Targeting Bcl-2 Proteins in Acute Myeloid Leukemia

Targeting Bcl-2 Proteins in Acute

Myeloid Leukemia

Yunxiong Wei

, Yaqing Cao

, Rui Sun

, Lin Cheng

, Xia Xiong

, Xin Jin

, Xiaoyuan He

Wenyi Lu

and Mingfeng Zhao

The First Central Clinical College of Tianjin Medical University, Tianjin, China,

Nankai University School of Medicine,

Tianjin, China,

Department of Hematology, Tianjin First Central Hospital, Tianjin, China

B cell lymphoma 2 (BCL-2) family proteins play an important role in intrinsic apoptosis.

Overexpression of BCL-2 proteins in acute myeloid leukemia can circumvent resistance to

apoptosis and chemotherapy. Considering this effect, the exploration of anti-apoptotic

BCL-2 inhibitors is considered to have tremendous potential for the discovery of novel

pharmacological modulators in cancer. This review outlines the impact of BCL-2 family

proteins on intrinsic apoptosis and the development of acute myeloid leukemia (AML).

Furthermore, we will also review the new combination therapy with venetocl ax that

overcomes resistance to venetoclax and discuss biomarkers of treatment response

identiﬁed in early-phase clinical trials.

Keywords: B cell lymphoma 2, AML—acute myeloid leukemia, venetoclax (ABT-199), intrinsic apoptosis, Bcl-

2 protein

INTRODUCTION

Acute myeloid leukemia (AML) is one of the most common hematological malignancies in adults,

with a median age at diagnosis of 68 ( 1). For the past 30 years, the main treatment has been

chemotherapy alone or combined with hematopoietic stem cell transplantation (HSCT). In 2017,

there was a br eakthrough, and several new drugs, i ncluding venetoclax and selective B cell

lymphoma-2 (BCL-2) inhibitors, seemed to reshape the therapeutic landscape of AML ( 2). The

BCL-2 family of proteins plays a signiﬁcant role in the intrinsic apoptotic pathway. They are also

critical for cell survival and are overexpressed in many tumors, including AML. In addition,

aberrant overexp ression of BCL-2 family proteins causes resistance to chemotherapy and is

associated with a poor prognosis (3–7). Recently, BCL-2 inhibitors, such as venetoclax and novel

MCL-1 inhibitors, have shown anti-leukemic activity in preclinical AML models. In 2018,

venetoclax in combination with azacitidine, decitabine, or low-dose cytarabine (LDAC) was

approved for use in untreated patients with AML aged 75 years or older or patients with

comorbidities that preclude the use of intensive induction chemotherapy (8). Here, we review

how they regulate apoptosis and how the targeting of BCL-2 has developed in AML. Furthermore,

we summarize the mechanism of resistance to venetoclax and provide some potential solutions.

Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 5849741

Edited by:

Naval Daver,

University of Texas MD Anderson

Cancer Center, United States

Reviewed by:

Luca Maurillo,

University of Rome Tor Vergata, Italy

Michael Diamantidis,

University Hospital of Larissa, Greece

*Correspondence:

Mingfeng Zhao

[email protected]

Specialty section:

This article was submitted to

Hematologic Malignancies,

a section of the journal

Frontiers in Oncology

Received: 19 July 2020

Accepted: 14 September 2020

Published: 05 November 2020

Citation:

Wei Y, Cao Y, Sun R, Cheng L,

Xiong X, Jin X, He X, Lu W and Zhao M

(2020) Targeting Bcl-2 Proteins in

Acute Myeloid Leukemia.

Front. Oncol. 10:584974.

doi: 10.3389/fonc.2020.584974

REVIEW

published: 05 November 2020

doi: 10.3389/fonc.2020.584974

APOPTOSIS AND THE BCL-2 FAMILY

There are two main pathways for apoptosis: the intrinsic pathway and

the extrinsic pathway (9). The extrinsic apoptotic pathway is initiated

by the interaction of an extracellular ligand and tumor necrosis factor

(TNF) family death receptors. Then, procaspase-8 is activated by the

binding of the deat h-inducing signali ng complex (DISC) to a series of

adaptors, and activated caspase-8 further initiates the caspase-3

cascade, which eventually leads to cell death via apoptosis (10, 11)

(Figure 1). The intrinsic pathway is initiated by internal cellular stress,

such as DNA damage, growth factor deprivation, and oxidative stress.

