Volume 29 Number xxx Month 2022 pp.

100798 1

Mcl-1 levels critically impact the Weilong Yao a,d ; Longchuan Bai b ; Shaomeng Wang b ;
Yifan Zha c ; Shi-Yong Sun d,∗
sensitivities of human colorectal
cancer cells to APG-1252-M1, a a
Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, Beijing,
PR China

novel Bcl-2/Bcl-XL dual inhibitor that b

c
Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
Ascentage Pharma (Suzhou) Co., Ltd, Suzhou, Jiangsu, PR China
d
induces Bax-dependent apoptosis Department of Hematology and Medical Oncology, Emory University School of Medicine and
Winship Cancer Institute, 1365-C Clifton Road, C3088, Atlanta, GA 30322, USA

Abstract

New treatment options, such as targeted therapies, are urgently needed for the treatment of colorectal cancer (CRC), the third leading
cause of cancer-related deaths worldwide. The current study focuses on demonstrating the therapeutic efficacies of APG-1252-M1
(an active form of the prodrug, APG-1252 or pelcitoclax), a highly potent Bcl-2/Bcl-XL dual inhibitor in clinical trials, against CRC
and understanding the underlying mechanisms. APG-1252-M1 effectively decreased the survival of CRC cell lines, particularly those
expressing relatively low levels of Mcl-1, with the induction of apoptosis. High levels of Mcl-1 were significantly correlated with
decreased sensitivity of CRC cell lines to APG-1252-M1. When combined with an Mcl-1 inhibitor, APG-1252-M1 synergistically
decreased the survival and induced apoptosis of APG-1252-M1-insensitive cell lines with high levels of Mcl-1. This combination
further decreased the survival and enhanced apoptosis even in sensitive cell lines with relatively low levels of Mcl-1, whereas enforced
expression of ectopic Mcl-1 in these cells abrogated APG-1252-M1’s effects on decreasing cell survival and inducing apoptosis, which
could be reversed by Mcl-1 inhibition. APG-1252-M1 rapidly induced cytochrome C and Smac release from mitochondria with
caspase-3 and PARP cleavage. Deficiency of Bax in CRC cells abolished APG-1252-M1’s ability to induce apoptosis, indicating
that APG-1252-M1 induces Bax-dependent apoptosis. The current study thus demonstrates the potential of APG-1252-M1 as a
monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with an Mcl-1 inhibitor,
warranting further evaluation in vivo and in the clinic.

Neoplasia (2022) 29, 100798

Keywords: Bcl-2, Bcl-XL , APG-1252-M1 (APG-1252), Mcl-1 apoptosis, Colorectal cancer

Introduction options, such as targeted therapies, are urgently needed. Fortunately, advances
in understanding the biology of the disease and in the development of cost-
In the United States, colorectal cancer (CRC) is the third most common effective technologies that can precisely determine the molecular profiling of
cancer diagnosed and the third leading cause of cancer-related deaths in the disease will allow us to develop various targeted therapies against CRC,
men and women, with about 52,980 deaths expected during 2021 [1]. For particularly metastatic CRC [3], some of which have been used to treat CRC
the past 2 decades, fluoropyrimidine-based chemotherapy has remained the in the clinic [2,4,5].
foundation of treatment in the metastatic setting [2]. Thus, new treatment Evasion of apoptosis, a form of programmed cell death that is essential
for tissue homeostasis, is known to be one of the major hallmarks of cancer.
Apoptosis is a particularly important process in the colon where it participates
Abbreviations: CRC, colorectal cancer; Cyt C, cytochrome C; KO, Knockout; SRB,
sulforhodamine B; CI, combination index. in intestinal turnover. Compromised apoptosis facilitates transformation,
∗
Corresponding author. tumor progression and therapeutic resistance [4,6]. Bcl-2 family proteins are
E-mail address: [email protected] (S.-Y. Sun). known for their key roles in regulation of the intrinsic apoptotic pathway.
Received 28 January 2022; received in revised form 8 March 2022; accepted 8 April 2022 Elevation of anti-apoptotic proteins such as Bcl-2 is commonly associated
with various cancers including CRC. Thus, targeting Bcl-2 family proteins
© 2022 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license
has emerged as a novel and promising cancer therapeutic strategy. As a
(http://creativecommons.org/licenses/by-nc-nd/4.0/) result, small molecule inhibitors named ‘BH3-mimetics’ (Bcl-2 homology
https://doi.org/10.1016/j.neo.2022.100798 3 mimetics) have been developed and some of these, such as ABT199 and
2 Mcl-1 negatively impacts APG-1252-M1 efficacy W. Yao et al. Neoplasia Vol. 29, No. xxx 2022

