Targeting Mitochondrial Apoptosis to Overcome Treatment Resistance in Cancer

Abstract

Deregulated cellular apoptosis is a hallmark of cancer and chemotherapy resistance. The B-cell lymphoma 2 (BCL-2) protein family members are sentinel molecules that regulate the mitochondrial apoptosis machinery and arbitrate cell fate through a delicate balance between pro- and anti-apoptotic factors. The recognition of the anti-apoptotic BCL2 gene as an oncogenic driver in hematological malignancies has directed attention toward unraveling the biological significance of each of the BCL-2 superfamily members in cancer progression and garnered interest in the targeting of apoptosis in cancer therapy. Accordingly, the approval of venetoclax (ABT-199), a small molecule BCL-2 inhibitor, in patients with chronic lymphocytic leukemia and acute myeloid leukemia has become the proverbial torchbearer for novel candidate drug approaches selectively targeting the BCL-2 superfamily. Despite the inspiring advances in this field, much remains to be learned regarding the optimal therapeutic context for BCL-2 targeting. Functional assays, such as through BH3 profiling, may facilitate prediction of treatment response, development of drug resistance and shed light on rational combinations of BCL-2 inhibitors with other branches of cancer therapy. This review summarizes the pathological roles of the BCL-2 family members in cancer, discusses the current landscape of their targeting in clinical practice, and highlights the potential for future therapeutic inroads in this important area.

Full text

cancers
Review
Targeting Mitochondrial Apoptosis to Overcome
Treatment Resistance in Cancer
Natalie Yan Li Ngoi 1, Clarice Choong 1, Joanne Lee 1, Gregory Bellot 2, Andrea LA Wong 1,3,
Boon Cher Goh 1,3 and Shazib Pervaiz 4,5,6,*
1Department of Haematology-Oncology, National University Cancer Institute, National University Health
System, Singapore 119228, Singapore; natalie_yl_ngoi@nuhs.edu.sg (N.Y.L.N.);
clarice_choong@nuhs.edu.sg (C.C.); joanne_lee@nuhs.edu.sg (J.L.);
Andrea_LA_WONG@nuhs.edu.sg (A.L.W.); boon_cher_goh@nuhs.edu.sg (B.C.G.)
2Department of Hand & Reconstructive Microsurgery, University Orthopedic, Hand & Reconstructive
Microsurgery Cluster, National University Health System, Singapore 119228, Singapore;
gregory_bellot@nuhs.edu.sg
3Cancer Science Institute, National University of Singapore, Singapore 117599, Singapore
4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore 117593, Singapore
5NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore,
Singapore 119077, Singapore
6National University Cancer Institute, National University Health System, Singapore 119228, Singapore
*Correspondence: phssp@nus.edu.sg; Tel.: +65-6516-6602
Received: 1 February 2020; Accepted: 27 February 2020; Published: 2 March 2020


Abstract:
Deregulated cellular apoptosis is a hallmark of cancer and chemotherapy resistance.
The B-cell lymphoma 2 (BCL-2) protein family members are sentinel molecules that regulate the
mitochondrial apoptosis machinery and arbitrate cell fate through a delicate balance between pro-
and anti-apoptotic factors. The recognition of the anti-apoptotic BCL2 gene as an oncogenic driver in
hematological malignancies has directed attention toward unraveling the biological significance of
each of the BCL-2 superfamily members in cancer progression and garnered interest in the targeting
of apoptosis in cancer therapy. Accordingly, the approval of venetoclax (ABT-199), a small molecule
BCL-2 inhibitor, in patients with chronic lymphocytic leukemia and acute myeloid leukemia has
become the proverbial torchbearer for novel candidate drug approaches selectively targeting the
BCL-2 superfamily. Despite the inspiring advances in this field, much remains to be learned regarding
the optimal therapeutic context for BCL-2 targeting. Functional assays, such as through BH3 profiling,
may facilitate prediction of treatment response, development of drug resistance and shed light
on rational combinations of BCL-2 inhibitors with other branches of cancer therapy. This review
summarizes the pathological roles of the BCL-2 family members in cancer, discusses the current
landscape of their targeting in clinical practice, and highlights the potential for future therapeutic
inroads in this important area.
Keywords: apoptosis; targeted therapy; cancer therapeutics; BCL-2; MCL-1; BCL-xL
1. Introduction—Apoptosis from the Chemotherapy Lens
Chemotherapy resistance in cancer has been attributed to multiple mechanisms, which often
act in concert [
1
,
2
]. This repertoire includes altering drug transport through influx/efflux pumps
such as ATP-binding cassette transporters [
3
] and P-glycoprotein overexpression [
1
]. Intracellularly,
well-described downstream mechanisms include activation of key signaling pathways, drug-target
alteration [
4
] and repair of drug-induced DNA damage [
2
]. Extrinsic to the cancer cell, cross-talk between
Cancers 2020,12, 574; doi:10.3390/cancers12030574 www.mdpi.com/journal/cancers

Cancers 2020,12, 574 2 of 29
tumor cells with the tumor microenvironment adds to chemo-resistance [
5
]. Tumor heterogeneity and
the existence of cancer stem cells, may further limit treatment response.
Increasingly, dysregulation of drug-induced autophagy and apoptosis has been recognized as
a key mechanism of carcinogenesis and chemotherapy resistance, whereby the surviving cancer
cell continues to accumulate oncogenic mutations which further propagate tumor progression [
6
].
Targeting apoptosis therefore holds promise in overcoming resistance to cancer therapy. Recently,
venetoclax (ABT-199) has successfully achieved USA Federal Drug Administration (FDA) approval
for the treatment of patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia
(AML), confirming that apoptosis-targeting strategies have finally come of age. In this article, we
discuss the roles of the BCL-2 superfamily in carcinogenesis and treatment resistance, and review the
successes and failures of strategies targeting the BCL-2 family members in cancer therapy.
2. The BCL-2 Superfamily and Its Role in Apoptosis
2.1. The BCL-2 Superfamily Controls the Intrinsic Apoptosis Pathway
Apoptosis is effected via the intrinsic and extrinsic pathways. The extrinsic, or death-receptor
mediated pathway, is initiated when cell death receptors such as Fas, TNFR1, TRAIL-R1, TRAIL-R2,
DR3 and DR6, interact with their ligands on the cell surface. Activation of Fas, TRAIL-R1 or TRAIL-R2
leads to the formation of a “death-inducing signaling complex” (DISC) and triggers a cascade of
caspase activation culminating in apoptosis, while the activation of other receptors induces apoptosis
by triggering different signaling pathways such as NF-
κ
B [
7
,
8
]. Detailed discussion of the extrinsic
pathway and its targeting is beyond the scope of this review. The intrinsic, or mitochondrial pathway,
responds to intracellular apoptotic stimuli such as viral infection, oxidative stress, calcium flux and
DNA damage caused by drug or radiation exposure [
9
,
10
]. When committed to apoptosis, mitochondria
outer membrane permeabilization (MOMP) is the decisive event through which cytochrome c and the
second mitochondria-derived activator of caspase (SMAC) are released into the cytoplasm, triggering
apoptosome assembly and caspase 9 activation [
7
]. Downstream executioner caspases 3, 6 and 7 cause
cellular dismantlement and cytoskeletal protein degradation, which lead to the classic morphological
features of crenation, DNA condensation and, ultimately, cell death [11].
The BCL-2 family members are central regulatory players in the intrinsic mitochondrial apoptotic
program, and their interplay controls cell fate [
12
] (Figure 1). More than 25 BCL-2 family members
have been identified. Advances in structural resolution of these members have categorized them
into three subfamilies—(1) the multidomain anti-apoptotic members (BCL-2, BCL-xL, MCL-1, BCL-w,
BCL-B/Boo and BFL-1/A1), (2) the multidomain pro-apoptotic members (BAX, BAK), and (3) the
BH3-only members (BAD, BID, NOXA, HRK, BMF, PUMA, BIM). Multi-domain members in (1) and (2)
have four BCL-2 homology (BH) domains (BH1, BH2, BH3 and BH4) each, while the BH3-only members
are comprised of only a single short BH3 domain [
13
–
15
]. The BH3 motif is composed of 9 to 15 amino
acids and is uniquely conserved across all BCL-2 family members [
16
]. BH3 interactions are responsible
for orchestrating the BCL-2 interactome via a BH3-into-hydrophobic groove mechanism [
17
,
18
], which
allows the formation of homo- and heterodimers that control apoptotic function [
19
]. Minor alterations
in the amino acid sequences of the binding grooves and BH3 domains control the specificity of
these interactions.
The balance between pro- and anti-apoptotic family members determines if intrinsic apoptosis will
proceed. When pro-apoptotic BAX or BAK are liberated, they are able to oligodimerize in the outer
mitochondrial membrane, leading to the formation of a mitochondrial membrane pore which commits
the cell to MOMP. The BH3-only proteins have complex roles as death sentinels that link apoptotic signals
to the intrinsic pathway, and are divided between two roles– either as “direct activators” (tBID, BIM,
PUMA) of BAX and BAK, exposing the BH3 domain of BAX and BAK to facilitate oligodimerization [
20
];
or as “inactivators” or “sensitizers” (BAD, BIK, BMF, HRK, NOXA) by binding with anti-apoptotic
BCL-2 thus allowing BAX/BAK to be unrestrained to trigger MOMP [21] (Figure 1).

