International Journal of
Molecular Sciences
Article
Bcl-2 Family Members Bcl-xL and Bax Cooperatively Contribute
to Bortezomib Resistance in Mantle Cell Lymphoma
Sudjit Luanpitpong 1, * , Montira Janan 1,† , Juthamas Yosudjai 1,† , Jirarat Poohadsuan 1 , Pithi Chanvorachote 2,3
and Surapol Issaragrisil 1,4,5
Citation: Luanpitpong, S.; Janan, M.;
Yosudjai, J.; Poohadsuan, J.;
Chanvorachote, P.; Issaragrisil, S.
Bcl-2 Family Members Bcl-xL and
Bax Cooperatively Contribute to
Bortezomib Resistance in Mantle Cell
Lymphoma. Int. J. Mol. Sci. 2022, 23,
14474. https://doi.org/10.3390/
ijms232214474
Academic Editor: Carmen
Cristina Diaconu
Received: 10 October 2022
Accepted: 19 November 2022
Published: 21 November 2022
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2022, 23, 14474. https://doi.org/10.3390/ijms232214474
1 Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok 10700, Thailand
2 Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330, Thailand
3 Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand
4 Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok 10700, Thailand
5 Bangkok Hematology Center, Wattanosoth Hospital, BDMS Center of Excellence for Cancer,
Bangkok 10310, Thailand
* Correspondence: sudjit.lua@mahidol.edu
† These authors contributed equally to this work.
Abstract: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor progno-
sis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome
inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a ma-
jor hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms
underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing
novel therapeutic strategies. Here, we established de novo BTZ-resistant human MCL-derived cells
with the highest resistance index of 300-fold compared to parental cells. We provided compelling
evidence that both Bcl-xL and Bax are key mediators in determining BTZ sensitivity in MCL cells.
Overexpression of antiapoptotic Bcl-xL and depletion of proapoptotic Bax cooperatively protected
MCL cells against BTZ-induced apoptosis, causing acquired BTZ resistance, likely by tilting the
balance of Bcl-2 family proteins toward antiapoptotic signaling. Bioinformatics analyses suggested
that high BCL2L1 (encoded Bcl-xL) and low BAX were, in part, associated with poor prognosis of
MCL patients, e.g., when combined with low OGT, which regulates cellular O-GlcNAcylation. Our
findings support recent strategies in small molecule drug discovery co-targeting antiapoptotic Bcl-2
family proteins using BH3 mimetics and Bax using Bax activators to overcome cancer drug resistance.
Keywords: mantle cell lymphoma; bortezomib resistance; Bcl-2 family proteins; Bcl-xL; Bax;
O-GlcNAcylation
1. Introduction
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma (NHL) arising
from the pre-germinal center of a mature B cell that is characterized by the presence of
translocation t(11;14)(q13;q32) resulting in an aberrant expression of cyclin D1 (encoded
by CCND1) and uncontrolled cell growth [1,2]. The disease accounts for 3% to 6% of
all NHL cases and is predominantly male, with a male-to-female ratio of approximately
3:1. Although some patients may follow an indolent clinical evolution, most have the
clinical pattern of poor response to conventional chemotherapy and frequent relapse. MCL
remains the worst prognosis among all NHL subtypes, with median overall survival (OS)
of approximately 3–5 years following initial induction therapy and median OS of 1–2 years
in patients with refractory or relapsed disease [3].
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 14474 2 of 21

Bortezomib (BTZ) is a Food and Drug Administration (FDA)-approved first-in-class

proteasome inhibitor for the treatment of relapsed/refractory MCL with an overall response
rate (ORR) of 30–50%, irrespective of their sensitivity to prior therapy [4–6], suggesting
little cross-resistance with conventional therapy. However, more than half of all MCL
patients are either intrinsically resistant to BTZ or subsequently acquire resistance [7,8].
Drug resistance is a manifestation of cancer and is associated with somatic mutations
and genomic plasticity. In this regard, numerous mechanisms associated with molecular
changes of drug efflux pumps, drug metabolism, cell death inhibition, enhanced DNA re-
pair, target gene amplification, and epigenetics have been shown to protect various cancers
against chemotherapy [9–11]. Although the information on BTZ resistance in MCL remains
largely unknown, attributable in part to its nature as a multifactorial phenomenon, certain
characteristics of BTZ-resistant MCL cells have previously been reported, namely uncon-
trolled plasmacytic differentiation [12], activation of B-cell receptor (BCR) signaling [13],
and alteration of lipid metabolism [14].
The aim of this study was to identify target candidates associated with BTZ resistance
in MCL cells that could also predict the apoptosis response to BTZ. We established de novo
BTZ-resistant MCL cells and profiled gene signatures involved in drug resistance and cell
survival of MCL using sets of quantitative polymerase chain reaction (qPCR). We identified
the involvement of Bcl-2 family members in BTZ response, stemming from the observed
increase in antiapoptotic Bcl-xL (encoded by BCL11A) and a decrease in proapoptotic Bax
(encoded by BAX) at both gene and protein levels in acquired BTZ-resistant MCL cells.
We also performed bioinformatics database analyses to find an association of BCL11A,
BAX and other reported genes involved in MCL aggressiveness with the clinical outcomes
of MCL patients. Our findings provide better understanding of the mechanisms of BTZ
resistance, which could be important in developing novel adjuvants for aggressive MCL.

2. Results
2.1. Establishment of BTZ-Resistant MCL Cells and Degrees of Resistance
To investigate the molecular mechanisms of BTZ resistance in MCL, BTZ-resistant cells
with acquired resistance were obtained from BTZ-sensitive human MCL-derived Z-138
(Z/Parent) cells after sequential treatments with increasing concentrations of BTZ, i.e., up
to 500 nM. BTZ-resistant cells at different degrees of resistance were designated Z/100R,
Z/250R, and Z/500R cells. Dose-response curves, which were generated from BTZ-induced
apoptosis as evaluated by Hoechst 33342 assay at 24 h, displayed the sensitivity of BTZ-
resistant cells compared to the parental Z/Parent cells (Figure 1A). The lethal concentration
50 (LC50), which caused half of the cells to undergo apoptosis, was calculated from the best-
fit curves to the pooled data, and the reported values were 28.11 nM in Z/100R, 396.10 nM
in Z/250R, and 628.3 nM in Z/500R cells versus 3.52 nM in Z/Parent cells. Resistance
index (R) was then calculated from the relationships of LC50 of resistant cells to the LC50
of parental cells. The degrees of resistance in established resistant cells were approximately
8, 100, and 300-fold more resistant to BTZ than the parental cells.
Representative micrographs of Z/Parent cells under the blue fluorescence of Hoechst
33342 clearly displayed greater numbers of fragmented and/or condensed nuclei, the
typical features of apoptosis, than those of Z/500R cells in all tested concentrations of
BTZ (Figure 2A). By comparing the percentage of apoptosis scored from the micrographs,
we confirmed that Z/500R cells were significantly more resistant than Z/Parent cells in
response to BTZ at 10–500 nM. In agreement with the data obtained from Hoechst 33342
assay, Annexin V/7-AAD assays using flow cytometry validated that Z/500R cells were
highly resistant to apoptosis induced by BTZ with a death rate of approximately 30% at
50 nM BTZ, the concentration that killed most, if not all, Z/Parent cells (Figure 2B). The
death rate of Z/500R cells remained at approximately 50% even when treated with an
extremely high concentration of BTZ at 500 nM. By comparing the percentage of total cell
death and percentage of apoptosis of Z/Parent cells under the same conditions, we found
that apoptosis was the mode of cell death induced by BTZ in these experimental settings.
Int.
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Mol.Sci.
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3 of2321

