Background
BCL-2 family proteins function through interactions with each other, and the balance between anti-apoptotic and pro-apoptotic members is critical for preventing or initiating apoptosis [
1‐
3]. This family of proteins share from one to four BCL-2 Homology (BH) motifs. The anti-apoptotic family members, BCL-2, BCL-X
L, BCL-W, A1 and MCL-1, promote survival by sequestering their pro-apoptotic counterparts such as the “BH3-only” proteins BIM, BAD, BID, HRK, NOXA and PUMA, and the multi-BH3 domain proteins BAK and BAX. BAK and BAX are the ultimate effectors of apoptosis. When free from anti-apoptotic proteins, they can become activated and subsequently oligomerize to form large pores in the mitochondrial outer membrane which enable the release of cytochrome
c and subsequent activation of the intrinsic apoptosis pathway through a caspase cleavage cascade.
The link between overexpressed anti-apoptotic BCL-2 family proteins and cancer is now well established [
4]. Enhanced expression of these proteins has been reported in numerous cancers, which permits cell growth and survival in the presence of apoptotic signals associated with the transformed phenotype, and can also lead to the failure of chemotherapeutic strategies. Navitoclax (ABT-263), an orally bioavailable small-molecule inhibitor of BCL-2, BCL-X
L, and BCL-W [
5], showed signs of clinical antitumor activity in chronic lymphocytic leukemia (CLL). However, most solid tumors are resistant to navitoclax due to high expression of MCL-1, to which the drug has a low affinity [
5,
6]. In addition it has been shown that high levels of MCL-1 co-related with resistance to ABT-263 in a panel of leukemia/lymphoma cell lines [
6]. Also as predicted by preclinical data, inhibition of BCL-X
L by navitoclax induces a rapid, concentration-dependent decrease in the number of platelets [
7‐
9]. This undesirable mechanism-based effect such as thrombocytopenia limited the ability to drive ABT-263 concentrations into a highly efficacious range.
Recently, a unique BCL-2–small molecule cocrystal structure was exploited to guide the rational design of venetoclax (ABT-199), a selective BCL-2 inhibitor intended to circumvent thrombocytopenia associated with BCL-X
L inhibition [
10]. Venetoclax is a first-in-class orally bioavailable BCL-2-selective inhibitor that has high binding affinity to BCL-2 (K
i = <0.01 nM) but not BCL-X
L, BCL-W or MCL-1 (K
i values = 48 nM, 21 nM and >440 nM, respectively). Venetoclax exhibits single-agent activity against a variety of leukemia/lymphoma cell lines
in vitro and
in vivo and clinical activity has been observed in CLL, non-Hodgkin lymphomas (NHL), acute myelogenous leukemia (AML) and multiple myeloma patients treated with venetoclax as a monotherapy [
11]. Venetoclax causes substantially less platelet killing
ex vivo and
in vivo as compared to navitoclax [
10]. In addition to showing preclinical efficacy in BCL-2–dependent cell lines and tumor xenograft models, venetoclax demonstrated immediate antileukemic activity after a single dose in three patients with refractory CLL while causing only minor changes in platelet counts [
11]. The results of that phase 1 study and a phase 2 study focused on CLL patients with the high-risk 17p deletion were recently published [
11,
12]. Of 116 patients in the phase 1 study, 79% exhibited objective responses to venetoclax, with 20% exhibiting complete responses (CR). Similar overall response rates (ORR) were observed in the 17p-deleted subset of patients in the phase 1 study (71 % ORR) and the phase 2 study dedicated to 17p-deleted CLL (79.4% ORR).
As with any targeted cancer therapy, it is important to identify potential mechanisms of venetoclax resistance, not only to inform patient selection but also to develop strategies to circumvent resistance as it emerges [
13]. Previously we demonstrated that MCL-1 overexpression is an inherent resistance factor for ABT-737, a potent BCL-2/BCL-X
L inhibitor, in a panel of SCLC cell lines, as well as an acquired resistance factor in H146 cells that had been selected for survival in the presence of ABT-737 [
14]. To more fully elucidate the potential mechanisms that may be involved in resistance to venetoclax, we undertook a study to assess potential changes in the expression levels of BCL-2 family members following extended treatment with venetoclax. We generated resistant variants from venetoclax-sensitive cell lines of different leukemia and lymphoma subtypes (two diffuse large B-cell lymphomas, two follicular lymphomas, one leukemia line, and two mantle cell lymphomas) by increasing exposure to venetoclax in a stepwise manner over time. We identified alterations in both pro-apoptotic and anti-apoptotic family members that account for resistance mechanisms. In several cases where the anti-apoptotic BCL-2 relatives MCL-1 or BCL-X
L were upregulated, sensitivity to venetoclax could be restored by co-treating with the appropriate MCL-1-selective or BCL-X
L-selective inhibitor, confirming both are causally related in mediating resistance to venetoclax. These data will help facilitate the rational design of combination strategies to circumvent venetoclax resistance and guide patient selection in future clinical trials.