These alterations then lead to mitochondrial depolarization, which

allows the release of cytochrome c, which is the hallmark of the

intrinsic apoptotic pathway. Cytochrome c binds to apoptosis

protease-activating factor 1 (APAF1) and procaspase-9, forming an

intracellular “apoptosome” that can activate caspase-9. Then, active

caspase-9 leads to executione r caspase-3 activation (11)(Figure 1).

The BCL-2 family of proteins regulates the intrinsic apoptotic

pathway by controlling mitochondrial outer membrane

permeabilization (MOMP) (12).

FIGURE 1 | The extrinsic and intrinsic pathways to apoptosis. The extrinsic pathway of apoptosis is activated when certain death receptor ligands of the tumour

necrosis factor (TNF) family (such as TNF) engage their cognate death receptors on the plasma membrane, leading to caspase-8 activation via the death-inducing

signalling complex (DISC), which results in apoptosis. The intrinsic pathway of apoptosis is activated by cellular stresses (such as DNA damage, growth factor

deprivation or oxidative stress) and is regulated by BCL-2 family proteins. BH3 proteins bind to and activate pro-apoptotic proteins BAX, BAK and possibly BOK,

which oligomerize in the mitochondrial membranes and release cytochrome c, then interact with apoptosis protease-activating factor 1(APAF1) and initiate caspase

activation that results in apoptosis. Futhermore, BH3-only activator proteins (BID, BIM, Puma and NOXA) directly bind to interact with BAX and BAK to promote

mitochondrial outer membrane permeabilization (MOMP). While the BH3-only sensitizer proteins (BAD, BIK, HRK, BMF, PUMA, NOXA) bind to anti-apoptotic

proteins with higher afﬁnity, freeing the activator proteins from the BH3 binding pockets in the anti-apoptotic proteins and executing cell death by binding to BAX or

BAK. The two pathways converge at activation of the effector caspase-3.

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Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 5849742

The BCL-2 gene, whose transcription can be upregulated, was

ﬁrst discovered as a part of t(14;18) chromosomal translocation

in follicular lymphoma and diffuse large B cell lymphoma (13,

14). It was ﬁrst recognized as a classical growth-driving

oncogene, but later proved that BCL-2 promotes malignant cell

survival by attenuating apoptosis (15, 16). The BCL-2 family

consists of more than 20 proteins, which may be functionally

classiﬁed as either anti-apoptotic or pro-apoptotic proteins (17).

The anti-apoptotic BCL-2 proteins, which include four BCL-2

homology domains (BH1–4), include BCL-2, BCL-XL (BCL-2-

like protein 1), MCL-1 (induced myeloid leukemia cell

differentiation protein MCL-1), BCL-W (Bcl-2-like protein 2),

BFL-1/A1 (BCL-2-related protein A1), and possibly BCL-B (Bcl-

2-like protein 10). The pro-apoptotic proteins can be divided

into effector proteins and BH3-only proteins. The former group

contains three BH domains and includes BAX (BCL-2 associated

X protein), BAK (BCL-2 antagonist killer), and possibly BOK

(BCL-2-related ovarian killer). The latter group possesses only a

single BH3 domain and includes BID (BH3-interacting domain

death antagonist), BIK (BCL2 interacting killer), BIM (BCL-

22L11; BCL2-interacting mediator of cell death), BAD (BCL2

antagonist of cell death), BMF (BCL-2 modifying factor), HRK

(activator of apoptosis hara-kiri), PUMA (p53 upregulated

modulator of apoptosis; also called BBC3 [BCL2 binding

component 3]), and NOXA (PMA induced protein 1; also

called PMAIP1 [phorbol-12-myristate-13-acetate-induced

protein 1]) (11, 17, 18). Once BAX and BAK are activated,

they oligomerize and form pores to induce MOMP, releasing

cytochrome c from mito chondria and ﬁnally inducing cell

apoptosis (19). The anti-apoptotic BCL-2 proteins can protect

cells from apoptosis by directly binding to BAX/BAK or

antag onizing pro-apoptotic BH3-only proteins. Furthermore,

the BH3-only proteins can be subdivided into activator and

sensitizer proteins. The activator proteins, which include BID,

BIM, Puma, and NOXA, directly bind to and interact with BAX

and BAK to promote MOMP. The sensitizer proteins bind to

anti-apoptotic proteins with higher afﬁnity, freeing the activator

proteins from the BH3 binding pockets in the anti-apoptotic

proteins and executing cell death by binding to BAX or BAK (20,

21)(Figure 1).