Increasing BTZ

Annexin V+ or PI+
Z/Parent Z/100R Z/250R Z/500R

B
100
Z/Parent

75
Z/100R
% Apoptosis

Z/250R
50 Z/500R

25

0
0 1 2 3 4
BTZ [Log](nM)

Figure 1. Generation of de novo BTZ-resistant MCL cells. (A) Schematic diagram for the establishment
Figure 1. Generation of de novo BTZ-resistant MCL cells. (A) Schematic diagram for the establish-
of BTZ-resistant Z-138 cells at different degrees from parental (Z/Parent) cells by stepwise selection
ment of BTZ-resistant Z-138 cells at different degrees from parental (Z/Parent) cells by stepwise
method.method.
selection (B) Parental Z/Parent
(B) Parental cells and
Z/Parent BTZ-resistant
cells Z/100R,
and BTZ-resistant Z/250R,
Z/100R, and Z/500R
Z/250R, cells cells
and Z/500R were
treated with various concentrations of BTZ (0–1200 nM), and apoptosis was determined
were treated with various concentrations of BTZ (0–1200 nM), and apoptosis was determined by the by the
Hoechst33342
Hoechst 33342assay
assayatat24
24h.h.Dose-response
Dose-response(best-fit)
(best-fit)curves
curveswere
weregenerated
generatedononthe
thelogarithmic
logarithmicscale
scale
ofofdrug
drugconcentrations
concentrations as opposed
opposed to tothe
thelinear
linearscale
scaleofofpercentage
percentageofof apoptosis.
apoptosis. Data
Data areare mean
mean ± SD±
SD
(n =(n3).
= 3).

2.2. Representative
Gene Expressionmicrographs
Profiling of BTZ-Resistant MCLunder
of Z/Parent cells Cells and
the Pathway Analysis of Hoechst
blue fluorescence
33342To clearly displayed
identify genes and greater numbers
pathways of fragmented
important and/or condensed
in the development of BTZ nuclei, the typ-
resistance, we
ical features
profiled of apoptosis,
putative than those
cancer-related genes of Z/500Rincells
involved druginresistance
all testedandconcentrations
cell survival ofof BTZ
MCL
using sets
(Figure 2A).ofBy
qPCR. These genes
comparing were grouped
the percentage into: (i) drug
of apoptosis scored efflux
frompumps ABCB1, ABCC1,
the micrographs, we
and ABCG2 [9]; (ii) drug metabolizing enzymes CYP1A2, CYP2D6,
confirmed that Z/500R cells were significantly more resistant than Z/Parent cells in re- and CYP3A4 [10];
(iii) apoptosis
sponse to BTZ regulators
at 10–500 nM. BCL2,In BCL2L1,
agreement MCL1,
withBAX, TP53,
the data and TP73
obtained from[11]; (iv) cell33342
Hoechst cycle
regulators
assay, CCND1,
Annexin V/7-AADCDK2,assays
CDK4,using
and CDKN2A [15]; (v)validated
flow cytometry cell survival
that and growth
Z/500R cellsfactors
were
NFKB2,
highly MYC, SOD1,
resistant STK38,induced
to apoptosis and IGF1Rby BTZ [16–18];
with (vi) Hippo
a death ratepathway components
of approximately 30%NF2,
at
LATS1,
50 nM BTZ,LATS2,
the STK3, and STK4
concentration [19],
that and (vii)
killed most,clonal
if notregulators
all, Z/ParentVIMcells OCT4 [20].
and(Figure 2B). Gene
The
expression
death rate oflevels
Z/500Rnormalized to theat
cells remained housekeeping
approximately GAPDH
50% even comparing parental
when treated Z/Parent
with an ex-
cells and BTZ-resistant Z/100R, Z/250R and Z/500R cells were aligned,
tremely high concentration of BTZ at 500 nM. By comparing the percentage of total cell clustered, and
represented in a heatmap. Notably, ABCB1, ABCG2, CYP1A2, and CYP3A4
death and percentage of apoptosis of Z/Parent cells under the same conditions, we found were expressed
at barely
that detectable
apoptosis was the levels
modein both parental
of cell death and BTZ-resistant
induced by BTZ incells,
these hence they were settings.
experimental excluded
from the analyses. Figure 3A shows that BCL2L1 and BAX are the top-ranked genes that
clearly distinguish Z/Parent and Z/100R cells from the highly resistant Z/250R and
Z/500R cells. Next, a volcano plot was created to better visualize the differential gene
expression in Z/Parent versus Z/500R cells using p < 0.05 and ± two-fold change filter
criteria. Figure 3B further confirms that only Bcl-2 family members BCL2L1 and BAX
passed the criteria—BCL2L1 was significantly upregulated, while BAX was conversely
downregulated in Z/500R cells when compared to Z/Parent cells. Therefore, BCL2L1 and
BAX were chosen as target candidates in the present study.
 Int. J.J. Mol.
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x FOR PEER REVIEW 44 of 23
of 21

A
Z/Parent
Z/500R 0 10 50 250 500

100 Z/Parent
Z/500R
% Apoptosis

***
50 ***
**
***

***
0
0 10 50 100 250 500
BTZ (nM)

B 0 10 50 250 500
Q1 Q2 Q1 Q2
1.1%
1.06
1.3%
1.28
Q1
2.6%
2.56
25.6%
Q2
25.6
Q1
3.4%
3.39
Q2
42.3%
42.3
Q1
3.4%
3.35
35.9%
Q2
35.9
5.8%
5.81
34.5%
34.5
Z/Parent

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
92.8%
92.8 4.9%
4.85 39.0%
39.0 32.9%
32.9 2.5%
2.46 51.9%
51.9 1.2%
1.18 59.6%
59.6 1.1%
1.14 58.5%
58.5

0.8%
Q1
1.9%
Q2
4.2%
Q5 2.2%
Q6 Q1
1.1%
1.09
7.9%
Q2
7.86
1.8%
Q1
9.8%
Q2 Q1
1.9% 12.5%
Q2
0.77 1.86 4.26 2.21 1.82 9.78 1.91 12.5
Z/500R
7-AAD

Q4 Q3 Q8 Q7 Q4 Q3 Q4 Q3 Q4 Q3
90.9%
90.9 6.5%
6.45 93.4%
93.4 0.2%
0.16 71.0%
71.0 20.1%
20.1 64.9%
64.9 23.5%
23.5 53.0%
53.0 32.6%
32.6