ABT263, are approved or under clinical investigation [4,7]. The Bcl-2 family system as described in our previous study [17]. HCT116/Bax-KO,
of proteins has been implicated in CRC initiation, progression and resistance HCT116/PUMA-KO, HCT116/Smac-KO, and HCT116/FADD-KO cell
to therapy and thus should be a valid target for the treatment of CRC as well lines were kindly provided by Drs. J. Yu and L. Zhang (University of
[4]. Pittsburgh, Pittsburgh, PA). HCT116/Bim-KO cell lines were established as
APG-1252-M1 (or APG-1244) is the in vivo active metabolite of APG- described previously [18]. These cell lines were not authenticated. The cell
1252 (pelcitoclax) developed by Ascentage Pharma and functions as a dual lines were cultured in RPMI 1640 or DMEM medium containing 5% fetal
inhibitor of Bcl-2 and Bcl-XL . In preclinical studies, it had remarkable single- bovine serum at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
agent antitumor effects in acute myeloid leukemia, small cell lung cancer and
gastric cancer [8–10] and in overcoming intrinsic and acquired resistance Cell survival and apoptosis assays
to ABT-199 in multiple myeloma cells [11]. It showed enhanced activity
against the growth of gastric cancer xenografts when combined with the Cells in 96-well plates were treated with the tested agents for 3 days.
chemotherapeutic agent 5-FU [10], and against nasopharyngeal carcinoma Cell numbers were then estimated with sulforhodamine B (SRB) assay as
when combined with gemcitabine [12]. Clinical trials are currently underway described previously [19] or by CellTiter-Glo Luminescent Cell Viability
to test the activity of APG-1252 as a single agent or in combination with Assay (Promega; Madison, WI). The combination index (CI) for drug
chemotherapeutic drugs (NCT03387332, NCT04354727, NCT04001777, interaction was calculated using CompuSyn software (ComboSyn, Inc;
NCT04210037, and NCT03080311). A serious concern of the Bcl-2/Bcl- Paramus, NJ). Apoptosis was detected with flow cytometry-based apoptotic
XL inhibitors is the platelet toxicity that causes thrombocytopenia in patients assay using an annexin V/7-AAD kit purchased from BD Biosciences (San
[13,14]. APG-1252 was synthesized based on the strategy of pro-drug design. Jose, CA) according to the manufacturer’s protocol. Protein cleavage was
As so, it has limited cell permeability during circulation when given through detected using Western blotting as an additional indication of apoptosis.
iv route and will be converted to the more active metabolite, APG-1252-
M1, once in tumors/tissues. This feature makes it a unique Bcl-2/Bcl-XL Western blot analysis
inhibitor with a favorable and manageable platelet toxicity profile in patients
as demonstrated in clinical trials [15,16]. Whole-cell protein lysates were prepared and Western blotting was
The current study focused on determining its activity against CRC conducted as previously described [20].
cells, revealing the factors that determine cell sensitivity to APG-1252-M1
and understanding the underlying molecular mechanisms. Consequently, Gene knockdown
we have demonstrated the potential of APG-1252-M1 as a monotherapy
in the treatment of CRC, particularly those with low Mcl-1 expression, or Lentiviruses carrying scramble and Mcl-1 shRNA, respectively, were
in combination with an Mcl-1 inhibitor via the potent induction of Bax- purchased from Dharmacon, Inc (Lafayette, CO) and used as instructed by
mediated apoptosis. the company. Knockdown efficiency was determined by Western blotting.

Materials and methods Mitochondrial isolation and Bax oligomerization assay

Reagents Digitonin-based mitochondrial isolation [21] was used to detect Cyt

C release and Bax oligomerization. In brief, cells were washed with PBS
APG-1252-M1, APG-1252 (prodrug of APG-1252-M1 for in vivo study) and then incubated with permeabilization buffer (20 mM HEPES/KOH,
and APG-3526 were provided by Ascentage Pharma Group Inc. (Suzhou, pH 7.5, 100 mM sucrose, 2.5 mM MgCl2 , 50 mM KCl and 0.025%
China). ABT263, ABT199 and S63845 (MIK665) were purchased from digitonin) with protease inhibitor cocktail for 5 min on ice. Supernatant
MedChemExpress (Monmouth Junction). AZD5991 was purchased from and membrane pellet fractions were separated by centrifugation at 13,000 g
Selleckchem (Houston, TX). The caspase inhibitors, CBZ-Val-Ala-Asp- for 5 min. The supernatant (cytosolic fraction) was prepared for Western
fluoromethyl ketone (z-VAD-fmk) and Z-lle-Glu(Ome)-Thr-Asp(OMe)- blot to detect Cyt C and Smac. The membrane fraction was treated with
fluoromethyl ketone (z-IETD-fmk), were purchased from Enzyme System crosslinker BMH (0.5 mM) in crosslinking buffer (20 mM HEPES/KOH,
Products (Livermore, CA). CYD-2-11 was provided by Dr. X. Deng (Emory pH 7.0, 100 mM sucrose, 2.5 mM MgCl2 and 50 mM KCl) for 30–60 min
University, GA). BMH was purchased from Thermo Scientific (Rockford, at room temperature. The reaction was quenched by the addition of reducing
IL). Rabbit antibodies against caspase-8, caspase-3, PARP, Smac, PUMA sample buffer, boiled for 5 min and then subjected to SDS-PAGE gels and
and DR5 (D4E9) were purchased from Cell Signaling Technology (Beverly, analyzed by western blotting using Bax antibody [22].
MA). Mouse antibodies against Bcl-2, Bax (N-20), Mcl-1, cytochrome C
(Cyt C) and rabbit anti-Bcl-XL antibodies were purchased from Santa Cruz Statistical analysis
Biotechnology Inc. (Dallas, Texas). Bim antibody was purchased from EMD
Millipore (Burlington, MA). Mouse monoclonal DR4 antibody (B-N28) Two-sided unpaired Student’s t-test that analyzes significant difference
was purchased from Cell Science (Newburyport, MA). Rabbit polyclonal between two groups, one-way ANOVA test that determines differences
microtubule-associated protein light chain 3 (LC3) antibody was purchased among multiple groups and correlation analysis were conducted with
from Novus Biologicals (Littleton, CO). GAPDH antibody was purchased GraphPad Prism 9 (GraphPad Software, San Diego, CA). Results were
from Trevigen (Gaithersburg, MD). Mouse monoclonal tubulin and actin considered statistically significant when P values were less than 0.05.
antibodies were purchased from Sigma Chemical (St. Louis, MO).
Results
Cell lines and cell culture
APG-1252-M1 effectively decreases survival and induces apoptosis in
Human colon cancer cell lines, HCT116, HCT15, SW480, SW620, CRC cell lines with low levels of Mcl-1
KM12, KM12SM, HT29, HCC2998 and DLD were obtained from
ATCC (Manassas, VA). HCT116/V, HCT116/Mcl-1, HCC2998/V and We first determined the effects of APG-1252-M1 on the growth of a
HCC2998/Mcl-1 cell lines were established using lentiviral Mcl-1 expression panel of CRC cell lines and found that the tested cell lines exhibited varied
Neoplasia Vol. 29, No. xxx 2022 Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL
dual inhibitor that induces Bax-dependent apoptosis W. Yao et al. 3