Cancers 2020,12, 574 3 of 29
Figure 1.
The intrinsic apoptotic pathway and interactions between pro- and anti-apoptotic B-cell
lymphoma 2 (BCL-2) family members. Intrinsic pathway apoptotic stimuli such as viral infection,
oxidative stress, calcium flux and DNA damage lead to changes in the balance of pro- and anti-apoptotic
BCL-2 family members. The anti-apoptotic proteins act to prevent BAX/BAK activation. Activator
BH3-only proteins (PUMA, tBID, BIM) inhibit all anti-apoptotic members, whereas sensitizer BH3-only
proteins interact and engage selective anti-apoptotic members, allowing BAX/BAK oligomerization
and indirect activation. Oligomerization of BAX/BAK in the mitochondrial membrane commits the
cell to mitochondria outer membrane permeabilization (MOMP), and triggers a downstream caspase
cascade which ends in apoptosis.
2.2. Regulation of BCL-2 Family Members
The importance of apoptosis in homeostasis requires that it is tightly regulated. The canonical
roles of the BCL-2 subfamilies suggest that apoptosis may be triggered through inactivation of the
anti-apoptotic multidomain subfamily proteins, or an increase in concentration of BH3-only proteins.
Yet the BCL-2 puzzle has proven far more complex and often unpredictable, contributed by pleiotropic
effects of multiple signaling controls, as well as post-transcriptional and post-translational modification
processes, in modifying the affinity between BCL-2 family members [
16
]. For example, PI3K-Akt
activation leads to phosphorylation and inactivation of BAD [
22
], leaving BCL-2 free to inhibit apoptosis,
while increasing the expression of anti-apoptotic genes [
11
]. Similarly, activation of the extracellular
signal regulated kinase (ERK) pathway results in increased transcription of the anti-apoptotic subfamily
of BCL-2 members, and increased ubiquitination and subsequent degradation of pro-apoptotic members,
leading to cell survival [
23
]. ERK-mediated phosphorylation of MCL-1 at T163 was further shown
to stabilize MCL-1, leading to suppression of apoptosis in various hematological malignancy cell
lines [24].
In the nucleus, genomic alterations such as chromosomal translocations and gene amplifications
may increase BCL-2 levels. A notable example would be in CLL, where deletion of chromosome 13q
in >50% of patients leads to silencing of the microRNAs miR-15 and miR-16, which are responsible
for degrading BCL-2 RNA, resulting in BCL-2 overexpression [
25
]. Post-translational modifications
moderate protein functions through ubiquitination, proteolysis, phosphorylation and proteasomal
degradation [
26
]. Phosphorylation of BCL-2 at S70 [
27
] has been described to alter its anti-apoptotic
ability [
28
] and confer resistance to taxane chemotherapy [
29
]. Specific BIM phosphorylation sites
have the ability to affect its BCL-2 binding capability, resulting in an anti-apoptotic phenotype, while

Cancers 2020,12, 574 4 of 29
mutations at other phosphorylation sites (Ser-55, -65 and -73) tag BIM for proteasomal degradation,
increasing therapy resistance [
30
]. Phosphorylation of BAX at specific residues (S184), mediated by Akt
activation, has been suggested to switch BAX from pro- to anti-apoptotic in phenotype, by allowing it
to sequester activator BH3 proteins [18].
2.3. Dysregulation of BCL-2 Family Members in Carcinogenesis and Treatment Resistance
Dysregulation of the anti-apoptotic members BCL-2, BCL-xL and MCL-1 have been widely
described in carcinogenesis, cancer progression and chemotherapy resistance [
31
]. Cancer cells
often upregulate anti-apoptotic BCL-2 proteins, thus tilting the ratio of anti- versus pro-apoptotic
members to fall in favor of apoptosis evasion, even in the presence of stimuli from chemotherapeutic
agents [
6
]. CLL is considered one of the classical hematological malignancies attributable to failure
of apoptosis. Nearly all CLL patients have increased BCL-2 expression. Repression of BCL-2 at
the post-transcriptional level allowed for the induction of apoptosis in CLL cell lines [
25
]. BCL-2
overexpression is a key event in follicular lymphoma (FL), driven by pathological chromosomal t(14;
18) translocation, whereby the BCL2 oncogene is pathogenically translocated to the immunoglobulin
heavy chain (IGHV) gene locus, leading to its amplification. In diffuse large B-cell lymphoma (DLBCL),
concomitant overexpression of BCL-2 and MYC is classified as a “double-hit” DLBCL, which is
associated with a dismal prognosis, high risk for relapse, resistance to standard chemotherapy and
justifies upfront escalation to more intensive treatment. These observations have fueled strategies
therapeutically targeting the anti-apoptotic BCL-2 members in cancer treatment.
An interesting and somewhat non-canonical aspect of the functional biology of BCL-2 is ability
to maintain a mild mitochondrial pro-oxidant milieu while preventing deleterious levels of reactive
oxygen species (ROS) production triggered by oxidative stressors through the regulation of cytochrome
c oxidase activity [
32
]. This mechanism appears to be the result of an interaction between BCL-2 and
the subunit COX Va that shifts the ratio of COX Va to COX Vb subunits, thus modulating cytochrome c
oxidase activity. The modulation of ROS production by BCL-2 expression is a critical component of its
anti-apoptotic activity as cells subjected to oxidative stress inducers modulate their mitochondrial redox
metabolism to buffer the excess ROS production, thereby promoting cell survival [
33
]. In addition,
the pro-oxidant milieu generated through superoxide anion production by an increased expression of
BCL-2 was shown to be linked to an interaction between BCL-2 and the small GTPase Rac1, a critical
regulator of NADPH oxidase, responsible for superoxide production [
34
]. Interestingly, a mild to
moderate increase in intracellular superoxide anion (pro-oxidant state) has also been shown to impact
the phosphorylation status of BCL-2, specifically at S70 via the generation of peroxynitrite (a reaction
product of superoxide and nitric oxide). This involves peroxynitrite mediated nitrative modification
of the regulatory subunit B56
δ
of the protein phosphatase 2A (PP2A), which prevents holoenzyme
assembly and results in the sustained S70 phosphorylation of BCL-2 to stabilize its anti-apoptotic
activity [
35
]. These findings provide evidence for an intricate crosstalk between BCL-2 and cellular
redox metabolism, thereby delineating a novel facet in the biology of this death regulatory protein
with potential therapeutic implications.
MCL1 is one of the most highly amplified genes in human cancers [
36
]. In hematological
malignancies, increased levels of MCL-1 have been described in multiple myeloma (MM) [
37
],
DLBCL [
38
], AML, chronic myeloid leukemia (CML) and mantle cell lymphoma (MCL). Many
chemotherapeutic agents affect apoptosis through the reduction of MCL-1 levels. In CLL cell
lines, up-regulation of MCL-1 after co-culture with stroma was linked to fludarabine resistance [
39
].
Conversely, knock-down of MCL1 in mice models not only triggered apoptosis of transformed AML
cells but also salvaged AML-afflicted mice from disease progression [40].
Finally, elevated BCL-xL expression has also been observed in MM [
41
] and non-Hodgkin’s
lymphoma (NHL), and is implicated in their progression. In one study, transgenic mice with
overexpression of BCL-xL readily developed lymphomas [
42
]. This is further supported by studies

