Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Cancer Res. 2017 Feb 15;77(9):2512–2521. doi: 10.1158/0008-5472.CAN-16-3242

Mcl-1 degradation is required for targeted therapeutics to eradicate colon cancer cells

Jingshan Tong 1, Peng Wang 1, Shuai Tan 1,3, Dongshi Chen 1, Zaneta Nikolovska-Coleska 4, Fangdong Zou 3, Jian Yu 2, Lin Zhang 1,*
PMCID: PMC5626525  NIHMSID: NIHMS909042  PMID: 28202514

Abstract

The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degradation has been associated with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degradation and anticancer drug responses has not been directly established, especially in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in determining effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degradation was induced by a variety of multi-kinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the anti-angiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degradation-resistant Mcl-1 bound and sequestered PUMA from other pro-survival proteins to maintain cell survival, which was abolished by small-molecule Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degradation in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance.

Keywords: Mcl-1, kinase inhibitors, apoptosis, PUMA, colon cancer

Introduction

Stress-induced apoptosis in mammalian cells is regulated by the Bcl-2 family proteins through a series of orderly events, including mitochondrial outer membrane permeabilization (MOMP), cytosolic release of mitochondrial proteins such as cytochrome c, and activation of caspases (1,2). Myeloid cell leukemia 1 (Mcl-1) is a pro-survival Bcl-2 family protein frequently overexpressed or amplified in human cancers (3). Mcl-1 inhibits cell death by binding to proapoptotic Bcl-2 family proteins to suppress MOMP and caspase activation (4). A number of recent studies suggest a critical role of Mcl-1 in tumor cell survival and therapeutic resistance (4).

Distinctive from other Bcl-2 family members, Mcl-1 is a very unstable protein, and degradation of Mcl-1 can be triggered by various stresses including anticancer agents (5). Mcl-1 degradation is regulated by its phosphorylation at several sites, leading to subsequent ubiquitination by E3 ligases such as F-box and WD repeat domain-containing 7 (FBW7), Mule, and β-TrCP (6–9). Mcl-1 depletion clearly contributes to apoptosis in hematopoietic cells (10,11). However, the role of Mcl-1 degradation in solid tumor cells is unclear, as Mcl-1 depletion typically occurs prior to the onset of apoptosis in response to therapeutic treatment, and by itself, is insufficient to trigger cell death (12).

Incorporation of targeted therapy has substantially improved cancer treatment. Most of clinically used targeted drugs are inhibitors of aberrantly activated oncogenic kinases. For example, the multi-kinase inhibitor regorafenib (Stivarga) and its analog sorafenib (Nexavar) are used to treat chemotherapy-resistant and metastatic colorectal cancer (CRC) and other gastrointestinal malignancies (13,14). Regorafenib and sorafenib inhibit kinases involved in mitogenic signaling and tumor angiogenesis, including CRAF, BRAF, VEGFRs, PDGFR, and c-KIT (15,16). Our recent study showed that intrinsic and acquired resistance of CRC cells to regorafenib is associated with blocked Mcl-1 degradation due to inactivating mutations in the tumor suppressor FBW7 (17), suggesting a critical role of Mcl-1 degradation in mediating response to targeted therapy in CRC cells.

In this study, we used a genetic knock-in (KI) approach to determine the role of Mcl-1 degradation in killing of cancer cells by targeted therapy. Our results indicate that Mcl-1 maintains CRC cell survival by sequestering the BH3-only Bcl-2 family protein PUMA, which explains why Mcl-1 degradation is necessary, but insufficient for cell death.

Materials and Methods

Cell culture

Human CRC cell lines were obtained from the American Type Culture Collection (Manassas, VA). FBW7-KO HCT116 cells were obtained from Horizon Discovery (Cambridge, UK). PUMA-KO HCT116 cells were previously described (18). Cell lines were authenticated in 2015 by genotyping, western blotting of protein expression, and routine detection of mycoplasma contamination by PCR. All cell lines were maintained at 37°C and 5% CO2 in McCoy’s 5A modified media (Invitrogen) supplemented with 10% defined FBS (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20–30% density 24 hr before treatment. Chemicals, including regorafenib, sorafenib, UCN-01, sunitinib, roscovitine, sulindac sulfide, etoposide, MG132, ABT-737, ABT-263, SB216736, TW37 (Selleck Chemicals), cycloheximide (CHX; Sigma), and UMI-77 (19,20), were solubilized in DMSO and diluted to appropriate concentrations with the cell culture medium. TRAIL (XcessBio, San Diego, CA) was diluted with distilled water.

MTS assay

Cells were seeded in 96-well plates at a density of 1×104 cells/well. After overnight incubation, various concentrations of agents were added into wells and incubated for additional 72 hr. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed using the MTS assay kit (Promega) according to the manufacturer’s instructions. Chemiluminescence was measured with a Wallac Victor 1420 Multilabel Counter (Perkin Elmer). Each assay was conducted in triplicate and repeated three times.

Western blotting

Western blotting was performed as previously described (21). The following antibodies were used: PUMA (18), phospho-Mcl-1 (Ser159/Thr163), cleaved caspases 3, 8, and 9, ERK, phospho-ERK (Thr202/Tyr204), GSK3β, phospho-GSK3β (Ser9), Bad (Cell Signaling), V5, cytochrome oxidase subunit IV (Invitrogen), cytochrome c (Sigma), Bak (Millipore), Bax, HA, Mcl-1 (Santa Cruz), Bcl-2 (Dako), Bim, Bid, Noxa, β-actin (EMD Biosciences), Bcl-XL (BD Biosciences), and FBW7 (Bethyl).

Transfection and siRNA knockdown

Adenoviruses expressing PUMA and expression construct of V5-tagged Bcl-XL are previously described (22,23). Mcl-1 expression construct was generated by cloning a PCR-amplified full-length human Mcl-1 cDNA fragment into pcDNA3.1/V5-His vector (Invitrogen). Mutations were introduced into Mcl-1 using QuickChange XL site-directed mutagenesis kit (Agilent Technologies).

Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Knockdown was performed 24 hr before regorafenib treatment by transfecting 200 pmol of siRNA for human CRAF (AAGCACGCTTAGATTGGAATA-dTdT), Mcl-1 (CGCCGAATTCATTAATTTATT-dTdT), or GSK3β (sc-35527; Santa Cruz Biotechnology), or control scrambled siRNA (GE Dharmacon).

Immunoprecipitation

Cells were harvested and suspended in 1 ml of EBC buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Applied Sciences). After disruption of cells by sonication, cell lysates were collected by centrifugation at 10,000×g for 10 min. For immunoprecipitation (IP), 1–2 μg of IP antibodies were mixed with protein G/A-agarose beads (Invitrogen) for 20 min at room temperature. The beads were washed twice with PBS containing 0.02% Tween 20 (pH 7.4), incubated with cell lysates on a rocker for 6 hr at room temperature, and then washed thrice with PBS (pH 7.4). Beads were then boiled in 2× Laemmli buffer and subjected to SDS-PAGE and western blotting.

Knock-in of mutant Mcl-1

The Mcl-1 targeting vector was constructed using the pUSER-rAAV (recombinant adeno-associated virus) System. Briefly, two homologous arms of ~1 kb each flanking the first intron of Mcl-1 were inserted between 2 USER sites in the AAV shuttle vector pTK-Neo-USER. The coding sequence for the targeted Mcl-1 mutant (S121A/E125A/S159A/T163A) was introduced into the left arm using the QuickChange XL Site-Directed Mutagenesis Kit (Agilent Technologies). For gene targeting, HCT116 cells were infected with the targeting rAAV and selected by G418 (0.5 mg/mL; Corning Mediatech) for 3 weeks. G418-resistant clones were pooled and screened by PCR for targeting events. To target the second allele, Neo flanked by 2 LoxP sites was excised from a heterozygous clone by infection with an adenovirus expressing Cre recombinase (Ad-Cre). The same targeting construct was used in the second round of gene targeting. After the second round, Neo was excised by Ad-Cre infection and targeting was verified by sequencing of genomic DNA and western blotting.

Reverse transcriptase (RT) PCR and genomic PCR

To identify knock-in cell lines, genomic DNA was isolated from 5–10×104 cells by using ZR-96 Quick-gDNA Kit (ZYMO Research) according to the manufacturer’s instructions. One μl out of 50 μl genomic DNA preparation was used for PCR using previously described conditions (24) and primers listed in Table S1. Cycle conditions are available upon request. For analysis of Mcl-1 mRNA expression, total RNA was isolated from cells using the Mini RNA Isolation II kit (ZYMO Research) according to the manufacturer’s protocol. One μg of total RNA was used to generate cDNA with SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed for Mcl-1 and β-actin using the primer pairs listed in Table S1.

Analysis of apoptosis

Adherent and floating cells were harvested, stained with Hoechst 33258 (Invitrogen), and analyzed for apoptosis by nuclear staining and counting cells with condensed and fragmented nuclei. At least 300 cells were analyzed for each treatment. Annexin V/propidium iodide (PI) staining was performed using Annexin-Alexa Fluor 488 (Invitrogen) and PI as described (24). For colony formation assays, equal numbers of cells were subjected to various treatments and plated into 12-well plates at different dilutions. Colonies were visualized by crystal violet staining 14 days after plating. Each experiment was performed in triplicate and repeated at least twice. For analysis of cytochrome c release, cytoplasmic and mitochondrial fractions were separated by Mitochondrial Fractionation Kit (Active Motif) according to the manufacturer’s instructions, followed by western blotting of cytochrome c in the cytoplasmic and mitochondrial fractions.

Xenograft tumor experiments

All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Female 5- to 6-week-old Nu/Nu mice (Charles River) were housed in a sterile environment with micro isolator cages and allowed access to water and chow ad libitum. Mice were injected subcutaneously in both flanks with 4×106 WT or Mcl-1-KI HCT116 cells. After tumor growth for 7 days, mice were treated daily with regorafenib at 30 mg/kg by oral gavage for 10 consecutive days. Regorafenib was dissolved in Cremephor EL/95% ethanol (50:50) as a 4× stock solution. Tumor size was monitored by calipers, and tumor volumes were calculated according to the formula 0.5 × length × width2. Mice were euthanized when tumors reached 1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL; Millipore), active caspase 3 (Cell Signaling), CD31 (Spring Bioscience), and Carbonic Anhydrase 9 (CA9; Santa Cruz) immunostaining was performed on 5-mm paraffin-embedded tumor sections as previously described (25), with an Alexa Fluor 488-conjugated secondary antibody (Invitrogen) for detection.

Interactions of FBW7/Mcl-1 and PUMA/Mcl-1 were detected by in situ proximity ligation assay (PLA) in paraffin-embedded sections of WT or Mcl-1-KI HCT116 tumor tissues from nude mice treated with or without regorafenib for 1 day. PLA was performed using the Duolink In Situ Redstarter Kit (Sigma) according to the manufacturers’ instructions. Incubation with primary antibodies for FBW7 (rabbit, Abcam, 1:100), PUMA (rabbit, Abcam, 1:50), and Mcl-1 (mouse, BD Biosciences, 1:100) was performed at 4°C overnight. The sections were mounted with Vectashield Mounting Medium (Vector Laboratories) with DAPI (Sigma) for nuclear counter staining. PLA signals were visualized by fluorescence microscopy (Olympus) and quantified by counting.

Statistical analysis

Statistical analyses were performed using GraphPad Prism V software. P values were calculated by the student’s t-test, and considered significant if P < 0.05. The means ± one standard deviation (s.d.) was displayed in the figures.