Fig. 1. APG-121252-M1 shows varied activities against the growth of human CRC cells (A and B), which have different expression levels of Bcl-2, Bcl-XL
and Mcl-1 (C) that impact cell responses to APG-1252-M1 (D), with induction of apoptosis (E and F). A, The indicated cell lines were exposed to varied
concentrations of APG-1252-M1 for 3 days. Cell numbers were estimated with the SRB assay. The data are means ± SDs of four replicate determinations. B.
IC50 s were generated from the concentration-dependent curves of the indicated cell lines exposed to the given inhibitors for 3 days presented in A. C, Basal
levels of the given proteins in the indicated cell lines with similar cell densities were detected with Western blotting. D, Correlation between the expression
levels of Mcl-1 and cell number decrease (CND) caused by 1.25 μM APG-1252-M1. E and F, The indicated cell lines were exposed to DMSO or 1 μM
APG-1252-M1 for about 24 h and then harvested for Western blotting to detect different proteins as indicated (E) and annexin V/flowcytometry (F). CF,
cleaved form. ∗∗ , P < 0.01; ∗∗∗ , P < 0.001; and ∗∗∗∗ , P < 0.0001 compared with DMSO control.

sensitivities to APG-1252-M1: some cell lines (e.g., HCC2998, HCT116 We further checked the effects of ABT199, a Bcl-2 inhibitor, and
and SW480) were very sensitive to APG-1252-M1 with IC50 s < 1 μM, ABT263, a Bcl-1/Bcl-XL /Bcl-w inhibitor [7, 25], on the growth of this panel
whereas others (e.g., HT29, KM12SM and HCT15) were very resistant of CRC cell lines. In general, both inhibitors, particularly ABT263, generated
with IC50 s > 10 μM (Fig. 1A and B). Given that APG-1252-M1 is a Bcl- similar cell response patterns to APG-1252-M1 (Fig. S2). However, APG-
2/Bcl-XL dual inhibitor, we then checked basal levels of Bcl-2 and Bcl-XL 1252-M1, in general, had even lower IC50 s than these agents in decreasing
in these cell lines in order to determine whether the levels of these proteins the survival of the sensitive cell lines although ABT263 showed better activity
impact cell responses to APG-1252-M1. In fact, we did not see a clear and than APG-1252-M1 against the insensitive cell lines (Fig. S2).
significant correlation between the expression of Bcl-XL or particularly Bcl-
2 and cell sensitivity to APG-1252-M1 (Fig. 1C and D and S1). Mcl-1 is
an anti-apoptotic protein that, functionally like Bcl-2 and Bcl-XL , negatively APG-1252-M1 combined with an Mcl-1 inhibitor synergistically
regulates intrinsic apoptotic pathway [23,24]. We also checked Mcl-1 levels decreases the survival and induces apoptosis of
in these cell lines and found that APG-1252-M1-senstive cell lines tended APG-1252-M1-insensitive cell lines with high levels of Mcl-1
to have lower levels of Mcl-1 than those insensitive to APG-1252-M1,
showing a significant negative correlation between Mcl-1 abundance and cell Given the finding that high baseline expression of Mcl-1 is significantly
response to APG-1252-M1 (Fig. 1C and D). Finally, we determined whether associated with cell resistance to APG-1252-M1, we asked whether Mcl-
APG-1252-M1 induces apoptosis of CRC cell lines. While HT29 cells 1 inhibition (e.g., with a Mcl-1 inhibitor) would sensitize the insensitive
were resistant, three sensitive cell lines, HCC2998, HCT116 and SW480, cell lines with high levels of Mcl-1 to APG-1252-M1. When combined
underwent rapid apoptosis after a 24 h treatment as evidenced by detection with any of the three Mcl-1 inhibitors, S63845, AZD5991 and APG3526,
of increased cleavage of caspase-3, caspase-8 and PARP (Fig. 1E) and annexin APG-1252-M1 synergistically decreased the survival of the tested insensitive
V-positive cell populations (Fig. 1F). Hence, APG-1252-M1 effectively CRC cell lines with CIs far smaller than 1 (Fig. 2A). The combination
decreases the survival of some sensitive CRC cell lines with the induction of of APG-1252-M1 with either S63845 or AZD5991 effectively enhanced
apoptosis. cleavage of caspase-3, caspase-8 and PARP and increased annexin V-positive
4 Mcl-1 negatively impacts APG-1252-M1 efficacy W. Yao et al. Neoplasia Vol. 29, No. xxx 2022