Cancers 2020,12, 574 5 of 29
showing that interactions between pro-apoptotic BCL-xL and anti-apoptotic BIM control the apoptosis
rate in MYC-related lymphoma [43].
Conversely, the loss of pro-apoptotic proteins appears to be relatively uncommon. Somatic
inactivation of BAX (and BAK) has been reported in both solid and hematological cancers [
44
]. Deletion
or silencing of NOXA, PUMA or BIM expression has been described in the pathogenesis of hematologic
cancers and their response to chemotherapy [
45
,
46
]. Of note, BIM is deleted in 17% of MCL [
47
],
while BAX mutations occur in 20% of hematologic cancers such as CLL, FL, MCL and NHL. In mouse
fibroblast models, loss of both BAX and BAK led to resistance to chemotherapy-induced apoptosis [
48
].
Additionally, loss of BAX in colon cancer cells led to 5-fluorouracil resistance [49].
Indeed, the complex roles of the BCL-2 family members have created immense potential for
targeting. Progressive and stepwise improvements in our mechanistic understanding of apoptosis
have since allowed for the identification of entry points into this network, toward the promise of
optimal therapeutic targeting in cancer. In the next section, we discuss the historical advancements
in BCL-2 family targeting that have led to the success of venetoclax in modern day hematological
malignancy treatment, and delve into upcoming novel strategies.
3. Targeting the BCL-2 Superfamily: A Summary of the Current Landscape
3.1. Antisense Oligonucleotides (ASO)
ASOs were the first approaches employed for BCL-2 inhibition. These are complementary strands
that hybridize with and silence anti-apoptotic BCL-2 subfamily mRNA, leading to hydrolysis of the
mRNA and promoting apoptosis [
50
,
51
]. Oblimersen is an 18-antisense oligonucleotide complementary
to the first six codons of BCL-2 mRNA that was evaluated in a variety of hematological malignancies.
Promising response rates were seen when combined with standard chemo-immunotherapy [
52
,
53
],
and also allowed lower doses of chemotherapy to be administered. Reduced BCL-2 mRNA and
protein levels were noted in AML patients who achieved a complete response (CR) with oblimersen,
providing proof-of-principle of its mechanism of action [
53
]. Common toxicities included fever, fatigue,
gastrointestinal side effects and night sweats. However, on several phase III studies, no survival
advantage could be shown for oblimersen addition [
52
]. Despite this, several patients treated with
oblimersen on study appeared to derive durable benefit from this drug [
52
]. Other ASOs under
evaluation include SPC2996, PNT2258 and bispecific ASOs targeting BCL-2/BCL-xL.
3.2. BH3-Mimetics
The recognition of BH3-only proteins as natural inhibitors of BCL-2 proteins led to the development
of BH3-mimetics. These small molecules are homologous to the BH3 domains of anti-apoptotic BH3-only
proteins, and bind competitively to the hydrophobic groove of anti-apoptotic proteins, displacing
BAX/BAK or pro-apoptotic BH3-only molecules, inducing apoptosis. Venetoclax (ABT-199, Abbvie
Inc, North Chicago, IL, USA), which targets BCL-2, was the front-runner inhibitor in developmental
pipelines for BH3-mimetics, and its FDA approval across four indications represents a major milestone
in this field. To date, BH3-mimetics specifically inhibiting BCL-2, MCL-1 and BCL-xL, respectively are
undergoing evaluation.
3.2.1. Gossypol and AT-101
Gossypol acts as a pan-BCL-2 family inhibitor [
54
], and has both BAX/BAK-dependent [
55
] and
-independent [
56
] mechanisms of action. In preclinical studies, gossypol demonstrated promising
activity through activation of the intrinsic apoptotic pathway in CML [
57
], NHL [
58
] and MM [
59
].
In-vivo studies in mouse models [
60
] showed significant slowing of tumor growth when gossypol was
combined with CHOP chemotherapy, compared to either CHOP or gossypol alone [
60
]. However,
gossypol has significant off-target side effects including dose-limiting thrombocytopenia, preventing
it from advancing into clinical trials. The more potent, and orally-available enantiomer of gossypol,

Cancers 2020,12, 574 6 of 29
AT-101 progressed further in its development. However, despite promising preclinical data, early
phase studies of AT-101 in combination with docetaxel for prostate cancer or non-small cell lung cancer
(NSCLC) did not show improved outcomes [
61
,
62
] (Table 1). In CLL, AT-101 with rituximab showed
only modest efficacy [
63
]. Gastrointestinal toxicities (such as nausea, vomiting and ileus), fatigue and
neutropenia were the most common side effects noted [
63
]. Newer analogues of AT-101 include TW37
and TM-1206, which have improved affinity to BCL-2, MCL-1 and BCL-xL.
3.2.2. Obatoclax
Obatoclax (GX15-070; Teva Pharmaceutical Industries Ltd, Parsippany, NJ, USA) is a relatively
weak polypyrrole pan-BCL-2 family inhibitor that is able to bind to anti-apoptotic BCL-2, BCL-xL, BCL-w,
BCL-B, BFL-1/A1 and also MCL-1 with sub-micromolar affinity, allowing BAK/BAX oligomerization
and cell death [
64
]. Obatoclax is also purported to have BCL-2 independent mechanisms, via its
effect on the Akt/mTOR signaling pathway [
65
], which increases the possibility of off-target toxicity.
In various hematological malignancy cell lines, and in-vivo mouse models, obatoclax monotherapy
showed anti-cancer activity [
65
,
66
]. However, raising obatoclax serum levels to clinically effective
concentrations in mice models was associated with severe neurotoxicity. Accordingly, when obatoclax
was tested in phase I/II trials for AML, CLL, acute lymphoblastic leukemia (ALL), myelodysplastic
syndrome, MCL and classical Hodgkin lymphoma (HL) [
67
–
70
], only limited clinical activity was
observed (Table 1). Common adverse events included mood disturbances and gastrointestinal side
effects, while high grade toxicities appeared to be mainly hematological [
68
]. Development of obatoclax
has been discontinued.
3.2.3. ABT-737
ABT-737 (Abbvie Inc, North Chicago, IL, USA) was the “first-in-class” small molecule inhibitor
designed as a BH3 mimetic of BAD. ABT-737 was shown to bind with a much higher affinity
(sub-nanomolar concentrations) and more selectively, compared to obatoclax, to BCL-2, BCL-xL and
BCL-w [
71
]. Activity of ABT-737 was shown in MM, and AML cell lines (Table 1). Notably, in CML
cell lines, ABT-737 plus imatinib reduced the development of BCL-2 driven imatinib-resistance [
72
].
The specific binding of ABT-737 to its intended targets resulted in an increase in MCL-1 expression
and phosphorylation thus bypassing the effect of ABT-737 and leading to ABT-737-resistance in AML
cells [
73
]. Compounding this, the unfavorable pharmacokinetic profile of ABT-737 further spurred the
development of newer generations of BH3 mimetics.
3.2.4. Navitoclax
Navitoclax (ABT-263; Abbvie Inc, North Chicago, IL, USA) is a second-generation, orally
bioavailable BH3-mimetic. Navitoclax binds preferentially to BCL-2, BCL-xL and BCL-w with
nanomolar affinity, specifically disrupting BCL-2 and BCL-xL interactions with pro-death BH3
members. However, navitoclax still lacks the ability to antagonize MCL-1 and BFL-1/A1 dependent
interactions. In-vivo xenograft models of small cell lung cancer (SCLC), ALL, NHL, MCL and MM
showed promising tumor regression [
74
]. In a phase I study of 29 patients with relapsed or refractory
CLL, navitoclax as a single-agent showed an overall response rate (ORR) of 35% in patients receiving
a daily dose of at least 110mg, although no CR was observed [
75
]. Despite this, durable responses
>12 months occurred, even in patients with the poor prognostic marker, deletion 17p. Furthermore, a
higher ratio of BIM to MCL-1 and BIM to BCL-2 correlated with improved efficacy of navitoclax [
75
].
As expected, patients with lower pre-treatment MCL-1 levels had improved response to navitoclax.
Prominent thrombocytopenia occurred early after treatment-initiation, this was often dose-limiting [
75
],
and consistent with previous data showing that the homeostasis of mature platelets is dependent
on BCL-xL. Currently, navitoclax is increasingly under investigation in solid tumors due to the
inherent risks of severe thrombocytopenia in patients with hematological malignancies who are already
myelosuppressed (Table 1).

