughout the specification to "some embodiments," "one embodiment," and so forth, means that a particular element described in connection with an embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. Furthermore, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. "combinations thereof are open and include any combination comprising at least one listed component or property optionally together with a similar or equivalent component or property not listed.

Unless specified to the contrary herein, all test criteria are the latest criteria validated since the filing date of the present application or, if priority is required, the filing date of the earliest priority application in which the test criteria appear.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.

Although particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may occur to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents.

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Claims (28)

1. A pharmaceutical combination comprising:

b-cell lymphoma 2 associated protein X (BAX) activating compounds; and

Anti-apoptotic protein inhibiting compounds.

2. The pharmaceutical combination of claim 1, wherein the anti-apoptotic protein inhibitory compound is a BCL-XL inhibitory compound, a BCL-2 inhibitory compound, a BCL-w inhibitory compound, a BFL-1 inhibitory compound, or an MCL-1 inhibitory compound.

3. The pharmaceutical combination according to claim 1 or 2, wherein the BAX activating compound is a compound having the structure BTSA1.2 or a pharmaceutically acceptable salt thereof

4. The pharmaceutical combination according to claim 1 or claim 2, wherein the anti-apoptotic protein inhibiting compound comprises naftopiramate or valnemulin.

5. The pharmaceutical combination according to claim 1 or 2, wherein the anti-apoptotic protein inhibiting compound is ABT-737, nadog, valnematox, AMG 176, AMG 397, AZD-4320, AZD-0466, AZD-5991, VU661013, S65487, MIK665, saibutoc, gambogic acid, obatuk mesylate, APG1252, DT2216, or a combination of any of the foregoing.

6. A pharmaceutical composition comprising the pharmaceutical combination according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

7. A method of treating cancer in a subject, comprising administering to the subject a B-cell lymphoma 2 associated X protein (BAX) activating compound in combination with a B-cell lymphoma oversized protein (BCL-XL) inhibiting compound, a BCL-2 inhibiting compound, a BCL-w inhibiting compound, a BFL-1 inhibiting compound, or an MCL-1 inhibiting compound in an amount effective to treat cancer in the subject.

8. The method of claim 7, wherein the BAX activating compound is a compound having the structure of BTSA1.2 or a pharmaceutically acceptable salt thereof

9. The method of claim 7 or 8, wherein the anti-apoptotic protein inhibiting compound comprises naviatox or vinatox.

10. The method of any one of claims 7-9, wherein the cancer is a hematologic cancer or a solid tumor.

11. The method according to any one of claim 7 to 10, wherein the cancer is breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, melanoma, malignant melanoma, ovarian cancer, brain or spinal cord cancer, primary brain epithelial carcinoma, medulloblastoma, neuroblastoma, glioma, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, gastric cancer, renal cancer, placenta cancer, gastrointestinal cancer, non-small cell lung cancer (NSCLC), head and neck epithelial cancer, breast epithelial cancer, endocrine cancer, eye cancer, genitourinary system cancer, vulval cancer, ovarian cancer, uterine or cervical cancer, hematopoietic cancer, myeloma, leukemia, lymphoma, ovarian epithelial cancer, lung epithelial cancer, small cell lung epithelial cancer, renal cell carcinoma, cervical epithelial cancer, testicular epithelial cancer, bladder epithelial cancer, pancreatic epithelial cancer gastric epithelial cancer, colonic epithelial cancer, prostate epithelial cancer, genitourinary epithelial cancer, thyroid epithelial cancer, esophageal epithelial cancer, myeloma, multiple myeloma, adrenal epithelial cancer, renal cell epithelial cancer, endometrial epithelial cancer, adrenal cortical epithelial cancer, malignant pancreatic insulinoma, malignant carcinoid epithelial cancer, chorionic epithelial cancer, mycosis mycotica, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, kaposi sarcoma, polycythemia vera, primary thrombocythemia, hodgkin's disease, non-hodgkin's lymphoma, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, sarcoma, primary macroglobulinemia, central nervous system carcinoma, and retinoblastoma.