Owing to subtle differences in their BH3 domains and in the

grooves of the anti-apoptotic proteins, the various BCL-2 family

proteins have differential speciﬁcity of binding to one another.

For example, BAX and BAK have high afﬁnities for BCL-XL, and

BAK has higher afﬁnities for MCL-1, as do BAX and BCL-2 (22).

Interestingly, some BH3-only proteins, such as BAD and NOXA,

are selective for subsets of their anti-apoptotic relatives, whereas

other BH3-only proteins, particularly BIM, BID, and PUMA,

probably neutralize all of the anti-apoptotic proteins (23)

(Figure 2). In healthy cells, anti-apoptotic proteins and pro-

apoptotic proteins maintain a delicate balance, while cancer cells

overexpress different anti-apoptotic proteins to facilitates

prolonged tumor cell survival (24). A good knowledge of the

roles of BCL-2 family proteins in promoting tumorigenesis

has contributed to the development of numerous novel drugs

targeting aberrant apoptotic pathways in cancer. Thus, targeting

BCL-2 family proteins may be a prominent strategy for

cancer therapy.

BCL-2 IN AML

A number of early studies showed that BCL-2 was overexpressed in

CD34+ AML cells and was associated with poor prognosis and

resistance to chemotherapy (25–27). Overexpression of MCL-1 and

BCL-XL also confers chemotherapy resistance in AML (4, 28, 29).

Thus, antisense oligonucleotides targeting BCL-2 were explored and

decreased the number of leukemia cells in vitro (30). A better

understanding of the intrinsic apoptosis pathway has contributed to

the focus on the development of novel small molecules that mimic

the BH3 domain found in all pro-apoptotic BCL-2 family proteins.

These drugs mimic the action of certain pro-apoptotic BH3-only

proteins by binding directly to the BH3-binding domains of anti-

apoptotic molecules, thereby displacing native BH3-only proteins

and thus inducing apoptosis (Figure 1). These protein-protein

interaction inhibitors that directly activate apoptosis represented a

milestone event in medicinal chemistry, as well as in the cell death

ﬁeld. With the inspired success in B cell lymphoid malignancies,

these new BH3 mimetics have also been used in AML.

EARLY ATTEMPTS TO TARGET

BCL-2 IN AML

Oblimersen

Oblimersen, which can target the ﬁrst six codons of the human

BCL-2 mRNA, is an 18-mer phosphorothioate Bcl-2 antisense

oligonucleotide. It can increase cancer cell apoptosis and

overcome chemotherapy resistance by binding to BCL-2

mRNA, thus downregulating BCL-2 protein expression (30,

31). The ﬁrst use in a clinical trial of AML was a phase I study

in relapsed or refractory acute leukemia in which patients

received a combi nation with ﬂudarabine, cytarabine, and

granulocyte colony-stimulating factor (FLAG) salvage

chemotherapy ( 32). Seventeen patients with relapsed and

refractory AML and 3 patients with acute lymphoblastic

leukemia (ALL) were recruited. Five of 17 (29%) achieved

complete response (CR), and 2 patients achieved complete

response with incomplete blood count recovery (CRi) (11%),

yielding an objective response of 41%. Furthermore, no dose-

limiting toxicity was observed in any of the cohorts examined.

Another phase 1 trial conducted by the same group combined

two different doses of oblimersen with chemothe rapy in

untreated older patients with AML (33). In this trial, 14 of 29

(48%) patients achieved CR, w ith no signi ﬁ cant increase

compared with the previously reported remission rates of 40%

in the absence of oblimersen. The side effects were similar to

those expected with chemotherapy alone and were not dose

limiting at either dose level, which proved that the drug was safe

and tolerable. The preclinical and early clinical data led the

Cancer and Leukemia Group B (CALGB) to conduct a large

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Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 5849743

phase III trial in which older adults with newly diagnosed AML

were randomized to standard induction chemotherapy with or

without oblimersen (34). Regrettably, there were no differences

observed in CR rates (48% vs 52%; p=0.75) or overall survival

rates (OS at 1 year 36% vs 40%; p=0.83), which may lead to no

further trials of this drug in AML. However, the failure of this

trial did not dispel the exploration of BCL-2 inhibitors in AML.

Obatoclax (GX15-070)

Obat oclax is a pan-Bcl-2 anta gonist and was the ﬁrst BH3

mimetic used in clinical trials of AML. It can bind to the BH3

domain of BCL-2 (as well as those of BCL-XL, MCL-1, BCL-w,

A1, and BCL-b) (35) and then prevent the anti-apoptotic

proteins from sequestering pro-apoptotic BH3-only proteins.