Cancers 2020,12, 574 7 of 29
Table 1.
Early-generation BH3-mimetics inhibiting BCL-2. R/R: relapsed/refractory; CLL: chronic lymphocytic leukemia; HL: hodgkin’s lymphoma; MDS:
myelodysplastic syndrome; ORR: objective response rate; FL: follicular lymphoma; FR: fludarabine plus rituximab; iwCLL: international workshop on CLL; MCL:
mantle cell lymphoma; CR: complete response; PR: partial response; PFS: progression-free survival; RP2D: recommended phase 2 dose; ALL: acute lymphoblastic
leukemia; SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; OS: overall survival.
Drug Mechanism of Action Phase Treatment Population Activity Reference
ABT-737
Binds and neutralizes
BCL-2, BCL-xL, and
BCL-w.
Preclinical CLL cell lines ABT-737 induced CLL cell death in a BAX/BAK
dependent manner. [76,77]
Obatoclax Inhibits BCL-xL, BCL-2,
MCL-1, BCL-w,
A1 and BCL-B.
Monotherapy
I R/R HL No objective responses observed. [78]
II Treatment-naïve MDS
ORR 8%, disease stabilization/response was maintained
≥
12 weeks in 50%. The study was terminated due to failure
to meet pre-specified response endpoint.
[68]
I R/R CLL PR 4%. Dose limiting neurologic toxicities including
somnolence, euphoria and ataxia were noticed on study. [79]
Combination therapy
II Treatment-naïve FL
Obatoclax single-agent or in combination with Rituximab,
no objective responses observed after 12 weeks of
single-agent obatoclax.
[80]
I R/R CLL
Obatoclax plus FR, ORR was 54% by IWCLL 2008 criteria.
Median time to progression was 20 months. [81]
I/II R/R MCL
Obatoclax plus bortezomib was feasible. ORR was 31%
(3 CR, 1 PR)
. Synergy observed in preclinical models was
not confirmed.
[70]

Cancers 2020,12, 574 8 of 29
Table 1. Cont.
Drug Mechanism of Action Phase Treatment Population Activity Reference
Navitoclax
(ABT-263)
Targets BCL-2, BCL- XL,
BCL-w.
Monotherapy
I R/R CLL
PR rate was 35%, median PFS was 25 months. Activity
was noted even in patients with chemotherapy-resistant
disease, bulky lymph nodes and deletion-17p.
Thrombocytopenia was the main dose-limiting toxicity
and was dose-dependent. RP2D determined as
250 mg daily.
[75]
Combination therapy
I R/R ALL
Combination of navitoclax, venetoclax, Peg-asparaginase,
vincristine, dexamethasone. ORR 67%. [82]
II Myelofibrosis Navitoclax and ruxolitinib: ongoing recruitment.
NCT03222609
Ib RAS-mutant advanced
solid tumors Navitoclax and trametinib: ongoing recruitment.
NCT02079740
Gossypol
compounds
(AT-101)
Natural phenol derived
from the cotton plant.
Specific antagonist of
BCL- XLand BCL-2.
Monotherapy
II Extensive stage,
chemo-sensitive SCLC Gossypol showed no clinical activity. [83]
Combination therapy
II R/R CLL AT-101 plus rituximab, only PR noted. [63]
II Metastatic NSCLC,
second-line therapy
AT-101 plus docetaxel versus docetaxel.
No difference in PFS or OS. [84]
II
Metastatic
castration-resistant
prostate cancer
AT-101 plus docetaxel/prednisolone versus placebo plus
docetaxel/prednisolone. No difference in OS. [62]

Cancers 2020,12, 574 9 of 29
3.2.5. Venetoclax: A Selective BCL-2 Inhibitor
Hydrogen bonds between venetoclax and Asp103 on BCL-2 result in the increased selectivity
of venetoclax for BCL-2 compared to previous compounds [
85
]. Venetoclax is an orally-available,
extremely potent and selective BCL-2 only inhibitor, and is platelet-sparing [
85
]. Due to its improved
therapeutic window, this drug emerged as the front-runner BH3-mimetic, particularly in malignancies
which are BCL-2 dependent.
As described earlier, the central role of the BCL2 super family in CLL has made this disease a key
substrate for studying and developing BCL-2-targeted therapy. In a phase I dose-escalation study of
116 relapsed/refractory CLL and NHL patients treated with venetoclax, ORR of 79% and CR rate of 20%
was seen in patients with CLL. This was particularly impressive as the target-population had included
heavily-pretreated CLL patients and 90% of patients harbored at least 2 poor prognostic markers, such
as chromosome 17p-deletion, 11q deletion, fludarabine-resistance, bulky lymphadenopathy and lack
of mutation in IGHV. This potent and rapid cell kill was further confirmed by the unexpectedly high
rate of tumor lysis (TLS) in 18% of patients leading to fatalities. Amending the dose schedule to feature
a risk-mitigating ramp-up dose, together with monitoring and adequate TLS prophylaxis, helped to
prevent this feared side effect. Despite rampant expression of BCL-2 in healthy tissues, other adverse
events (AEs) were manageable, such as diarrhea, nausea and neutropenia. Treatment with venetoclax
in the dose-escalation cohort resulted in an estimated 2-year overall survival (OS) rate of 84% [
86
]
(Table 2).
These impressive results led to a pivotal phase II study of 107 patients with relapsed/refractory
deletion-17p CLL treated with venetoclax. ORR with venetoclax was 79%, including CR 8%, and
responses were seen regardless of the presence of poor-prognostic markers [
86
] (Table 2). Specifically
in CLL patients treated with venetoclax after progressing on the B cell receptor inhibitors (BCRis)
ibrutinib or idelalisib, the phase II M14-032 study reported ORR 67% and time-to-response of 2.5
months. Even in a small exploratory subgroup of 28 patients who had previously received more than 1
previous BCRi, encouraging activity was noted [87] (Table 2).
Venetoclax therapy is made even more convincing by its ability to result in unprecedented
phenomenon of undetectable minimal residual disease (uMRD), which is defined when there is <1 CLL
cell per 10,000 lymphocytes in marrow or peripheral blood. Low or uMRD has been shown to correlate
with improvements in OS [
88
]. In a pooled analysis of 2 phase II studies of relapsed/refractory CLL
patients treated with venetoclax, the PFS rate was 92.8% in patients achieving uMRD at 24 months on
treatment [
89
,
90
]. The first approval for venetoclax in patients with CLL came in 2016, where patients
with 17p deletion were approved to receive venetoclax in the subsequent-line setting. This was later
extended to patients with CLL or small lymphocytic lymphoma (SLL) in June 2018, regardless of 17p
deletion, in the subsequent-line setting.
The combination of rituximab, an anti-CD20 antibody, to venetoclax has also shown to be highly
effective and able to achieve high uMRD rates in relapsed/refractory CLL. Preclinical data showed
that this combination was able to counteract micro-environmental signals that were contributing to
venetoclax resistance in CLL [
91
]. The phase III MURANO study [
92
] compared venetoclax-rituximab
for 6 cycles followed by a 2 year-maintenance treatment, to 6 cycles of bendamustine-rituximab, and
showed remarkable improvements in 2-year progression-free survival of 84.9% versus 36.3% [
92
], as
well as 3-year uMRD rate (62% versus 13%) [
93
]. Another phase III study recently reported results
comparing venetoclax-obinutuzumab versus chlorambucil-obinutuzumab in previously untreated
CLL patients. Venetoclax-obinutuzumab was associated with significantly improved PFS at 24 months
(24-month PFS rate 88.2% versus 64.1%), and this benefit was extended to patients with poor prognostic
factors [
94
]. These impressive results relating to uMRD, together with pooled analysis data suggesting
that venetoclax should be sequenced earlier in treatment paradigms, ultimately led to the FDA
indication being expanded to all adult patients with CLL or SLL in May 2019.
Venetoclax monotherapy is modestly active in relapsed/refractory AML. Of note, patients harboring
IDH1/2mutations appeared to perform better with venetoclax therapy, with CR rate of 33% [
95
,
96
].

