ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 2009-2026 © Pleiades Publishing, Ltd., 2025.
Published in Russian in Biokhimiya, 2025, Vol. 90, No. 12, pp. 2119-2138.
2009
Mechanisms and Ways to Overcome Acquired
Resistance of Cancer Cells to Mcl-1 Antagonists
Nikolay V. Pervushin
1,2
, Bertha Y. Valdez Fernandez
2
, Vyacheslav V. Senichkin
2
,
Maria A. Yapryntseva
1,2
, Vladislav S. Pavlov
1
, Boris Zhivotovsky
1,2,3,a
*,
and Gelina S. Kopeina
1,2,b
*
1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
2
Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia
3
Institute of Environmental Medicine, Karolinska Institutet, 17177 Stockholm, Sweden
a
e-mail: boris.zhivotovsky@ki.se
b
e-mail: lirroster@gmail.com
Received August 24, 2025
Revised October 22, 2025
Accepted October 29, 2025
Abstract— Acquired drug resistance reduces the effectiveness of anticancer therapy and leads to cancer pro-
gression. Selective inhibition of anti-apoptotic proteins of the Bcl-2 family using BH3-mimetics is a promising
treatment strategy for cancer patients. Recently, antagonists of the anti-apoptotic protein Mcl-1 have been
actively studied in clinical trials. However, like other BH3-mimetics, they can lose their effectiveness due to
the development of acquired resistance. We have found that cancer cells develop resistance to Mcl-1 inhibition
through increased gene expression of other anti-apoptotic proteins, such as Bcl-2 or Bcl-xL, thereby becom-
ing less Mcl-1-dependent. Alterations in cellular metabolism have also accompanied the development of this
resistance. We have shown that combining the Mcl-1 antagonist S63845 with various anticancer compounds
can overcome the resistance of malignant cells to its action.
DOI: 10.1134/S0006297925602710
Keywords: drug resistance, Mcl-1, BH3 mimetics, apoptosis, cancer cells
* To whom correspondence should be addressed.
INTRODUCTION
During carcinogenesis, tumor cells undergo vari-
ous adaptations that allow them to evade the body’s
defense mechanisms. One of the distinctive features
of malignant clones, which enables them to actively
divide and invade surrounding tissues and organs,
is their avoidance of various types of programmed
cell death (PCD), including apoptosis, a process that
plays a vital role in protection against cancer [1-3].
The limited sensitivity of tumor cells to apoptosis un-
derlies the progression of many oncological diseases;
therefore, triggering this type of PCD is a rational
strategy for anticancer therapy [4, 5].
Apoptosis is a strictly genetically controlled pro-
cess. There are two primary pathways for initiating
apoptosis: the extrinsic (receptor-dependent) pathway
and the intrinsic (mitochondrial) pathway. The key
event in the intrinsic apoptotic pathway is mitochon-
drial outer membrane permeabilization (MOMP),
which leads to the release of cytochrome c and other
proapoptotic factors from the intermembrane space,
thereby promoting cell death [4, 5]. The proteins of
the Bcl-2 (B-cell leukemia/lymphoma protein-2) family
control MOMP. The functional activity of the mem-
bers of this family is accomplished through the for-
mation of various protein–protein complexes. Some
proteins are responsible for MOMP activation, while
other proteins inhibit this process [6-10]. According
to their properties, the proapoptotic proteins of the
Bcl-2 family are divided into two categories: regula-
tory BH3-only (Bcl-2 homology domain) and effector
proteins [8, 10-12]. The latter include the multidomain
proteins Bak (Bcl-2 homologous antagonist killer) and
Bax (Bcl-2 associated X protein), which, upon activa-
tion, are capable of forming pores in the outer mito-
chondrial membrane (OMM) through the formation
of homo- and hetero-oligomers. In the absence of
PERVUSHIN et al.2010
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
apoptotic stimuli, Bax is located in the cytoplasm, and
Bak is located on the OMM in monomeric form [13,
14]. The regulatory BH3-only proteins contain only
one BH domain in their structure, possess various
intracellular localizations (cytoplasm, OMM, cytoskel-
eton, etc.), and can perform two functions. Firstly, all
proteins in this subgroup are capable of neutralizing
the action of anti-apoptotic proteins; however, their
interaction profiles and inhibitory capacities vary
significantly among the different members of the
Bcl-2 family. Secondly, some BH3-only proteins (Bim
[Bcl-2-interacting mediator of cell death], tBid [trun-
cated form of the Bid protein]) can also activate the
effector proteins Bak and Bax with varying degrees
of efficiency [15-18]. The anti-apoptotic proteins (Bcl-2,
Mcl-1, Bcl-xL, etc.) localized on the OMM and in the
cytoplasm promote cell survival by binding both ef-
fector and regulatory proapoptotic members of the
Bcl-2 family [8, 15, 19, 20]. Thus, the proteins of the
Bcl-2 family participate in forming a three-component
control system of MOMP, which determines the sub-
sequent fate of cells [8, 15, 21].
The anti-apoptotic proteins of the Bcl-2 family
represent attractive targets for anticancer therapy,
as an increase in their levels often underlies the re-
sistance of tumor cells to apoptosis, leading to a de-
crease in the effectiveness of many chemotherapeu-
tic agents [8, 22-24]. Over the past decades, highly
selective small-molecule inhibitors of anti-apoptotic
proteins of the Bcl-2 family have been actively de-
veloped. According to their mechanism of action,
these inhibitors serve as “exogenous” analogs of the
regulatory BH3-only proteins, which is why they are
referred to as BH3-mimetics [8, 25]. To date, one drug
from this group of compounds (Venetoclax, a selec-
tive antagonist of the Bcl-2 protein) has already been
approved for clinical use in many countries to treat
chronic lymphocytic leukemia (CLL) [8, 26].
Among all proteins of the Bcl-2 family respon-
sible for cell survival, Mcl-1 (Myeloid cell leukemia
protein-1) attracts special attention. This protein is
important for maintaining embryogenesis and ho-
meostasis in various cell types, as well as for regu-
lating the cell cycle [9, 27]. Its gene is expressed in
all tissues of the human body [20]. It is worth noting
that Mcl-1 is important for mitochondrial homeosta-
sis [28]. The molecular weight of Mcl-1 (37.2 kDa) is
significantly higher than that of other anti-apoptotic
proteins due to the presence of an N-terminal region
containing its mitochondrial localization sequence, as
well as PEST sequences (proline [P], glutamic acid [E],
serine [S], and threonine [T]-rich sequences), which
are regions enriched in residues of proline, glutam-
ic acid, serine, and threonine. The presence of PEST
sequences is a feature of short-lived proteins, which
also distinguishes Mcl-1 [20, 29, 30]. In recent years,
various negative modulators of Mcl-1 have been dis-
covered at the transcriptional, translational, and post-
translational levels [20, 29]. In particular, we have
previously found that Mcl-1 in tumor cells undergoes
proteasomal degradation under conditions of nutrient
limitation, independent of autophagy [31]. Addition-
ally, there are peculiarities in the Mcl-1 interactions
with other members of the Bcl-2 family. For example,
Mcl-1 has low affinity for the effector protein Bax
and higher affinity for Bak [30, 32]. We have also
demonstrated a key role of Bak in apoptosis induced
by selective Mcl-1 antagonists [33]. In addition, the
BH3-only protein Noxa binds to Mcl-1, facilitating its
proteasomal degradation [34].
Various Mcl-1 protein antagonists (S64315/MIK665,
AZD5991, AMG176, PRT1419, etc.) have been actively
studied in clinical trials over the past few years [35].
However, to date, they have not been able to replicate
the success of Venetoclax for several reasons. Firstly,
suppression of the anti-apoptotic proteins of the Bcl-2
family can lead to serious side effects, since all of
them perform multiple physiological functions. In
particular, the use of Bcl-xL antagonists caused severe
thrombocytopenia [8, 36], and recently it was found
that Mcl-1 suppression results in impaired hemato-
poiesis and cardiotoxicity [35]. Secondly, Mcl-1 antag-
onists often possessed low efficacy in clinical trials,
which may be due to the development of acquired
resistance by tumor cells to Mcl-1 inhibition [35].
The development of drug resistance with subse-
quent loss of treatment efficacy is a serious problem
arising with the use of most modern drugs, partic-
ularly antitumor agents [37]. This phenomenon has
also been observed with the use of BH3-mimetics. For
example, possible causes of cancer cell resistance to
Venetoclax have been characterized in detail, includ-
ing metabolic and proliferative adaptations of malig-
nant clones, emergence of mutations in the genes of
the target protein Bcl-2 and its proapoptotic partner
Bax, compensatory effects (increased levels of other
anti-apoptotic proteins), etc. [38, 39]. However, in the
case of Mcl-1 antagonists, this issue has not been suf-
ficiently studied. Therefore, this work aims to con-
duct a detailed study of the possible causes of the
emergence of drug resistance of tumor cells to the
action of selective Mcl-1 antagonists using S63845 as
an example (its derivative S64315/MIK665 is being
studied in the clinic [40]), as well as potential ways
to overcome it.
MATERIALS AND METHODS
Cell lines and culture conditions. HeLa cervi-
cal adenocarcinoma, H23 lung adenocarcinoma, and
SK-N-Be(2)c neuroblastoma cell lines (all obtained
RESISTANCE TO MCL-1 INHIBITION 2011
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
from the Department of Toxicology at Karolinska In-
stitutet, Stockholm, Sweden) were used in this study.
Cells were grown in a CO
2
incubator (5% CO
2
) at 37°C
using a Dulbecco’s Modified Eagle Medium (DMEM)
(#C410p, PanEco, Russia) supplemented with gluta-
mine and glucose (4.5 g/L), 10% bovine calf serum
(#11965092, Gibco, USA), an antibiotic (penicillin,
100 U/mL), and an antifungal agent (streptomycin,
100 U/mL) (#15240062, Gibco). Cells were routinely
checked for the absence of mycoplasma. Cells with
70-80% confluency were used in the experiments.