Annexin V-PE

BTZ (nM) 0 10 50 100 250 500

100
Late Apop/Nec
Early Apop
Cellls (%)

Viable
50

Figure 2.
Figure 2. Resistant
Resistant phenotype
phenotype of
of the
the highly
highly BTZ-resistant
BTZ-resistant MCLMCL cells
cells in
in comparison
comparison toto parental
parental cells.
cells.
(A) Parental Z/Parent and highly BTZ-resistant Z/500R cells were treated with BTZ (0–500
(A) Parental Z/Parent and highly BTZ-resistant Z/500R cells were treated with BTZ (0–500 nM) and nM) and
apoptosis was evaluated by the Hoechst 33342 assay at 24 h. (Upper) Representative micrographs
apoptosis was evaluated by the Hoechst 33342 assay at 24 h. (Upper) Representative micrographs of
of Hoechst 33342 nuclear staining under an inverted fluorescence microscope. (Lower) Percentages
Hoechst 33342 nuclear staining under an inverted fluorescence microscope. (Lower) Percentages of
of apoptotic cells, which displayed condensed and/or fragmented nuclei, were scored from the mi-
apoptotic
crographscells,
andwhich displayed
plotted. condensed
Data are mean ± SDand/or fragmented
(n = 3). ** p < 0.01,nuclei,
*** p <were
0.001scored
versusfrom the micrographs
Z/Parent cells; two-
Int. J. Mol. Sci. 2022, 23, 14474 5 of 21

and plotted. Data are mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 versus Z/Parent cells; two-sided
Student’s t-test. (B) Cells were similarly treated with BTZ (0–500 nM) and cell death was determined
by Annexin V/7-AAD assay. Percentages of Annexin V single-positive cells defining early apoptosis,
and Annexin V and 7-AAD double-positive cells defining late apoptosis/necrosis6combined
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW of 24 with
7-AAD defining necrosis were plotted.

A
G/Parent G/100R G/250R G/500R
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

BCL2L1
BAX
NFKB2
LATS2
CDK4
CDKN1B
MYC
TP73
SOD1
BCL2
CYP2D6
STK3
VIM
ABCC1
TP53
MCL1
CCND1
NF2
LATS1
Low STK4
IGF1R
CDK2
OCT4
High STK38
CTTNB1

B
G/Parent versus G/500R

BAX

0.000001 BCL2L1

NFKB2

CTTNB1
0.0001
p-Value

MYC
CYP2D6
TP53

OCT4 VIM MCL1

0.01 TP73
CDK2 CDKN1B
STK38 BCL2 p = =0.05
P value 0.05
SOD1 LATS2
IGF1R ABCC1
CDK4
LATS1 STK3
CCND1 NF2
1 STK4

-3 -2 -1 0 1 2 3

Log 2 Fold Change

148
Figure 3. qPCR analysis of mRNA expression of putative cancer-related genes involved in drug
Figure 3. Analysis of mRNA expression of putative cancer-related genes involved in drug resistance 149
resistance
and andof
cell survival cell
MCLsurvival of MCL.
by qPCR. (A) Gene
(A) Gene expression
expression data in data in parental
parental G/ParentZ/Parent cells and
cells and BTZ- BTZ-
150
resistant
resistant Z/100R,
G/100R, Z/250R,
G/250R, and Z/500R
and G/500R cells
cells from from
four four independent
independent experimentsexperiments were normalized
were normalized to 151
housekeeping GAPDH,
to housekeeping aligned,aligned,
GAPDH, clustered,clustered,
and represented in a heatmap.in(B)
and represented Volcano plot
a heatmap. of Volcano
(B) differ- plot
152 of
entially expressedexpressed
differentially genes comparing G/Parent and
genes comparing G/500R cells
Z/Parent andwith fold change
Z/500R > 2 and
cells with foldp change
< 0.05; >153
2 and
two-sided Student’s t-test. 154
p < 0.05; two-sided Student’s t-test.
Int. J. Mol. Sci. 2022, 23, 14474 6 of 21

To gain better insight into the pathways associated with BTZ resistance, we input
the significantly changed genes between Z/Parental and Z/500R cells (p < 0.05) into
the database for Annotation, Visualization and Integrated Discovery (DAVID) functional
annotation tool, and BioCarta pathway analysis was performed [21,22]. Figure 4A shows a
list of top-ranked pathways, including the p53 signaling pathway and apoptosis signaling,
in response to DNA damage, which are schematically illustrated in Figure 4B. The limitation
of the analyses was the use of a predefined gene list in which only those genes known to
be involved in drug resistance and cell survival were included. It is worth noting that our
Int. J. Mol. Sci. 2022,studies
23, x FOR PEER REVIEW
do not exclude the involvement of additional signaling pathways. This7 requires
of 24

further investigation.

A
p53 Signaling Pathway BioCarta

Apoptotic Signaling in Response to

DNA Damage
Telomeres, Telomerase, Cellular Aging,
and Immortality

Role of Mitochondria in Apoptotic Signaling

First Multivalent Nuclear Factor

00 0.001
0.0 01 0.002
0.0 02 0.003
0.0 03 0.004
0.0 04 0.005
0.0 05

p-Value
B

p = 3.26×10-5

p = 7.32×10-5

155
Figure 4. Top-ranked pathways associated with BTZ resistance in MCL cells. (A) Lists of top-ranked
Figure 4. Top-ranked pathways associated with BTZ resistance in MCL cells. (A) Lists of top-ranked 156
BioCarta pathways
BioCartagenerated from lists
pathways generated fromof lists
significantly changed
of significantly changed genes
genesbetween parental
between parental Z/Parent
G/Parent 157
and BTZ-resistant Z/500R cells.
and BTZ-resistant G/500R (B)cells.
Schematic diagrams
(B) Schematic of the
diagrams p53
of the p53signaling pathway
signaling pathway (upper)
(upper) and and
158
the apoptosis
apoptosis signaling signaling
in response toinDNA
response to DNA
damage damage
(lower) (lower) generated
generated by BioCarta.
by BioCarta. YellowYellow
starsstars 159
indicate
indicate listed genes. 160
listed genes.
2.3. BTZ-resistant MCL cells displayed elevated Bcl-xL and repressed Bax levels 161
Int. J. Mol. Sci. 2022, 23, 14474 7 of 21

2.3. BTZ-Resistant MCL Cells Displayed Elevated Bcl-xL and Repressed Bax Levels
Having demonstrated that BCL2L1 and BAX were differentially expressed in the highly
resistant Z/500R cells when compared to Z/Parent cells (Figure 3B), we next evaluated
whether Bcl-xL and Bax protein levels correlated well with the degree of BTZ resistance.
Figure 5B shows a remarkable increase in Bcl-xL level when the cells acquired higher
resistance, which was correlated well with its gene expression (Figure 5A), suggesting
that the regulation of Bcl-xL in BTZ-resistant cells occurs mainly at the transcriptional
Int. J. Mol. Sci. 2022, level. ByPEER
23, x FOR contrast, Bax level decreased by approximately 30–40% in all BTZ-resistant cells
REVIEW 8
regardless of its gene expression and degree of resistance, suggesting that post-translational
modifications might be involved in the regulation of Bax in BTZ-resistant cells.