Methods
Reagents
Venetoclax (ABT-199/GDC-0199), navitoclax (ABT-263), BCL-X
L-selective inhibitor A-1155463 [
15], and MCL-1-selective inhibitor A-1208746 [
16] were synthesized at AbbVie (North Chicago, IL). All antibodies were purchased from Epitomics (Burlingame, CA).
Cell culture and cell-based assays
NHL cell lines HBL1, SC-1, U2932, and OCI-Ly1 were cultured in IMDM (Invitrogen Corp., Grand Island, NY) supplemented with 10% human serum (Sigma). All other cell lines were cultured in RPMI supplemented with 10% fetal bovine serum (Invitrogen), 1% sodium pyruvate, and 4.5 g/L glucose (Invitrogen Corp., Grand Island, NY). All cell lines were maintained in a humidified chamber at 37oC containing 5% CO2. Cells resistant to venetoclax were generated by chronically exposing the parental cells to venetoclax starting at a sub-lethal concentration and subsequently step-wise increasing the concentration over several weeks. Selection pressure was maintained by growing the resistant cells in the presence of at least 1 μM venetoclax.
Sequencing analysis
For the 14 cell lines, genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen). DNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher, Waltham, MA). A total of four amplicons covering
BCL2 exons 1 and 2 were sequenced using the primers and cycling conditions (Additional file
1: Table S1). 10 μl of the PCR products were purified using AMPure magnetic beads (Beckman Coulter, Inc., Brea, CA) and eluted in 40 μl of sdH2O. 4 μl of the purified product was cycle sequenced using Big Dye Terminator Mix v3.1(Applied Biosystems, Grand Island, NY) at 1/16 chemistry. Both forward and reverse orientations of the PCR products were sequenced and applied to an Applied Biosystems 3130xl DNA Sequencer (Applied Biosystems, Grand Island, NY). Base calling was performed using Sequence Analysis v5.2 (Applied Biosystems, Grand Island, NY) with the KB basecaller v1.2. Sequence data was aligned to the reference
BCL2 sequence (NM_000633.2) using Sequencher (Gene Codes Corp., Ann Arbor, MI).
Western blot analysis
Cell lysates were prepared in RIPA buffer (Sigma) plus protease inhibitor cocktail (Roche). 20 μg of total protein was resolved on Bis-Tris gels and transferred to PVDF membranes. Blots were probed overnight at 4oC with the appropriate primary antibodies and for 1 hr with the secondary antibodies the following day. Blots were imaged using a LI-COR Biosciences (Lincoln, Nebraska) Odyssey imager.
Cytotoxicity assay
Cells were treated with indicated agents in 96-well tissue culture plates in RPMI supplemented with 10% FBS for 1 day before assessing viability using the CellTiter Glo Luminescent cell viability assay according to the manufacturer’s protocol (Promega, Madison, WI). Synergistic activities of venetoclax and specific MCL-1 or BCL-X
L inhibitors were determined using the Bliss additivity model, whereby the combined response (C) of both agents with individual effects A and B is C = A + B – (A × B), where A and B represent the fractional inhibition between 0 and 1. Drug interactions were considered synergistic if the combined Bliss scores were >0 and antagonistic if <0 [
17]. Student’s t-test was used to determine statistical significance.
Microarrays
RNA was converted to biotinylated antisense RNA and hybridized to Affymetrix human U133A 2.0 gene expression arrays. Data were analyzed using Rosetta Resolver software comparing each venetoclax-resistant cell line to its untreated parental line. Genes/probe sets with >1.5-fold change relative to parental and meeting a 5% False Discovery Rate were considered significant. Zero signifies no significant change from baseline.
DNA copy number determination
DNA was amplified, biotin labeled, and then hybridized to Affymetrix SNP 6.0 arrays. Gene copy numbers were calculated after segment smoothing of raw data using Partek software.
Flow cytometry
Approximately 1-2×10
6 cells were pelleted by centrifugation, resuspended in Lyse Fix Buffer I (BD BioSciences) for 10 minutes, washed once with PBS-D, then permeabilized with 1x BD Phosflow Perm Wash Buffer I. BAX antibody or isotype control was added to 1×10
5 fixed/permeabilized cells. Samples were incubated for 30 min at room temperature in the dark and washed, followed by the addition of an APC-conjugated F(ab’)2. After incubation for 30 min at room temperature, the cells were washed twice and the samples stored in Fix buffer I. Samples were then analyzed using a BD FACSCalibur and CellQuest Pro software [
18].