Cells were counted using a Beckman Z2 Coulter
counter (Beckman Coulter Life Sciences, USA).
Reagents. Following cell cultivation for 24-36 h,
the culture medium was replaced with a fresh medi-
um and the following chemical compounds were add-
ed: Mcl-1 antagonist S63845 (Active Biochem, China),
DNA-damaging agent cisplatin (Teva Pharmaceutical,
Israel), Bcl-2 and Bcl-xL antagonists – Venetoclax
and A1331851, respectively (both from Selleckchem,
USA), P-glycoprotein inhibitors Zosuquidar and Ver-
apamil, GSK3 (Glycogen synthase kinase 3) inhibitor
CHIR99021, ATP synthase inhibitor oligomycin, proto-
nophore carbonyl cyanide-m-chlorophenylhydrazone
(CCCP), inhibitors of electron transport chain (ETC)
complexes I (rotenone) and II (thenoyltrifluoroace-
tone, TTFA), and the MDM2 [Mouse double minute 2
homolog] antagonist Nutlin-3a (all from Sigma, USA).
All drugs were used at concentrations indicated in
the respective figures. Control cells were supplement-
ed with the solvent that corresponds to the used re-
agent.
Mcl-1 downregulation by RNA interference.
After washing with phosphate-buffered saline (#P061,
PBS, PanEco), the cells were incubated with 1 ml of
transfection mixture prepared by mixing Opti-MEM
transfection medium (#31985070, Gibco), 3 μl of Li-
pofectamine RNAiMAX transfection agent (Invitrogen,
USA), and siRNA to Mcl-1 (50 μM) in an amount re-
quired to achieve working concentration (100 nM).
The sense and antisense sequences of the strands of
siRNA to Mcl-1 are 5′-GCATCGAACCATTAGCAGAdTdT-3′
and 5′-TCTGCTAATGGTTCGATGCdTdT-3′. Control siRNA
#1 D-001810-01 (Dharmacon Reagents, UK) was used
as a non-targeting siRNA. Incubation time was 24 h.
Western blot analysis. The cell pellet obtained
after cell detachment by scraping was washed with
cold PBS and centrifuged. Next, cells were resuspend-
ed in 30-60 µl of a RIPA lysis buffer (Bio-Rad, USA)
and incubated for 20 min on ice. After centrifugation
(20,000g, 20 min, 4°C), a portion of the supernatant
was collected to measure protein concentration in
the cell lysates using a Pierce BCA Protein Assay Kit
(#23225, Thermo Fisher Scientific, USA), and samples
for Western blot analysis were prepared from the
remaining supernatant as described previously [41].
Antibodies. Primary antibodies for detection
of the following proteins were used in the experi-
ments: Bcl-2 (sc-7382) from Santa Cruz (USA); PARP
[poly(ADP-ribose) polymerase] (ab74290), tubulin
(ab4074), and vinculin (ab123002) (all from Abcam,
UK); p89 PARP cleaved (#5625), caspase-3 (#9662),
p19/17 caspase-3 cleaved (#9661), Bak (#6947), Bax
(#2772), Bcl-xL (#2764), Mcl-1 (#5453), MDR1 [multi-
drug resistance protein 1] (#12683), GAPDH (#2118),
GSK3a (#4337), and GSK3b (#9315) (all from Cell Sig-
naling, USA). Secondary antibodies used were anti-
mouse (#515-035-062) or anti-rabbit IgG (#111-035-144)
horseradish peroxidase-conjugated antibodies from
Jackson ImmunoResearch (USA). Dilutions of all anti-
bodies were chosen according to the manufacturer’s
recommendations.
The changes in protein levels were determined
using densitometric analysis. Image processing was
performed with ImageJ 1.53t. Protein levels were
normalized to the corresponding gel loading proteins
(tubulin, vinculin, GAPDH); statistical analysis was
performed using the Mann–Whitney U-test.
Flow cytometry. During sample preparation,
cells were trypsinized, washed twice with cold PBS,
and centrifuged (500g, 5 min, 4°C).
(a) Annexin V-FITC [fluorescein isothiocyanate]
(annexin) and propidium iodide (PI) double stain-
ing assay. After resuspending the cell pellet in PBS
(700 µl/1,000,000 cells), approximately 200,000 cells
were removed and added to 200 µl of 1x annexin-
binding buffer (BD Biosciences, USA). Next, cell pop-
ulations were analyzed using BD FACSCanto II flow
cytometer (BD Biosciences, USA) and BD FACSDiva
(BD Biosciences, USA) and FlowJo (FlowJo LLC, USA)
software, as described previously [42]. The percent-
age of dead cells was calculated from all cells that
were stained with annexin (apoptosis), PI (necrosis),
or both dyes (late apoptosis); the percentage of viable
cells was calculated from the cell population that was
not stained with either annexin or PI [43].
(b) SubG1 test. Cells were fixed in a cold 70% eth-
anol solution in PBS at −20°C for 24 h, washed, and
resuspended in PBS. Next, PI (50 μg/mL) and RNase
A (100 μg/mL) were added, and the cells were incu-
bated in the dark at room temperature for 10 min.
The analysis was performed similarly to that for the
annexin and PI double staining (a). The proportion of
cells in the subG1 phase correlates with the intensity
of apoptosis induction and reflects the percentage of
cell death [43, 44]. For both flow cytometry methods,
10,000 cells were analyzed in all samples during ex-
periments.
Real-time polymerase chain reaction (RT-PCR).
Total RNA was isolated from the cell pellet using
TRIzol reagent (#15596018, Thermo Fisher Scientific),
and cDNA was obtained by reverse transcription
PERVUSHIN et al.2012
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
using the MMLV RT kit (Eurogen, Russia) according
to the manufacturer’s instructions. The qPCRmix-HS
SYBR (Eurogen), specific primers for Bcl-2, Bcl-xL, and
the transcription factor TATA-binding protein were
added to the cDNA samples. The primer sequences
are listed in Table S1, the Online Resource 1. Next,
mRNA expression was assessed using the CFX96
Real-Time PCR Detection System (Bio-Rad). Tran-
script analysis was performed using the Pfaffl meth-
od [45]. Bcl-2 and Bcl-xL mRNA concentrations were
normalized to the mRNA level of the TATA-binding
protein.
Cell metabolism analysis. Cells were seeded
in 96-well plates in a standard DMEM culture medi-
um. After reaching 70-80% confluency, the cells were
washed and incubated in an assay medium (DMEM
without phenol red, calf serum, sodium pyruvate,
and glucose) for 1-3 h at 37°C. To analyze respira-
tion, sodium pyruvate (1 mM) and glucose (10 mM)
were added to the same medium. Experiments were
performed in real time using a Seahorse XF analyz-
er (Agilent, USA). To test respiration (the first three
measurements), oligomycin (1 μM), carbonyl cyanide
m-chlorophenylhydrazone (CCCP, 1 μM), and rotenone
(1 μM) were added to the cells. To assess glycolysis,
D-glucose (10 mM), oligomycin (1 μM), and 2-deoxy-
glucose (50 mM) were added to the wells. Data were
normalized to the protein levels in each well.
Clonogenic activity assay. Cells (1000 per well)
were seeded in triplicate in 6-well plates (Nunc, The
Netherlands). After culturing (10-14 days), cells were
washed twice with PBS, fixed with paraformaldehyde
(4% in PBS), and stained with crystal violet (0.5% in
aqueous solution). Plates were visualized using the
ChemiDoc XRS+ system (Bio-Rad) and analyzed using
ImageJ 1.53t.
Gene sequencing. To identify potential mutations
in the BAK1 and MCL1 genes, Sanger sequencing and
next-generation sequencing (NGS) were employed, re-
spectively.
(a) Sanger sequencing. For BAK1 sequencing, ge-
nomic DNA was isolated from the original and Mcl-1
inhibition-resistant HeLa, H23, and SK-N-Be(2)c cell
lines using the QIAamp DNA Mini Kit (#56304, Qia-
gen, Germany). Fragments of exons 2-6 of BAK1 were
amplified by RT-PCR and sequenced using the Sanger
method. The primer sequences are listed in Table S1,
the Online Resource 1.
(b) NGS of MCL1 was performed as described pre-
viously [46].
Cell viability assessment. For the experiments,
cells (5,000) were seeded in 96-well plates. Cell cultiva-
tion and induction were performed using a standard
DMEM culture medium with appropriate reagents.
Cell viability was analyzed 24 h after induction using
the MTS assay or Alamar Blue assay.
(a) MTS assay. After induction, cells were treat-
ed with 20 µl of MTS reagent (CellTiter 96 AQueous
One Solution Cell Proliferation Assay, #G3580, Prome-
ga, USA), incubated for 2.5-3 h at 37°C, and analyzed
spectrophotometrically using Varioskan Flash instru-
ment (Thermo Fisher Scientific) at 480 nm.
(b) Alamar Blue assay. After induction, the cul-
ture medium was replaced with a fresh medium con-
taining 10% Alamar Blue reagent (#DAL1100, Thermo
Fisher Scientific). Samples were incubated for 3-4 h
at 37°C, and fluorescence was measured using
Varioskan Flash instrument (Thermo Fisher Scientif-
ic) at wavelengths of 560 nm (excitation) and 590nm
(emission).
Statistical analysis was performed using the
Mann–Whitney U-test. Data processing and statistical
analysis were performed using Microsoft Excel and
GraphPad Prism 6 (GraphPad Software, USA). Data in
histograms are presented as mean ± standard devia-
tion (n = 4); * p < 0.05.
RESULTS
Generation of cancer cells with acquired re-
sistance to the BH3 mimetic S63845. To study po-
tential mechanisms underlying the development of
acquired resistance to selective Mcl-1 antagonists in
cancer cells, three cell lines of different origins (HeLa
cervical adenocarcinoma, H23 lung adenocarcinoma,
and SK-N-Be(2)c neuroblastoma) were selected based
on their inherent sensitivity to Mcl-1 inhibition [33].