A B
6 1.5
Relative BCL2L1

Relative BAX
*** *** 1.0
**
3 **
*** 0.5

0 0.0

t
nt

nt
0R

0R

0R

0R
0R

0R
re

re
10

50

10

50
25

25
Pa

Pa
Z/

Z/

Z/

Z/
Z/
Z/
Z/

Z/
C D
kDa kDa
Bcl-xL — 30 Bax — 20

β-actin — 45 β-actin — 45
0R

0R

0R

0R

0R
0R
nt

tn
re

re
10

25

10

25

50
50
Pa

Pa

Z/

Z/

Z/
Z/

Z/

Z/
Z/

Z/

6 1.5
Relative Bcl-xL Level

Relative Bax Level

*** 1.0
*** *
** **
3
**
0.5

0 0.0
t

t
nt

0R

0R

nt
0R

0R

0R
0R
re

re
10

50
25

10

50
25
Pa

Pa
Z/

Z/
Z/

Z/

Z/
Z/
Z/

Z/

Figure 5. Determination of Bcl-2 and Bax levels in BTZ-resistant cells in comparison to their gene
Figure 5. Determination of Bcl-2 and Bax levels in BTZ-resistant cells in comparison to their
expression. (A,B) mRNA expression
expression. (A,B) mRNA BCL2L1 (encodes
of expression of BCL2L1Bcl-xL)
(encodes BAX inand
and Bcl-xL) parental Z/Parent
BAX in parental Z/Paren
cells and BTZ-resistant Z/100R, Z/250R,
and BTZ-resistant Z/100R,and Z/500R
Z/250R, andcells using
Z/500R qPCR.
cells using Data are mean
qPCR. ± SD
Data are (n =±3).
mean SD (n = 3).
** p < 0.01, *** p 0.01,
< 0.001
***versus
p < 0.001Z/Parent
versus cells; two-sided
Z/Parent Student’s t-test.
cells; two-sided (C,D)t-test.
Student’s Western blotWestern
(C,D) analysisblot analy
of Bcl-xL and Bax levels
Bcl-xL andin Z/Parent,
Bax levels Z/100R, Z/250R,
in Z/Parent, and Z/250R,
Z/100R, Z/500R andcells.Z/500R
Data are mean
cells. ± SD
Data are (n = 3).± SD (n =
mean
* p < 0.05, ** p <<0.01, ** p << 0.01,
0.05,*** 0.001*** p < 0.001
versus versuscells;
Z/Parent Z/Parent cells; two-sided
two-sided Student’s t-test.
Student’s t-test.

2.4. Overexpression of Bcl-xL Contributes

2.4. Overexpression to Contributes
of Bcl-xL Acquired BTZ ResistanceBTZ Resistance
to Acquired
The Bcl-2 family
TheisBcl-2
the best characterized
family family of proteins
is the best characterized involved
family in the
of proteins regulation
involved in the regul
of apoptosis, consisting
of apoptosis, consisting of antiapoptotic and proapoptotic members.function
of antiapoptotic and proapoptotic members. The main The main functi
of antiapoptotic Bcl-2 family
antiapoptotic proteins
Bcl-2 familyisproteins
to restrain
is toproapoptotic Bcl-2 family
restrain proapoptotic proteins
Bcl-2 family protein
primarily by direct binding interactions, thus preserving mitochondrial outer membrane
marily by direct binding interactions, thus preserving mitochondrial outer membrane
permeabilization (MOMP) and
meabilization preventing
(MOMP) the subsequent
and preventing release of cytochrome
the subsequent release of C [23,24].
cytochrome C [2
To first evaluate whether antiapoptotic Bcl-xL rendered MCL cells apoptosis-resistant to
To first evaluate whether antiapoptotic Bcl-xL rendered MCL cells apoptosis-resista
BTZ, we introduced Bcl-xL transgene into parental Z/Parent cells using retroviral particles,
BTZ, we introduced Bcl-xL transgene into parental Z/Parent cells using retroviral p
cles, hereafter designated as Z/Parent-Bcl-xL cells, and evaluated their apoptosis i
sponse to BTZ in comparison to their counterpart control Z/Parent cells and the h
resistant Z/500R cells. All cells were treated with BTZ (0–500 nM), and apoptosis was
uated by Hoechst 33342 and Annexin V/7-AAD assays at 24 h. Western blot analysis
Int. J. Mol. Sci. 2022, 23, 14474 8 of 21

hereafter designated as Z/Parent-Bcl-xL cells, and evaluated their apoptosis in response

to BTZ in comparison to their counterpart control Z/Parent cells and the highly resistant
Z/500R cells. All cells were treated with BTZ (0–500 nM), and apoptosis was evaluated by
Hoechst 33342 and Annexin V/7-AAD assays at 24 h. Western blot analysis was performed
to ascertain an increase in Bcl-xL level in Z/Parent-Bcl-xL cells (Figure 6A). Bax level
was also checked and was found to be minimally changed in Z/Parent-Bcl-xL cells. By
nuclear staining, the percentage of apoptosis was significantly lower in Z/Parent-Bcl-xL and
Z/500R cells when compared to Z/Parent cells. Representative micrographs that clearly
exhibit the lesser extent of cells with condensed and/or fragmented nuclei in Z/Parent-Bcl-
xL cells when compared to Z/Parent cells are shown in the lower panel. Further, the results
from Annexin V/7-AAD assay validated that Bcl-xL overexpression protected the Z/Parent
cells from cell death induced by BTZ (10–500 nM) (Figure 6B). Notably, we observed that
overexpression of Bcl-xL alone could cause the Z/Parent cells to acquire the BTZ-resistant
phenotype to a degree that was almost comparable to those of Z/500R cells, although we
did not make a direct comparison considering other confounding variables. Altogether,
these results indicate the important role of Bcl-xL in BTZ sensitivity of MCL cells.

2.5. Depletion of Bax Similarly Leads to BTZ Resistance

Bax is a proapoptotic Bcl-2 family protein that, together with its homolog protein
Bak, is considered a core regulator of the intrinsic pathway of apoptosis [24,25]. Bax and
Bak, which form pores in the mitochondrial outer membrane upon their conversion from
harmless monomers into deadly oligomers, are regulated by various antiapoptotic Bcl-2
family proteins, including Bcl-xL. To identify the functional role of Bax in BTZ response in
MCL cells, the CRISPR/Cas9 system was used to deplete Bax in the parental Z/Parent cells,
and their effect on apoptosis was evaluated using Hoechst 33342 and Annexin V/7-AAD
assays. Similar to the overexpression of Bcl-xL (Figure 6), depletion of Bax conferred BTZ
resistance to Z/Parent cells to a degree that resembled the highly resistant Z/500R cells
(Figure 7A,B), indicating that loss of Bax efficiently protected the Z/Parent cells from
apoptosis by BTZ (10–500 nM) and that acquired BTZ resistance was regulated in part
by Bax, although its protein level was not correlated well with the degree of resistance in
BTZ-resistant cells.