Obatoclax potently induced apoptosis and decreased leukemia

cell proliferation in AML cell lines and primary AML samples

(36). In a phase I trial of 44 patients with advanced hematological

malignancies, which included 25 AML, 14 myelodysplasia

(MDS), 4 chronic lymphocytic leukemia (CLL), and 1 ALL,

patients showed good tolerance but modest efﬁcacy in the

clinic (37). Only one patient with AML with mixed lineage

leukemia t(9;11) rearrangement achieved complete remission.

In addition, obato clax had neurological side effects, which

further limited its clinical development. In addition, another

phase I/II study of obatoclax in older patients with previously

untreated AML showed similar results in that obatoclax did not

FIGURE 2 | The selectivity of BCL-2 family proteins. (A) BAK is inhibited predominantly by BCL-XL, MCL-1 and BFL-1, while BCL-2 contributes in some situations.

However, BAX is probably inhibited by all of the pro-survival proteins. (B) Some BH3-only proteins, such as BAD and NOXA, are selective for subsets of their anti-

apoptotic relatives, whereas other BH3-only proteins, particularly BIM, BID and PUMA, probably neutralize all of the anti-apoptotic proteins.

Wei et al. Targeting Bcl-2 Proteins in AML

Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 5849744

seem to be associated with an objective response (38). The failure

of this drug may encourage a future study on its combination

with other new drugs, such as histone deacetylase inhibitors or

sorafenib (39, 40). Furthermore, the insolubility of this

compound also limits its function, so new BH3 mimetics

should be explored.

DUAL BCL-2/BCL-XL INHIBITORS

ABT-737

ABT-737 is a BH3 mimetic with high potency activity against

BCL-2, BCL-XL, BCL-W, and, to a lesser extent, MCL-1. It was

discovered as the ﬁrst high-afﬁnity inhibitor of BCL-2 family

protein s by using nuclear magnetic resonance (NMR)-based

screening (41). In a preclinical study, ABT-737, which can

effectively trigger Bax/Bak-mediated apoptosis, induced AML

cell apoptosis in vitro, and the same activity was demonstrated in

a murine xenograft model in vivo (42–44). High expression of

MCL-1 and phosphorylation of BCL-2 are associated with

resistance to ABT-737, as they lead to a reduction in MCL-1.

Thus, targeting these changes may be a potential way to enhance

ATB-737 response. For example, ABT-737 could be combined

with pan-Bcl-2 famil y inhibitors, MEK inhibitors, or PI3K/

mTOR inhibitors (45–47). However, owing to its poor oral

bioavailability and solubility in water, further studies on this

drug are likely to be limited.

Navitoclax (ABT-263)

Navitoclax, which is a derivative of ABT-737, is an improved

orally bioavailable BH3 mimetic with high afﬁnity for BCL-2,

BCL-XL, and BCL-w and substantially lower afﬁnity for MCL-1

than ABT-737 (48). Navitoclax also showed efﬁcacy in AML

preclinical studies (49–51). In early clinical trials, it has shown

activity in CLL and small-cell lung cancer (52, 53). However, it

can cause thrombocytopenia due to its effect on BCL-XL (54–56).

This on-target toxicity of navitoclax has undoubtedly limited its

further development, which prompted AbbVie’s development of

venetoclax (ABT-199).

SELECTIVE BCL-2 INHIBITORS

Venetoclax

Venetoclax is a highly selective small molecule BH3 mimetic

with subn anomolar afﬁnity (Ki<0.01 nM) for BCL-2 and

nanomolar afﬁnity (Ki<245 nM) for BCL-XL. Based on this

feature, it circumvents signiﬁ cant thrombocytopenia due to

concomitant inhibition of BCL-XL, making it clinically

available for the treatment of AML, a disease typically

associated with thrombocytopenia (57, 58). In preclinical

studies, venetoclax induced rapid cell death in AML cell lines

and primary patient samples in vitro and in a mouse xenograft

model in vivo (58). In the study of this drug, mitochondrial BH3

proﬁling could b e used as a biomarker for predicting the

response of AML primary cells to venetoclax in vitro and in a

patient-derived xenograft model in vivo.Consideringthe

preclinical data for ABT-767 and navitoclax and the efﬁciency

and safety of venetoclax in CLL, venetoclax was immediately

moved into clinical trials.