It is worth noting that all cancer cells used in this
study are characterized by impaired functional ac-
tivity of the transcription factor p53. The H23 [47]
and SK-N-Be(2)c [48] cell lines contain mutant p53
protein. In contrast, in HeLa cells, the functional ac-
tivity of the wild-type p53 protein is suppressed due
to the presence of the E6 protein of human papil-
lomavirus (HPV) type 18, which leads to rapid deg-
radation of p53 [49]. The presence of wild-type p53
protein could maximize the effectiveness of BH3-mi-
metics [50]; however, its absence or altered function-
al activity does not prevent the initiation of cancer
cell death upon exposure to these drugs due to their
mechanism of action. Moreover, Venetoclax, which is
approved for clinical use, can also be used as a ther-
apeutic option for patients with CLL and 17p deletion
or TP53 mutation [51].
A “pulse” method was used to generate resistant
cells reproducing effects of chemotherapeutic courses
in patients that determine its clinical significance [52].
This method includes culturing of the original cell
lines with gradually increasing concentrations of the
drug, alternating with drug-free cell culture. Nano-
molar concentrations of S63845 (from 125 to500 nM)
RESISTANCE TO MCL-1 INHIBITION 2013
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 1. Generation of HeLa, H23, and SK-N-Be(2)c cancer cells with acquired resistance to the Mcl-1 antagonist S63845.
HeLa-Res, H23-Res, and SK-N-Be(2)c-Res are cancer cells resistant to S63845 (hereinafter). The figure was created using
Servier Medical Art (https://smart.servier.com/) under a CC BY4.0 license (https://creativecommons.org/licenses/by/4.0/).
were used in the first stage, and micromolar (up to
1 μM for H23 and up to 3 μM for HeLa and SK-N-
Be(2)c) concentrations of this BH3-mimetic were used
in the second stage. Both stages consisted of three
cycles. Each cycle sequentially included incubation
of cells with S63845 for 24 h, culturing cells without
S63845 addition for 96 h, and the reseeding of sur-
viving cells (Fig. 1).
Resistance to Mcl-1 inhibition in the selected
models was tested using two independent approach-
es: Western blot (WB) analysis and flow cytometry.
Both parental and developed cells were treated with
S63845 at concentrations ranging from 250 nM to
3 μM. According to the results of WB analysis, in
all cases, the developed cells were characterized by
lower sensitivity to the BH3-mimetic as compared to
parental cells (Fig. 2a; Fig. S1a; Fig. S2a in the On-
line Resource 1). Cell death was assessed during WB
analysis based on the degree of cleavage of two key
apoptosis markers: the effector caspase-3 and its sub-
strate, the repair protein PARP [43, 53]. The greatest
differences in the response to S63845 were observed
in cells treated with micromolar concentrations (1
and 3 μM) in all lines. In resistant cells, there was a
marked decrease in the cleavage of full-length forms
of caspase-3 and PARP and accumulation of their
fragments – p19/17 caspase-3 and p89 PARP (Fig. 2a;
Fig. S1a; Fig. S2a in the Online Resource 1). Flow cy-
tometry results confirmed the presence of acquired
resistance to S63845 in the derived HeLa (Fig. S1b in
the Online Resource 1), H23 (Fig. S2b in the Online
Resource 1), and SK-N-Be(2)c cells (Fig. 2b). In partic-
ular, upon incubation with S63845 (3 μM), the num-
ber of subG1 fractions was more than 2-fold lower in
resistant SK-N-Be(2)c cells compared to the wild-type
cells (Fig. 2b).
It is worth noting that the increased concentra-
tion of the Mcl-1 antagonist S63845 from 1 to 3 μM
did not influence the level of H23 cell death. More-
over, the reduced sensitivity to S63845 in resistant
H23 cells persisted up to a concentration of 1 μM
BH3-mimetic. In contrast, the use of 3 μM S63845 led
to overcoming this type of resistance, which distin-
guishes this cell model from HeLa and SK-N-Be(2)c
cell lines (Fig. S2, a, b in the Online Resource 1).
Assessment of the contribution of the an-
ti-apoptotic proteins Bcl-xL and Bcl-2 to the devel-
opment of acquired tumor cell resistance to the
BH3-mimetic S63845. We have previously shown that
not only the initial insensitivity but also the acquired
resistance of tumor cells to Mcl-1 inhibition can be
mediated by high levels of the anti-apoptotic protein
Bcl-xL [33]. Here, densitometric analysis of the WB
data revealed that not only could Bcl-xL levels be sig-
nificantly increased in resistant cells compared to pa-
rental cells, as was observed in the HeLa-Res cell line
(Fig. S1c in the Online Resource 1), but also that the
level of another anti-apoptotic protein, Bcl-2, could be
increased, as was observed in SK-N-Be(2)c-Res cells
(Fig. 2c). Moreover, the development of resistance
to Mcl-1 inhibition may not be accompanied by the
changes in the levels of other anti-apoptotic proteins,
as was observed in H23-Res cells (Fig. S2c in Online
Resource 1). Furthermore, in all three cell models,
the Mcl-1 protein level in resistant cells remained
unchanged compared to parental tumor lines (Fig. S3
in the Online Resource 1). It was hypothesized that,
as tumor cell lines develop resistance to Mcl-1 inhi-
bition, cells with increased expression of Bcl-xL and
Bcl-2 protein genes are accumulated compared to the
original cells. The data of RT-PCR analysis of mRNA
levels for these anti-apoptotic proteins confirmed this
PERVUSHIN et al.2014
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 2. Analysis of the efficacy of the BH3-mimetic S63845 (0.25-3μM, 24h) in SK-N-Be(2)c and SK-N-Be(2)c-Res cells. Results
of WB (a), subG1 (b), and densitometric analysis of Bcl-xL and Bcl-2 in SK-N-Be(2)c and SK-N-Be(2)c-Res cells (c). Data are
presented as mean ± standard deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
hypothesis: the increased Bcl-xL and Bcl-2 mRNA lev-
els compared to the original cell lines were observed
in HeLa-Res and SK-N-Be(2)c-Res cells, respectively
(Fig. 3, a, b).
The observed changes in the Bcl-2 protein pro-
file in resistant cells may indicate a reduced role
of Mcl-1 in maintaining their viability compared to
other anti-apoptotic proteins. This was confirmed by
assessing cell death triggered by Mcl-1 suppression
using RNA interference. The WB and flow cytometry
analysis data revealed that two cell models with ac-
quired resistance to S63845, where increased levels
of Bcl-xL (HeLa) or Bcl-2 (SK-N-Be(2)c) were observed,
and were characterized by lower sensitivity to the ge-
netic Mcl-1 suppression (Fig.4,a, b, d,e). At the same
time, compared with the parental H23 line, in H23-
Res cells, where the profile of Bcl-2 family proteins
remained unchanged, the proportion of dead cells
upon Mcl-1 silencing did not change (Fig. 4, c, f).
The results obtained clearly demonstrate a
compensatory phenomenon observed among differ-
ent members of the Bcl-2 family: inhibition of one
RESISTANCE TO MCL-1 INHIBITION 2015
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 3. Analysis of Bcl-xL and Bcl-2 mRNA levels in HeLa and HeLa-Res (a) and SK-N-Be(2)c and SK-N-Be(2)c-Res (b) cells.
Data are presented as mean ± standard deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
anti-apoptotic protein could lead to an increase in
the expression of genes encoding other cell surviv-
al proteins, resulting in the emergence of acquired
resistance [8, 35]. However, the example of H23 cells
clearly illustrates the multifactorial nature of drug
resistance development: an increase in gene expres-
sion encoding Bcl-2 or Bcl-xL proteins is a probable,
but not the only reason, underlying resistance to the
action of selective Mcl-1 antagonists.
Assessing the contribution of P-glycoprotein to
the development of acquired cancer cell resistance
to the BH3-mimetic S63845. The development of
drug resistance could be accompanied by increased
expression of genes encoding multidrug resistance
proteins, such as P-glycoprotein (MDR1). Proteins of
this group are located on the plasma membrane and
mediate the ATP-dependent reverse transport of many
substances from cells [54, 55]. This phenomenon has
also been demonstrated for Mcl-1 antagonists, partic-
ularly for S63845, which was used in this study [56].
To prevent the development of tumor cell re-
sistance to the BH3-mimetic S63845 due to its ex-
port during the development of resistance (Fig. 1),
the MDR1 inhibitor Verapamil (50 μM) was added
to the cells, combined with the Mcl-1 antagonist.
In addition, comparative WB analysis of cell death
was performed in the original and the Mcl-1 inhibi-
tion-resistant cells using a combination of S63845 and
another MDR1 inhibitor, Zosuquidar (Fig. S4 in the
Online Resource 1). MDR1 was undetectable in both
HeLa/H23 and HeLa-Res/H23-Res cells. Furthermore,
Zosuquidar alone did not induce cell death in either
wild-type or resistant HeLa and H23 cells, nor did
it enhance S63845-mediated cell death in these cell
models (Fig. S4, a, b in the Online Resource 1).
In SK-N-Be(2)c neuroblastoma cells, MDR1 was
present in both parental and S63845-resistant cells;
however, its content in the latter cells did not in-
crease, as determined by densitometric analysis. As
in other cell types, Zosuquidar did not induce death
as a single agent, but it equally effectively enhanced
S63845-dependent apoptosis (~2-fold decrease in cell
viability according to the MTS assay) in both SK-N-
Be(2)c and SK-N-Be(2)c-Res cells without overcom-
ing resistance of neuroblastoma cells to the Mcl-1
antagonist (Fig. S4, c-e in the Online Resource 1).
Thus, an increase in MDR1 does not underlie ac-
quired resistance of tumor cells to the BH3-mimetic
S63845.