Discussion
In this study, we generated leukemia and lymphoma cell line populations resistant to venetoclax by selection in the presence of gradually increasing drug concentrations. These cells were then compared to their parental counterparts for obvious changes in BCL2 family gene sequences or copy number, BCL2 family transcript levels, and/or the expression of BCL-2 family proteins to gain a better understanding of the mechanisms of resistance to venetoclax.
Notably, we found that a variety of distinct mechanisms had the potential to confer venetoclax resistance. While no single change accounted for resistance in all the cell pairs, some common themes emerged, such as increases in redundantly acting anti-apoptotic proteins like MCL-1 (OCI-Ly1, RS4;11, Granta-519, HBL2 and U2932) or BCL-X
L (SC-1). In these populations, resistance to venetoclax could be reversed by concurrent treatment with the MCL-1-selective inhibitor A-1208746 or the BCL-X
L-selective inhibitor A-1155463, demonstrating that these proteins are directly involved in mediating the resistance and highlighting the importance of developing BCL-X
L and MCL-1 inhibitors for clinical use. It should be noted that A-1208746 typically shows cell killing around 2.5-10 μM for MCL-1-dependent cell lines [
23]. Therefore, the concentrations at which combination effects are observed are consistent with the on-target activity of A-1208746. Once MCL-1 is sufficiently neutralized, the EC
50 for venetoclax shifted to below 0.1 μM (Fig.
5). Changes in pro-apoptotic proteins were also observed in multiple resistant cell lines. In these cases, decreases were observed. Multiple cell lines showed reductions in the BH3-only protein BIM (Granta-519, HBL1, OCI-Ly1) or the effector protein BAX relative to their parental counterpart. A striking absence of BAX was observed in venetoclax-resistant RS4;11 cells and subsequent flow cytometry analyses identified a pre-existing sub-population of BAX-deficient cells amongst the parental RS4;11 population, thus representing a possible case of innate resistance to venetoclax.
Changes in the expression of apoptotic proteins were sometimes associated with gene copy number gains or losses and/or changes in mRNA levels. In the most striking examples, venetoclax-resistant SC-1 cells showed amplification of the gene locus encoding BCL-X
L (
BCL2L1) and a corresponding elevation in
BCL2L1 mRNA and BCL-X
L protein, whereas venetoclax-resistant HBL1 cells showed a loss in the gene encoding BIM (
BCL2L11) with corresponding reduction in
BCL2L11 transcript and absence of detectable BIM protein. Recently, it was shown that PI3Kδ inhibitor-induced apoptosis is BIM-mediated as Bim
-/- Eμ-Tcl1 Tg leukemias demonstrated resistance [
24]. In some cases,
BCL2 family transcripts were elevated without evidence of a corresponding gene gain or amplification, suggesting that altered signaling pathways may account for elevated protein levels. Elevated MCL-1 levels were sometimes associated with elevated
MCL1 mRNA (OCI-Ly1), and sometimes not (U2932, HBL2 and Granta-519), indicating that alterations in pathways impacting MCL-1 stability may also be involved. Although the originating source of the resistance signal was not immediately apparent in these cases, the most target-proximal cause of resistance is likely to be the altered expression of the BCL-2 family protein furthest downstream in the intrinsic cell death pathway.
Most proximal of all, mutations in the BH3-binding groove of BCL-2 itself were also identified in venetoclax-resistant cell populations, including the F104L mutation, which was previously shown to reduce venetoclax binding affinity and confer resistance [
21]. Such mutations have yet to be reported in association with clinical resistance to venetoclax. Nevertheless, should this mechanism arise as a significant factor in the development of venetoclax resistance, it should be possible to synthesize BCL-2 inhibitors that maintain high affinity for F104L-mutated BCL-2, akin to the second- and third-generation tyrosine kinase inhibitors that were designed in response to the Abl kinase gatekeeper mutations found to confer imatinib resistance[
25].
It should also be noted that the concentrations up to which these cells show resistance to venetoclax are clinically relevant. Venetoclax can achieve and maintain plasma exposures ~1-3 μM at daily doses ranging from 400 mg to 800 mg [
12]. Finally, although numerous ABT-199-resistant cell line populations were isolated in this study, we cannot make distinctions between cells that acquired resistance by “rewiring” and cases where pre-existing sub-clones with constitutive resistance were selected for. The identification of a pre-existing sub-population of BAX-deficient cells amongst the parental RS4;11 population may represent an example of the latter; however, this could not be formally demonstrated as cell fixation was required to identify the population lacking BAX.
Acknowledgements
We thank past and present members of the BCL-2 project team for helpful discussions.