Results

Induction of Mcl-1 degradation by kinase inhibitors in CRC cells

After analyzing over 50 different anticancer agents, we identified several kinase inhibitors that induced depletion of Mcl-1 protein at a concentration that triggered apoptosis in HCT116 colon cancer cells, including FDA-approved drugs regorafenib, sorafenib, and sunitinib, the broad-range kinase inhibitor UCN-01, and the CDK inhibitor roscovitine (Fig. 1A). In contrast, Mcl-1 stability was not affected by other agents such as the death receptor ligand TRAIL, the DNA damaging agent etoposide, and the non-steroidal anti-inflammatory drug sulindac sulfide. We found that regorafenib and sorafenib induced Mcl-1 degradation in a dose- and time-dependent manner (Fig. 1B) (17), and in different CRC cells regardless of their KRAS, BRAF, PIK3CA or p53 status (Fig. 1C). The half-life of Mcl-1 protein, but not its mRNA expression, was reduced following regorafenib or sorafenib treatment (Fig. 1D; Supplementary Fig. S1, A and B). Mcl-1 depletion was blocked by the proteasome inhibitor MG 132 (Fig. 1E), suggesting ubiquitin/proteasome-dependent protein degradation.

Figure 1. Induction of Mcl-1 proteasomal degradation by inhibition of oncogenic kinase signaling.

Figure 1

Open in a new tab

(A) Western blotting of Mcl-1 in HCT116 colon cancer cells treated for 24 hr with 10 nM TRAIL, 20 μM etoposide, 120 μM sulindac sulfide (sulindac), 20 μM sorafenib, 40 μM regorafenib, 1 μM UCN-01, 15 μM sunitinib, or 10 μM roscovitine. (B) Western blotting of Mcl-1 in HCT116 cells treated with 40 μM regorafenib or 20 μM sorafenib at indicated time points. (C) Western blotting of Mcl-1 in indicated colon cancer cell lines treated with 40 μM regorafenib for 24 hr. (D) Western blotting of Mcl-1 in HCT116 cells treated with control vehicle or 40 μM regorafenib for 1 hr, and then with 10 μg/ml of the translation inhibitor cyclohemide (CHX) at indicated time points. (E) Western blotting of Mcl-1 in HCT116 cells treated with 40 μM regorafenib or 20 μM sorafenib for 4 hr, with or without pretreatment with 5 μM MG132 for 30 min.

Mcl-1 degradation requires phosphorylation by GSK3β and ubiquitination by FBW7

Stress-induced Mcl-1 degradation is mediated by its phosphorylation (26). Regorafenib treatment promoted Ser159/Thr163 phosphorylation of Mcl-1, coinciding with a sharp decline in Mcl-1 protein (Fig. 2A). Consistent with the requirement of FBW7 for Mcl-1 degradation (17), regorafenib treatment promoted binding of FBW7 with phosphorylated Mcl-1 (p-Mcl-1) (Fig. 2B), and ubiquitination of Mcl-1, which was absent in FBW7-knockout (KO) HCT116 cells (Fig. 2C). GSK3β, which is activated by suppression of its inhibitory Ser9 phosphorylation (Fig. 2D), is responsible for the effect of regorafenib on Mcl-1, as regorafenib-induced Mcl-1 phosphorylation, ubiquitination and degradation was abrogated by the GSK3β inhibitor SB216736 (Fig. 2, E-G), or GSK3β knockdown by siRNA (Supplementary Fig. S1C). We then dissected the upstream kinases responsible for GSK3β activation and Mcl-1 degradation. Knockdown of CRAF or inhibition of ERK alone was sufficient to induce GSK3β de-phosphorylation and Mcl-1 degradation (Fig. 2, H and I), while knockdown of other regorafenib targets, including c-KIT, PDGFR, VEGFR, AKT, and BRAF, did not affect Mcl-1 stability (data not shown). These results suggest that inhibition of CRAF/ERK signaling by regorafenib promotes GSK3β-mediated Mcl-1 phosphorylation, which facilitates its binding to FBW7 and subsequent ubiquitination and proteasomal degradation.

Figure 2. GSK3β-dependent Mcl-1 phosphorylation mediates its interaction with FBW7 and proteasomal degradation.

Figure 2

Open in a new tab

(A) Western blotting of total and phosphorylated Mcl-1 (p-Mcl-1; Ser159/Thr163) in HCT116 cells treated with 40 μM regorafenib at indicated time points. (B) HCT116 cells pretreated with 5 μM of the proteasome inhibitor MG132 for 30 min were treated with 40 μM regorafenib for 24 hr. Immunoprecipitation (IP) was performed to pull down Mcl-1, followed by western blotting of indicated proteins. (C) WT and FBW7-KO HCT116 cells transfected with HA-ubiquitin and pretreated with 5 μM MG132 for 30 min were treated with 40 μM regorafenib for 4 hr. IP was performed to pull down Mcl-1, followed by western blotting of indicated proteins. (D) Western blotting of total and phosphorylated GSK3 (p-GSK3β; Ser9) and ERK (p-ERK; Thr202/Tyr204) in HCT116 cells treated with 40 μM regorafenib at indicated time points. (E) HCT116 cells pretreated with 5 μM MG132 for 30 min were treated with 40 μM regorafenib with or without the GSK3 inhibitor SB216736 for 24 hr. IP was performed to pull down Mcl-1, followed by western blotting of indicated proteins. (F) HCT116 cells transfected with HA-ubiquitin and pretreated with 5 μM of MG132 for 30 min were treated with 40 μM regorafenib with or without SB216736 for 4 hr. IP was performed to pull down Mcl-1, followed by western blotting of indicated proteins. (G) Western blotting of indicated proteins in HCT116 cells treated with 40 μM regorafenib with or without SB216736 for 48 hr. (H) Western blotting of indicated proteins in HCT116 cells treated with 25 μM of the ERK inhibitor PD98059 for 24 hr. (I) Western blotting of Mcl-1 in HCT116 cells with siRNA knockdown of CRAF, which was verified by RT-PCR.