12. The method of any one of claims 7-11, wherein the cancer is colon cancer, rectal cancer or colorectal cancer.

13. The method of any one of claims 7-12, wherein the route of administration comprises oral, rectal, sublingual, buccal, intravenous, intramuscular, transdermal, dermal, subcutaneous, intrathecal, nasal, vaginal, or a combination thereof.

14. The method of any one of claims 7-13, wherein the route of administration is oral.

15. A method of treating cancer in a subject in need thereof, the method comprising:

obtaining a biological sample comprising cancer cells from the subject;

detecting BAX immunoprecipitated from the cancer cells: the level of anti-apoptotic protein complex and/or detecting that the cancer cell is anti-apoptotic protein dependent or non-apoptotic; and

administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptosis inhibiting compound in an amount effective to treat the cancer.

16. The method of claim 15, wherein the anti-apoptotic protein inhibiting compound is a BCL-XL inhibiting compound, a BCL-2 inhibiting compound, a BCL-w inhibiting compound, a BFL-1 inhibiting compound, or an MCL-1 inhibiting compound.

17. The method of claim 15, wherein the BAX: an anti-apoptotic protein complex is formed by co-immunoprecipitation of BAX and anti-apoptotic proteins from the cancer cells.

18. The method of claim 17, wherein the BAX: the anti-apoptotic protein complex is BAX: BCL-XL, and the anti-apoptotic protein is BCL-XL; the BAX: the anti-apoptotic protein complex is BAX: BCL-2, and said anti-apoptotic protein is BCL-2; the BAX: the anti-apoptotic protein complex is BAX: BCL-w, and said anti-apoptotic protein is BCL-w; the BAX: the anti-apoptotic protein complex is BAX: BFL-1, and said anti-apoptotic protein is BFL-1, or, said BAX: the anti-apoptotic protein complex is BAX: MCL-1, and the anti-apoptotic protein is MCL-1.

19. The method of claim 17 or claim 18, wherein the method further comprises BAX in the cancer cell when compared to a normal cell of the same type: BCL-XL, BAX: BCL-2, BAX: BCL-w, BAX: BFL-1 or BAX: determining said cancer cells that are sensitive to said anti-cancer agent when the level of MCL-1 complex increases.

20. The method of any one of claims 16-19, wherein the detection of cancer cells that are anti-apoptotic BCL-XL, BCL-2, BCL-w, BFL-1, or MCL-1 dependent or non-triggering apoptosis comprises BH3 profiling.

21. The method of claim 20, wherein the BH3 profiling comprises contacting the cancer cell with a BH3 domain peptide, measuring the amount of BH3 domain peptide-induced mitochondrial depolarization in the cancer cell, and comparing the amount of BH3 domain peptide-induced mitochondrial depolarization in the cancer cell to a control cell population of the same type.

22. A method of treating cancer in a subject in need thereof, the method comprising:

obtaining a biological sample from the subject;

measuring the expression level of at least one gene in the biological sample, wherein the gene comprises MUC13, EPS8L3, IGFBP7, or a combination thereof; and

administering to the subject an anti-cancer agent comprising a B-cell lymphoma 2 associated protein X (BAX) activating compound and an anti-apoptotic protein inhibiting compound in an amount effective to treat the cancer.

23. The method of claim 22, wherein the anti-apoptotic protein inhibiting compound is a BCL-XL inhibiting compound, a BCL-2 inhibiting compound, a BCL-w inhibiting compound, a BFL-1 inhibiting compound, or an MCL-1 inhibiting compound.

24. The method of claim 23, further comprising determining that the expression level of the genes MUC13, EPS8L3, IGFBP7, or a combination thereof is increased in the biological sample as compared to a control sample prior to administration of the sample, and the anti-apoptotic protein inhibitory compound is a BCL-XL inhibitory compound.

25. The method of claim 24, wherein the biological sample comprises cancer cells and the control sample comprises normal cells of the same type.

26. The method of any one of claims 23-25, wherein the detecting comprises quantitative reverse transcription PCR to determine the level of gene messenger RNA in the biological sample.

27. The method of any one of claims 15-26, wherein the BAX activating compound is a compound having the structure of BTSA1.2 or a pharmaceutically acceptable salt thereof

28. The method of any one of claims 15-27, wherein the anti-apoptotic inhibiting compound comprises nadir or valnemtock.