2.6. Overexpression of Bcl-xL and Depletion of Bax Cooperatively Caused More BTZ-Resistant
MCL Cells
Previous studies have reported that cancer cells commonly evade apoptosis by upreg-
ulation of the antiapoptotic Bcl-2 family proteins. In more resistant cells, downregulation
or inactivation of proapoptotic Bcl-2 family proteins, particularly BH3-only proteins, also
occurs to suppress apoptosis [26,27]. To test whether depletion of Bax, the proapoptotic
pore-former, directly synergized with the protective effect of Bcl-xL on BTZ-induced apopto-
sis, Bax was inhibited in Z/Parent-Bcl-xL cells using the CRISPR/Cas9 system. Figure 8A,B
shows that Z/Parent-Bcl-xL + BAXi cells were significantly less responsive to apoptosis in
response to BTZ (10–500 nM) when compared to the counterpart control Z/Parent-Bcl-xL
cells, indicating that Bax worked in concert with Bcl-xL in evading BTZ-induced apoptosis
and acquiring BTZ resistance.
To substantiate the findings on the functional role of Bcl-xL and Bax in determining
BTZ sensitivity in MCL cells, BTZ-sensitive human MCL-derived Jeko-1 cells were used,
and genetic manipulation of Bcl-xL and Bax alone or in combination, was performed as
previously described in Z-138 cells. Western blot analysis was performed to ascertain the
successful overexpression of Bcl-xL and/or depletion of Bax (Figure 9A) prior to treatment
with BTZ (10–500 nM). Cell death was then determined by the Annexin V/7-AAD assay
at 24 h. The results showed that overexpression of Bcl-xL and, to a much lesser extent,
depletion of Bax alone, could render Jeko-1 cells resistant to apoptosis induced by BTZ
(10–500 nM), suggesting the generality of their roles in BTZ-induced apoptosis in MCL
cells (Figure 9B). We, however, noticed that depletion of Bax in Bcl-xL-overexpressed cells
 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 23

Int. J. Mol. Sci. 2022, 23, 14474 9 of 21

the Z/Parent cells from cell death induced by BTZ (10–500 nM) (Figure 6B). Notably, we
observed that overexpression of Bcl-xL alone could cause the Z/Parent cells to acquire the
BTZ-resistant phenotype to a degree that was almost comparable to those of Z/500R cells,
minimally although
improvedweBTZ
did response
not make when
a directcompared
comparison
to considering other confounding
their counterpart variables.
control Bcl-xL cells,
Altogether, these results indicate the important role of Bcl-xL in BTZ sensitivity
which is likely due to the already very high protection of Bcl-xL alone against BTZ-induced of MCL
cells.
apoptosis in Jeko-1 cells.

A
kDa
100
— 30

% Apoptosis
Bcl-xL
***
Bax — 20
** *** *** ***
50
*** *** ***
β-actin — 45 ***
***
0
0 10 50 100 250 500
BTZ (nM)
Z/Parent-WT Z/Parent-Bcl-xL Z/500R

0 10 50 250 500
Z/Parent-WT
Z/Parent-Bcl-xL

B Z/Parent-WT Z/Parent-Bcl-xL Z/500R

Q1
0.5% 0%
Q2 0.6%
Q1
0%
Q2 Q1
2.0% 0%
Q2 Q1
0.2% 0%
Q2
0.7%
Q1
0%
Q2
0.45 0.030 0.62 0.025 1.99 5.63E-3 0.24 0 0.71 0

0 nM 0 nM 100 nM 0 nM 100 nM

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
85.5%
85.5 14.0%
14.0 90.7%
90.7 8.6%
8.61 61.6%
61.6 36.4%
36.4 94.9%
94.9 4.9%
4.83 69.8%
69.8 29.5%
29.5

3 4

1.5%
Q1 0%
Q2 Q1
2.3% 0%
Q2 2.0%
Q1 0%
Q2 Q1
0.6% 0%
Q2 0.6%
Q1 Q20%
1.49 0.026 2.32 0 1.95 0 0.63 5.13E-3 0.64 5.19E-3

50 nM 50 nM 250 nM 50 nM 250 nM
7-AAD

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
91.3%
7.17 7.2%
91.3 65.4%
65.4 32.3%
32.3 61.0%
60.9 37.0%
37.1 72.9%
72.9 26.4%
26.4 61.7%
61.7 37.7%
37.7

Annexin V-PE
BTZ (nM) 0 50 100 250
100
Late Apop/Nec
Early Apop
Cellls (%)

Viable
50

Z/Parent

Figure 6. Bcl-xL
Figureis 6.
a key mediator
Bcl-xL is a keyofmediator
BTZ-induced apoptosis.apoptosis.
of BTZ-induced Parental Z/Parent cells were
Parental Z/Parent transduced
cells were trans-
with Bcl-xLduced
retroviral particles
with Bcl-xL or control
retroviral particles
particles (WT)
or control and treated
particles with
(WT) and BTZwith
treated (0–500BTZnM) fornM)
(0–500 24 h.
for
24 h. (A)
(A) Upper left: Upperblot
Western left: analysis
Western blot analysislevel,
of Bcl-xL of Bcl-xL level,
along along
with with
Bax Bax level,
level, in Z/Parent-WT
in Z/Parent-WT and
and
Z/Parent-Bcl-xL cells in comparison to the highly BTZ-resistant Z/500R cells prior to experiments.
Upper, right: Percentages of apoptosis as evaluated by Hoechst 33342 assay were scored and plotted.
Data are mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 versus Z/Parent-WT cells; two-sided Student’s
t-test. Lower: Representative micrographs of Hoechst 33342 nuclear staining under an inverted
fluorescence microscope. (B) Analysis of cell death by Annexin V/7-AAD assay. Percentages of
Annexin V single-positive cells defining early apoptosis, and Annexin V and 7-AAD double-positive
cells defining late apoptosis/necrosis combining with 7-AAD single-positive cells defining necrosis
were plotted.
 cells, and their effect on apoptosis was evaluated using Hoechst 33342 and Annexin V/7-
AAD assays. Similar to the overexpression of Bcl-xL (Figure 6), depletion of Bax conferred
BTZ resistance to Z/Parent cells to a degree that resembled the highly resistant Z/500R
cells (Figure 7A,B), indicating that loss of Bax efficiently protected the Z/Parent cells from
apoptosis by BTZ (10–500 nM) and that acquired BTZ resistance was regulated in part by
Int. J. Mol. Sci. 2022, 23, 14474 Bax, although its protein level was not correlated well with the degree of resistance in10 of 21
BTZ-resistant cells.

A
kDa
100
— 20
Bax

% Apoptosis
Bcl-xL — 30 ***
***
50 ** *
*** * ** *
β-actin — 45 ** **
*
*** **
***
0
0 10 50 100 250 500
BTZ (nM)
Z/Parent-WT Z/Parent-BAXi Z/500R

0 10 50 250 500
Z/Parent-WT
Z/Parent-BAXi

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 11 of 23

B 0 10 50 250 500
Q1 Q2 Q1 Q2 Q1 Q2
0.3%
0.35
1.5%
1.48
8.3%
6.32
18.4%
18.4
Q1
3.3%
3.31
Q2
36.8%
36.8
4.3%
4.27
33.8%
33.8
3.8%
Q1
3.81
34.8%
Q2
34.8
Z/Parent-WT

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
93.5%
93.5 4.7%
4.71 44.1%
44.1 31.2%
31.2 3.3%
3.27 56.6%
56.6 1.6%
1.61 60.3%
60.3 1.3%
1.34 60.1%
60.1

1 2 3 4 5 6

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2
0.7%
0.68
0.9%
0.87
1.2%
1.21
3.4%
3.36
0.5%
0.53
3.1%
3.07
0.5%
0.48
3.3%
3.28
0.5%
0.50
4.2%
4.23
Z/Parent-BAXi
7-AAD