Fig. 2. APG-1252-M1 in combination with an Mcl-1 inhibitor synergistically decreases the survival (A) and enhances apoptosis (B and C) of APG-1252-M1-
insensitive CRC cell lines; this is in agreement with the data generated with Mcl-1 knockdown (D and E) A, The indicated cell lines were exposed to varied
concentrations of APG-1252-M1 (M1) alone, S63845 alone, AZD5991 alone, APG-3526 alone, APG-1252-M1 plus S63845, APG-1252-M1 plus AZD5991
or APG-1252-M1 plus APG-3526 for 3 days. Cell numbers were estimated with the SRB assay. The data are means ± SDs of four replicate determinations.
The numbers inside the graphs are CIs. B and C, The indicated cell lines were treated with DMSO, 5 μM APG-1252-M1, 0.5 μM S63845 or AZD5991, or
APG-1252-M1combined with S64845 or AZD5991. After about 25 h (B) or around 30 h (C), the cells were harvested for Western blotting (B) or annexin
V/flow cytometry (C). CF. cleaved from. ∗∗ , P < 0.01; and ∗∗∗ , P < 0.001 at least compared with either single agent alone. D and E, HT29 cells were infected
with lentivisuses carrying shMcl-1 for 40 h followed by treatment with 0.5 μM APG1252-M1 for 16 h (D) or different concentrations of APG1252-M1 for
3 days (E). The data in E are the means ± SDs of triplicate determinations.

cell populations while each agent alone at the tested concentrations did so generated in SW620 cells (data not shown). Thus genetic inhibition of Mcl-1
minimally or not detectably (Fig. 2B and C), indicating that Mcl-1 inhibition further demonstrate the critical role of Mcl-1 in conferring cell resistance to
combined APG-1252-M1 restores the sensitivity of insensitive CRC cell lines APG-1252-M1.
to undergo APG-1252-M1-induced apoptosis. We further knocked down Interestingly, we found that ABT263, but not ABT199, when combined
Mcl-1 expression in HT29 cells (Fig. 2D) and then checked their responses with S63845 synergistically decreased the survival of APG-1252-M1-
to APG-1252-M1. In agreement with the effects of Mcl-1 inhibitors, Mcl-1 insensitive CRC cells (Fig. S3), suggesting that APG-1252-M1 and
knockdown made the cells very sensitive to APG-1252-M1 as determined by ABT263 may share similar mechanisms in inducing the death of CRC
cell viability (Fig. 2E) and PARP cleavage (Fig. 2D). Similar results were also cells.
Neoplasia Vol. 29, No. xxx 2022 Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL
dual inhibitor that induces Bax-dependent apoptosis W. Yao et al. 5

Fig. 3. APG-1252-M1 combined with an Mcl-1 inhibitor synergistically decreases the survival (A and B) and enhances apoptosis (C and D) of APG-1252-
M1-senstive CRC cell lines, whereas enforced expression of ectopic Mcl-1 in the sensitive cells abrogates APG-1252-M1’s ability to decrease cell survival (E
and F), which can be reversed by Mcl-1 inhibition (G). A and B, The indicated cell lines were treated with varied concentrations of APG-1252-M1 (M1)
alone, S63845 alone, APG3526 alone, APG-1252-M1 plus S63845, or APG-1252-M1 plus APG3526 for 3 days. Cell numbers were estimated with the SRB
assay. The data are means ± SDs of four replicate determinations. C and D, The indicated cell lines were treated with DMSO, 0.25 μM APG-1252-M1, 0.5
μM S63845 or APG-1252-M1 combined with S64845. After around 20 h, the cells were harvested for Western blotting (C) or annexin V/flow cytometry
(D). CF. cleaved from. E, Ectopic Mcl-1 expression in the given cell lines was detected with Western blotting. F and G, The different cell lines were exposed
to varied concentrations of APG-1252-M1 as indicated for 3 days or treated with indicated concentrations of APG-1252-M1 alone, S63845 alone or their
combination for 3 days. Cell numbers were estimated with the SRB assay. The data are means ± SDs of four replicate determinations.
6 Mcl-1 negatively impacts APG-1252-M1 efficacy W. Yao et al. Neoplasia Vol. 29, No. xxx 2022