Cancers 2020,12, 574 10 of 29
Further phase Ib studies have also combined venetoclax with hypomethylating agents based on
preclinical models demonstrating synergy [
97
]. When combined with low dose cytarabine, decitabine
or azacytidine in untreated elderly patients, CR/CRi rates ranged between 54–68% across studies with a
median time to response of 1.2–1.4 months, with tolerable toxicity [
98
,
99
] (Table 2). This led to a further
FDA breakthrough status in November 2018 for venetoclax in combination with hypomethylating
drugs for newly-diagnosed elderly AML patients ineligible for intensive chemotherapy.
Venetoclax has also shown promising activity in relapsed/refractory MCL. In a phase I trial of
106 patients with relapsed/refractory NHL, patients with MCL had particularly high response rates
(ORR of 75%, CR 21% [
100
]. Venetoclax plus ibrutinib was evaluated on a phase II study, which
recruited a majority of relapsed/refractory MCL patients, again showing high response rates of ORR
71%, CR 63% [
101
], and this is being explored further on a phase III study (Table 2). Venetoclax
monotherapy appears to be less active in other NHL, in particular relapsed/refractory DLBCL, where
only modest response rates of around 18% were noted [
100
]. Similarly, in relapsed/refractory MM,
ORR for venetoclax monotherapy was 21% [
102
]. In cell lines, the t(11; 14) (q13;q32) translocation was
shown to increase BCL-2:MCL-1 ratio and lead to lower BCL-xL levels, and patients harboring this
translocation may benefit the most [103].
As alluded to, venetoclax has heralded the way for the development of other BCL-2 inhibitors.
Newer BCL-2 inhibitors in the pipeline include S55746 (Servier, Suresnes, France) which has dual
BCL-2/BCL-xL inhibiting capabilities [
104
,
105
] (Table 2). It is likely that the role for BCL-2 inhibitors is
likely to expand in cancer therapy, and further results are awaited.
3.2.6. BCL-xL—Selective BH3-Mimetics
BCL-xL dependency has been described across tumor types, aggregating mainly in solid
tumors [
106
]. This makes selective BCL-xL inhibition an attractive target, especially in the treatment
of venetoclax- resistant cancers. As described earlier, BCL-xL expression in AML, MM and some
solid tumor models, is associated with chemotherapy and venetoclax resistance [
107
,
108
]. WEHI-539
(The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) was the first selective
BCL-xL inhibitor published. When bound to BCL-xL, WEHI-539 induced BAK-mediated cell death in
SCLC cell lines. When used in osteosarcoma cells that overexpressed BCL-xL, WEHI-539 was able to
potentiate the effect of low-dose doxorubicin [
109
]. However, further development of this compound
has been halted due to in-vivo toxicity. On-target toxicities of such inhibitors include thrombocytopenia,
which occurs rapidly and reversibly, similar to what was observed with navitoclax. Further BCL-xL
selective inhibitors under pre-clinical evaluation include A-1155463 and A-1331852 (Abbvie Inc., North
Chicago, IL, USA) (Table 3).
3.2.7. MCL-1 Antagonists
In healthy tissues, MCL-1 regulates neural and cardiac cell survival. In cancer, not only has the
MCL-1 protein been shown to regulate cell survival in myeloid and lymphoid cancers including MM,
AML and NHL [
36
,
110
], but MCL1 amplifications have been found in >10% of solid tumor cancer
types [
106
]. In triple-negative breast cancer, MCL1 amplification correlates with poor prognosis [
111
].
Drug development in MCL-1 inhibition is ongoing with several candidate compounds in early
phase testing (Table 3). A-1210477 (Abbvie Inc., North Chicago, IL, USA) was the first inhibitor able
to disrupt MCL-1-NOXA and MCL-1-BIM2A interactions selectively [
107
]. When used in MM and
NSCLC cell lines that showed MCL-1 dependency, A-1210477 triggered MOMP and apoptosis [
107
].
A more potent MCL-1 inhibitor, S63845 (Servier, Suresnes, France) [
112
], is also undergoing evaluation,
and has been shown to have sub-molar affinity to the MCL-1 BH3 binding groove with BCL-2/
BCL-xL binding.
In vitro
, S63485 induced BAX/BAK-mediated apoptosis in solid tumors, as well
as elicited intriguing synergism with tyrosine kinase inhibitors(TKIs) [
112
]. Several other MCL-1
inhibitors [AZD5991(AstraZeneca), AMG-176, AMG-397(Amgen), S64315/MIK665(Novartis)] are
currently undergoing phase I clinical trials in a variety of hematological malignancies (Table 3). Despite

Cancers 2020,12, 574 11 of 29
these advancements, the concern for the development of side effects of MCL-1-targeting agents on
cardiac and neurological systems may pose challenges to clinical development of these agents, and
further results are awaited.
3.3. Targeting the BH4 Domain
Similar to BH3, the BH4 domain is conserved amongst the members of the BCL-2 superfamily.
Aside from its crucial role in the anti-apoptotic activity of BCL-2, the BH4 domain also is required
for other non-canonical functions of the BCL-2 superfamily, such as in calcium homeostasis at the
ER [
113
]. Notably, losing the BH4 domain greatly diminishes the anti-apoptotic function of BCL-2 [
114
].
Targeting BH4 is therefore emerging as a novel strategy in cancer therapy (Table 3).
3.4. Interference Technology
Interference technologies at the DNA and RNA level utilize a nucleic acid-based approach to
block transcription and translation of BCL2 respectively. Silencing BCL2 by utilizing RNA interference
(RNAi) technology is still in its infancy. Early data regarding the efficacy of this approach have been
generated using ALL cell lines and xenografts [
115
]. PNT2258 (ProNAi Therapeutics Inc., Vancouver,
Canada) a first-in-class DNAi drug that consists of a 24-base sequence complementary to regions of
DNA that are upstream from sites of gene transcription, thus preventing BCL-2 transcription [
116
].
In pre-clinical studies, PNT2258 was active in BCL-2 driven xenografts, including in NHL, prostate
cancer and melanoma [
117
]. Differential activity was seen in different NHL cell lines according to their
levels of BCL-2 overexpression. Initial phase I studies of PNT2258 confirmed a safe toxicity profile
with tolerable lympho- and thrombocytopenia [
118
]. Initial interesting responses, especially in DLBCL
patients, were noted on a phase I study of PNT2258 in relapsed/refractory NHL. However, these results
were not corroborated in a phase II study of relapsed/refractory DLBCL, and the development of
PNT2258 was subsequently discontinued [119] (Table 3).
4. Navigating Anti-Apoptotic BCL-2 Dependency to Tackle Therapy Resistance
4.1. BCL2 Dependency in Intrinsic and Acquired Therapy Resistance
Despite the excellent results obtained from the use of venetoclax, many patients progress after
a period of treatment (acquired resistance), while others do not respond at all (intrinsic resistance).
Although BCL-2 is overexpressed in a multitude of solid tumors and hematological malignancies,
significant responses to venetoclax monotherapy are limited to only a handful of cancer types. This is
because, although BCL-2 may be over-expressed, this may not be reflective of its pathological function.
Concurrent or dominant expression of MCL-1 or BCL-xL may indicate dependency on these family
members instead, leading to intrinsic resistance to and limited utility of venetoclax monotherapy.
Acquired resistance is also linked to the concept of BCL-2 dependency. Functional redundancy
within the BCL-2 superfamily allows acquired resistance to develop by switching reliance on other
anti-apoptotic members as a result of treatment pressure. The exact mechanisms of these dynamic
inhibitory responses have yet to be defined. In CLL cells, compensatory BCL-xL and BFL-1/A1
upregulation was associated with acquired venetoclax resistance [
120
,
121
]. In NHL cell lines, prolonged
venetoclax treatment also resulted in increased BCL-xL and MCL-1 expression, mediated by Akt
signaling [
122
]. Similarly, in lymphoma experiments, resistance to ABT-737 was affected through a
shift in BCL-2 family member dependency by the upregulation of MCL-1 or BFL-1/A1 [
123
]. Aberrant
NF-kB signaling has been shown to affect resistance to ibrutinib plus venetoclax combination therapy
in CLL cells by increasing MCL-1 and BCL-xL expression [124].

Cancers 2020,12, 574 12 of 29
Table 2.
Key venetoclax trials including upcoming novel combinations. MRD: minimal residual disease; TLS: tumor-lysis syndrome; CRi: complete remission
with incomplete marrow recovery; SLL: small lymphocytic lymphoma; AML: acute myeloid leukemia; IDH2: isocitrate dehydrogenase 2; NHL: non-hodgkin’s
lymphoma; DLBCL: diffuse large B-cell lymphoma; WM: waldenstrom macroglobulinemia; MZL: marginal zone lymphoma; MM: multiple myeloma; FCR:
fludarabine/cyclophosphamide/rituximab; BR: bendamustine/rituximab.
Phase Treatment Population Activity Reference
Monotherapy
II R/R CLL
ORR of 79% was noted, including CR 8%. Grade 4 neutropenia occurred in 23% of patients and
were managed with dose reductions and growth factor support. [90]
II R/R CLL after progressing on
ibrutinib or idelalisib ORR 67%, rapid time to response. [87]
I R/R NHL
Venetoclax monotherapy in MCL, FL, DLBCL, WM and MZL. ORR was 44% and highest in MCL
patients (ORR of 75%). Median PFS was 6 months (14 months in MCL patients). [100]
Ib/II R/R AML ORR of 19%, rapid responses were noted with 20% of responders achieving >50% reduction in
the percentage of marrow blasts at the first disease assessment. [96]
Combination therapy – CLL/SLL
Ib R/R CLL Venetoclax and Rituximab: ORR 86% (including CR 51%). 2-year PFS rate was 82%. Negative
marrow MRD attained in 57% of patients overall. Clinical TLS occurred in 2/49 patients. [125]
III R/R CLL
MURANO: venetoclax-rituximab for 6 cycles followed by a 2 year-maintenance treatment versus
6 cycles of bendamustine-rituximab. Improved 2-year PFS and uMRD rate. [92]
Ib R/R CLL Venetoclax-obinutuzumab for 6 cycles followed by 6 additional venetoclax cycles. 100% of
patients achieved uMRD, and 100% PFS at 1 year [126]
Ib/II Relapsed and previously untreated CLL
Venetoclax, obinutuzumab and ibrutinib in sequential administration. ORR was 92%, including
42% CR/CRi. [127]
III Previously untreated CLL
Venetoclax-obinutuzumab versus chlorambucil-obinutuzumab showed improved 24-month PFS
rate favoring venetoclax-obinutuzumab (88.2% versus 64.1%). [94]
I/II R/R CLL or Richter’s syndrome Venetoclax plus duvelisib: ongoing recruitment. NCT03534323
I/II Symptomatic CLL
Venetoclax, ublituximab and umbralisib, starting with umbralisib-ublituximab debulking
followed by umbralisib-venetoclax from cycle 4 onwards. ORR was 100% after cycle 7.
100% of patients who received ≥12 cycles of treatment, attained undetectable MRD.
[128]
III Previously untreated CLL or SLL Venetoclax plus ibrutinib versus chlorambucil plus obinutuzumab: active, not recruiting.
GLOW/CLL3011
NCT03462719