A search for possible mutations in BAK1
and MCL1 as causes of acquired cancer cell re-
sistance to the BH3-mimetic S63845. Investigation
of the Bcl-2 antagonist Venetoclax revealed, among
other factors, that mutations in the target protein
gene or the Bax effector protein gene could lead
to the development of tumor cell resistance to this
BH3-mimetic [38, 39]. Taking this data into account,
we searched for possible mutations in the Bak ef-
fector protein gene, since Mcl-1 has a higher affin-
ity for Bak, while Bcl-2 preferentially interacts with
Bax [15]. Using Sanger sequencing, it was found that
in two of the three S63845-resistant cell populations
(HeLa-Res and H23-Res), there is a single mutation,
BAK1(NM_001188.4):c.309G>A p.(Thr103=), compared
to the original lines (HeLa and H23, respectively).
This mutation is located in exon 4 of BAK1 and is a
synonymous single-nucleotide substitution that does
not affect the protein structure. No changes in BAK1
were observed in resistant SK-N-Be(2)c neuroblasto-
ma cells.
PERVUSHIN et al.2016
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 4. Analysis of cell death induced by genetic silencing of Mcl-1 using RNA interference. Results of WB(a-c), flow cytom-
etry with annexin and PI double staining (d, f), and subG1 (e) in HeLa/HeLa-Res (a, d), SK-N-Be(2)c/SK-N-Be(2)c-Res (b, e),
and H23/H23-Res (c, f) cells. Transfection period: 24 h. NT (non-target) – non-target siRNA. Data are presented as mean
± standard deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
HeLa/HeLa-Res and H23/H23-Res cell lines were
subsequently examined for mutations in MCL1 using
NGS; however, no changes in MCL1 were detected.
Additionally, not only an increase in the anti-apop-
totic proteins but also a decrease in the proapoptotic
proteins (primarily the Bak and Bax effector proteins)
of the Bcl-2 family could mediate acquired chemo-
therapy resistance, as has been demonstrated, for ex-
ample, after treatment with the monoclonal antibody
drug Rituximab [57]. In our study, WB analysis of
all three models did not reveal any significant chang-
es in Bak and Bax protein levels in resistant cells
compared to parental cells (Fig. S5 in the Online Re-
source 1).
Comparative assessment of proliferative ac-
tivity and metabolism in parental and resistant
to the BH3-mimetic S63845 cells. Malignant clones
are capable of altering their proliferative activity and
developing various metabolic adaptations during the
development of resistance to chemotherapy. We ini-
tially analyzed potential changes in proliferation rate
in cell populations resistant to Mcl-1 suppression us-
ing a clonogenic assay. In all three models, there was
no statistically significant change in the proliferation
RESISTANCE TO MCL-1 INHIBITION 2017
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 5. Assessment of proliferative activity. Results of clonogenic assay for parental and S63845-resistant HeLa (a), H23 (b),
and SK-N-Be(2)c (c) cells. Data are presented as mean ± standard deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
Fig. 6. Basal glycolysis (a-c) and respiration (d-f) in parental and S63845-resistant HeLa (a, d), H23 (b, e), and SK-N-Be(2)
c(c,f) cells. Data are presented as mean ±standard deviation, n = 4(e); n=3(a-d,e); *p<0.05, n.s. – not significant (U-test).
rate of resistant cells compared to the wild-type cells
(Fig. 5, a-c).
Next, parental and S63845-resistant cells from
all three lines were examined for possible metabolic
changes using the Seahorse technology (Fig. 6).
In all three experimental models, basal glycolytic
activity was unchanged in resistant clones compared
to the original cell lines (Fig. 6, a-c). Basal respiration
also remained unchanged in the HeLa/HeLa-Res and
SK-N-Be(2)c/SK-N-Be(2)c-Res pairs (Fig. 6, d, f). How-
ever, a significant increase in cellular respiration
was observed in resistant H23 cells compared to the
parental H23 line (Fig. 6e), which likely contributes
to the development of acquired resistance to Mcl-1
inhibition.
Analysis of the efficiency of cisplatin and other
BH3-mimetics in overcoming cancer cell resistance
to Mcl-1 inhibition. To overcome acquired cancer cell
resistance to the BH3-mimetic S63845, it is worthwhile
to combine this compound with other chemothera-
peutic agents. We previously demonstrated that com-
bining the DNA-damaging agent cisplatin or selective
PERVUSHIN et al.2018
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 7. Evaluation of the efficacy of cisplatin (a, c) and Venetoclax (b, d) in overcoming resistance of SK-N-Be(2)c cells to
S63845. WB (a, b), MTS assay (c), and subG1 (d) results. Incubation period: 24 h. Data are presented as mean ± standard
deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
antagonists of the anti-apoptotic proteins Bcl-2/Bcl-xL
with S63845 can effectively overcome cancer cell
resistance to the latter in HeLa and H23 cells [33].
In our work, similar results were obtained in SK-N-
Be(2)c cells (Fig. 7; Fig. S6 in the Online Resource 1).
Thus, cisplatin (Fig. 7, a, c), Venetoclax (Fig. 7, b, d),
and the BH3-mimetic to Bcl-xL A1331851 (Fig. S6, a-c
in the Online Resource 1), when used in combination
with S63845, enhanced the cytotoxicity of the latter
and led to the comparable induction of cell death
in both parental and Mcl-1 inhibition-resistant neu-
roblastoma cells, overcoming this type of resistance.
Furthermore, as we have previously shown in other
models [33], co-inhibition of Mcl-1 and Bcl-xL using
the corresponding BH3-mimetics at low concentra-
tions (100 nM) led to the pronounced activation of
apoptosis in SK-N-Be(2)c and SK-N-Be(2)c-Res cells af-
ter just 6 h (Fig. S6a in the Online Resource 1).
Analysis of the efficiency of the GSK3 inhib-
itor to overcome cancer cell resistance to Mcl-1
inhibition. GSK3 is a serine–threonine protein ki-
nase involved in regulating multiple intracellular
signaling pathways. For example, in addition to its
primary function of controlling glycogen metabolism,
GSK3 plays a key role in carcinogenesis by activating
the NF-κB signaling pathway, one of the key path-
ways that promotes cell survival. Activation of the
NF-κB-dependent cascade could lead to inhibition of
apoptosis, promote survival of malignant clones, and
underlie resistance to chemotherapy. Therefore, GSK3
is a promising target for anticancer therapy [58-60].
Earlier studies have demonstrated the ability of GSK3
to regulate Mcl-1 [61, 62]. Therefore, a combination
of inhibitors of both proteins could be effective in
the induction of cancer cell death. Here, we assessed
the feasibility of using CHIR99021, an inhibitor of
the two major GSK3 isoforms GSK3α and GSK3β, to
overcome cancer cell resistance to the BH3-mimetic
S63845 (Fig. 8, a, b).
WB and flow cytometry analyses revealed that
CHIR99021, used as a single agent, did not induce
cell death in either parental or S63845-resistant HeLa
cells. However, it did enhance S63845-mediated cell
death in wild-type and, especially, in resistant HeLa
clones, thereby overcoming resistance of this line to
Mcl-1 inhibition (Fig. 8). Similar results were observed
in H23/H23-Res (Fig. S7,a,c in the Online Resource1)
and SK-N-Be(2)c/SK-N-Be(2)c-Res (Fig. S7, b, d in the
Online Resource 1) cells, as determined by WB and
the MTS assay.
RESISTANCE TO MCL-1 INHIBITION 2019
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 8. Evaluation of the efficacy of the GSK3 inhibitor CHIR99021 in overcoming resistance of HeLa cells to S63845. Results
of WB (a) and flow cytometry with annexin and PI double staining (b). Incubation period: 24 h. Data are presented as
mean ± standard deviation, n = 4; * p < 0.05, n.s. – not significant (U-test).
Analysis of the efficiency of MDM2 antagonist
and inhibitors of cellular respiration in overcom-
ing resistance of H23 cells to Mcl-1 inhibition. The
transcription factor p53 is a critical tumor suppressor;
however, its functional activity is typically suppressed
in tumor cells due to either MDM2, a negative p53
regulator, or mutations in the structure of the p53
gene. In the first case, MDM2 antagonists, which
have been actively studied in clinical trials over the
past few years, could be utilized. In the second case,
reactivators of mutant p53 could be used [63-65].
However, it has been found that MDM2 antagonists
can also exhibit antitumor activity in cells regardless
of the presence of p53 and its status [66-68]. Here,
using the H23 cells, we found that the MDM2 antago-
nist Nutlin-3a, progenitor of this group of compounds
[69], alone did not cause death of the original and
S63845-resistant cells. However, it enhanced the cy-
totoxicity of S63845 in resistant clones, thereby over-
coming their resistance to Mcl-1 inhibition (Fig. S8 in
the Online Resource 1).
Next, we analyzed the feasibility of targeting cel-
lular respiration to overcome the resistance of H23
cells to Mcl-1 inhibition. For this purpose, the ATP
synthase inhibitor oligomycin, the protonophore CCCP,
and inhibitors of ETC complexes I (rotenone) and II
(TTFA) were used (Fig. 9, Fig. S9 in the Online Re-
source 1).
According to the results of WB analysis and
the Alamar Blue assay, we found that only rotenone
alone reduced the viability of the original H23 cells
(Fig. 9, b, d). At the same time, rotenone, TTFA, and
oligomycin statistically significantly reduced viability
of resistant H23 cells (by 10-25%) according to the
Alamar Blue assay; however, according to the WB
results, they did not cause cleavage of the apoptotic
markers (caspase-3 and/or PARP protein) (Fig. 9,
Fig. S9 in the Online Resource 1). Since the Alamar
Blue assay, like the MTT/MTS tests, evaluates not only
the direct viability of cells, but also their metabol-
ic and proliferative activities [70], it can be assumed
that the above-mentioned agents in H23-Res cells
suppressed their metabolism, but did not cause ini-
tiation of their death at the indicated concentrations.
