Genomic analysis and selective small molecule inhibition identifies BCL-XL as a critical survival factor in a subset of colorectal cancer
© Zhang et al. 2015
Published: 2 July 2015
Defects in programmed cell death, or apoptosis, are a hallmark of cancer. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins, including BCL-2, BCL-XL, and MCL-1 have been characterized as key survival factors in multiple cancer types. Because cancer types with BCL2 and MCL1 amplification are more prone to inhibition of their respectively encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification would have greater dependency on BCL-XL for survival.
To identify tumor subtypes that have significant frequency of BCL2L1 amplification, we performed data mining using The Cancer Genome Atlas (TCGA) database. We then assessed the dependency on BCL-XL in a panel of cell lines using a selective and potent BCL-XL inhibitor, A-1155463, and BCL2L1 siRNA. Mechanistic studies on the role of BCL-XL were further undertaken via a variety of genetic manipulations.
We identified colorectal cancer as having the highest frequency of BCL2L1 amplification across all tumor types examined. Colorectal cancer cell lines with BCL2L1 copy number >3 were more sensitive to A-1155463. Consistently, cell lines with high expression of BCL-XL and NOXA, a pro-apoptotic protein that antagonizes MCL-1 activity were sensitive to A-1155463. Silencing the expression of BCL-XL via siRNA killed the cell lines that were sensitive to A-1155463 while having little effect on lines that were resistant. Furthermore, silencing the expression of MCL-1 in resistant cell lines conferred sensitivity to A-1155463, whereas silencing NOXA abrogated sensitivity.
This work demonstrates the utility of characterizing frequent genomic alterations to identify cancer survival genes. In addition, these studies demonstrate the utility of the highly potent and selective compound A-1155463 for investigating the role of BCL-XL in mediating the survival of specific tumor types, and indicate that BCL-XL inhibition could be an effective treatment for colorectal tumors with high BCL-XL and NOXA expression.
Cancer is a genetic disease that arises from the accumulation of somatic gene alterations. One way to identify key genes in cancer is to examine genomic regions that undergo frequent alterations. Recent technological advances in characterizing these alterations make it possible to identify genes that are essential for the initiation and/or survival of cancer, and thus encode potential therapeutic targets.
Evasion of apoptosis is a hallmark of cancer cells. One mechanism of apoptotic pathway deregulation is via upregulation of the anti-apoptotic BCL-2 family members. Apoptotic pathway proteins belong to a family of BCL-2 Homology (BH)-domain-containing proteins comprising three classes: 1) multi-domain anti-apoptotic (BCL-2, BCL-XL, BCL-W, BFL-1/A1, and MCL-1), 2) multi-domain pro-apoptotic (BAX, BAK), and 3) BH3-only pro-apoptotic (BID, BIM, BAD, BIK, NOXA, PUMA, BMF, and HRK). The BH3-only proteins contain a single BH3 domain and are bound by specific anti-apoptotic proteins . For example, BCL-2 and BCL-XL bind and antagonize BIM but not NOXA. In contrast, MCL-1 and A1 bind and antagonize NOXA but not BAD protein. Other BH3 domain proteins such as BIM and PUMA are bound and antagonized by all anti-apoptotic proteins. BAX and BAK are known as the “effectors”. Once activated, these proteins oligomerize on the outer mitochondrial membrane and induce pore formation; this results in the release of cytochrome c and other pro-apoptotic proteins that ultimately carry out the cell death mechanism.
The role of anti-apoptotic BCL-2 family proteins in various cancers has been well studied . BCL-2 was initially identified from the breakpoint of the t(14;18) chromosomal translocation found in over 60 % of indolent B cell non-Hodgkin’s lymphoma [3–6]. In addition to the vast majority of follicular lymphomas, many germinal center B cell (GCB) subtype diffuse large B cell lymphomas (DLBCL) also exhibit the t(14;18) chromosomal translocation [7–9]. BCL2 amplification is also detected in many hematologic malignancies such as the activated B cell-like (ABC) subtype of DLBCL . Not surprisingly, cell lines with the translocation or amplification are more sensitive to the selective BCL-2 inhibitor ABT-199 .