Cancers 2020,12, 574 13 of 29
Table 2. Cont.
Phase Treatment Population Activity Reference
III Previously untreated CLL or SLL
without del(17p) or TP53
Venetoclax plus acalabrutinib (AV) versus AV plus obinutuzumab versus chemoimmunotherapy
(FCR or BR): ongoing recruitment. NCT03836261
Combination therapy—AML
Ib Untreated older (≥65years) AML,
ineligible for intensive chemotherapy.
Venetoclax plus decitabine or azacitidine, CR/CRi: 67%. In patients ≥75 years old or with
poor-risk cytogenetics, CR/CRi was 65% and 60% respectively. Median OS was 17.5 months.
Treatment was well tolerated.
[98]
ITreatment-naÏve AML Venetoclax in combination with intensive chemotherapy: ongoing recruitment. NCT03709758
I R/R AML Venetoclax plus CDK inhibitors alvocidib, CYC065, dinaciclib: all studies ongoing recruitment.
NCT03441555
NCT04017546
NCT03484520
I/II R/R AML in older (≥60years) patients
not suitable for cytotoxic chemotherapy Venetoclax plus idasanutlin or venetoclax plus cobimetinib: ongoing recruitment. NCT02670044
I/II R/R AML with IDH2 (R140 or R172)
mutations Venetoclax plus enasidenib: planned, not yet recruiting. NCT04092179
I R/R CD33+AML Venetoclax plus gemtuzumab ozogamicin: planned, not yet recruiting. NCT04070768
Combination therapy - NHL
III Treatment-naïve MCL Venetoclax plus ibrutinib versus placebo plus ibrutinib: active, not recruiting NCT03112174
Combination therapy - MM
Ib R/R MM
Venetoclax plus bortezomib and dexamethasone. 39% of patients were previously refractory to
bortezomib. ORR was 67%, 42% achieved very good PR or better. Patients with high BCL2
expression had higher ORR compared to patients with low BCL2 expression.
[129]
II R/R MM
Venetoclax plus carfilzomib and dexamethasone. Of 17 patients evaluated after completing 2 or
more cycles, 3/17 had CR. [130]
III R/R MM
BELLINI: Venetoclax plus bortezomib/dexamethasone versus placebo plus
bortezomib/dexamethasone. Improved PFS, ORR and MRD for venetoclax arm, however 13
treatment-emergent deaths occurred in the venetoclax-containing arm. Trend towards improved
OS in patients with t(11;14). Study suspended for safety.
[131]
I/II R/R MM Venetoclax plus daratumumab, bortezomib, dexamethasone: planned, not yet recruiting. NCT03701321

Cancers 2020,12, 574 14 of 29
Table 3.
Other BCL2 family inhibitors under investigation. SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; R/R: relapsed/refractory; MM:
multiple myeloma; AML: acute myeloid leukemia; NHL: non-hodgkin’s lymphoma; DLBLC: diffuse large B cell lymphoma; ORR: objective response rate; PFS:
progression-free survival.
Drug Mechanism of Action Phase Treatment Population Activity Reference
Dual BCL-2/BCL-xL inhibitors
S44563 Inhibitor of both BCL-2
and BCL-xL. Preclinical Uveal melanoma and SCLC models.
In-vivo activity in uveal melanoma and
SCLC models. [104,105]
BCL-xL -selective inhibitors
A-1155463 Selective BCL-xL
inhibitor. Preclinical SCLC xenografts
More potent against BCL- XL-
dependent cell lines compared to
WEHI-539. Inhibited SCLC xenograft
tumor growth in-vivo.
[132]
A-1331852 Selective BCL-xL
inhibitor. Preclinical
Cell lines and xenograft models of seven
different solid tumors such as breast cancer,
ovarian cancer and NSCLC
Enhances the efficacy of docetaxel
in-vitro and in-vivo.[107]
MCL-1 inhibitors
AZD5991
Selective MCL-1 inhibitor.
Preclinical R/R hematological malignancies Preclinically, preferential activity was
noted in hematological cell lines. [133]
AMG-176
Selective MCL-1 inhibitor.
I R/R MM and AML First-in human study, recruitment
suspended.
NCT02675452
AMG-397
Selective MCL-1 inhibitor.
I R/R hematological malignancies Recruitment suspended due to cardiac
toxicity signal.
NCT03465540
S64315/MIK665
Selective MCL-1 inhibitor.
Preclinical and
phase I R/R hematological malignancies Potent activity in-vitro and in-vivo.
Phase I studies are ongoing.
[134,135]
NCT02992483
NCT02979366
MCL-1 inhibitor plus venetoclax combination therapy
AZD5991 plus venetoclax I R/R hematological malignancies
Phase I study of AZD5991 in
combination with venetoclax: ongoing
recruitment.
NCT03218683
S64315 plus venetoclax I R/R AML
Phase I study of S64315 in combination
with venetoclax: active, not recruiting.
NCT03672695

Cancers 2020,12, 574 15 of 29
Table 3. Cont.
Drug Mechanism of Action Phase Treatment Population Activity Reference
AMG-176 plus venetoclax I R/R AML, NHL, DLBCL
Phase I study of AMG-176 in
combination with venetoclax:
suspended to evaluate safety.
NCT03797261
Targeting BH4 domain
BDA-366
BCL2 BH4 domain
antagonist. Converts
BCL-2 into a
pro-apoptotic molecule.
Preclinical MM cell lines and mouse models BDA-366 inhibited MM tumor growth
in-vitro and in-vivo.[136]
BCL2 converting peptides
NuBCP-9
20 amino acid peptide
that acts as a molecular
switch to expose the BH3
domain of BCL-2.
Preclinical Breast cancer cell lines and Ehrlich tumor
mouse models
Synergistic potential of paclitaxel with
NuBCP-9 loaded nanoparticles in
reducing tumor burden.
[137]
DNA interference
PNT2258
24 base single-stranded
DNA
oligodeoxynucleotide
wrapped in liposomes,
inhibits BCL-2 promoter
activity.
I R/R NHL
13 patients were enrolled. Notable
responses were observed in DLBCL
patients (4/4 DLBCL patients).
NCT01733238
II R/R DLBCL
ORR 8.1%; all partial metabolic
responses. Median PFS was
1.9 months.
NCT02226965