SELECTIVE BCL-XL INHIBITORS

To date, many studies on BCL-XL inhibitors have focused on

solid tumors (such as breast cancer, non-SCLC, ovarian

cancer, colorectal cancer, and multiple myeloma) because

solid tumors rely on BCL-XL for their survival. Selective

BCL-XL inhibitors include WEHI-539, A-1155463, and

A-1331852 (59–63), and further study may focus on the use

of these inhibitors in AML.

SELECTIVE MCL-1 INHIBITORS

MCL-1 has been shown to play a signiﬁcant role in promoting

cell survival in AML cell lines ( 64), and its overexpression in

tumor cells may be associated with resistance to radiotherapy,

chemotherapy, and BH3-mimetics targeting BCL-2/BCL-XL

(65–67). Overexpression of MCL-1 has been identiﬁed in many

primary AML cells and as a major factor in the development of

resistance to venetoclax (68, 69). Thus, it led to the development

of MCL-1 inhibitors. There are a number of MCL-1 inhibitors,

and we reviewed several MCL-1 inhibitors that have entered

clinical trials.

To date, S63845 has been identiﬁed as the most promising

selective MCL-1 inhibitor. It showed low nanomolar cytotoxic

activity in multiple hematological cancer-derived cell lines in

vitro and potent efﬁcacy in vivo in preclinical mouse models of

diverse hematological malignancies (70). A side-by-side

comparison of BH3 mimetics showed that MCL-1 may be a

more potent therapeutic target than BCL-2 in AML (71).

Therefore, the utilization of S63845 should be the ﬁrst line of

choice in future clinical trials.

Very little information on S64315 (MIK665) has been disclosed,

with two clinical trials under investigation in patients with AML or

myelodysplastic syndrome (NCT02979366, NCT03672695) (72).

AZD5991, which has high selectivity and af ﬁnity for MCL-1, has

been shown to cause an effective apoptotic response in AML cell

lines at a low nanomolar range. Furthermore, AZD5991 binds

directly to the MCL-1 and BAK interaction and was shown to have

potent antitumor activity in vivo,asdemonstratedbyhightumor

regression in an AML xenograft model. Based on these promising

data, a phase I clinical trial is undergoing relapsed or refractory

hematological malignancies (NCT03218683) (73, 74).

AMG176 is a ﬁrst-in-class selective MCL-1 inhibitor that is

being studied in humans. It targets the BH3-binding groove of

MCL-1 and thus frees BAX, resulting in activation of the intrinsic

apoptotic pathway. In preclinical studies, it showed potent effects on

AML cell lines, xenograft models, and primary patient samples.

Based on this background, AMG176 is under several clinical

investigations for AML (NTC02675452, NCT03797261) (75, 76).

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CLINICAL USES OF VENETOCLAX

Single-Agent Venetoclax in Patients

With Relapsed/Refractory AML

The ﬁrst clinical trial of venetoclax in AML was a phase 2 single-

agent study in patients with relapsed/refractory disease or

untreated patients ineligible for intensive chemotherapy (77).

In this study, they recruited 32 patients, with a median age of 71

years, which consisted of 30 relapsed/refractory patients and 2

treatment-naïvepatientsthatwereineligibleforintensive

chemotherapy. This study showed a modest overall response

rate of 19% (6/32), with 6% of patients (2/32) achieving CR and

13% of patients (4/32) achieving CRi; another 19% of patients

had anti-leukemic activity that did not meet the criteria for

response. The median duration of remission was only 48 days,

and the median time spent on the study was only 63.5 days. Most

patients had high-risk features, including pre-existing

myelodysplastic syndrome or m yeloproliferative neoplasm

(MDS or MPN, 41%), FLT3-ITD mutations (13%), and older

age (median 71 years).

Interestingly, patients with IDH1/2 mutations had a

stronger response, with 4 of 12 (33%) patients achieving

CR/CRi. Moreover, another 2 patients with IDH mutations

demonstrated anti-leukemic activity that did not meet formal

criteria for response due to a lack of hematological recovery.

This is consistent with preclinical studies, in which IDH1/2

mutant AML suppress es the activity of cytochrome c oxidase

and lowers the mitochondrial threshold to trigger apoptosis

upon BCL-2 inhibition ( 78). Another impressive discovery was

that all 6 patients who achieved CR/CRi r eceived prior

hypomethylating agents, which may lead to the exploration of

its combination with hypomethylating agents. The most

common grade 3/4 adverse events (AEs) were febrile

neutropenia (31%), hypokalemia (22%), and pneumonia

(19%), and no tumor lysis syndrome (TLS) events were

reported. Overall, this study showed that single-agent

venetoclax demonstrated antitumor activity in AML with

good tolerability.