As noted above, the level of respiration in resistant
H23 cells is higher than in parental H23 cells, and,
therefore, suppression of respiration affected their
ability to proliferate. In combination with the Mcl-1
antagonist S63845, all compounds, except CCCP, slight-
ly enhanced cytotoxicity in parental H23 cells and
more significantly increased S63845-induced death in
H23-Res cells (Fig. 9, Fig. S9 in the Online Resource 1),
thereby overcoming resistance to Mcl-1 inhibition
and confirming the contribution of metabolic alter-
ations to this type of resistance.
DISCUSSION
The development of acquired drug resistance to
chemotherapeutic agents is one of the most important
problems in oncology, as decreased sensitivity of ma-
lignant clones to therapy leads to cancer progression
and subsequent death of patients. As previously noted,
PERVUSHIN et al.2020
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Fig. 9. Evaluation of the efficacy of TTFA (a,c) and rotenone (b,d) in overcoming resistance of H23 cells to S63845. Results
of WB (a,b) and Alamar Blue assay(c,d). Incubation period: 24 h. Data are presented as mean ± standard deviation, n=4;
* p < 0.05, n.s. – not significant (U-test).
inhibition of anti-apoptotic proteins of the Bcl-2 fam-
ily represents a promising approach for treating can-
cer patients. Approximately 20 years ago, the first
non-selective BH3-mimetic, ABT-737, was developed,
followed by the development of selective antagonists
of Bcl-2, Bcl-xL, and Mcl-1. However, limited effica-
cy and observed side effects have not allowed these
BH3-mimetics, except for Venetoclax, to be approved
for clinical use to date [9]. It should be noted that
various approaches to inhibit anti-apoptotic proteins
of the Bcl-2 family, including Mcl-1, are in progress
[10]. For example, in 2025, a study was launched
to evaluate the new compound S227928 in patients
with myelodysplastic syndrome, acute myeloid leu-
kemia (AML), and chronic myelomonocytic leukemia,
both as an individual agent and in combination with
Venetoclax (NCT06563804). S227928 is the Mcl-1 an-
tagonist S64315/MIK665 conjugated to a monoclonal
antibody that targets the membrane protein CD74,
the gene of which is actively expressed in AML cells
[40]. Establishing potential reasons for resistance to
Mcl-1 inhibition and identifying approaches to over-
come it may contribute to the successful completion
of clinical trials for this group of compounds in the
future. To generate cell populations resistant to Mcl-1
inhibition, we selected three cell lines of different or-
igins (HeLa, H23, and SK-N-Be(2)c), which are high-
ly sensitive to the suppression of this protein [33].
There are two main strategies for obtaining resistant
clones. The first involves culturing tumor cells with
high concentrations of chemotherapeutic agents for
several months. In this case, the clones most adapted
to therapy survive; however, this scheme could lead
to artifactual results, since such conditions are not
present in the patient’s body. Another strategy in-
volves alternating short periods of incubation of the
original cells with gradually increasing concentra-
tions of anticancer agents and drug-free periods. This
“pulse” approach was chosen in our study, as it mim-
ics the course of treatment for cancer patients [52,
70, 71].
Subsequent analysis revealed that in all three
cell lines, the observed resistance to the Mcl-1 an-
tagonist S63845 is not associated with MDR1-linked
drug efflux systems. In two of the three cell models
of this type of resistance, clones with increased ex-
pression of the genes encoding other anti-apoptotic
proteins, Bcl-2 in SK-N-Be(2)c cells or Bcl-xL in HeLa
RESISTANCE TO MCL-1 INHIBITION 2021
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
cells, were generated. Moreover, these resistant cells
became less sensitive to death triggered by the genet-
ic silencing of Mcl-1. Thus, survival of resistant SK-
N-Be(2)c and HeLa cells depends less on Mcl-1 and
more on other Bcl-2 family anti-apoptotic proteins,
clearly demonstrating the aforementioned compen-
satory phenomenon of these proteins. Earlier studies
have revealed that the loss of cancer cell sensitivity
to Venetoclax is accompanied by increased levels of
the Bcl-xL and Mcl-1 proteins [8, 20, 35].
While studying the mechanisms of malignant
clones’ resistance to Venetoclax, it was suggested that
the Bcl-2 protein could mutate and alter its struc-
ture, thus preventing binding of Venetoclax to its
target and neutralizing the anti-apoptotic activity
of Bcl-2 [8, 72]. Novel Bcl-2 antagonists, triggering
cell death in Venetoclax-resistant cells, have already
been developed [73]. Furthermore, mutations could
occur in the Bax protein gene, which primarily in-
teracts with Bcl-2, disrupting functional activity of
the effector protein and blocking initiation of apop-
tosis [38]. In our study, no mutations were detected
that affected the structure and function of either
Mcl-1 or its proapoptotic partner Bak. Currently, lit-
tle data on mutations in the Mcl-1 structure have
been found that could interfere with its inhibition
by selective antagonists. Mutations in the MCL1 and
BAK genes, which are rare in tumors [35, 74], ap-
pear to be an unlikely cause of this type of resis-
tance; however, this issue requires further clarifica-
tion [35].
The presence of drug resistance is often charac-
terized not only by the decrease in cell death activa-
tion but also by metabolic adaptations and changes
in proliferation rates, and both modifications could
change in opposite directions. Indeed, previously we
found that resistance to cisplatin is accompanied by
a decrease in proliferation and metabolism of mutant
clones [70], while the opposite pattern was observed
in the case of resistance to the MDM2 antagonist
RG7388 [46]. In other models of cisplatin resistance,
resistant clones could switch either to the predomi-
nantly anaerobic type of metabolism, increasing gly-
colysis and slowing oxidative phosphorylation, or to
an aerobic type with opposite metabolic changes [75].
Here, proliferative activity of wild-type and S63845-re-
sistant cells has not changed, and no changes in gly-
colysis occurred. Only in resistant H23 cells was an
increase in cellular respiration detected. Since the
profile of anti-apoptotic proteins of the Bcl-2 family
remained unchanged, the detected metabolic adapta-
tion could determine resistance to Mcl-1 inhibition.
Its importance for the survival of H23 cells was con-
firmed by us using various inhibitors of cellular res-
piration, which enhanced the cytotoxicity of S63845
and led to overcoming the resistance to its action.
Finally, combining various chemotherapeutic
agents is a rational approach in clinical practice, al-
lowing both to prevent the development of acquired
resistance by using reduced concentrations of each
drug, thereby reducing the toxic effect on healthy
cells, and to overcome the formed resistance by
enhancing the cytotoxic effect compared to mono-
therapy. A promising direction is the combined use
of various BH3-mimetics, which could reduce the
above-mentioned compensatory phenomenon. In ad-
dition, combinations of BH3-mimetics and DNA-dam-
aging agents, or MDM2 antagonists, have the poten-
tial for high therapeutic effect [35]. The feasibility of
combining S63845 with Venetoclax, a Bcl-xL antag-
onist, cisplatin, and MDM2 antagonists was demon-
strated by us in various experimental models, both
in this study and in several previous studies [33, 46].
For example, it has recently been shown that the
MDM2 antagonist RG7388 could induce both apopto-
sis and pyroptosis, mediated by, among other factors,
reactive oxygen species (ROS) in H23 cells expressing
mutant p53 protein [76]. Since we have demonstrat-
ed that H23-Res cells exhibit increased cellular res-
piration, potentially resulting in higher ROS levels,
it is likely that the mechanism described above for
RG7388 explains the effect of overcoming H23 cell
resistance to S63845 when combined with the MDM2
antagonist Nutlin-3a. In addition, we demonstrated for
the first time the effectiveness of the combination of
selective GSK3 and Mcl-1 antagonists in overcoming
cancer cell resistance to the action of the latter.
CONCLUSION
Three cell types, HeLa, H23, and SK-N-Be(2)c, were
used to study possible reasons for acquired resistance
of cancer cells to the BH3-mimetic S63845, the Mcl-1
antagonist. Various potential mechanisms for avoid-
ing cell death during Mcl-1 inhibition were identified
in resistant populations: increased expression of the
Bcl-xL or Bcl-2 genes was found in HeLa and SK-N-
Be(2)c cells, respectively, and no changes in the levels
of anti-apoptotic Bcl-2 family proteins were observed
in H23 cells. However, an increased cellular respira-
tion was detected in H23 cells, clearly demonstrating
multifactorial mechanisms underlying acquired drug
resistance. No mutations that could alter the structure
or function of Mcl-1 or its proapoptotic partner, Bak,
were found in all selected cell lines. Cell proliferative
activity was unchanged in S63845-resistant cell lines,
and metabolic adaptations were detected only in H23
cells. Use of the MDM2 antagonist Nutlin-3a or inhibi-
tors of cellular respiration (oligomycin, rotenone, and
TTFA) enhanced cytotoxicity of S63845 in resistant H23
cells, which led to overcoming resistance to its action.
PERVUSHIN et al.2022
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Finally, in addition to combining S63845 with cispla-
tin or BH3 mimetics, the feasibility of combining the
Mcl-1 antagonist with the GSK3 inhibitor CHIR99021
was demonstrated in all models.
Abbreviations
Bak Bcl-2 homologous antagonist killer
Bax Bcl-2-associated X protein
Bcl-2 B-cell leukemia/lymphoma protein-2
BH-domain Bcl-2 homology domain
CCCP carbonyl cyanide-m-chlorophenylhy-
drazone
FITC fluorescein isothiocyanate
GSK3 glycogen synthase kinase 3
MDM2 mouse double minute 2 homolog
Mcl-1 myeloid cell leukemia protein-1
MDR1 multidrug resistance protein 1
MOMP mitochondrial outer membrane perme-
abilization
NGS next-generation sequencing
p19/17
caspase-3
catalytically active fragments of effec-
tor caspase-3
PARP poly(ADP-ribose) polymerase
p89 PARP cleaved fragment of the PARP protein
PCR polymerase chain reaction
TTFA thenoyltrifluoroacetone
Supplementary information
The online version contains supplementary material
available at https://doi.org/10.1134/S0006297925602710.