MCL1 was reported to be amplified in 10.9 % of tumor samples analyzed, spanning multiple cancer subtypes . Fluorescence in situ hybridization (FISH) of the MCL1 region identified lung and breast cancers as having significantly higher frequencies of focal amplification, suggesting that these tumors depend on MCL-1 for survival. This is supported by multiple studies demonstrating that cell lines with MCL1 amplification are sensitive to siRNA knockdown of MCL1 [12, 13].
BCL-XL has been implicated as a key survival factor in numerous solid tumors . Based on the evidence that cancer types with BCL2 and MCL1 amplification are more prone to inhibition of their encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification are more dependent on BCL-XL for survival. In this study, we identified colorectal cancer as having a significant incidence of BCL2L1 amplification. We then dissected the role of BCL-XL in colorectal cancer cell lines using a selective small-molecule inhibitor of BCL-XL and a variety of genetic manipulations.
Materials and methods
BCL-XL inhibitor A-1155463 and navitoclax were synthesized at AbbVie, Inc. (North Chicago, IL). All the siRNAs were purchased from Dharmacon (Lafayette, CO).
Cell culture, transfection, and cell-based assays
Colorectal cell lines (ATCC) were cultured in RPMI (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen), 1 % sodium pyruvate (Invitrogen), and 4.5 g/L glucose (Sigma, MO), or DMEM (Invitrogen) supplemented with 10 % FBS. All the lines were maintained in a humidified chamber at 37 °C containing 5 % CO2.
LS1034, SW1417, GEO, and RKO cells were transfected in 6-well plates with siRNAs using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). A final concentration of 20 nM siRNA was used in all cases. The sense sequences of the BCL-XL siRNA used is ACAAGGAGAUGCAGGUAUUUU (Dharmacon). The sense sequences of the MCL-1 siRNAs used is GCATCGAACCATTAGCAGATT (Dharmacon). The cells were then grown in medium without antibiotic before harvesting for western blotting analysis. LS1034 cells were transfected at 1.5–2.5 × 104 cells/100 μl in 96-well tissue culture plates with 20 nM Noxa siRNA pool (Dharmacon). The cells were grown in medium without antibiotic before harvesting.
Cells were treated with increasing concentration of A-1155463. Cells were assayed for viability after 72 h using the CellTiter-Glo luminescent cell viability assay according to the manufacturer’s protocol (Promega, Madison, WI). Results were normalized to cells without treatment. EC50 was calculated using the GraphPad Prism software (La Jolla, CA).
Western blot analysis
Cell lysates were prepared in RIPA buffer (Sigma) plus protease inhibitor cocktail (Roche). 20 μg of total protein was resolved on a 12 % SDS polyacrylamide gel and probed with anti-BCL-XL (Epitomics, Burlingame, CA), anti-MCL-1 (Epitomics), anti-BCL-2 (BD), anti-BIM (Epitomics), anti-actin and anti-NOXA (Abcam, Cambridge, MA). Antibody against tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as a loading control.
Fluorescence-activated cell sorting (FACS) analysis
LS1034 cells were treated with DMSO or 200 nM A-1155463, with or without 50 μM Z-VAD caspase inhibitor (Santa Cruz Biotechnology, Inc.) for 72 h. DNA content was measured by flow cytometry to determine the effect of the inhibitors on the cell cycle and cell death. Following treatment, cells were spun down, the medium was removed and the cells were resuspended in staining solution (50 μg propidium iodide, 40 U/ml RNase, 0.1 % triton X-100 in PBS) at a cell concentration of 1 × 106 cells/ml. The cells were stored in the dark at room temperature for at least 30 min or up to 24 h at 4 °C before the DNA content was determined by flow cytometry using a FACS Calibur (BD Biosciences, San Jose, CA).