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
96.1%
96.1 2.4%
2.39 86.7%
86.7 8.7%
8.70 88.0%
88.0 8.4%
8.37 89.2%
89.2 7.0%
6.99 88.3%
88.3 6.9%
6.94

Annexin V-PE
BTZ (nM) 0 10 50 100 250 500
100
Late Apop/Nec
Early Apop
Cellls (%)

Viable
50

0
Z/Parent

Figure 7. Bax
Figure is also
7. Bax a key
is also mediator
a key mediatorof of
BTZ-induced
BTZ-inducedapoptosis.
apoptosis.Parental
ParentalZ/Parent cells were
Z/Parent cells weretrans-
transduced
withduced
lentiviral
with particles
lentiviral carrying Cas9 and
particles carrying sgRNA
Cas9 againstagainst
and sgRNA humanhuman BAX or BAXcontrol particles
or control (WT) and
particles
(WT)with
treated and BTZ
treated with nM)
(0–500 BTZ (0–500
for 24 nM) forUpper
h. (A) 24 h. (A) Upper
left: left: Western
Western blot analysis
blot analysis of Bax of Bax level,
level, along with
along with Bcl-xL level, in Z/Parent-WT and Z/Parent-BAXi cells in comparison to the highly BTZ-
Bcl-xL level, in Z/Parent-WT and Z/Parent-BAXi cells in comparison to the highly BTZ-resistant Z/500R
resistant Z/500R cells prior to experiments. The dashed line separates juxtaposed lanes taken from
cellsthe
prior to experiment
same experiments. andThe
blotdashed line separates
image. Upper juxtaposed
right: Percentages lanes taken
of apoptosis from the same
as evaluated experiment
by Hoechst
and 33342
blot image. Upper
assay were Percentages
right:and
scored of apoptosis
plotted. Data are meanas ± evaluated
SD (n = 3).by** pHoechst 33342
< 0.01, *** assayversus
p < 0.001 were scored
Z/Parent-WT cells; two-sided Student’s t-test. Lower: Representative micrographs of Hoechst 33342
nuclear staining under an inverted fluorescence microscope. (B) Analysis of cell death by Annexin
V/7-AAD assay. Percentages of Annexin V single-positive cells defining early apoptosis, and An-
nexin V and 7-AAD double-positive cells defining late apoptosis/necrosis combined with 7-AAD
single-positive cells defining necrosis were plotted.
Int. J. Mol. Sci. 2022, 23, 14474 11 of 21

and plotted. Data are mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 versus Z/Parent-WT cells;
two-sided Student’s t-test. Lower: Representative micrographs of Hoechst 33342 nuclear staining
under an inverted fluorescence microscope. (B) Analysis of cell death by Annexin V/7-AAD assay.
Percentages of Annexin V single-positive cells defining early apoptosis, and Annexin V and 7-AAD
double-positive cells defining late apoptosis/necrosis combined with 7-AAD single-positive cells
defining necrosis were plotted.

2.7. High BCL2L1 and Low BAX Are in Part Associated with Poor Prognosis
Having shown that Bcl-xL activation and/or Bax depletion dictates the BTZ-resistant
phenotype in MCL cells, this section outlines how Bcl-xL and Bax might correlate with the
clinical outcome. Since it is well-accepted that drug resistance is dependent on multiple
mechanisms, we performed mRNA expression analysis of BCL2L1, BAX, and other genes
reported to be important regulators of BTZ resistance in MCL cells, including OGT, CD36,
and ZEB1, in human MCL microarrays available on Gene Expression Omnibus (GEO;
accession number GSE10793) [28] to examine the association between their expression and
survival of MCL patients. We first grouped patients according to their Bcl-2 family gene co-
expression into patients with high BCL2L1 (cutoff: above Q3) and low BAX (cutoff: below
Q3), and patients with the opposite gene co-expression of low BCL2L1 and high BAX. After
which, an additional gene of interest was added to the co-expression analysis. Figure 10A
shows a similar outcome between the two groups of Bcl-2 family gene co-expression.
O-GlcNAcylation is an essential posttranslational modification and an ideal metabolic
sensor in the hexosamine biosynthetic pathway that is regulated by two cycling enzymes
O,-GlcNAc transferase (OGT; encoded by OGT) and O-GlcNAcase (OGA encoded by
MGEA5) [29,30]. We recently reported that an increase in cellular O-GlcNAcylation, which
could be endorsed by an inhibition OGA or alternatively an activation of OGT, sensitizes
MCL cells to BTZ-induced apoptosis and reverses their apoptosis resistance via the sta-
bilization of truncated Bid (tBid) [31]. Herein, we showed that BTZ-resistant Z-138 cells
exhibited a decrease in cellular O-GlcNAcylation when compared to the parental cells, in
association with the degree of BTZ resistance (Figure 10B). Our survival analysis revealed
that MCL patients with low OGT (cutoff: below Q3) together with high BCL2L1 and low
BAX have shorter survival than those patients with high OGT: the hazard ratio increased
by approximately three-fold. As the overall survival in patients with high OGT versus low
OGT alone was not significantly different (p = 0.1700), our data suggest that high Bcl-xL and
low Bax could be cooperative factors that. with decreased O-GlcNAcylation, are involved
in controlling aggressive MCL (Figure 10C).
Dysregulated lipid metabolism in MCL cells via CD36 has been shown to be linked
to BTZ resistance [14]. Likewise, Zeb-1, which was highly expressed in a cancer stem cell
(CSC) subpopulation of MCL cells [32], induced lipid accumulation and correlated with
BTZ resistance in MCL cells [14]. Figure 10D,E, however, shows that the overall survival
between patients with high CD36 or ZEB1 (cutoff: above Q3) together with high BCL2L1
and low BAX, and patients with low CD36 or ZEB1 (cutoff: below Q3) alone, was not
significantly different, suggesting that Bcl-2 family proteins and CD36 or Zeb-1 might not
cause a synergistic effect.
Int. J. Mol. Sci. 2022, 23, 14474
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 12 of 23 12 of 21

A
kDa
100
Bcl-xL — 30

% Apoptosis
### ### ###
— 20 ###
Bax ***
50 ### *** ***
***
β-actin — 45
***
#

0
0 10 50 100 250 500
BTZ (nM)
Z/Parent
Z/Parent-WT Z/Parent-Bcl-xL + BAXi

0 10 50 250 500
Z/Parent-Bcl-xL
+ BAXi

B 0 50 100 250 500

Q1 Q2 Q1 Q2
0.3%
Q1
0.32
0.8%
Q2
0.76
Q1
0.8%
0.80
Q2
9.6%
9.63
Q1
1.2%
1.20 10.8%
10.8
Q2
1.3%
1.32
12.2%
12.2
1.7%
1.70
12.5%
12.5
Z/Parent-Bcl-xL

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
96.5%
96.5 2.5%
2.46 64.3%
64.3 25.3%
25.3 58.5%
58.5 29.5%
29.5 53.6%
53.6 32.8%
32.8 47.7%
47.7 38.0%
38.0