Contributions
N. V. Pervushin, B. Zhivotovsky, and G. S. Kopeina
conceived and supervised the study; N. V. Pervushin,
B. Y. Valdez Fernandez, V. V. Senichkin, M. A. Yaprynt-
seva, and V. S. Pavlov performed the experiments;
N. V. Pervushin, V. V. Senichkin, B. Zhivotovsky, and
G. S. Kopeina discussed the study results; N. V. Per-
vushin wrote the text and prepared the figures;
B. Zhivotovsky and G. S. Kopeina edited the text.
Funding
This work was supported by a grant from the
non-commercial organization “Russian Science Foun-
dation” (project no.23-74-30006). Work in the authors’
laboratories (for B. Zhivotovsky) was also supported
by the Swedish (222013) and Stockholm (181301) can-
cer foundations.
Ethics approval and consent to participate
This work does not contain any studies involving
human and animal subjects.
Conflict of interest
The authors of this work declare that they have
noconflicts of interest.
REFERENCES
1. Hanahan,D., and Weinberg, R.A. (2011) Hallmarks of
cancer: the next generation, Cell, 144, 646-674, https://
doi.org/10.1016/j.cell.2011.02.013.
2. Hanahan, D. (2022) Hallmarks of cancer: new
dimensions, Cancer Discov., 12, 31-46, https://
doi.org/10.1158/2159-8290.CD-21-1059.
3. Mustafa, M., Ahmad, R., Tantry, I. Q., Ahmad, W.,
Siddiqui, S., Alam, M., Abbas, K., Moinuddin, Has-
san, M.I., Habib,S., and Islam,S. (2024) Apoptosis: a
comprehensive overview of signaling pathways, mor-
phological changes, and physiological significance
and therapeutic implications, Cells, 13, 1838, https://
doi.org/10.3390/cells13221838.
4. Elmore,S. (2007) Apoptosis: a review of programmed
cell death, Toxicol. Pathol., 35, 495-516, https://
doi.org/10.1080/01926230701320337.
5. Moyer, A., Tanaka, K., and Cheng, E. H. (2025) Apop-
tosis in cancer biology and therapy, Annu. Rev.
Pathol., 20, 303-328, https://doi.org/10.1146/annurev-
pathmechdis-051222-115023.
6. Czabotar, P. E., Lessene, G., Strasser, A., and Adams,
J. M. (2014) Control of apoptosis by the Bcl-2 pro-
tein family: implications for physiology and thera-
py, Nat. Rev. Mol. Cell Biol., 15, 49-63, https://doi.org/
10.1038/nrm3722.
7. Czabotar, P. E., and Garcia-Saez, A. J. (2023) Mecha-
nisms of Bcl-2 family proteins in mitochondrial apop-
tosis, Nat. Rev. Mol. Cell Biol., 24, 732-748, https://
doi.org/10.1038/s41580-023-00629-4.
8. Senichkin, V. V., Pervushin, N. V., Zuev, A. P., Zhi-
votovsky, B., and Kopeina, G. S. (2020) Targeting
Bcl-2 family proteins: what, where, when? Biochem-
istry (Moscow), 85, 1210-1226, https://doi.org/10.1134/
S0006297920100090.
9. Croce, C. M., Vaux, D., Strasser, A., Opferman, J. T.,
Czabotar, P. E., and Fesik, S.W. (2025) The Bcl-2 pro-
tein family: from discovery to drug development, Cell
Death Differ., 32, 1369-1381, https://doi.org/10.1038/
s41418-025-01481-z.
10. Vogler, M., Braun, Y., Smith, V. M., Westhoff, M.-A.,
Pereira, R. S., Pieper, N. M., Anders, M., Callens, M.,
Vervliet, T., Abbas, M., Macip, S., Schmid, R.,
Bultynck, G., and Dyer, M. J. (2025) The BCL2 family:
from apoptosis mechanisms to new advances in tar-
geted therapy, Signal Transduct. Targeted Ther., 10,
91, https://doi.org/10.1038/s41392-025-02176-0.
11. Adams, J. M., and Cory, S. (1998) The Bcl-2 protein
family: arbiters of cell survival, Science, 281, 1322-
1326, https://doi.org/10.1126/science.281.5381.1322.
12. Burlacu, A. (2003) Regulation of apoptosis by Bcl-2
family proteins, J.Cell. Mol. Med., 7, 249-257, https://
doi.org/10.1111/j.1582-4934.2003.tb00225.x.
13. Westphal, D., Kluck, R. M., and Dewson, G. (2014)
Building blocks of the apoptotic pore: how Bax and
RESISTANCE TO MCL-1 INHIBITION 2023
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
Bak are activated and oligomerize during apoptosis,
Cell Death Differ., 21, 196-205, https://doi.org/10.1038/
cdd.2013.139.
14. Peña-Blanco,A., and García-Sáez, A.J. (2018) Bax, Bak
and beyond – mitochondrial performance in apop-
tosis, FEBS J., 285, 416-431, https://doi.org/10.1111/
febs.14186.
15. Kale, J., Osterlund, E. J., and Andrews, D. W. (2018)
Bcl-2 family proteins: changing partners in the dance
towards death, Cell Death Differ., 25, 65-80, https://
doi.org/10.1038/cdd.2017.186.
16. Glab, J. A., Mbogo, G. W., and Puthalakath, H. (2017)
BH3-only proteins in health and disease, Int. Rev.
Cell Mol. Biol., 328, 163-196, https://doi.org/10.1016/
bs.ircmb.2016.08.005.
17. Doerflinger,M., Glab, J.A., and Puthalakath,H. (2015)
BH3-only proteins: a 20-year stock-take, FEBS J.,
282, 1006-1016, https://doi.org/10.1111/febs.13190.
18. Pervushin, N. V., Nilov, D. K., Zhivotovsky, B., and
Kopeina, G. S. (2025) Bcl-2 modifying factor (Bmf):
“a mysterious stranger” in the Bcl-2 family proteins,
Cell Death Differ., https://doi.org/10.1038/s41418-
025-01562-z.
19. Pervushin, N. V., Kopeina, G. S., and Zhivotovsky, B.
(2023) Bcl-B: an “unknown” protein of the Bcl-2 fam-
ily, Biol. Direct, 18, 69, https://doi.org/10.1186/s13062-
023-00431-4.
20. Pervushin, N.V., Senichkin, V.V., Zhivotovsky,B., and
Kopeina, G. S. (2020) Mcl-1 as a “barrier” in cancer
treatment: can we target it now? Int. Rev. Cell Mol. Biol.,
351, 23-55, https://doi.org/10.1016/bs.ircmb.2020.01.002.
21. Kaloni, D., Diepstraten, S. T., Strasser, A., and Kelly,
G. L. (2023) Bcl-2 protein family: attractive targets
for cancer therapy, Apoptosis, 28, 20-38, https://
doi.org/10.1007/s10495-022-01780-7.
22. Garner, T. P., Lopez, A., Reyna, D. E., Spitz, A. Z.,
and Gavathiotis, E. (2017) Progress in targeting the
Bcl-2 family of proteins, Curr. Opin. Chem. Biol., 39,
133-142, https://doi.org/10.1016/j.cbpa.2017.06.014.
23. Cavallo, M. R., Yo, J. C., Gallant, K. C., Cunanan, C. J.,
Amirfallah,A., Daniali,M., Sanders, A.B., Aplin, A.E.,
Pribitkin, E.A., and Hartsough, E.J. (2024) Mcl-1 me-
diates intrinsic resistance to RAF inhibitors in mutant
BRAF papillary thyroid carcinoma, Cell Death Discov.,
10, 175, https://doi.org/10.1038/s41420-024-01945-0.
24. Dolnikova,A., Kazantsev,D., Klanova,M., Pokorna,E.,
Sovilj, D., Kelemen, C. D., Tuskova, L., Hoferkova, E.,
Mraz,M., Helman,K., Curik,N., Machova Polakova,K.,
Andera, L., Trneny, M., and Klener, P. (2024) Block-
age of Bcl-xL overcomes venetoclax resistance across
BCL
2+
lymphoid malignancies irrespective of BIM sta-
tus, Blood Adv., 8, 3532-3543, https://doi.org/10.1182/
bloodadvances.2024012906.
25. Townsend, P. A., Kozhevnikova, M.V., Cexus, O.N. F.,
Zamyatnin, A.A., and Soond, S.M. (2021) BH3-mimet-
ics: recent developments in cancer therapy, J. Exp.
Clin. Cancer Res., 40, 355, https://doi.org/10.1186/
s13046-021-02157-5.
26. Seymour,J. (2019) Venetoclax, the first Bcl-2 inhibitor
for use in patients with chronic lymphocytic leuke-
mia, Clin. Adv. Hematol. Oncol., 17, 440-443.
27. Brinkmann,K., McArthur,K., Malelang,S., Gibson,L.,
Tee, A., Elahee Doomun, S. N., Rowe, C. L., Arand-
jelovic, P., Marchingo, J. M., D’Silva, D., Bachem, A.,
Monard, S., Whelan, L. G., Dewson, G., Putoczki,
T. L., Bouillet, P., Fu, N. Y., Brown, K. K., Kueh, A. J.,
Wimmer, V. C., Herold, M. J., Thomas, T., Voss, A. K.,
and Strasser, A. (2025) Relative importance of the
anti-apoptotic versus apoptosis-unrelated functions
of Mcl-1 in vivo, Science, 389, 1003-1011, https://
doi.org/10.1126/science.adw1836.
28. Perciavalle, R. M., Stewart, D. P., Koss, B., Lynch, J.,
Milasta, S., Bathina, M., Temirov, J., Cleland, M. M.,
Pelletier, S., Schuetz, J. D., Youle, R. J., Green, D. R.,
and Opferman, J. T. (2012) Anti-apoptotic Mcl-1 lo-
calizes to the mitochondrial matrix and couples mi-
tochondrial fusion to respiration, Nat. Cell Biol., 14,
575-583, https://doi.org/10.1038/ncb2488.