Cancers 2020,12, 574 16 of 29
4.2. BH3 Profiling to Define BCL2 Dependency
BH3 profiling is a functional assay technique that has helped to provide clarity on BCL-2
dependency. This technology is able to determine (1) the degree of ‘mitochondrial priming’ of a
cell [
138
], and (2) BCL-2 member dependency, and hence predict response and resistance to therapies
targeting the BCL-2 family [139].
Briefly, an array of functionally-distinct BH3-only proteins are added to isolated mitochondria
or permeabilized cells taken from a fresh cancer sample, and allowed to interact with other BCL-2
superfamily proteins at the mitochondrial surface, inducing MOMP. MOMP is measured indirectly by
the amount of cytochrome release or by the loss of inner mitochondrial membrane potential (MMP).
In cells that have highly-primed mitochondria, BH3-only proteins rapidly induce MMP loss, compared
to cells that have low priming. Importantly, the differing specificity of some BH3-only proteins for
anti-apoptotic members e.g.: NOXA for MCL-1 will allow the BCL-2 dependency of the cell to be
inferred by the degree of MOMP triggered when different BH3-only proteins are added [140].
MM and AML cells with dominant MCL-1 dependency or heterogenous dependency on multiple
members—BCL-2, MCL-1, BCL-xL—were predicted to be resistant to BCL-2 inhibitor monotherapy,
unless the other members are able to be abrogated [
76
]. In MM and NSCLC, the presence of MCL-1/BAK
complexes predicted sensitivity to MCL-1 inhibition with A-1210477 [
107
]. The level of mitochondrial
priming inferred from BH3 profiling also provides information regarding the depth of response. In CLL
cells, high mitochondrial priming pre-treatment was associated with deeper venetoclax responses [
141
].
In AML cell lines, low mitochondrial priming has been correlated with chemotherapy resistance [
142
].
New developments in BH3 profiling include whole cell JC-1 based technology that allows easier
measurements of cytochrome c release through the use of JC-1, a fluorescent probe. FACS technology
now allows BH3 profiling to be performed in polyclonal cell populations [
120
], potentially providing
insight into BCL-2 dependency despite tumor heterogeneity. Furthermore, given the described
compensatory upregulation of other anti-apoptotic BCL-2 family members such as MCL-1 and
BFL-1/A1 in response to ABT-737 treatment in lymphoma [
123
] and BCL-xL in response to venetoclax in
CLL, identification of these changes via BH3 profiling may allow for sequential use of novel inhibitors
such as those against MCL-1 or BCL-xL to combat acquired resistance. Therefore, systematic sequential
BH3 profiling has promise as a dynamic biomarker to allow us to document changes in a tumor’s
anti-apoptotic BCL-2 dependency longitudinally, predict depth of response, and even select the right
therapeutic strategy to target specific molecular vulnerability in a personalized approach.
5. Expanding Clinical Contexts for BCL-2 Targeting
5.1. Promising Combination Strategies in Hematological Malignancies
Despite the gains we have made through venetoclax in specific clinical contexts, rational
combinations of BCL2-targeting therapy with chemotherapeutics and other targeted therapy hold
promise to advance treatment paradigms. Venetoclax is currently being combined with different
branches of cancer therapy in different hematological and solid malignancies, chosen based on
known pathways that are aberrant in specific tumor types (Table 2). Aside from combination with
chemotherapy, other important combinations under investigation include those with proteasome
inhibitors (bortezomib, carfilzomib), PI3K inhibitors (idelalisib), BTK inhibitors (ibrutinib), CDK
inhibitors (dinaciclib, palbociclib), MEK inhibitors (cobimetinib), MDM2 inhibitors (idasanutlin) and
other novel agents (Table 2). Co-targeting of different BCL-2 family members to overcome resistance,
such as concurrent BCL-2 and MCL-1 targeting or BCL-2/BCL-xL targeting is also under study (Table 2).
The rationale to combine BCL-2 targeted drugs with chemotherapy is based on the understanding
of mitochondrial priming. Treatment with BH3-mimetics is expected to raise the mitochondrial
priming state, thereby allowing them to act as “chemosensitizers” for synergism with cytotoxic
chemotherapy [
143
] (Table 2). Furthermore, this approach holds benefit not just in enhancing cell kill,
but also may reduce treatment doses, thus reducing toxic side effects.

Cancers 2020,12, 574 17 of 29
Venetoclax in combination with BTK inhibitors in CLL and MCL treatment is actively being
explored. Samples taken from CLL patients receiving ibrutinib were analyzed in-vitro with the addition
of venetoclax, and proved synergism of this combination [
144
]. Ibrutinib appears to downregulate
MCL-1 and BCL-xL, potentiating venetoclax’s effect [
144
]. Adding venetoclax to obinutuzumab and
ibrutinib in combination is being evaluated on a phase Ib study (NCT02427451), and a phase III
study (GLOW/CLL3011) is studying ibrutinib plus venetoclax versus obinutuzumab plus chlorambucil
(Table 2).
BAK, BAX and other pro-apoptotic members are degraded by ubiquitination and the proteasomal
pathway. Therefore, proteasomal inhibition allows for their stabilization and accumulation in
mitochondria, increasing the pro- to anti-apoptotic protein ratio [
145
]. In relapsed/refractory MM
treatment, venetoclax, bortezomib and dexamethasone combination therapy initially showed a high
ORR. Patients achieving PR or better had higher levels of BCL-2 [
129
]. However, on the phase III
BELLINI study randomizing patients with relapsed/refractory MM to bortezomib combined with
venetoclax or matched placebo, although the study showed improved PFS, ORR and uMRD for
the venetoclax-containing arm, 13 treatment-emergent deaths occurred in the venetoclax-containing
arm. Most deaths were attributable to infection, and this risk strengthened the urge toward a
biomarker-driven approach. Authors suggested that this combination could be most relevant in
patients with t(11; 14), where a trend towards improved OS was also noted, limiting exposure of
toxicity to a smaller group of patients [
131
,
146
]. An additional phase II study investigating venetoclax
with carfilzomib and dexamethasone (NCT02899052) is underway, interim results describe no new
safety signals [130].
Strategies inhibiting important cyclin dependent kinases (CDKs) are also promising. CDK9
is a key component of positive transcription elongation factor (pTEFb) which is a transcriptional
regulator complex. Inhibition of CDK9 blocks transcription resulting in MCL-1 repression [
147
]. CDK9
inhibition also down-regulates miRNAs that in turn negatively regulates pro-apoptotic BCL-2 family
members, leading to a net activation of pro-apoptotic members. Voruciclib, a CDK1, 4, 6 and 9 inhibitor,
synergized with venetoclax in DLBCL models to induce tumor remission [
148
]. In MM, several
pre-clinical studies have similarly described how CDK inhibition down-regulates MCL-1 in cell lines.
In AML, inhibition of CDK9 was demonstrated to transcriptionally silence MCL1, and thus overcome
MCL-1 dependent drug resistance [
149
]. In venetoclax-resistant AML cell lines and mouse xenografts,
voruciclib combined with venetoclax were synergistic in triggering BIM-dependent apoptosis [
150
].
Several early phase clinical trials investigating combinations of venetoclax with CDK inhibitors are
ongoing (Table 2).
Promising results are also emerging from the combination of PI3K inhibitors (PI3Ki) with
venetoclax and other therapies, particularly in CLL. Recent data released from a phase I/II study
of umbralisib (a PI3Ki), ublituximab (a CD-20 antibody) and venetoclax in relapsed/refractory CLL
included 27 patients, starting with umbralisib-ublituximab debulking to reduce the risk for tumor lysis
syndrome, followed by umbralisib-venetoclax starting from the fourth cycle onwards. In 13 patients
treated for >7 cycles of triple combination treatment, the ORR was 100% after cycle 7, and in 9 patients
who received 12 or more cycles of treatment, 100% of patients attained uMRD. At short follow up of
6.4 months, none of the 27 patients had experienced disease progression [128] (Table 2).
Finally, novel therapies are being combined with BCL-2 inhibition. MDM2 inhibition has been
shown to promote MCL-1 degradation in preclinical AML models [
151
]. Early results from a phase Ib
study combining idasanutlin with venetoclax in relapsed/refractory AML have shown a response rate
of 35.9%, with manageable toxicity [
152
]. Further studies combining venetoclax with novel therapies
such as gemtuzumab ozogamicin, enasidenib and liposomal cytarabine and daunorubicin are ongoing
(Table 2).