Inhibition of Mcl-1 further enhances APG-1252-M1’s effects on The same category of compound, ABT263, basically exerted very similar
decreasing the survival and inducing apoptosis of effects as APG-1252-M1 did on facilitating release of Cyt C and Smac,
APG-1252-M1-sensitive cells expressing low levels of Mcl-1, whereas inducing cleavage of caspase-8, caspase-3 and PARP, and modulating the
enforced expression of ectopic Mcl-1 in the cells abrogates levels of Mcl-1 even at 4 h post treatment. We detected clear release of Cyt
APG-1252-M1’s therapeutic efficacy, which can be reversed by Mcl-1 C and Smac, but weak or no cleavage of caspase-3, caspase 8 and PARP in
cells exposed to ABT199 under the same tested conditions (Fig. S4A and B),
inhibition
suggesting that ABT199 may have relatively weak activity in the induction of
apoptosis. Similarly, ABT263 and ABT199 did not alter the levels of other
We were also curious to know whether Mcl-1 inhibition further sensitizes
apoptosis-regulating proteins including Bcl-2, Bax, Bim, DR4 and DR5 (Fig.
the sensitive CRC cells to APG-1252-M1. As we observed in the resistant
S4A).
CRC cells, APG-1252-M1 combined with either S63845 (Fig. 3A) or APG-
3526 (Fig. 3B) synergistically decreased the survival of the tested sensitive
CRC cell lines. Consistently, the combination of APG-1252-M1 and S63845
was much more potent than either agent alone in inducing the cleavage APG-1252-M1 or its combination with Mcl-1 inhibition induces
of caspases and PARP (Fig. 3C) and in increasing annexin V-positive cells Bax-dependent apoptosis
(Fig. 3D) in each tested sensitive CRC cell line. To further leverage the
critical role of Mcl-1 in impacting cell sensitivity to APG-1252-M1, we To determine whether APG-1252-M1 indeed initiates apoptosis through
enforced the elevation of Mcl-1 levels in two sensitive cell lines (HCT116 activation of the intrinsic apoptotic pathway, we first checked the effect of
and HCC2998) via lentiviral overexpression of ectopic Mcl-1 as confirmed caspase inhibition on induction of apoptosis by APG-1252-M1. The presence
by Western blotting (Fig. 3E). Here, HCT116/Mcl-1 #1 and #2 represent two of the pan caspase inhibitor, VAD-FMK, or the caspase-8 inhibitor, IETD-
different cell populations expressing ectopic Mcl-1 generated from HCT116 FMK, abrogated the ability of APG-1252-M1 to induce cleavage of caspase-
cells duplicated for Mcl-1 lentiviral infection. These Mcl-1-expressing cell 3, caspase-8 and PARP (Fig. 5A), indicating caspase-dependent apoptosis.
lines were fully resistant to APG-1252-M1 (Fig. 3F). However, the presence Similar results were also generated with ABT263 (Fig. S5). Following this
of S63835 restored the sensitivities of both HCT116/Mcl-1 #1 and #2 cells to study, we then checked the effect of deficiency of Bax, Bim, Smac, Puma
APG-1252-M1 (Fig. 3G). These results together convincingly demonstrate or FADD, which are critical components in the regulation of both intrinsic
the critical role of Mcl-1 in determining the sensitivity of CRC cells to APG- and extrinsic apoptotic pathways [26], on protection of cells from APG-
1252-M1. 1252-M1-induced killing or apoptosis using a panel of isogenic cell lines
derived from HCT116. In both cell survival and apoptotic assays, we found
that BAX-KO cells were fully resistant to APG-1252-M1, whereas FADD-
APG-1252-M1 activates caspase-8 and caspase-3 and induces release of
KO cells behaved like the parental cells to fully respond to APG-1252-M1.
Cyt C and Smac from mitochondria, accompanied with varied effects on
Deficiency in Smac, Puma or Bim was partially resistant to APG-1252-M1
Mcl-1 levels in APG-1252-M1-sensitive CRC cells with varied degrees (Fig. 5B and C). By comparing cleavage of caspases and
PARP between HCC116 and either of the deficient cell lines, we generated
APG-1252-M1 obviously induces caspase- 8 activation while activating similar results (Fig. 5D). Beyond APG-1252-M1 alone, the combination
caspase-3 as shown above in Fig. 1E. To better understand the role of of APG-1252-M1 with S63845 showed enhanced effect on decreasing the
caspase-8 activation, we conducted detailed time-course analyses of caspase-8 survival of HCT116 cells, but this effect was lost in HCT116/Bax-KO
cleavage in comparison with caspase-3 activation in the sensitive CRC cells. cells (Fig. 5E). Hence, it is clear that APG-1252-M1 or its combination
We detected clear cleavage of caspase-8, caspase-3 and PARP with similar with Mcl-1 inhibition induces Bax-dependent apoptosis. To further support
degrees early at 4 h post APG-1252-M1 treatment in both HCT116 and this conclusion, we also determined the effect of APG-1252-M1 on Bax
HCC2998 cells (Fig. 4A and B). We also clearly detected the release of Cyt activation by assessing the formation of Bax oligomerization including dimers
C and Smac from mitochondria in HCT116 cells exposed to APG-1252- and trimers and found that increased levels of Bax dimers and trimers were
M1 at 2 h post APG-1252-M1 treatment (Fig. 4C). To gain insight into detected in cells exposed to APG-1252-M1, particularly at early times starting
the dynamic activation of caspases and release of Cyt C and Smac, we then at 4 h (Fig. 5F). Here we used CYD-2-11, a Bax activator known to activate
performed an even shorter time-course analyses of these events within a 0–3 Bax [27], as a positive control. Thus, APG-1252-M1 apparently activates Bax
h treatment period in HCT116 as shown in Fig. 4D. We started to observe in CRC cells.
clear cleavage of caspase-8, caspase-3 and PARP at 1.5 h post APG-1252-M1
treatment while detecting clear Cyt C and Smac in the cytosol early at 0.5 h
post treatment, indicating the release of Cyt C and Smac ahead of caspase-8, Caspase-8 activation is likely secondary to mitochondrial activation of
caspase-3 and PARP cleavage. caspase-3 and further facilitates mitochondria-dependent apoptosis
We also determined whether APG-1252-M1 exerts any effects on the
induced by APG-1252-M1
levels of several apoptosis-regulating proteins and on inducing autophagy. We
found that APG-1252-M1 treatment did not apparently alter the levels of
We found that enforced overexpression of ectopic Mcl-1 in HCT116
Bcl-2, Bcl-XL , Bim, Bax, DR4, DR5 and LC3 II in HCT116 cells, but we
prevented caspase-8 from being cleaved or activated with treatment with
clearly detected increased levels of Mcl-1 and Bid from 2 h to 24 h (Fig. 4A).
either APG-1252-M1 or ABT263 (Fig. 6A), suggesting an event downstream
APG-1252-M1 did not alter LC3 II levels in HCC2998 cells either (Fig. 4B),
of mitochondrial apoptosis. Thus, we further determined the effects of
suggesting that APG-1252-M1 does not induce autophagy while inducing
caspase inhibition on Cyt C and Smac release from mitochondria induced
apoptosis. Interestingly, we found that APG-1252-M1 deceased Mcl-1 levels
by APG-1252-M1 or ABT263, which are events that lead to activation of
in the sensitive HCC2998 cells starting at 8 h post treatment (Fig. 4B). Given
caspase-3. Interestingly, we found that both the pan-caspase inhibitor, Z-
the varied effects of APG-1252-M1 on modulation of Mcl-1 levels, we further
VAD-FMK, and the caspase-8 inhibitor, Z-IETD-FMK, partially attenuated
checked its effects on Mcl-1 in additional CRC cell lines and found that APG-
the ability of either APG-1252-M1 or ABT263 to induce the release of Cyt
1252-M1 decreased Mcl-1 levels in sensitive SW480 cells, but elevated Mcl-1
C and Smac from mitochondria (Fig. 6B), suggesting an impact of caspase-8
levels in resistant HT29 cells (Fig. 4E). Hence, it appears that APG-1252-M1
activation on induction of Cyt C and Smac release and subsequent apoptosis.
exerts cell line-dependent effects on modulation of Mcl-1 levels.
Neoplasia Vol. 29, No. xxx 2022 Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL
dual inhibitor that induces Bax-dependent apoptosis W. Yao et al. 7