Venetoclax-Based Combinations in

Treatment-Naïve Patients With AML

Preclinical models have proven a synergistic effect upon

combining venetoclax and hypomethylating agents (HMAs) in

AML cell lines and primary AML patient samples (79, 80).

Moreover, studies have shown that azacitidine can reduce the

protein levels of MCL-1, an anti-apoptotic pro tein t hat is

associated with potential mechanism of resistance to

venetoclax (81, 82). Based on these data, the combination of

venetoclax with HMAs for the treatment of AML may be a

promising therapeutic approach.

AbbVie launched a phase 1b clinical study (NCT02203773)

that recruited 212 patients over the age of 60 years with

treatment-naïve AML. They conducted a dose escalation and

expansion study of venetoclax (83 , 84). At ﬁrst, a total of 45

patients were enrolled in the dose escalation, a dose of 400 mg of

venetoclax was administered to 10 patients (4 in the azacitidine

group, and 6 in the decitabine group), a dose of 800 mg of

venetoclax was administered to 24 patients (12 each in the

azacitidine and decitabine groups), and a dose of 1200 mg of

venetoclax was administered to 11 patients (6 in the azacitidine

group, and 5 in the decitabine group). Although the maximum

tolerated dose was not reached in any group, the 1200 mg dose

led to a high frequency of gastrointestinal AEs (nausea in 82%

and diarrhea in 64% of patients) (83). As a result, the 400 mg and

800 mg dose cohorts for both azacitidine and decitabine were

expanded, and 50 patients were added to each group. This study

demonstrated a remarkable overall response rate of 68% and

included CR and CRi rates of 37% and 30%, respectively. The

median follow-up was 15.1 months, and the median OS for all

groups was 17.5 months. In addition, the patients who received

400 mg venetoclax plus HMA achieved a remarkable CR/CRi

rate of 73% (76% for azacitidine and 74% for decitabine), which

revealed that the combinations with azacitidine or decitabine

were not signiﬁcantly different and that 400 mg of venetoclax

might be a favorable dose (84, 85). Furthermore, patients with

IDH1/2 mutations, FLT3 mutations, or NPM1 mutations had

CR/CRi rates of 71%, 72%, and 91.5%, respectively. Even patients

with poor risk features showed impressive responses, including

those with high-risk cytogenetics (60% CR/CRi) and TP53

mutant patients (47% CR/CRi). The most common grade 3/4

adverse effects for all groups were thrombocytopenia (47%),

febrile neutropenia (42%), and neutropenia (40%). No

laboratory or clinical TLS was observed. Further studies

explained how this treatment executed anti-leukemia activity.

Leukemic stem cells (LSCs) rely on amino acid metabolism for

oxidative phosphorylation and survival. Venetoclax with

azacitidine was able to induce LSC toxicity in vitro by

decreasing amino acid uptake, as conﬁrmed by decreased a-

ketoglutarate and increased succinate levels, suggesting

inhibition of electron transport chain complex II. These

metabolic perturbation s suppress oxidative phosphorylation ,

which in turn efﬁciently and selectively targets LSCs (86, 87).

Similarly, preclinical studies demonstrated that cytarabine

can boost venetoclax activity i n AML by reducing MCL-1

levels (88, 89). An open-label, multicenter phase trial phase

1b/2 study of venetoclax in combination with LDAC recruited

82 patients over the age of 60 years (median age 74 years) with

treatment-naïve AML (90). In the dose escalation phase, most

patients needed dose interruption to permit blood count

recovery and had a higher rate of hematological toxicity.

Therefore, a 600 mg dose was recommended for the phase 2

study of the trial. The CR/CRi rate was 54% (CR, 26%; CRi, 28%).

The median OS was 10.1 months, and the median duration of

response was 8.1 months. Fifty-eight patients were treatment

naïve; the CR/CRi rate for this group was 62%, which is similar to

the 67% observed with venetoclax+ HMAs, whereas the group of

patients who had prior HMA exposure only obtained a CR/CRi

rate of 33%. Patients with mutations in NPM1 or IDH1/2

mutations had CR/CRi rat es of 89% and 72%, resp ectiv ely,

which are higher than the average CR/CRi rate. However,

patients with TP53 or FLT3 mutations had worse CR/CRi rates

(30% and 44%, respectively). The most common grade 3/4 AEs

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were febrile neutropenia (42%), thrombocytopenia (38%), and

neutropenia (27%). There were 2 cases of laboratory TLS and no

evidence of clinical TLS.