Cancers 2020,12, 574 18 of 29
5.2. Targeting BCL2 Pathways in Solid Tumor Therapy
Currently, results from targeting the BCL-2 superfamily in solid tumors, using venetoclax or
navitoclax have been disappointing [
153
]. On a wide study of multiple solid tumor cell lines, MCL-1
mRNA was the anti-apoptotic BCL-2 member with the highest levels in glioma, lung, renal, prostate,
ovarian and breast cancer lines. In comparison, BCL-2 and BFL-1/A1 mRNA levels were highest
in leukemia/lymphoma and melanoma cell lines [
154
]. This may explain why therapeutic success
with venetoclax monotherapy has been thus far limited to hematological malignancies. Strategies
targeting MCL-1 in solid tumors, or combinations including MCL-1 could achieve more success [
154
].
In cervical cancer cell lines, resistance to venetoclax, the BCL-xL selective inhibitor A1331852 or the
MCL-1 inhibitor A-1210477 was noted when these agents were used individually. However, combining
MCL-1 and BCL-xL inhibitors, or MCL-1 and BCL-2 inhibitors led to inhibition of proliferation in the
same cell lines [155].
In other solid tumors, BCL-2 pathway targeting could sensitize to standard therapy, possibly
related to its effects on mitochondrial priming. In hormone receptor (HR)-positive breast cancer
xenografts, the BH3 mimetics venetoclax and ABT-737 potentiated tumor responses to tamoxifen.
Further synergy was seen when the BH3 mimetics were combined with PI3K/mTOR inhibitors, which
are already approved therapy in HR-positive advanced breast cancer, in addition to tamoxifen [
156
].
Currently, a randomized phase II study is comparing fulvestrant versus fulvestrant plus venetoclax in
advanced HR-positive breast cancer (NCT03584009), and a phase Ib study of combination letrozole,
palbociclib and venetoclax in metastatic ER-positive breast cancer is planned (NCT03900884).
BH3 mimetics were also shown to potentiate chemotherapy efficacy in basal-like HR-negative
breast cancer xenografts. Immunocompromised mouse xenografts were treated with either ABT-737,
docetaxel or both [
157
]. As expected, treatment with ABT-737 alone was ineffective, but treatment
with combination therapy led to significant improvements in tumor response and OS in-vivo in breast
cancer xenografts which overexpressed BCL-2. This finding correlated with a marked increase in
apoptosis and BIM-BCL-2 dissociation, and suggests a role for BH3 mimetics to sensitize breast cancers
to docetaxel chemotherapy. These results further corroborate with in-vitro experiments showing
that endogenous BCL-2 phosphorylation occurs with spindle poison treatment which then leads to
increased endogenous BCL-2/BIM binding. The addition of BCL-2 inhibitors was able to disrupt mitotic
BCL-2/BIM binding in-vitro, enhancing paclitaxel cytotoxicity [29].
New strategies are exploiting the signaling pathways that induce dependency on BCL-2-like
proteins [
158
]. Oncogenic addiction of a cell to RAS, HER2 or EGFR inhibits apoptosis by
downregulating BH3-only activator proteins through the MAPK/ERK pathway [
158
], however, this may
also trigger a second oncogenic signal through MYC which promotes BIM expression. Overall, this may
lead to increased BCL-2-like protein dependency and increase sensitivity of oncogene addicted cells to
apoptosis induced by BH3 mimetics. Two studies have reported on the upregulation of BCL-2-like
members in EGFR-TKI-resistant NSCLC which harbor oncogenic EGFR mutations [
159
,
160
]. In one
study, erlotinib-resistant EGFR mutant lung cancer cells showed increased MCL-1 expression, and
were sensitive to EGFR TKIs when combined with navitoclax [161].
In melanoma, low BCL-xL expression was shown to bias the anti-apoptotic pool towards MCL-1.
The combination of MCL-1 inhibition using AZD5991 with MEK1/2 inhibitors (MEKi) was noted to
induce synthetic lethality by BAX/BAK-dependent cell death in-vivo [
162
]. AZD5991 was also shown
to delay the development of acquired BRAFi/MEKi resistance, and enhanced the efficacy of ERKi
in previously-resistant models [
162
]. Similar observations were made in patient-derived xenograft
models of high-grade serous ovarian cancer which were resistant to the MEKi, cobimetinib. Proteomic
interrogation showed that cobimetinib upregulated BIM, increasing mitochondrial ‘priming’, and
sensitized models to synergistic targeting with the dual BCL-2/X
L
inhibitor navitoclax [
163
]. A phase
Ib study combining navitoclax with trametinib in RAS-mutant advanced solid tumors is underway
(Table 1).

Cancers 2020,12, 574 19 of 29
6. Future Directions and Challenges
The recognition of the BCL-2 protein superfamily in regulating intrinsic apoptosis has brought
attention to its targeting in overcoming treatment-related resistance in cancer therapy. Progressive
refinement in the development of selective BCL-2 inhibitors has led to the successful approval
of venetoclax, and significant improvement in clinical outcomes of CLL and AML therapy, while
minimizing off-target toxicities. This success has catalyzed the progressive development of other BH3
mimetics, which is likely to change practice in the coming decade. Thus far, the limited success seen in
other hematological malignancies and solid tumors only serves to underscore the following challenges
we face in harnessing the benefit of BCL-2 inhibitors more broadly.
Firstly, though active development of BCL-xL and MCL-1 inhibitors is ongoing, it is uncertain if
these inhibitors will maintain sufficient safety profile for widespread use [
164
]. Glaringly, no selective
BFL-1/A1 inhibitors have been developed, although the ML214 probe may be useful to evaluate
potential interaction sites for BFL-1/A1 inhibition [
165
]. Successful efforts targeting the pro-apoptotic
family members are also notably missing from this space; however, apoptotic modulators such as
BAM7 which are able to engage the BAX trigger site toward functional oligomerization are under
investigation [166].
The selectivity of venetoclax has undeniable benefit in allowing off-target toxicity to be minimized.
However, this selectivity itself promotes resistance and compensatory upregulation of non-target
anti-apoptotic members such as MCL-1, which may necessitate combination or sequential targeting
approaches. At the juncture, it remains to be seen if multiple BH3 mimetics can be successfully used in
combination due to overlapping toxicity, and clinical trials evaluating the safety of these combinations
are underway.
Thirdly, increasing data is emerging regarding the regulatory role that the mitochondrial membrane
itself exerts on the BCL-2 superfamily. Membrane insertion and BAX oligomerization are the rate
limiting steps for intrinsic apoptosis to proceed. Changes in the mechanical properties of the
mitochondrial membrane may regulate BCL-2 proteins, or the membrane itself may have direct effects
in modulating BCL-2 family member function [
19
]. One study has reported increased resistance for
BAX-BCL-xL complexes when membrane inserted, and it is proposed that the inhibition of BAX
oligomerization by BCL-2 proteins in the context of cellular membranes may be an effective means
to allow the cell to avoid BAX activation [
167
]. Examining BCL-2 family member interactions in the
presence of membranes appears imperative to forward our efforts.
The invention of BH3 profiling technology has made it plausible that a means of examining
functional BCL-2 protein dependency and its dynamism during cancer development and progression
is now available. Its predictive benefit should be consistently evaluated on prospective clinical trials.
Additionally, BH3 profiling was developed in and has immense potential in the current era of BH3
mimetics. However, it is not clear if this technology will also help predict benefit to other anti-BCL-2
therapies such as interference strategies [168].
Finally, the BCL-2 family members have numerous non-canonical functions such as cross-talk
with metabolic pathways, cellular redox status, involvement in ER calcium homeostasis and autophagy.
These pleiotropic effects mean that targeting BCL-2 and BCL-2-like proteins may have a multitude of
effects on cancer cell fate, and these consequences on anti-cancer therapy remain under investigation.
Recent data have also shown that the BCL-2 family members have an unexpected immunological
role, which is provocative for development. In melanoma cells with strong BCL-2 expression, the
addition of ABT-737 to co-culture with expanded peripheral blood cytotoxic T-lymphocytes amplified
tumor cell kill. As postulated, the addition of BCL-2 inhibitors may sensitize the target tumor cells to
perforin/granzyme-B mediated cell kill [
169
]. More recently, Brokatzky and colleagues have described
that activation of BAX/BAK induces mitochondrial DNA release [
170
], which can go on to trigger the
innate immune system through the cGAS-STING signaling pathway. This may have the potential
to increase the immunogenicity of immunologically “cold” tumors. In this way, the mitochondrial

Cancers 2020,12, 574 20 of 29
apoptosis pathway may serve as a modulator of anti-tumor immunity, therefore paving the way for
novel combinations of drugs targeting the BCL-2 family together with immune-checkpoint blockade.
7. Conclusions
Despite the exciting advances made in our understanding of the BCL-2 proteins’ role in controlling
cell fate and treatment resistance, these observations indicate that the benefit of BCL-2 targeting therapy
is not yet fully exploited. The promise of personalized biomarker technology and rational combinations
of BCL-2 inhibitors with other branches of cancer therapy are imminent, and will certainly add to our
therapeutic arsenal to improve outcomes in a wider group of patients.
Author Contributions:
Conceptualization, N.Y.L.N., G.B. and S.P.; data curation, N.N, G.B.; writing—original draft
preparation, N.Y.L.N.; writing—review and editing, N.Y.L.N., C.C., J.L., G.B., A.L.W., B.C.G., S.P.; visualization,
N.Y.L.N., G.B., S.P.; supervision, S.P.; project administration, N.Y.L.N. All authors have read and agreed to the
published version of the manuscript.
Funding:
N.Y.L.N. is supported by the National Medical Research Council, Singapore (MOH-FLWSHP19may-0006),
A.L.W. is supported by the National Medical Research Council, Singapore (NMRC/TA17nov003). B.C.G. is
supported by the National Medical Research Council, Singapore (NMRC/CSA-SI/0006/2016). S.P. is supported by
the National Medical Research Council, Singapore (NMRC CIRG/1433/2015 and OFIRG/0041/2017).
Conflicts of Interest: The authors declare no conflict of interest.
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