Knock-in (KI) of a phosphorylation site mutant blocks Mcl-1 phosphorylation, ubiquitination and degradation

To determine the role and mechanism of Mcl-1 degradation, we sought to specifically inhibit Mcl-1 degradation by blocking its phosphorylation. Previous studies identified 4 phosphorylation sites on Mcl-1, including S121, E125, S159 and T163, which can be phosphorylated by various kinases to promote Mcl-1 degradation in response to different stimuli (8,9). A transfected quadruple mutant in these 4 sites (S121A/E125A/S159A/T163A) was resistant to regorafenib-induced Mcl-1 ubiquitination and degradation (Supplementary Fig. S1, D and E), which prompted us to design a targeting strategy to knock-in (KI) this mutant in HCT116 cells (Fig. 3A). After two rounds of homologous recombination, we identified HCT116 cell lines with KI of the Mcl-1 phosphorylation site mutant (Mcl-1-KI) (Fig. 3B). KI of the Mcl-1 mutant did not affect its steady-state expression and mitochondrial localization (Fig. 3, C and D), but substantially increased its half-life (Fig. 3E). We then compared Mcl-1-KI and wild-type (WT) HCT116 cells for their response to regorafenib and sorafenib. In stark contrast to WT cells, Mcl-1-KI cells almost completely lacked regorafenib-induced Mcl-1 degradation, FBW7 binding, and ubiquitination (Fig. 3, F-H), indicating complete dependence of Mcl-1 phosphorylation and ubiquitination on these 4 sites.

Figure 3. Selectively blocking Mcl-1 phosphorylation by knock-in of a phosphorylation site mutant suppresses Mcl-1 degradation.

Figure 3

Open in a new tab

(A) Schematic representation of the Mcl-1 genomic locus and the knock-in vector highlighting the putative phosphorylation sites of Mcl-1 that are involved in its degradation. (B) DNA sequencing of the targeted genomic regions in WT and Mcl-1 knock-in (Mcl-1-KI) HCT116 cells highlighting WT and corresponding mutant sequences. (C) Western blotting of Mcl-1 in untreated WT and two independent Mcl-1-KI cell lines. (D) Mcl-1 localization was analyzed by western blotting of mitochondrial and cytosolic fractions isolated from indicated cells. β-Actin and cytochrome oxidase subunit IV (COX IV) were used as a control for loading and fractionation. (E) Western blotting of Mcl-1 in WT and Mcl-1-KI cells treated with control vehicle or 40 μM regorafenib for 1 hr, and then with 10 μg/ml of the translation inhibitor cyclohemide (CHX) at indicated time points. (F) Western blotting of Mcl-1 in WT and Mcl-1-KI HCT116 cells treated with 40 μM regorafenib at indicated time points. (G) WT and Mcl-1-KI HCT116 cells pretreated with 5 μM MG132 for 30 min were treated with 40 μM regorafenib for 24 hr. IP was used to pull down Mcl-1, followed by western blotting of indicated proteins. (H) WT and Mcl-1-KI HCT116 cells transfected with HA-ubiquitin and pretreated with 5 μM MG132 for 30 min were treated with 5 μM regorafenib for 4 hr. IP was used to pull down Mcl-1, followed by western blotting of indicated proteins.

Mcl-1 KI causes resistance to kinase inhibitors by blocking the intrinsic pathway

Mcl-1-KI cells were found to be highly resistant to regorafenib and sorafenib, showing markedly increased viability (Fig. 4A), enhanced clonogenic survival (Fig. 4B), and reduced apoptosis measured by nuclear fragmentation (Supplementary Fig. S2A), annexin V/propidium iodide (PI) staining (Supplementary Fig. S2B), and activation of caspases 3, 8 and 9 (Fig. 4C). The Mcl-1 dependence was verified by knockdown of Mcl-1, which restored regorafenib sensitivity in Mcl-1-KI cells (Fig. 4A and Supplementary Fig. S2A). Mcl-1-KI cells were defective in regorafenib-induced mitochondrial events (1,2), including release of cytochrome c and collapse of MOMP (Fig. 4D and data not shown), and Bax conformational changes and oligomerization (Fig. 4, E and F). Inhibiting Mcl-1 upstream GSK3β or knockout of its downstream BAX also suppressed apoptosis induced by regorafenib or sorafenib (Supplementary Fig. S2, C-E).

Figure 4. Blocking Mcl-1 degradation renders resistance to regorafenib and sorafenib by blocking the mitochondrial apoptotic pathway.

Figure 4

Open in a new tab

(A) MTS analysis of viability of WT, Mcl-1-KI, and Mcl-1-KI with knockdown of Mcl-1 HCT116 cells treated with regorafenib or sorafenib at different concentrations for 72 hr. (B) Colony formation assay was done by seeding an equal number of WT and Mcl-1-KI HCT116 cells treated with 40 μM regorafenib or 20 μM sorafenib for 48 hr in 12-well plates, and staining of the attached cells with crystal violet after 14 days. Left, representative pictures of colonies; right, enumeration of colony numbers. ***, P < 0.001. (C) Western blotting of Mcl-1 and cleaved (C) caspases 3, 8 and 9 in WT and Mcl-1-KI HCT116 cells treated with 40 μM regorafenib or 20 μM sorafenib for 48 hr. (D) Cytochrome c release in cells treated with 40 μM regorafenib or 20 μM sorafenib was analyzed by western blotting of mitochondrial or cytosolic fractions isolated from treated cells. β-Actin and cytochrome oxidase subunit IV (COX IV) were used as a control for loading and fractionation, respectively. (E) Bax conformational change was detected by immunoprecipitation (IP) with anti-Bax 6A7 (activated) antibody followed by western blotting. (F) Bax multimerization in isolated mitochondria was analyzed by western blotting under non-denaturing conditions following DSP cross-link.