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2
0.4%
0.44
0.5%
0.53 0.5%
0.47 1.3%
1.27 0.4%
0.42 2.6%
2.63 1.3%
0.46 2.0%
2.00 0.3%
0.30 2.2%
2.18
+ BAXi
7-AAD

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
97.7%
97.7 1.3%
1.33 94.1%
94.1 4.1%
4.13 92.4%
92.4 4.5%
4.52 92.7%
92.7 4.9%
4.86 93.3%
93.3 4.2%
4.17

Annexin V-PE
BTZ (nM) 0 50 250 500
100
Late Apop/Nec
Early Apop
Cellls (%)

Viable
50

0
Z/Parent

Figure 8. Overexpression of Bcl-xL and depletion of Bax cooperatively induces acquired BTZ resistance
in MCL cells. Bcl-xL-overexpressed Z-138 cells were transduced with lentiviral particles carrying Cas9
and sgRNA against human BAX (Z/Parent-Bcl-XL + BAXi) or control particles (Z/Parent-Bcl-xL) and
treated with BTZ (0–500 nM) for 24 h. (A) Upper left: Western blot analysis of Bcl-xL and Bax levels in
Z/Parent-Bcl-xL + BAXi and Z/Parent-Bcl-xL cells in comparison to the parental Z/Parent-WT cells
prior to experiments. The dashed line separates juxtaposed lanes taken from the same experiment and
blot image. Upper right: Percentages of apoptosis as evaluated by Hoechst 33342 assay were scored and
plotted. Data are mean ± SD (n = 3). *** p < 0.001 versus Z/Parent-WT cells; # p < 0.05, ### p < 0.001
versus Z/Parent-Bcl-xL cells; two-sided Student’s t-test. Lower: Representative micrographs of Hoechst
33342 nuclear staining under an inverted fluorescence microscope. (B) Analysis of cell death by Annexin
V/7-AAD assay. Percentages of Annexin V single-positive cells defining early apoptosis, and Annexin V
Int. J. Mol. Sci. 2022, 23, 14474 13 of 21

and 7-AAD double-positive cells defining late apoptosis/necrosis combining with 7-AAD single-
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 14 of 23
positive cells defining necrosis were plotted.

A Jeko-1 kDa

Bcl-xL — 30

Bax — 20

β-actin — 45

B 0 10 50 250 500
Q1
0.3%
0.32
1.4%
Q2
1.37
2.2%
Q1
2.19
28.0%
Q2
28.0
1.1%
Q1 46.9%
Q2
1.0%
Q1 48.7%
Q2 Q1
0.8% 49.6%
Q2
1.09 46.9 0.97 48.7 0.77 49.6
WT

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
93.7%
93.7 4.6%
4.56 15.0%
15.0 54.8%
54.8 1.9%
1.86 50.0%
50.2 1.4%
1.38 49.0%
49.0
1.4%
1.41 48.3%
48.3

Q1
0.3% 0.9%
Q2
2.5%
Q1
8.9%
Q2 Q1
2.3%
2.33
Q2
25.6%
25.6
Q1
2.0%
1.95
Q2
29.5%
29.5
Q1
1.6%
Q2
37.5%
0.31 0.86 2.48 8.90 1.55 37.5
BAXi

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
95.3%
95.3 3.5%
3.50 55.0%
55.0 33.6%
33.6 25.1%
25.1 47.0%
47.0 20.4%
20.4 48.1%
48.1 17.7%
17.7 43.3%
43.3

0.4%
Q1
0.7%
Q2
0.6%
Q1 Q2
5.9% Q1
1.1%
Q2
22.6% Q1
1.2% Q2
17.1% Q1
1.1%
Q2
21.3%
0.40 0.71 0.62 5.87 1.11 13.4 1.24 17.1 1.12 21.3
Bcl-xL

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
96.2%
96.2 2.7%
2.67 78.5%
78.5 15.0%
15.0 62.9%
62.9 13.4%
22.6 58.4%
58.4 23.2%
23.2 55.4%
55.4 22.2%
22.2

0.4%
Q1 1.1%
Q2
1.1%
Q1
3.6%
Q2 Q1
1.3% Q2
8.8% Q1
1.1% Q2
13.0% Q1
2.0% Q2
11.0%
0.40 1.07 1.12 3.55 1.26 8.76 1.10 13.0 1.95 10.9
+ BAXi
7-AAD

Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
95.7%
95.7 2.9%
2.88 83.3%
83.3 12.0%
12.0 66.2%
66.2 23.8%
23.8 59.9%
59.9 25.9%
25.9 63.0%
62.9 24.2%
24.2

Annexin V-PE

BTZ (nM) 0 50 250 500

100
Late Apop/Nec
Early Apop
Cellls (%)

Viable
50

9. Generality
Figure Figure ofofthe
9. Generality observed
the observed Bcl-xL
Bcl-xL and
and Bax Baxineffects
effects in BTZ-induced
BTZ-induced apoptosis
apoptosis in MCL cells. in MCL
cells. Genetic manipulation
Genetic manipulation of Bcl-xL
of Bcl-xL and Baxand
was Bax wasperformed
similarly similarlyinperformed in human
human MCL-derived Jeko-1MCL-derived
cells using transgene and/or CRISPR/Cas9 system. After which, cells were treated with BTZ (0–500
Jeko-1 cells using transgene and/or CRISPR/Cas9 system. After which, cells were treated with
BTZ (0–500 nM) for 24 h. (A) Western blot analysis of Bcl-xL and Bax levels in control Jeko-1 (WT),
Bax-depleted (BAXi), Bcl-xL-overexpressed alone (Bcl-xL) or in combination with Bax depletion
(Bcl-xL + BAXi) cells prior to experiments. The dashed line separates juxtaposed lanes taken from the
same experiment and blot image. (B) Analysis of cell death by Annexin V/7-AAD assay. Percentages
of Annexin V single-positive cells defining early apoptosis, and Annexin V and 7-AAD double-
positive cells defining late apoptosis/necrosis combining with 7-AAD single-positive cells defining
necrosis were plotted.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 16 of 23
Int. J. Mol. Sci. 2022, 23, 14474 14 of 21

A
Lo BCL2L1 / Hi BAX
1.0
kDa

Probability of Survival
Hi BCL2L1 / Lo BAX

0.5
p = 0.6908
HR = 1.165

0.0
0 2 4 6 8 10
Overall Survival (years)

B C
kDa

— 130
Hi OGT
1.0
kDa
Probability of Survival
Lo OGT
— 45 Hi BCL2L1 / Lo BAX
O-GlcNAc
— 30
0.5
p = 0.0192
HR = 3.497
— 45
β-actin 0.0
0 5 10 15
Overall Survival (years)
0R
nt

0R

0R
re

50
10

25
Pa

Z/
Z/

Z/
Z/

D E
Lo CD36
1.0 kDa 1.0 Lo ZEB1
Probability of Survival

Probability of Survival

Hi CD36 Hi ZEB1
Hi BCL2L1 / Lo BAX Hi BCL2L1 / Lo BAX

0.5 0.5
p = 0.2927 p = 0.2245
HR = 1.494 HR = 1.767

0.0 0.0
0 5 10 15 0 2 4 6
Overall Survival (years) Overall Survival (years)