Previously, we and others have shown that copy number (CN) gain, protein and mRNA expression of BCL-2 family members is a crucial determinant of sensitivity to BH3 mimetics such as ABT-737, an inhibitor of BCL-2, BCL-XL, and BCL-W, and ABT-199, a specific inhibitor of BCL-2 [11, 16–18]. Herein we report for the first time that cell lines with BCL2L1 gains are sensitive to selective BCL-XL inhibition. Among the 11 lines with BCL2L1 gain (CN > 3), 9 are sensitive to A-1155463 (EC50 < 0.5 μM). In contrast, only 3 out of 11 lines with no CN gain of BCL2L1 are sensitive to A-1155463 (Fig. 2b).
Two recent manuscripts reported that BCL-XL is regulated by the β-catenin pathway. In an unbiased screen of 242 genomically characterized cancer cell lines with an Informer Set of 354 small molecules, cell lines with β-catenin mutation were reported to be amongst the most sensitive to navitoclax (ABT-263), an inhibitor of BCL-2, BCL-XL and BCL-W . In a separate study, β-catenin-active cancers were reported to be dependent on a signaling pathway involving the transcriptional regulator YAP1 and the transcription factor TBX5, both of which form a complex with β-catenin . This complex was further shown to be important for regulating BCL-XL and the anti-apoptotic gene BIRC5. APC is part of a destruction complex that mediates the ubiquitin-targeted proteolysis of β-catenin. Mutations in APC destabilize this complex and lead to the accumulation of β-catenin, which can then translocate to the nucleus and aberrantly activate the transcription of key drivers of oncogenesis . Interestingly, a small subgroup of colorectal tumors with wild-type APC has point mutations in β-catenin that allow it to escape degradation . Since BCL-XL was described to be regulated by the β-catenin pathway and multiple components of the pathway are highly mutated in colorectal cancer, we determined whether a correlation exists between these mutations and the activity of inhibitor A-1155463 in the colorectal cancer cell lines. While the majority of these lines have mutations in APC, only two lines have β-catenin mutations according to the Cancer Cell Line Encyclopedia (CCLE) . Contrary to recent reports, we found no correlation between APC or β-catenin mutation status and dependence on BCL-XL for survival as revealed by sensitivity to A-1155463 (Fig. 2b) or navitoclax (data not shown). This could be explained by differences in cell lines between the studies. In particular, our studies focus on colon cell lines only. It is also possible that the disparity results from the lack of appropriately selective small molecules in the study by Basu et al. .
To assess whether the BCL-XL inhibitor induced cell death in sensitive colorectal cell lines via apoptosis, we evaluated the cell cycle distribution of LS1034 and SW1417 cells after treatment with A-1155463 in the presence or absence of the caspase-3/7 inhibitor Z-VAD. As shown in Fig. 4c, A-1155463 treatment led to substantial accumulation of sub-G0/apoptosis cells (>50 %). This effect was completely abrogated in the presence of Z-VAD, indicating that cell killing occurs via the activation of caspase-3/7 pathway.
NOXA is a pro-apoptotic protein that antagonizes MCL-1 activity. Because NOXA expression is higher in the sensitive lines (Fig. 3a), we sought to assess its role in determining sensitivity to BCL-XL inhibitors. We thus asked whether silencing NOXA would rescue sensitive colorectal lines from treatment with A-1155463. Transfection of LS1034 cells with NOXA siRNA rendered the cells insensitive to A-1155463 treatment (Fig. 5b), indicating that NOXA function, likely involving its inhibition of MCL-1, is required to sensitize this cell line to BCL-XL inhibition. Similar results were observed in another BCL-XL inhibitor-sensitive line, SW1417 (data not shown).
In this study, we set out to first identify cancer types that show high levels of BCL2L1 amplification using genomic mining. Our second major aim was to investigate the dependency of corresponding cell lines on BCL-XL for survival. To this end, we used not only siRNA but a recently disclosed small molecule inhibitor of BCL-XL that shows high potency and selectivity against other pro-survival proteins. Our study identified colorectal and cervical cancers as having the most frequent BCL2L1 amplification. Further studies showed that the majority of colorectal cancer cell lines with a gain in BCL2L1 are sensitive to the selective BCL-XL inhibitor A-1155463 as well as to siRNA-medited knockdown of BCL2L1. Although the gains are typically modest (3–4.4 copies), it should be noted that CN gain of other anti-apoptotic genes such as MCL1 is also modest in other tumors that nevertheless become dependent on these genes for survival . In addition, the sensitive lines exhibit higher protein and mRNA expression of BCL-XL and NOXA, and a trend towards lower protein and mRNA expression of MCL-1 than the resistant lines.