Mcl-1-KI cells were defective in Mcl-1 degradation and apoptosis induced by other kinase inhibitors, including UCN-01, sunitinib, and roscovitine, but remained sensitive to the agents that did not induce Mcl-1 degradation (Fig. 5, A and B). Interestingly, all of the kinase inhibitors that relied on Mcl-1 degradation were found to induce the expression of the BH3-only Bcl-2 family protein PUMA (Fig. 5C). These kinase inhibitors, including regorafenib, sorafenib, UCN-01 and sunitinib, have been shown to be dependent on PUMA to induce apoptosis in CRC cells (24,25,27,28). In contrast, regorafenib treatment did not affect the expression of other Bcl-2 family members, except for a slight induction of Bim (Supplementary Fig. S3A).

Figure 5. Mcl-1 degradation mediates sensitivity of CRC cells to kinase inhibitors through PUMA-dependent apoptosis.

Figure 5

Open in a new tab

(A) Western blotting of Mcl-1 in WT and Mcl-1-KI HCT116 cells treated with indicated agents for 24 hr. (B) Apoptosis in WT and Mcl-1-KI HCT116 cells treated with indicated agents for 48 hr. (C) Western blotting of Mcl-1 and PUMA in HCT116 cells treated with indicated agents for 24 hr. (D) WT and Mcl-1-KI HCT116 cells were treated with 40 μM regorafenib for 24 hr. IP was performed to pull down Mcl-1, followed by western blotting of indicated proteins. (E) WT and Mcl-1-KI HCT116 cells transfected with V5-Bcl-XL were treated with 40 μM regorafenib for 24 hr. IP was performed to pull down V5 (Bcl-XL), followed by western blotting of indicated proteins. (F) Apoptosis in WT and Mcl-1-KI HCT116 cells treated with indicated agents with or without a combination with 5 μM UMI-77 or TW-37. (G) Western blotting of cleaved (C) caspase 3 in WT and Mcl-1-KI HCT116 cells treated as in (F). (H) Apoptosis in WT and Mcl-1-KI HCT116 cells infected with a low dose of adenovirus expressing full-length (Ad-PUMA) or BH3 domain-deleted PUMA (ΔBH3), and then treated with indicated agents. (I) Western blotting of cleaved (C) caspase 3 in WT and Mcl-1-KI HCT116 cells treated as in (H). In (A)-(H): UCN-01, 1 μM; sunitinib 15 μM; roscovitine, 10 μM; TRAIL, 10 nM; etoposide, 20 μM; sulindac sulfide, 120 μM. In (B), (F) and (H), apoptosis was analyzed by counting cells with condensed and fragmented nuclei after nuclear staining with Hoechst 33258.

Degradation-resistant Mcl-1 binds to PUMA to inhibit cell death, which is abrogated by small-molecule Mcl-1 inhibitors and enhanced PUMA expression

The concordance of Mcl-1 degradation and PUMA induction prompted us to determine the functional relationship between Mcl-1 and PUMA. Mcl-1 degradation and PUMA induction did not affect each other and had similar impact on regorafenib-induced apoptosis (Supplementary Fig. S3, A-C), indicating they are two independent events in response to regorafenib. In Mcl-1-KI cells, the degradation-resistant Mcl-1 had markedly enhanced binding to PUMA (Fig. 5D), which led to reduced interaction of Bcl-XL with PUMA, but increased interaction of Bcl-XL with Bax (Fig. 5E). Furthermore, treating cells with the Mcl-1 inhibitor UMI-77 (19,20), or the pan-Bcl-2 inhibitor TW37 (29), almost completely restored regorafenib-induced apoptosis and caspase activation in Mcl-1-KI cells, as well as apoptosis induced by other kinase inhibitors that required Mcl-1 degradation (Fig. 5, F and G). In contrast, the Bcl-2/Bcl-XL inhibitor ABT-737 or ABT-263, which mimics the BH3 domain of Bad that is unable to bind to Mcl-1 (30), was unable to restore apoptosis induced by kinase inhibitors in Mcl-1-KI cells (Supplementary Fig. S3D). Similarly, infecting Mcl-1-KI cells with a low, non-apoptotic dose of PUMA-expressing adenovirus (Ad-PUMA) restored regorafenib-induced apoptosis and caspase activation, while a control adenovirus lacking the BH3 domain of PUMA (Ad-ΔBH3) was unable to do so (Fig. 5, H and I; Supplementary Fig. S3E). These results suggest that Mcl-1 degradation is a key event that allows PUMA, upon its induction, to bind to other pro-survival Bcl-2 family proteins such as Bcl-XL to promote apoptosis, explaining why Mcl-1 degradation is necessary but insufficient for apoptosis induction.

Mcl-1 degradation mediates the in vivo anti-tumor effects of regorafenib

We used xenograft tumors to analyze the in vivo effects of blocking Mcl-1 degradation. The growth inhibitory effect of regorafenib was statistically significant (P<0.05) in WT tumors, but not (P>0.05) in Mcl-1-KI tumors (Fig. 6A). Compared to WT tumors, Mcl-1-KI tumors were significantly more resistant to regorafenib (Fig. 6, A and B, P<0.05). Apoptosis analyzed by TUNEL and active caspase 3 staining was significantly reduced in Mcl-1-KI tumors relative to WT tumors (Fig. 6, C and D). Staining of tumor vasculature by CD31 revealed an attenuated anti-angiogenic effect of regorafenib in Mcl-1-KI tumors (Fig. 6E). The anti-hypoxic effect of regorafenib analyzed by Carbonic Anhydrase 9 (CA9) staining was also reduced in Mcl-1-KI tumors relative to WT tumors (Fig. 6F). Consistent with the in vitro findings, Mcl-1-KI tumors lacked regorafenib-induced Mcl-1 degradation, but had intact GSK3β de-phosphorylation (Fig. 6G). Probing in situ protein-protein interactions by proximity ligation assay (PLA) (31) detected enhanced interaction between WT Mcl-1 to FBW7 in WT tumors, and between mutant Mcl-1 and PUMA in Mcl-1-KI tumors (Fig. 6, H and I). These results demonstrate a pivotal role of Mcl-1 phosphorylation and degradation in mediating the in vivo antitumor effects of regorafenib.