Fig. 4. APG-1252-M1 rapidly induces cleavage of caspase-8, caspase-3 and PARP and release of Cyt. C and Smac from mitochondria (A-D) with different
effects on Mcl-1 levels (A, B and E) in APG-1252-M1-senstive cells. A-D, HCT116 or HCC2998 cells were exposed to DMSO or 1 μM APG-1252-M1
(M1) for different times as indicated and then harvested for preparation of whole-cell protein lysates or cytosolic fraction and subsequent Western blotting.
CF, cleaved from. E, The tested cell lines were exposed to different concentrations of APG-1252-M1 as indicated for 24 h. Proteins of interest were detected
with Western blotting.

Discussion role of Mcl-1 in determining the sensitivity of CRC cells came from the
following findings: (1) enforced overexpression of ectopic Mcl-1 in the
The success of targeted cancer therapies largely relies on the identification sensitive CRC cell lines rendered them resistant to APG-1252-M1; and (2)
of patient populations who are most likely to respond to a given targeted addition of an Mcl-1 inhibitor to these Mcl-1-expressing cell lines fully
therapy. In this end, it is essential to identify the biomarker(s) that can be restored APG-1252-M1 sensitivity. These data strongly support the notion
used to select these patients. As a novel Bcl-2/Bcl-XL dual inhibitor, APG- that Mcl-1 is a critical molecular determinant for CRC cells to respond to
1252-M1 effectively decreased the survival and induced apoptosis of a few APG-1252-M1 monotherapy. Fortunately, APG-1252-M1 combined with
CRC cell lines with IC50 s < 1 μM, while weakly decreasing the survival an Mcl-1 inhibitor exerted synergistic effects on decreasing the survival
of most other CRC cell lines (IC50 s > 10 μM). Both Bcl-2 and Bcl-XL and inducing apoptosis of CRC cells regardless of Mcl-1 level. Therefore,
were universally expressed in the tested CRC cell lines and the baseline developing this combination regimen may have potential to maximize the
expression levels of either Bcl-XL or particularly Bcl-2 did not correlate with therapeutic efficacy of APG-1252-M1, and likely other similar inhibitors such
cell sensitivities to APG-1252-M1. The interesting and important finding as ABT263, against CRCs. Our findings with APG-1252-M1 in CRC cells
is that the baseline levels of Mcl-1 varied among these CRC cell lines are in agreement with previous reports demonstrating the negative impact of
with a significant negative correlation with cell responses to APG-1252- Mcl-1 on cell sensitivity to other Bcl-2 inhibitors such as ABT263 [28–31].
M1: i.e., low baseline expression was significantly correlated with increased Another interesting finding is that APG-1252-M1 modulated Mcl-1 levels
cell sensitivity to APG-1252-M1. Hence, our findings suggest that baseline in a cell context-dependent manner. i.e., it elevated Mcl-1 in some cell lines
Mcl-1 expression may be a biomarker to predict CRCs that may respond (e.g., HCT116) while decreasing Mcl-1 levels in others (e.g., HCC2998).
to APG-1252-M1 monotherapy and likely other similar Bcl-2/Bcl-XL dual This is likely also the case for other similar inhibitors such as ABT263. The
inhibitors such as ABT263 since both APG-1252-M1 and ABT263 shared underlying mechanisms accounting for the cell-dependent modulation of
some similarities in suppressing the growth of CRC cells. Mcl-1 are unclear and thus deserve further investigation in the future. Indeed,
In relation to this finding, APG-1252-M1, when combined with an Mcl- recent studies have also shown the induction of Mcl-1 due to inhibition of
1 specific inhibitor, synergistically decreased the survival of the insensitive Bcl-2 or Bcl-XL with BH3 mimetics, resulting in a rescue loop triggering
CRC cell lines with high levels of Mcl-1 with enhanced induction of cell protective effect [32,33]. It is likely that APG-1252-M1-induced Mcl-1
apoptosis. The activity of APG-1252-M1 against those sensitive CRC cell elevation in some cell lines may evade apoptosis and counteract its therapeutic
lines with low baseline levels of Mcl-1 could be even further augmented efficacy. Hence, this finding further supports the rationale of co-targeting
when combined with an Mcl-1 inhibitor. The direct evidence for the critical Mcl-1 to enhance the therapeutic efficacies of APG-1252-M1 and possibly
8 Mcl-1 negatively impacts APG-1252-M1 efficacy W. Yao et al. Neoplasia Vol. 29, No. xxx 2022