In addition, a “real-world” report of venetoclax combined with

azacitidine for the same patient populations at the same institution

has been published (91). Thirty-three patients who received

venetocl ax + azacitidi ne off-trial were retrospec tively analyze d and

compared with 33 patients who received the same therapy on trial.

The CR/CRi rate was 63.3% for off-trial patients who received

treatment and 84.9% for trial patients, with a median OS periods of

381 days and 880 days, respectively. Although the responses and

survival with venetoclax in real-world AML were inferior to those

treated in a clinical trial, it still remained effective and tolerable

compared with induction chemoth erapy.

In summary, venetoclax in combination with HMAs or

LDAC seemed to achieve a positive outcome in similar patient

populations. On the basis of the above-described clinical data,

two phase 3 trials comparing venetoclax + azacitidine/LDAC

with azacitidine/LDAC alone are ongoing (NCT02993523,

NCT03069 352). The promising responses ob serv ed in t hese

trials led to the FDA approval of venetoclax in combination

with HMAs or LDAC for treatment-naïve AML patients who

were at least 75 years old or ineligible for intensive induction

chemotherapy because of comorbidities.

Venetoclax-Based Combinations in

Relapsed/Refractory Patients With AML

Less than 10% of patients with relapsed/refractory AML are

cured with current standard chemotherapy. Allogeneic HSCT is

the only realistic hope of a cure for these patients, but only a

minority of patients consider this option (92). There is an urgent

need to explore new strategies for these patients. Several

retrospective studies of relapsed or refractory patients receiving

venetoclax-based therapies have been reported. A single

institution retrospectively analyzed 33 consecutive adults with

relapsed/refractory AML in the real world, and the combination

with HMAs achieved a CR/CRi rate of 33%, which included

patients with no prior HMAs or allogeneic stem cell transplants

(93). How ever, another series of 39 patients with relapsed/

refractory AML achieved a dismal CR/CRi rate of 12% (8/39)

(94). Moreover, in a series of 90 patients analyzed after either

HMA treatment (51%) or allogeneic stem cell transplant (29%),

the combination of venetoclax and HMAs achieved a CR/CRi

rate of 46% in patients at a younger median age than the former

study (95). Hence, the data revealed that venetoclax-based

combinations may be a potential treatment strategy for

patients with relapsed/refractory AML.

VENETOCLAX RESISTANCE AND

COMBINATION STRATEGIES TO

OVERCOME

Although numerous reports have shown that venetoclax-based

therapies exert a promising effect on AML, they still remain

partly resistant to venetoclax. It is necessary to explore how

resistance evolves and ho w this can b e overc ome. The

upregulation of MCL-1 protein in AML cells is one of the

most well-known reasons for res istance to treatment with

venetoclax (69). Therefore, targeting MCL-1 directly or

indirectly is a new way to solve the resistanc e. Moreover,

mitochondrial cristae structure may be another reason for

resistance. Targeting mitochondrial architecture may provide a

promising approach to circumvent it (96). Directly targeting

MCL-1 has been introduced previously.

Here, we introduce several combinations that can reduce the

MCL-1 level indirectly to circumvent the resistance. Indirect

MCL-1 inhibitors include the following compounds:

bromodomain extra-terminal protein inhibitors (BETis), which

reduce MCL-1 and BCL-XL levels while increasing BIM levels

and enhance the lethal effects of venetoclax on AML (97); cyclin-

dependen t kin ase 9 (CDK 9) i nhibitors, which inhibi t the

transcription of MCL-1 (98); midostaurin or gilteritinib, FLT3

inhibitors that induce downregulation of MCL-1 to increase

venetoclax activity (99); CUDC-907, a dual PI3K and histone

deacetylase inhibitor that downregulates MCL-1, upreg ulates

BIM, and induces DNA damage (100); MEK inhibitors (101);

MDM2 inhibitors (102); PI3K inhibitors (103); and selinexor, an

XPO1-selective inhibitor (104). In addition, an inhibitor of the

Nedd8-activating enzyme (MLN4924) can upregulate Noxa, and

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR)

inhibitors (statins) can upregulate PUMA. These two pro-

apoptotic proteins, NOXA and PUMA, can neutralize Mcl-1 to

boost the activity of venetoclax (105, 106). Ibrutinib, a Burton’s

tyrosine kinase inhibitor, and ArQule 531, a multi-kinase

inhibitor of Src family kinases and Burton’s tyrosine kinase,

can also synergize with venetoclax (107, 108). Pharmacologic

inhibition of mitochondrial protein synthesis with antibiotics

that target the ribosome, including tedizolid and doxycycline,

can potently reverse venetoclax resistance. Thus, inhibition of

mitochondrial translation may be a new approach to overcoming

venetoclax resistance (109). Owing to these preclinical results, an

increasing number of clinical trials are ongoing.