Figure 6. Mcl-1 degradation contributes to the in vivo antitumor activity of regorafenib.

Figure 6

Open in a new tab

(A) Nude mice were injected s.c. with 4 × 106 WT or Mcl-1-KI HCT116 cells. After 1 week, mice were treated with 30 mg/kg regorafenib daily by oral gavage (indicated by arrows), or the vehicle control cremephor EL/ethanol for 10 consecutive days. Tumor volume at indicated time points after treatment was calculated and plotted with statistical significance for indicated comparisons (n=7 in each group). (B) Representative tumors at the end of the experiment. (C–F) Nude mice with WT or Mcl-1-KI HCT116 tumors were treated with regorafenib as in (A) for 4 consecutive days. Tissue sections were analyzed by staining for TUNEL (C), active caspase 3 (D), CD31 (E), and Carbonic Anhydrase 9 (CA9) (F). Left, representative staining pictures; right, quantification of positive cells. (G) Western blotting of indicated proteins in WT and Mcl-1-KI HCT116 tumors from (C). (H) Tissue sections from WT HCT116 xenograft tumors with or without regorafenib treatment as in (A) for 1 day were analyzed by proximity ligation assay (PLA) to detect the interaction of Mcl-1 and FBW7. Left, representative PLA pictures, with a section without primary antibody staining as a negative control; right, quantification of PLA signals per field. (I) Tissue sections from WT and Mcl-1-KI HCT116 xenograft tumors treated with regorafenib as in (A) for 1 day were analyzed by PLA for the interaction of Mcl-1 and PUMA. Left, representative PLA pictures, with a section without primary antibody staining as a negative control; right, quantification of PLA signals per field. In (C), (D), (E), (F), (H), and (I), results were expressed as means ± s.d. of three independent experiments. Arrows indicate example cells with positive staining. Scale bars, 25 μm. NS, P >0.05, *, P <0.05, **, P <0.01, ***, P <0.001.

Discussion

One of the hallmarks of cancer is resistance to apoptosis, which maintains survival of cells en route to oncogenic transformation (32). Overexpression or amplification of Mcl-1 is one of the most frequent alterations in human cancers (33). Defective Mcl-1 degradation allows tumor cells to evade the fate of death and underlies development of therapeutic resistance. Distinguished from other Bcl-2 family members, Mcl-1 contains two PEST (proline/glutamic acid/serine/threonine) sequences, which are enriched in putative phosphorylation sites that mediate its degradation. Our results from genetic knock-in experiments demonstrate that the previously described 4 phosphorylation sites (S121/E125/S159/T163) are essential for Mcl-1 ubiquitination and degradation induced by inhibition of oncogenic kinases, accounting for most, if not all, of phosphorylation that engages Mcl-1 to bind to the E3 ubiquitin ligase FBW7. Upon GSK3β-mediated phosphorylation, Mcl-1 is likely recruited by FBW7 to the SCF ubiquitin ligase complex composing of FBW7, CUL1, SKP1 and ROC1 (9). This complex then covalently links ubiquitin chains to Mcl-1, leading to its degradation in the 26S proteasome. In addition to GSK3β, several other kinases have also been implicated in regulating Mcl-1 turnover, including p38, JNK, CDK1, and casein kinase II (8,9,26). Mcl-1 stability can be regulated by other E3 ubiquitin ligases including Mule and β-TrCP (6–9), and by the deubiquitinase USP9X (34). Depending on apoptotic stimuli and cell types, these proteins may also be involved in therapeutic resistance caused by Mcl-1 stabilization.

Our results clarify the functional role of Mcl-1 in apoptosis in solid tumor cells. Unlike hematopoietic cells, solid tumor cells such as CRC cells are not highly primed to cell death, and do not die in response to Mcl-1 inhibition or depletion alone (10). At least two independent events are necessary for initiating apoptotic response to regorafenib, including early Mcl-1 degradation and later PUMA induction (24,25). Mcl-1 degradation unleashes the full pro-apoptotic activity of PUMA, and allows it to bind to and neutralize other pro-survival factors such as Bcl-XL (23). In Mcl-1-KI and FBW7-mutant CRC cells that are deficient in Mcl-1 degradation, Mcl-1 sequesters PUMA from Bcl-XL and other antiapoptotic proteins. Blocking Mcl-1 degradation not only abolishes apoptosis induction, but also attenuates the anti-angiogenic and anti-hypoxic activities of regorafenib. This can probably be explained by suppression of the release of anti-angiogenic and anti-hypoxic signals by apoptotic cells into microenvironment (35). Our findings further indicate that the effects of targeted therapy on cancer cells and their microenvironment are closely related and influence each other via cell death (25,36).

Inhibiting pro-survival Bcl-2 family proteins in tumor cells has emerged as an attractive therapeutic strategy, leading to recent FDA approval of the Bcl-2-selective inhibitor ABT-199 (Venetoclax) for the treatment of chronic lymphocytic leukemia (CLL) (37). However, ABT-199 and related agents do not bind to Mcl-1, and resistance to these agents quickly emerges due to overexpression of Mcl-1 (38–40). There is an urgent need for developing small-molecule Mcl-1 inhibitors. Mcl-1 binds to the BH3 domains of proapoptotic proteins through a surface grove containing 4 hydrophobic pockets (p1-p4) (41,42). Several different chemical classes of Mcl-1 inhibitors have been described (19,20,43–49). However, the existing Mcl-1 inhibitors lack sufficient potency and specificity for further development. Targeting Mcl-1, especially for solid tumor cells, has been hampered by lack of a reliable system for evaluating specificity. Our data suggest that Mcl-1-binding BH3 mimetics or enhancing PUMA expression can be used to overcome therapeutic resistance caused by Mcl-1 stabilization in CRC cells. In this context, the previously identified Mcl-1 inhibitors exhibited on-target activity in the isogenic Mcl-1-KI cells, which are potentially useful for identifying new inhibitors with improved potency, specificity and in vivo activity to overcome therapeutic resistance in colon and other solid tumor cells.