Fig. 5. APG-1252-M1 induces Bax-dependent, but FADD-independent, apoptosis, in which Smac, Puma and Bim are partially involved. A, HCT116 cells
were pre-treated with 40 μM Z-VAD or Z-IETD for 30 min and then co-treated with 1 μM APG-1252-M1 for additional 4 h. Proteins of interest were
detected with Western blotting. B and C, The indicated isogeneic cell lines derived from HCT116 were exposed to varied concentrations of APG-1252-M1
for 3 days (B) or to DMSO or 1 μM APG-1252-M1 for 24 h (C). Cell numbers were estimated with the SRB assay (B) and apoptosis was detected with
annexin V/ flow cytometry (C), respectively. The data are means ± SDs of four replicate (B) or duplicate (C) determinations. D, the given cell lines were
exposed to DMSO or 1 μM APG-1252-M1 (M1) for 4 h. Different proteins were detected with Western blotting. E, The tested cell lines were treated with
indicated concentrations of APG-1252-M1 alone, S63845 alone or their combination for 3 days. Cell numbers were estimated with the SRB assay. The data
are means ± SDs of four replicate determinations. F, HCT116 cells were exposed to 1 μM APG-1252-M1 for different times as indicated. Moreover, A549
cells were exposed to 5 μM CYD-2-11 (CYD) for 24 h. The cells were then harvested for detection of Bax oligomerization. CYD-2-11 treatment here serves
as a positive control.

other similar inhibitors in the treatment of CRCs, particularly those with negative regulation of Bax-mediated apoptosis, whereas BH3-only proteins
treatment-induced Mcl-l elevation. such as Bim and Puma facilitate Bax-dependent apoptosis through interacting
We noted that, in contrast to APG-1252-M1 and ABT263, the and sequestering these Bcl-2-like family proteins [26, 36]. Smac, as a protein
combination of ABT199 and S63845 did not have or had very weak enhanced released from mitochondria during apoptosis, promotes caspase-9/caspase-
effects on decreasing the survival of CRC cell lines. Similar finding was also 3 activation via suppression of the function of the inhibitors of apoptosis
observed in cervical cancer cells [31]. Beyond, synergy between co-targeting (IAPs) such as XIAP [26,36]. Therefore, we can speculate that APG-1252-
Bcl-XL and Mcl-1 in decreasing the survival of different types of cancer cells M1, as a Bcl-2/Bcl-XL dual inhibitor, should induce apoptosis via eventual
was also reported [34,35]. Thus, it is very likely that Bcl-XL inhibition plays activation of Bax-dependent apoptosis. Indeed, our results have demonstrated
a dominant role in mediating synergistic effects of APG1252-M1 combined that APG-1252-M1 or its combination with Mcl-1 inhibition induces Bax-
with an Mcl-1 inhibitor in decreasing the survival and inducing apoptosis of dependent apoptosis since Bax deficiency in CRC cells abolished the ability
CRC cells. of APG-1252-M1 or its combination with a Mcl-1 inhibitor to decrease
It is well established that Bcl-2-like family proteins such as Bcl-2, Bcl- cell survival and induce apoptosis. Consistently, deficiency of Puma, Bim or
XL and Mcl-1 inhibit Bax activation via direct interaction, resulting in the Smac partially protected the tested CRC cells from APG-1252-M1-induced
Neoplasia Vol. 29, No. xxx 2022 Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL
dual inhibitor that induces Bax-dependent apoptosis W. Yao et al. 9

Fig. 6. Caspase-8 activation induced by APG-1252-M1 is likely secondary to Bax-dependent activation of caspase-3 and facilitates activation of the
mitochondrial apoptotic pathway. A, The given cell lines were exposed to DMSO, 1 μM APG-1252-M1, 3 μM ABT199 and 3 μM ABT263 for 4 h.
The proteins of interest were detected with Western blotting. CF, cleaved from. B, HCT116 cells were pre-treated with 40 μM Z-VAD or Z-IETD for 30
min and then co-treated with 1 μM APG-1252-M1 or 3 μM ABT263 for additional 4 h. The cells were then harvested for preparation of cytosolic fraction
and subsequent Western blotting. C, A schema for a working model suggesting feedback activation of caspase-8 by caspase-3 and subsequent feedforward
facilitation of the mitochondrial apoptotic pathway during APG-1252-M1-induced apoptosis. The combination of APG-1252-M1 with Mcl-1 inhibition has
maximal effect on the induction of Bax-mediated apoptosis.