29. Senichkin, V. V., Streletskaia, A. Y., Gorbunova, A. S.,
Zhivotovsky, B., and Kopeina, G. S. (2020) Saga of
Mcl-1: regulation from transcription to degradation,
Cell Death Differ., 27, 405-419, https://doi.org/10.1038/
s41418-019-0486-3.
30. Senichkin, V. V., Streletskaia, A. Y., Zhivotovsky, B.,
and Kopeina, G. S. (2019) Molecular comprehen-
sion of Mcl-1: from gene structure to cancer ther-
apy, Trends Cell Biol., 29, 549-562, https://doi.org/
10.1016/j.tcb.2019.03.004.
31. Pervushin, N. V., Senichkin, V. V., Kapusta, A. A.,
Gorbunova, A. S., Kaminskyy, V. O., Zhivotovsky, B.,
and Kopeina, G. S. (2020) Nutrient deprivation pro-
motes Mcl-1 degradation in an autophagy-indepen-
dent manner, Biochemistry (Moscow), 85, 1235-1244,
https://doi.org/10.1134/S0006297920100119.
32. Srivastava, S., Sekar, G., Ojoawo, A., Aggarwal, A.,
Ferreira, E., Uchikawa, E., Yang, M., Grace, C. R.,
Dey,R., Lin, Y.-L., Guibao, C.D., Jayaraman,S., Mukher-
jee,S., Kossiakoff, A.A., Dong, B., Myasnikov, A., and
Moldoveanu,T. (2025) Structural basis of BAK seques-
tration by Mcl-1 in apoptosis, Mol. Cell, 85, 1606-1623.
e10, https://doi.org/10.1016/j.molcel.2025.03.013.
33. Senichkin, V. V., Pervushin, N. V., Zamaraev, A. V.,
Sazonova, E. V., Zuev, A. P., Streletskaia, A. Y.,
Prikazchikova, T. A., Zatsepin, T. S., Kovaleva, O. V.,
Tchevkina, E. M., Zhivotovsky, B., and Kopeina,
G. S. (2021) Bak and Bcl-xL participate in regulating
sensitivity of solid tumor derived cell lines to Mcl-1
inhibitors, Cancers, 14, 181, https://doi.org/10.3390/
cancers14010181.
34. Gomez-Bougie, P., Ménoret, E., Juin, P., Dousset, C.,
Pellat- Deceunynck,C., and Amiot,M. (2011) Noxa con-
trols Mule-dependent Mcl-1 ubiquitination through
PERVUSHIN et al.2024
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
the regulation of the Mcl-1/USP9X interaction, Bio-
chem. Biophys. Res. Commun., 413, 460-464, https://
doi.org/10.1016/j.bbrc.2011.08.118.
35. Tantawy, S. I., Timofeeva, N., Sarkar, A., and
Gandhi, V. (2023) Targeting Mcl-1 protein to treat
cancer: opportunities and challenges, Front. On-
col., 13, 1226289, https://doi.org/10.3389/fonc.2023.
1226289.
36. Mason, K.D., Carpinelli, M.R., Fletcher, J.I., Collinge,
J. E., Hilton, A. A., Ellis, S., Kelly, P. N., Ekert, P. G.,
Metcalf, D., Roberts, A. W., Huang, D. C. S., and Kile,
B. T. (2007) Programmed anuclear cell death delim-
its platelet life span, Cell, 128, 1173-1186, https://
doi.org/10.1016/j.cell.2007.01.037.
37. Vasan,N., Baselga,J., and Hyman, D.M. (2019) A view
on drug resistance in cancer, Nature, 575, 299-309,
https://doi.org/10.1038/s41586-019-1730-1.
38. Moujalled, D.M., Brown, F.C., Chua, C.C., Dengler, M.A.,
Pomilio, G., Anstee, N. S., Litalien, V., Thompson, E.,
Morley, T., MacRaild, S., Tiong, I. S., Morris, R.,
Dun,K., Zordan,A., Shah,J., Banquet,S., Halilovic,E.,
Morris, E., Herold, M. J., Lessene, G., Adams, J. M.,
Huang, D. C. S., Roberts, A. W., Blombery, P., and
Wei, A. H. (2023) Acquired mutations in BAX confer
resistance to BH3-mimetic therapy in acute myeloid
leukemia, Blood, 141, 634-644, https://doi.org/10.1182/
blood.2022016090.
39. Zielonka, K., and Jamroziak, K. (2024) Mechanisms
of resistance to venetoclax in hematologic malig-
nancies, Adv. Clin. Exp. Med., 33, 1421-1433, https://
doi.org/10.17219/acem/181145.
40. Maragno, A.-L., Seiss, K., Newcombe, R., Mistry, P.,
Schnell, C. R., Von Arx, F., Koenig, J., Malamas, A. S.,
Le Toumelin-Braizat, G., Bresson, L., Rocchetti, F.,
Demarles, D., Kurth, E., Renteria, L., Hainzl, D.,
Engelhardt, V., Liot, C., Madelain, V., Kostova, V.,
Starck, J.-B., Valour, D., Franzetti, G.-A., Colland, F.,
Vostiarova,H., Tschantz, W.R., Bachmann,N., Palacio-
Ramirez, S., Burger, M. T., Campbell, J. M., Chen, Z.,
Koenig,R., McNeill,E., Mo,R., Nakajima,K., Palermo,
M. G., Shen, Y., Yu, B., Zambrowski, M., Zhang, A.,
Zecri, F., Broniscer, A., Geneste, O., Horton, K., and
D’Alessio, J.A. (2024) S227928: A novel anti-CD74 ADC
with Mcl-1 inhibitor payload for the treatment of
acute myeloid leukemia (AML) and other hematologic
malignancies, Blood, 144, 1381, https://doi.org/10.1182/
blood-2024-210048.
41. Gongalsky, M. B., Tsurikova, U. A., Kudryavtsev,
A. A., Pervushin, N. V., Sviridov, A. P., Kumeria, T.,
Egoshina, V. D., Tyurin-Kuzmin, P. A., Naydov,
I. A., Gonchar, K. A., Kopeina, G. S., Andreev, V. G.,
Zhivotovsky, B., and Osminkina, L. A. (2025) Amphi-
philic photoluminescent porous silicon nanoparticles
as effective agents for ultrasound-amplified cancer
therapy, ACS Appl. Mater. Interf., 17, 374-385, https://
doi.org/10.1021/acsami.4c15725.
42. Gongalsky, M. B., Pervushin, N. V., Maksutova, D. E.,
Tsurikova, U. A., Putintsev, P. P., Gyuppenen, O. D.,
Evstratova, Y. V., Shalygina, O. A., Kopeina, G. S.,
Kudryavtsev, A. A., Zhivotovsky, B., and Osminkina,
L. A. (2021) Optical monitoring of the biodegrada-
tion of porous and solid silicon nanoparticles, Nano-
materials (Basel, Switzerland), 11, 2167, https://
doi.org/10.3390/nano11092167.
43. Crowley, L.C., Marfell, B.J., Scott, A.P., Boughaba, J.A.,
Chojnowski, G., Christensen, M. E., and Waterhouse,
N. J. (2016) Dead cert: measuring cell death, Cold
Spring Harb. Protocols, 2016, pdb.top070318, https://
doi.org/10.1101/pdb.top070318.
44. Plesca, D., Mazumder, S., and Almasan, A. (2008)
DNA damage response and apoptosis, Methods
Enzymol., 446, 107-122, https://doi.org/10.1016
/S0076-6879(08)01606-6.
45. Pfaffl, M. W. (2001) A new mathematical model for
relative quantification in real-time RT-PCR, Nucleic
Acids Res., 29, e45, https://doi.org/10.1093/nar/29.9.e45.
46. Pervushin, N. V., Nilov, D. K., Pushkarev, S. V.,
Shipunova, V. O., Badlaeva, A. S., Yapryntseva, M. A.,
Kopytova, D. V., Zhivotovsky, B., and Kopeina, G. S.
(2024) BH3-mimetics or DNA-damaging agents in
combination with RG7388 overcome p53 mutation-
induced resistance to MDM2 inhibition, Apoptosis, 29,
2197-2213, https://doi.org/10.1007/s10495-024-02014-8.
47. Leroy,B., Girard,L., Hollestelle,A., Minna, J.D., Gazdar,
A.F., and Soussi, T. (2014) Analysis of TP53 mutation
status in human cancer cell lines: a reassessment,
Hum. Mutat., 35, 756-765, https://doi.org/10.1002/
humu.22556.
48. Al-Ghabkari, A., and Narendran, A. (2019) In vitro
characterization of a potent p53-MDM2 Inhibitor,
RG7112 in neuroblastoma cancer cell lines, Can-
cer Biother. Radiopharmaceut., 34, 252-257, https://
doi.org/10.1089/cbr.2018.2732.
49. Ajay, A. K., Meena, A. S., and Bhat, M. K. (2012) Hu-
man papillomavirus 18 E6 inhibits phosphorylation
of p53 expressed in HeLa cells, Cell Biosci., 2, 2,
https://doi.org/10.1186/2045-3701-2-2.
50. Diepstraten, S.T., Yuan, Y., La Marca, J. E., Young, S.,
Chang, C., Whelan, L., Ross, A. M., Fischer, K. C.,
Pomilio, G., Morris, R., Georgiou, A., Litalien, V.,
Brown, F. C., Roberts, A. W., Strasser, A., Wei, A. H.,
and Kelly, G. L. (2024) Putting the STING back into
BH3-mimetic drugs for TP53-mutant blood cancers,
Cancer Cell, 42, 850-868.e9, https://doi.org/10.1016/
j.ccell.2024.04.004.
51. Gentile, M., Petrungaro, A., Uccello, G., Vigna, E.,
Recchia, A. G., Caruso, N., Bossio, S., De Stefano, L.,
Palummo,A., Storino,F., Martino,M., and Morabito,F.