Supplementary Material

Supplemental table and figures

Acknowledgments

We thank our lab members for critical reading and discussion. This work is supported by U.S. National Institute of Health grants (R01CA106348, R01CA172136, and R01CA203028 to LZ; U01DK085570 and U19AI068021 to JY; R01CA149442 to ZNC) and National Natural Science Foundation of China (81672942 to FZ). This project used the UPCI shared facilities that were supported in part by award P30CA047904.

Footnotes

Conflict of interest: The authors have no conflict of interest.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

References

  • 1.Moldoveanu T, Follis AV, Kriwacki RW, Green DR. Many players in BCL-2 family affairs. Trends in biochemical sciences. 2014;39:101–11. doi: 10.1016/j.tibs.2013.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bhola PD, Letai A. Mitochondria-Judges and Executioners of Cell Death Sentences. Mol Cell. 2016;61:695–704. doi: 10.1016/j.molcel.2016.02.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Thomas LW, Lam C, Edwards SW. Mcl-1; the molecular regulation of protein function. FEBS Lett. 2010;584:2981–9. doi: 10.1016/j.febslet.2010.05.061. [DOI] [PubMed] [Google Scholar]
  • 4.Perciavalle RM, Opferman JT. Delving deeper: MCL-1’s contributions to normal and cancer biology. Trends in cell biology. 2013;23:22–9. doi: 10.1016/j.tcb.2012.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mojsa B, Lassot I, Desagher S. Mcl-1 ubiquitination: unique regulation of an essential survival protein. Cells. 2014;3:418–37. doi: 10.3390/cells3020418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Zhong Q, Gao W, Du F, Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell. 2005;121:1085–95. doi: 10.1016/j.cell.2005.06.009. [DOI] [PubMed] [Google Scholar]
  • 7.Ding Q, He X, Hsu JM, Xia W, Chen CT, Li LY, et al. Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol. 2007;27:4006–17. doi: 10.1128/MCB.00620-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Inuzuka H, Shaik S, Onoyama I, Gao D, Tseng A, Maser RS, et al. SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature. 2011;471:104–9. doi: 10.1038/nature09732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, et al. Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature. 2011;471:110–4. doi: 10.1038/nature09779. [DOI] [PubMed] [Google Scholar]
  • 10.Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K, et al. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science. 2005;307:1101–4. doi: 10.1126/science.1106114. [DOI] [PubMed] [Google Scholar]
  • 11.Fernandez-Marrero Y, Spinner S, Kaufmann T, Jost PJ. Survival control of malignant lymphocytes by anti-apoptotic MCL-1. Leukemia. 2016 doi: 10.1038/leu.2016.213. [DOI] [PubMed] [Google Scholar]
  • 12.Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M. Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther. 2008;7:3129–40. doi: 10.1158/1535-7163.MCT-08-0013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Grothey A, Van Cutsem E, Sobrero A, Siena S, Falcone A, Ychou M, et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381:303–12. doi: 10.1016/S0140-6736(12)61900-X. [DOI] [PubMed] [Google Scholar]
  • 14.Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007;356:125–34. doi: 10.1056/NEJMoa060655. [DOI] [PubMed] [Google Scholar]
  • 15.Wilhelm SM, Dumas J, Adnane L, Lynch M, Carter CA, Schutz G, et al. Regorafenib (BAY 73–4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer. 2011;129:245–55. doi: 10.1002/ijc.25864. [DOI] [PubMed] [Google Scholar]
  • 16.Abou-Elkacem L, Arns S, Brix G, Gremse F, Zopf D, Kiessling F, et al. Regorafenib inhibits growth, angiogenesis, and metastasis in a highly aggressive, orthotopic colon cancer model. Mol Cancer Ther. 2013;12:1322–31. doi: 10.1158/1535-7163.MCT-12-1162. [DOI] [PubMed] [Google Scholar]
  • 17.Tong J, Tan S, Zou F, Yu J, Zhang L. FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation. Oncogene. 2016 Jul 11; doi: 10.1038/onc.2016.247. Epub ahead of print. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L. PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A. 2003;100:1931–36. doi: 10.1073/pnas.2627984100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Abulwerdi F, Liao C, Liu M, Azmi AS, Aboukameel A, Mady AS, et al. A novel small-molecule inhibitor of mcl-1 blocks pancreatic cancer growth in vitro and in vivo. Mol Cancer Ther. 2014;13:565–75. doi: 10.1158/1535-7163.MCT-12-0767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Abulwerdi FA, Liao C, Mady AS, Gavin J, Shen C, Cierpicki T, et al. 3-Substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: structure-based design, synthesis, SAR, and biological evaluation. J Med Chem. 2014;57:4111–33. doi: 10.1021/jm500010b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Peng R, Tong JS, Li H, Yue B, Zou F, Yu J, et al. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation. Cell Death Differ. 2013;20:744–54. doi: 10.1038/cdd.2013.4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yu J, Yue W, Wu B, Zhang L. PUMA sensitizes lung cancer cells to chemotherapeutic agents and irradiation. Clin Cancer Res. 2006;12:2928–36. doi: 10.1158/1078-0432.CCR-05-2429. [DOI] [PubMed] [Google Scholar]
  • 23.Ming L, Wang P, Bank A, Yu J, Zhang L. PUMA dissociates Bax and BCL-XL to induce apoptosis in colon cancer cells. J Biol Chem. 2006;281:16034–42. doi: 10.1074/jbc.M513587200. [DOI] [PubMed] [Google Scholar]