Despite a recent report that navitoclax-sensitive cell lines are enriched with β-catenin mutations , we found no correlation between BCL-XL dependence and the presence of β-catenin or APC mutations. Possible reasons for this discrepancy could be differences in the colorectal cell lines used in the two studies, or alternatively, the fact that the highly selective BCL-XL inhibitor was not available at the time of the earlier study by Basu et al. and the study was thus confounded by the mixed selectivity of the compound used . Our data is consistent with those of Rosenbluh et al., who reported that β-catenin-active cancers regulate BCL-XL as a survival factor . In this regard, we found that sensitive lines are enriched with an active β-catenin pathway as determined by a β-catenin transcriptional reporter assay. Although this is a small sample size, preliminary data show that five out of six of the sensitive lines display active β-catenin pathway versus three out of six in the resistant group (data not shown). Collectively, our data indicate that BCL-XL may be regulated through multiple pathways in colorectal cancer, including CN gain and active β-catenin signaling. The role of other pathways cannot be ruled out.
Similar to navitoclax, we found that MCL-1 is a resistance factor for the BCL-XL-selective inhibitor A-1155463. Mechanistically, A-1155463 and navitoclax displace pro-apoptotic protein BIM from BCL-XL, which leads to the activation of apoptosis [2, 25]. In resistant cells, it is likely that the BIM released by BCL-XL inhibitor is readily sequestered by MCL-1, thereby inhibiting the activation of the apoptotic pathway. Indeed, it has been shown that MCL-1 and BCL-XL play compensatory roles in regulating apoptosis in most solid tumor cell lines [13, 16, 26]. Our experiments further demonstrate that silencing MCL-1 can sensitize solid tumor cell lines to selective BCL-XL inhibition, expanding on earlier studies with the BCL-2/BCL-XL inhibitors ABT-737 and ABT-263 [27–30]. By targeting MCL-1 via mTOR inhibition, colorectal cancers with Kras or BRAF mutations are sensitized to BCL-2/BCL-XL inhibition . Our observation that silencing NOXA could rescue sensitive cells from A-1155463 also supports the role of MCL-1 as a resistance factor. NOXA is known to interact with MCL-1 and neutralize its activity by targeting this protein for degradation . Thus, silencing NOXA decreases the antagonism of MCL-1, leaving it free to sequester pro-apoptotic proteins and prevent the activation of apoptosis .
Other gene amplifications have served as therapeutic targets for other cancers. For example, the MET and ALK tyrosine kinase inhibitor crizotinib (PF-02341066) shows differential antitumor effects in non-small cell lung and gastric cancer according to MET and ALK alterations . By utilizing genomic data to identify tumors with BCL2L1 gain, we identified colorectal cancers as being potentially more dependent on BCL-XL and more susceptible to BCL-XL inhibition. Indeed, by using a potent and selective BCL-XL inhibitor, A-1155643, we have shown that BCL-XL inhibition is sufficient to kill colon cancer cell lines expressing high levels of BCL-XL and the MCL-1-neutralizing protein NOXA. Of particular interest, sensitive lines are enriched in a gain of BCL2L1, suggesting BCL2L1 gain could be used as a patient stratification biomarker. This finding demonstrates the power of identifying potential new targets by examining genomic regions that undergo frequent alterations as well as the utility of highly potent and selective tool molecules. Finally, BCL-XL selective inhibitors may have utility as therapeutics for the treatment of colorectal cancer, a disease for which there is significant unmet medical need.
BCL-XL inhibition could be an effective treatment for colorectal tumors with high BCL-XL and NOXA expression. Additionally, BCL2L1 gain may represent stratification biomarker for colorectal cancer patients undergoing treatment with a BCL-XL inhibitor. Future studies should investigate the utility of BCL2L1 gain as a stratification biomarker.
We thank the AbbVie Oncology Biomarkers group for discussion and critical review of the manuscript.
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