PREDICTORS OF RESPONSE TO

VENETOCLAX

The speciﬁc population that is most suitable for venetoclax

remains unknown. However, recent studies have shown that

mutations in NPM1, RAD21, MLL, or IDH1/IDH2 may predict

venetoclax sensitivity (110, 111). In addition, a FLT3 internal

tandem duplication gain or TP53 loss confers cross-resistance to

both venetoclax and c ytotoxicity-based therapies (112).

According to a retrospective analysis, HMA plus venetoclax

produces better results than traditional standard-of-care

regimens in older patients with NPM1+ AML (113). To date,

an increasing number of studies have shown that tumor cells

with an NPM1 or IDH1/IDH2 mutation may more sensitive to

venetoclax. Regarding other mutation s, including FLT3 and

TP53, VEN-based therapy may be a more e ffective therapy

than traditional chemotherapy. Furthermore, blast cells of FAB

Wei et al. Targeting Bcl-2 Proteins in AML

Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 5849747

M0/1 AML show higher sensitivity to venetoclax, while

differentiated monocytic cells abundantly present in M4/5

subtypes show resistance to BCL-2 inhibition (114, 115). This

may be associated with decreased expression of BCL-2 and a

reliance on MCL-1 to mediate oxidative phosphorylation and

survival. Therefore, as the number of patients treated with

venetoclax increases, precise predic tors of the respon se will

be revealed.

CONCLUSIONS AND PROSPECTS

Targeting the BCL-2 protein has shown compelling clinical promise

in AML. Venetoclax, a selective BCL-2 inhibitor that was approved

by the FDA for treatment-naïve elderly AML patients, has shown

potential to be further explored. According to a recent study,

previously untreated patients aged 75 years or older who were

ineligible for intensive chemotherapy experienced a longer overall

survival and a higher incidence of remission after treatment with

azacitidine plus venetoclax than patients who received azacitidine

alone (116). In addition, venetoclax+HMA has also been conﬁrmed

to be safe and ef fective in younger patients (117). Based on the

encouraging results achieved with venetoclax, the scope of

application of venetoclax is expected to be expanded in the near

future for patients who are 75 years of age or older who are not

suitable for standard chemotherapy, and potentially even for

younger patients. In our center, we administer this treatment to

elderly patients and even considered administering it to some young

patients who are sensitive to venetoclax in a previous study. Of

course, more standardized and larger clinical trials are needed to

conﬁrm the safety and ef fectiveness of VEN in younger patients.

Chimeric antigen receptor T cells, which have shown exciting

results in B cell lymphocytic leukemia, are another a novel

strategy for AML treatment. What outcomes would occur if

this or other immune therapies was combined with venetoclax?

Moreover, venetoclax-based therapies have shown effects on

some high-risk AML subtypes. Could these therapies be

combined with allogeneic HSCT or be used as a consolidation

therapy after HSCT? Will these therapies affect graft versus-host

disease or graft anti-leukemia? All of these questions remain

unanswered. Furthermore, preclinical studies with MCL-1

inhibitors have demonstrated effective antitumor activity in

AML. Will MCL-1 inhibitors be another promising strategy to

cure AML? In summary, continued efforts to explore BCL-2

family proteins are necessary.

AUTHOR CONTRIBUTIONS

MZ designed the research. YW, YC, RS, LC, XX, XJ, XH and WL

performed the research and analyzed the data. YW wrote the

manuscript. All authors contributed to the article and approved

the submitted version.

FUNDING

This work was supported by grants from the National Natural

Sciences Foundation of China (81970180; to MZ), the National

Natural Sciences Foundation of China (81800105; to WL), and

Tianjin First Central Hospital.

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Conﬂict of Interest: The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be construed as a

potential conﬂict of interest.

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Wei et al. Targeting Bcl-2 Proteins in AML

Frontiers in Oncology | www.frontiersin.org November 2020 | Volume 10 | Article 58497411