  • 24.Dudgeon C, Peng R, Wang P, Sebastiani A, Yu J, Zhang L. Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3beta and NF-kappaB to suppress tumor cell growth. Oncogene. 2012;31:4848–58. doi: 10.1038/onc.2011.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chen D, Wei L, Yu J, Zhang L. Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis. Clin Cancer Res. 2014;20:3472–84. doi: 10.1158/1078-0432.CCR-13-2944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell. 2006;21:749–60. doi: 10.1016/j.molcel.2006.02.009. [DOI] [PubMed] [Google Scholar]
  • 27.Dudgeon C, Wang P, Sun X, Peng R, Sun Q, Yu J, et al. PUMA induction by FoxO3a mediates the anticancer activities of the broad-range kinase inhibitor UCN-01. Mol Cancer Ther. 2010;9:2893–902. doi: 10.1158/1535-7163.MCT-10-0635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sun J, Sun Q, Brown MF, Dudgeon C, Chandler J, Xu X, et al. The Multi-Targeted Kinase Inhibitor Sunitinib Induces Apoptosis in Colon Cancer Cells via PUMA. PLoS One. 2012;7:e43158. doi: 10.1371/journal.pone.0043158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wang G, Nikolovska-Coleska Z, Yang CY, Wang R, Tang G, Guo J, et al. Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins. J Med Chem. 2006;49:6139–42. doi: 10.1021/jm060460o. [DOI] [PubMed] [Google Scholar]
  • 30.Billard C. BH3 mimetics: status of the field and new developments. Mol Cancer Ther. 2013;12:1691–700. doi: 10.1158/1535-7163.MCT-13-0058. [DOI] [PubMed] [Google Scholar]
  • 31.Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature methods. 2006;3:995–1000. doi: 10.1038/nmeth947. [DOI] [PubMed] [Google Scholar]
  • 32.Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
  • 33.Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature. 2010;463:899–905. doi: 10.1038/nature08822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Schwickart M, Huang X, Lill JR, Liu J, Ferrando R, French DM, et al. Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature. 2010;463:103–7. doi: 10.1038/nature08646. [DOI] [PubMed] [Google Scholar]
  • 35.Medina CB, Ravichandran KS. Do not let death do us part: ‘find-me’ signals in communication between dying cells and the phagocytes. Cell Death Differ. 2016;23:979–89. doi: 10.1038/cdd.2016.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ricotta R, Sartore-Bianchi A, Verrioli A, Vanzulli A, Siena S. Regorafenib for metastatic colorectal cancer. Lancet. 2013;381:1537. doi: 10.1016/S0140-6736(13)60976-9. [DOI] [PubMed] [Google Scholar]
  • 37.Del Poeta G, Postorino M, Pupo L, Del Principe MI, Dal Bo M, Bittolo T, et al. Venetoclax: Bcl-2 inhibition for the treatment of chronic lymphocytic leukemia. Drugs of today. 2016;52:249–60. doi: 10.1358/dot.2016.52.4.2470954. [DOI] [PubMed] [Google Scholar]
  • 38.Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006;10:375–88. doi: 10.1016/j.ccr.2006.10.006. [DOI] [PubMed] [Google Scholar]
  • 39.Tahir SK, Yang X, Anderson MG, Morgan-Lappe SE, Sarthy AV, Chen J, et al. Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737. Cancer Res. 2007;67:1176–83. doi: 10.1158/0008-5472.CAN-06-2203. [DOI] [PubMed] [Google Scholar]
  • 40.van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE, et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell. 2006;10:389–99. doi: 10.1016/j.ccr.2006.08.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Day CL, Smits C, Fan FC, Lee EF, Fairlie WD, Hinds MG. Structure of the BH3 domains from the p53-inducible BH3-only proteins Noxa and Puma in complex with Mcl-1. J Mol Biol. 2008;380:958–71. doi: 10.1016/j.jmb.2008.05.071. [DOI] [PubMed] [Google Scholar]
  • 42.Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9:47–59. doi: 10.1038/nrm2308. [DOI] [PubMed] [Google Scholar]
  • 43.Friberg A, Vigil D, Zhao B, Daniels RN, Burke JP, Garcia-Barrantes PM, et al. Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design. J Med Chem. 2013;56:15–30. doi: 10.1021/jm301448p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Bruncko M, Wang L, Sheppard GS, Phillips DC, Tahir SK, Xue J, et al. Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity. J Med Chem. 2015;58:2180–94. doi: 10.1021/jm501258m. [DOI] [PubMed] [Google Scholar]
  • 45.Pelz NF, Bian Z, Zhao B, Shaw S, Tarr JC, Belmar J, et al. Discovery of 2-Indole-acylsulfonamide Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods. J Med Chem. 2016;59:2054–66. doi: 10.1021/acs.jmedchem.5b01660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Cohen NA, Stewart ML, Gavathiotis E, Tepper JL, Bruekner SR, Koss B, et al. A competitive stapled peptide screen identifies a selective small molecule that overcomes MCL-1-dependent leukemia cell survival. Chemistry & biology. 2012;19:1175–86. doi: 10.1016/j.chembiol.2012.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Nguyen M, Marcellus RC, Roulston A, Watson M, Serfass L, Murthy Madiraju SR, et al. Small molecule obatoclax (GX15–070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis. Proc Natl Acad Sci U S A. 2007;104:19512–7. doi: 10.1073/pnas.0709443104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Wei J, Stebbins JL, Kitada S, Dash R, Zhai D, Placzek WJ, et al. An optically pure apogossypolone derivative as potent pan-active inhibitor of anti-apoptotic bcl-2 family proteins. Frontiers in oncology. 2011;1:28. doi: 10.3389/fonc.2011.00028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Kotschy A, Szlavik Z, Murray J, Davidson J, Maragno AL, Le Toumelin-Braizat G, et al. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature. 2016;538:477–82. doi: 10.1038/nature19830. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental table and figures
NIHMS909042-supplement-Supplemental_table_and_figures.pdf (337.8KB, pdf)

RESOURCES