(2017) Venetoclax for the treatment of chronic
lymphocytic leukemia, Expert Opin. Invest. Drugs,
26, 1307-1316, https://doi.org/10.1080/13543784.2017.
1386173.
RESISTANCE TO MCL-1 INHIBITION 2025
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
52. McDermott,M., Eustace, A.J., Busschots,S., Breen,L.,
Crown, J., Clynes, M., O’Donovan, N., and Stordal, B.
(2014) In vitro development of chemotherapy and
targeted therapy drug-resistant cancer cell lines: a
practical guide with case studies, Front. Oncol., 4, 40,
https://doi.org/10.3389/fonc.2014.00040.
53. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S.,
Poirier, G. G., and Earnshaw, W. C. (1994) Cleavage
of poly(ADP-ribose) polymerase by a proteinase with
properties like ICE, Nature, 371, 346-347, https://
doi.org/10.1038/371346a0.
54. Gottesman, M. M., Pastan, I., and Ambudkar, S. V.
(1996) P-glycoprotein and multidrug resistance, Curr.
Opin. Genetics Dev., 6, 610-617, https://doi.org/10.1016/
s0959-437x(96)80091-8.
55. Dong,J., Yuan,L., Hu,C., Cheng,X., and Qin, J.-J. (2023)
Strategies to overcome cancer multidrug resistance
(MDR) through targeting P-glycoprotein (ABCB1):
an updated review, Pharmacol. Ther., 249, 108488,
https://doi.org/10.1016/j.pharmthera.2023.108488.
56. Bolomsky,A., Miettinen, J.J., Malyutina,A., Besse,A.,
Huber, J., Fellinger, S., Breid, H., Parsons, A.,
Klavins, K., Hannich, J. T., Kubicek, S., Caers, J.,
Hübl,W., Schreder,M., Zojer,N., Driessen,C., Tang,J.,
Besse, L., Heckman, C. A., and Ludwig, H. (2021)
Heterogeneous modulation of Bcl-2 family members
and drug efflux mediate Mcl-1 inhibitor resistance in
multiple myeloma, Blood Adv., 5, 4125-4139, https://
doi.org/10.1182/bloodadvances.2020003826.
57. Olejniczak, S. H., Hernandez-Ilizaliturri, F. J., Cle-
ments, J. L., and Czuczman, M. S. (2008) Acquired
resistance to rituximab is associated with chemother-
apy resistance resulting from decreased Bax and Bak
expression, Clin. Cancer Res., 14, 1550-1560, https://
doi.org/10.1158/1078-0432.CCR-07-1255.
58. Dolcet, X., Llobet, D., Pallares, J., and Matias-Guiu, X.
(2005) NF-kB in development and progression of hu-
man cancer, Virchows Arch., 446, 475-482, https://
doi.org/10.1007/s00428-005-1264-9.
59. Lin, J., Song, T., Li, C., and Mao, W. (2020) GSK-3β
in DNA repair, apoptosis, and resistance of chemo-
therapy, radiotherapy of cancer, Biochim. Biophys.
Acta, 1867, 118659, https://doi.org/10.1016/j.bbamcr.
2020.118659.
60. Duda, P., Akula, S. M., Abrams, S. L., Steelman,
L. S., Martelli, A. M., Cocco, L., Ratti, S., Candido, S.,
Libra, M., Montalto, G., Cervello, M., Gizak, A.,
Rakus,D., and McCubrey, J.A. (2020) Targeting GSK3
and associated signaling pathways involved in can-
cer, Cells, 9, 1110, https://doi.org/10.3390/cells9051110.
61. Wang, R., Xia, L., Gabrilove, J., Waxman, S., and
Jing, Y. (2013) Downregulation of Mcl-1 through
GSK-3β activation contributes to arsenic trioxide-in-
duced apoptosis in acute myeloid leukemia cells,
Leukemia, 27, 315-324, https://doi.org/10.1038/leu.
2012.180.
62. Kang, X.-H., Zhang, J.-H., Zhang, Q.-Q., Cui, Y.-H.,
Wang, Y., Kou, W.-Z., Miao, Z.-H., Lu, P., Wang, L.-F.,
Xu, Z.-Y., and Cao, F. (2017) Degradation of Mcl-1
through GSK-3β activation regulates apoptosis in-
duced by bufalin in non-small cell lung cancer H1975
cells, Cell. Physiol. Biochem., 41, 2067-2076, https://
doi.org/10.1159/000475438.
63. Fallatah, M. M. J., Law, F. V., Chow, W. A., and
Kaiser, P. (2023) Small-molecule correctors and sta-
bilizers to target p53, Trends Pharmacol. Sci., 44,
274-289, https://doi.org/10.1016/j.tips.2023.02.007.
64. Hassin, O., and Oren,M. (2023) Drugging p53 in can-
cer: one protein, many targets, Nature Reviews. Drug
Discovery, 22, 127-144, https://doi.org/10.1038/s41573-
022-00571-8.
65. Levine, A. J. (2020) p53: 800 million years of evolu-
tion and 40 years of discovery, Nat. Rev. Cancer, 20,
471-480, https://doi.org/10.1038/s41568-020-0262-1.
66. Shuvalov,O., Kizenko,A., Shakirova,A., Fedorova,O.,
Petukhov, A., Aksenov, N., Vasileva, E., Daks, A., and
Barlev, N. (2018) Nutlin sensitizes lung carcinoma
cells to interferon-alpha treatment in MDM2-depen-
dent but p53-independent manner, Biochem. Biophys.
Res. Commun., 495, 1233-1239, https://doi.org/10.1016/
j.bbrc.2017.11.118.
67. Lau, L. M. S., Nugent, J. K., Zhao,X., and Irwin, M. S.
(2008) HDM2 antagonist Nutlin-3 disrupts p73-HDM2
binding and enhances p73 function, Oncogene, 27,
997-1003, https://doi.org/10.1038/sj.onc.1210707.
68. Ambrosini, G., Sambol, E. B., Carvajal, D., Vassilev,
L. T., Singer, S., and Schwartz, G. K. (2007) Mouse
double minute antagonist Nutlin-3a enhances chemo-
therapy-induced apoptosis in cancer cells with mu-
tant p53 by activating E2F1, Oncogene, 26, 3473-3481,
https://doi.org/10.1038/sj.onc.1210136.
69. Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D.,
Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U.,
Lukacs,C., Klein, C., Fotouhi,N., and Liu, E.A. (2004)
In vivo activation of the p53 pathway by small-
molecule antagonists of MDM2, Science, 303, 844-848,
https://doi.org/10.1126/science.1092472.
70. Sazonova, E. V., Yapryntseva, M. A., Pervushin, N. V.,
Tsvetcov, R. I., Zhivotovsky, B., and Kopeina, G. S.
(2024) Cancer drug resistance: targeting prolifera-
tion or programmed cell death, Cells, 13, 388, https://
doi.org/10.3390/cells13050388.
71. Amaral, M. V. S., de Sousa Portilho, A. J., da Silva,
E. L., de Oliveira Sales, L., da Silva Maués, J. H., de
Moraes, M.E. A., and Moreira-Nunes, C.A. (2019) Es-
tablishment of drug-resistant cell lines as a model in
experimental oncology: a review, Anticancer Res., 39,
6443-6455, https://doi.org/10.21873/anticanres.13858.
72. Ong, F., Kim, K., and Konopleva, M. Y. (2022) Vene-
toclax resistance: mechanistic insights and future
strategies, Cancer Drug Resist., 5, 380-400, https://
doi.org/10.20517/cdr.2021.125.
PERVUSHIN et al.2026
BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025
73. Liu, J., Li, S., Wang, Q., Feng, Y., Xing, H., Yang, X.,
Guo, Y., Guo, Y., Sun, H., Liu, X., Yang, S., Mei, Z.,
Zhu, Y., Cheng, Z., Chen, S., Xu, M., Zhang, W.,
Wan, N., Wang, J., Ma, Y., Zhang, S., Luan, X.,
Xu, A., Li, L., Wang, H., Yang, X., Hong, Y., Xue, H.,
Yuan,X., Hu,N., Song,X., Wang,Z., Liu,X., Wang, L.,
and Liu, Y. (2024) Sonrotoclax overcomes BCL2
G101V mutation-induced venetoclax resistance
in preclinical models of hematologic malignancy,
Blood, 143, 1825-1836, https://doi.org/10.1182/blood.
2023019706.
74. Sakamoto, I., Yamada, T., Ohwada, S., Koyama, T.,
Nakano, T., Okabe, T., Hamada, K., Kawate, S.,
Takeyoshi, I., Iino, Y., and Morishita, Y. (2004) Mu-
tational analysis of the BAK gene in 192 advanced
gastric and colorectal cancers, Int. J. Mol. Med., 13,
53-55, https://doi.org/10.3892/ijmm.13.1.53.
75. Pervushin, N.V., Yapryntseva, M.A., Panteleev, M.A.,
Zhivotovsky, B., and Kopeina, G. S. (2024) Cisplatin
resistance and metabolism: simplification of com-
plexity, Cancers, 16, 3082, https://doi.org/10.3390/
cancers16173082.
76. Tang, G., Cao, X., Chen, J., Hui, F., Xu, N., Jiang, Y.,
Lu, H., Xiao, H., Liang, X., Ma, M., Qian, Y., Liu, D.,
Wang,Z., Liu,S., Yu,G., and Sun,L. (2025) Repurpos-
ing MDM2 inhibitor RG7388 for TP53-mutant NSCLC:
a p53-independent pyroptotic mechanism via ROS/
p-p38/NOXA/caspase-3/GSDME axis, Cell Death Dis.,
16, 452, https://doi.org/10.1038/s41419-025-07770-2.
Publisher’s Note. Pleiades Publishing remains
neutral with regard to jurisdictional claims in published
maps and institutional affiliations. AI tools may have
been used in the translation or editing of this article.