apoptosis. Thus, we have convincingly demonstrated that APG-1252-M1 same cells exposed to TRAIL that is known to induce caspase-8 activation and
activates the intrinsic apoptotic pathway, resulting in Bax activation-mediated Bid cleavage, we did not detect cleaved band of Bid either albeit substantial
apoptosis in CRC cells. reduction of Bid levels (Fig. S6). It is likely that Bid antibody we used does
In this study, APG-1252-M1 as well as ABT263 quickly and robustly not capture cleaved band well. Hence, Bid reduction in cells exposed to APG-
induced cleavage of caspase-8, indicating activation of caspase-8 in CRC cells; 1252-M1 is likely due to Bid cleavage,
this event may also play a critical role in mediating or enhancing APG-1252- One interesting observation is that we clearly detected release of Cyt C and
M1-induced apoptosis since the caspase-8 inhibitor, IETD-FMK, effectively Smac from mitochondria, but not increased cleavage of caspases and PARP in
abrogated cleavage of caspase-3 and PARP induced by APG-1252-M1. CRC cells exposed to ABT199 for a short time (4 h; Fig. S4). It is possible that
FADD plays an essential role in mediating death receptor-induced activation other unknown factors, which negatively regulate events leading to activation
of extrinsic apoptosis via recruitment and activation of caspase-8 [37,38]. of caspases downstream of Cyt C and Smac, may not be effectively removed
However, FADD knockout failed to provide any protective effect on APG- by ABT199 during the short period of treatment. It is also possible that it
1252-M1-induced apoptosis, suggesting no involvement of activation of the may take a longer time for ABT199 to induce caspase activation and PARP
extrinsic apoptotic pathway. It is known that caspase-3 can activate caspase-8, cleavage although it quickly triggers Cyt C and Smac release. Nonetheless,
leading to facilitation or amplification of Bax-dependent apoptosis through this deserves a further investigation.
Bid cleavage [39–41]. In this study, release of Cyt C and Smac occurred early In summary, the current study has demonstrated the therapeutic potential
before cleavage of caspase 3 and caspase-8 in the sensitive CRC cells exposed of the new Bcl-1/Bcl-XL dual inhibitor, APG-1252-M1, either alone or in
to APG-1252-M1. Moreover, caspase-8 inhibition such as with a caspase combination with Mcl-1 inhibition in the treatment of CRC via effective
inhibitor compromised the ability of APG-1252-M1 to induce the release induction of the intrinsic apoptotic pathway. It is clear that the level of Mcl-l
of both Cyt C and Smac. Hence, it is plausible to speculate that caspase- critically determines the response of CRC cells to APG-1252-M1 when used
8 activation induced by APG-1252-M1 in CRC cells is likely secondary to as a monotherapy. Findings in this study warrant further in vivo studies of the
Bax-mediated activation of caspase-3; this feedback activation of caspase-8 therapeutic efficacy against CRC.
will feed forward further activation of the mitochondrial apoptotic signaling,
enhancing apoptotic cell death (Fig. 6C). Bid is known to be a substrate Funding
of caspase-8 and facilitate Bax activation via its truncate form [42,43]. In
this study, we detected Bid reduction in cells exposed to APG1252-M1 in This study was supported by a research fund from Ascentage Pharma Corp
parallel to caspase 8 cleavage albeit without cleaved band detected. In the Ltd (to SYS).
10 Mcl-1 negatively impacts APG-1252-M1 efficacy W. Yao et al. Neoplasia Vol. 29, No. xxx 2022

Declaration of Competing Interest BCL-2/BCL-XL inhibitor pelcitoclax (APG-1252) overcomes intrinsic and
acquired resistance to venetoclax in multiple myeloma cells. Blood 2021;138:
Y. Zhai is an equity shareholder of Ascentage Pharma Group International 2655.
and a full-time employee of one or more of its affiliates. S. Wang is a co- [12] Luo F, Lu FT, Qiu MZ, Zhou T, Ma WJ, Luo M, Zeng KM, Luo QY, Pan WT,
founder, chief-scientific advisor and board member of Ascentage Pharma Zhang L, et al. Gemcitabine and APG-1252, a novel small molecule inhibitor
Group, which has licensed APG-1252 from the University of Michigan. He is of BCL-2/BCL-XL, display a synergistic antitumor effect in nasopharyngeal
carcinoma through the JAK-2/STAT3/MCL-1 signaling pathway. Cell Death Dis
also a paid consultant of Ascentage, owns shares in Ascentage, is a co-inventor
2021;12:772.
of APG-1252 and receives royalty from the University of Michigan. S-Y. Sun
[13] Schoenwaelder SM, Jarman KE, Gardiner EE, Hua M, Qiao J, White MJ,
received research fund from Ascentage Pharma Group Corp Ltd, an affiliate of
Josefsson EC, Alwis I, Ono A, Willcox A, et al. Bcl-xL-inhibitory BH3 mimetics
Ascentage Pharma Group International. Others disclose no potential conflicts can induce a transient thrombocytopathy that undermines the hemostatic
of interest. function of platelets. Blood 2011;118:1663–74.
[14] Vogler M, Hamali HA, Sun XM, Bampton ET, Dinsdale D, Snowden RT,
CRediT authorship contribution statement Dyer MJ, Goodall AH, Cohen GM. BCL2/BCL-X(L) inhibition induces
apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.
Weilong Yao: Conceptualization, Visualization, Investigation, Data Blood 2011;117:7145–54.
curation, Formal analysis, Writing – original draft, Writing – review & [15] Lakhani NJ, Rasco DW, Zeng Q, Tang Y, Liang Z, Wang H, Lu M, Chen J,
editing. Longchuan Bai: Conceptualization, Visualization, Investigation, Fu L, Wang C, et al. First-in-human study of palcitoclax (APG-1252), a novel
Data curation, Formal analysis, Writing – original draft, Writing – review dual Bcl-2/Bcl-xL inhibitor, demonstrated advantages in platelet safety while
& editing. Shaomeng Wang: Conceptualization, Visualization, Project maintaining anticancer effect in U.S. patients with metastatic solid tumors. J Clin
administration, Writing – original draft, Writing – review & editing. Yifan Oncol 2020;38:3509.
Zha: Conceptualization, Visualization, Project administration, Writing – [16] Zhang L, Zhao H, Ma Y, Cheng Y, Zhao Y, Cui J, Yang C, JZhang J,
original draft, Writing – review & editing. Shi-Yong Sun: Conceptualization, Wang P, Xu L, et al. Phase 1b study of pelcitoclax (APG-1252) in combination
Visualization, Project administration, Writing – original draft, Writing – with osimertinib in patients with EGFR TKI-resistant NSCLC. J Thor Oncol
2021;16:S891.
review & editing.
[17] Ren H, Zhao L, Li Y, Yue P, Deng X, Owonikoko TK,
Chen M, Khuri FR, Sun SY. The PI3 kinase inhibitor
Acknowledgements NVP-BKM120 induces GSK3/FBXW7-dependent Mcl-1 degradation,
contributing to induction of apoptosis and enhancement
We are thankful to Drs. J. Yu and L. Zhang (University of Pittsburgh, of TRAIL-induced apoptosis. Cancer Lett 2013;338:229–
Pittsburgh, PA) for providing some KO cell lines. We are also grateful to Dr. 238.
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