Figure 10. Kaplan-Meier survival plot of MCL patients from the gene microarrays (GSE10793)
Figure 10. Kaplan-Meier survival plot of MCL patients from the gene microarrays (GSE10793) ac-
according to the levels of BCL2L1, BAX, OGT, CD36, and ZEB1 expression. Values above the upper
cording to the levels of BCL2L1, BAX, OGT, CD36, and ZEB1 expression. Values above the upper
quartile
quartile (Q3)
(Q3)were
weredefined
definedasashigh
highexpression.
expression. (A)(A)
Overall
Overallsurvival of patients
survival withwith
of patients BCL2L1
highhigh and
BCL2L1
low BAX (red line) is compared to patients with low BCL2L1 and high BAX (green
and low BAX (red line) is compared to patients with low BCL2L1 and high BAX (green line). (B) line). (B) Western
blot analysis
Western blot of
analysis O-GlcNAc
global of level in parental
global O-GlcNAc level inZ/Parent
parental and BTZ-resistant
Z/Parent Z/100R, Z/250R,
and BTZ-resistant Z/100R,
Z/250R,
and and cells
Z/500R Z/500R cells showing
showing a dose-dependent
a dose-dependent decreasedecrease inO-GlcNAcylation
in cellular cellular O-GlcNAcylation
when thewhencells
the cells acquired
acquired higher BTZ higher BTZ resistance.
resistance. (C) Overall (C)survival
Overallofsurvival
patientsofwith
patients with together
low OGT low OGTwith together
high
with high
BCL2L1 andBCL2L1
low BAX and (red
low line)
BAX is(red line) is compared
compared to patientstowith
patients
highwith
OGThigh OGT
(green (green
line). (D)line). (D)
Overall
Overall survival
survival of patientsof with
patients
highwith
CD36 high CD36 with
together together
highwith
BCL2L1highand
BCL2L1 and (red
low BAX low line)
BAXis(red line) is
compared
compared to patients with low CD36 (green line). (E) Overall survival of patients with high ZEB1
to patients with low CD36 (green line). (E) Overall survival of patients with high ZEB1 together
together with high BCL2L1 and low BAX (red line) is compared to patients with low ZEB1 (green
with high BCL2L1 and low BAX (red line) is compared to patients with low ZEB1 (green line). HR,
line). HR, hazard ratio.
hazard ratio.
Int. J. Mol. Sci. 2022, 23, 14474 15 of 21

3. Discussion
MCL is a particularly deadly subtype of NHL with poor prognosis, attributable to its
innate and acquired resistance to current chemotherapy regimens. Despite being the first-in-
class proteasome inhibitor for relapsed/refractory MCL, resistance to BTZ in MCL patients
remains a major hurdle of effective therapy, and relapse following BTZ is frequent [7,8,33].
Therefore, a better understanding of the mechanisms underlying BTZ resistance is crucial
for developing novel strategies for better outcomes. Bcl-2 family proteins have been shown
to be important in regulating apoptosis, including anoikis, during tumor progression and
metastasis, and in regulating cellular responses to various chemotherapeutic drugs [34–36].
Activation of antiapoptotic Bcl-2 family proteins, such as Bcl-2, Mcl-1, and Bcl-xL, are
frequently observed in chemoresistant tumor cells, offsetting the functions of proapoptotic
Bcl-2 family proteins and suppressing apoptosis [37,38]. In MCL cells, dysregulation of
Bcl-2 family proteins, including BCL2 overexpression and BIM repression caused by genetic
abnormalities, and BCL2L1 upregulation caused by microenvironmental interactions, are
detected [39,40]. Accordingly, antiapoptotic Bcl-2 family proteins have become important
therapeutic targets for which several ongoing clinical trials have been conducted with Bcl-2
inhibitors, either alone or in combination with existing chemotherapeutic drugs.
Upon screening of several genes involved in drug resistance and cell survival of MCL,
we found the striking upregulation of BCL2L1 (encodes Bcl-xL) and downregulation of BAX
in highly BTZ-resistant cells (Figure 3). Subsequent Western blot analyses suggested that the
elevated Bcl-xL level in BTZ-resistant cells, which correlates well with its gene expression
and the degree of BTZ resistance, is likely mediated via transcriptional program, while Bax
level is additionally mediated at the posttranscriptional level (Figure 5). Reported PTMs of
Bax include the phosphorylation at S184 and T167, which inactivates Bax, the dephospho-
rylation at T172, T174, and T176, which downregulates Bax, and the polyubiquitination at
a non-specific site, which targets Bax for proteasomal degradation [24].
Overexpression of Bcl-xL has been reported in the majority of various hematologic
malignancies, including Burkitt’s lymphoma (BL), diffuse large B cell lymphoma (DL-
BCL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple
myeloma (MM) [41,42]. Although little is known about the role of Bcl-xL in BTZ response
in hematologic malignancies, a gradual loss of Bcl-xL, in concert with Noxa upregulation
and its increased binding to Bcl-xL, was shown to be necessary for BTZ-induced apoptosis
in neuroblastoma cells [43]. In murine hematopoietic cells, expression of bcl-xL caused mul-
tidrug resistance to chemotherapeutic agents, including bleomycin, cisplatin, vincristine,
and etoposide [44]. Additionally, Bcl-xL has been linked to the acquired resistance of
chronic lymphocytic leukemia (CLL) cells to ABT-199 (venetoclax), an orally bioavailable
and selective Bcl-2 inhibitor approved for both treatment-naïve and relapsed CLL [45].
We herein report that overexpression of Bcl-xL desensitized various BTZ-sensitive MCL
cells to BTZ-induced apoptosis (Figures 6 and 9), highlighting the contribution of Bcl-xL to
BTZ sensitivity.
Activation of the proapoptotic pore-formers Bax and Bak is an indispensable gateway
to MOMP and consequently apoptosis. Downregulation and loss-of-function mutation
of BAX were found to be associated with chemoresistance in various cancers [46]. A
missense G179E BAX mutation, which abrogates Bax anchoring to mitochondria and blocks
apoptosis, was found in ABT-199-resistant MCL cells. Interestingly, G179E BAX mutation
could possibly explain the partial cross-resistance of ABT-199-resistant MCL cells to other
cytotoxic drugs, including taxol, cisplatin, doxorubicin, temsirolimus, TRAIL, and BTZ [47].
In the present study, depletion of Bax in BTZ-sensitive MCL cells exerted protective effects
against BTZ-induced apoptosis (Figures 7 and 9). We earlier showed that BTZ treatment
in MCL cells caused the induction of tBid [31], which could somehow recruit inactive Bax
from the cytosol and induce its oligomerization and MOMP [24]. Thereby, depletion of Bax
might interfere with the function of tBid in mediating BTZ-induced apoptosis.
The relative ratios of pro- and antiapoptotic Bcl-2 family proteins have been shown
to determine the sensitivity of cells to various apoptotic stimuli, including loss of cell
Int. J. Mol. Sci. 2022, 23, 14474 16 of 21

anchorage, growth factor deprivation, radiation, and chemotherapeutic drugs [48]. We

showed that overexpression of Bax, together with depletion of Bcl-xL, mimicking the highly
BTZ-resistant MCL cells, strongly tilted the balance toward antiapoptotic signaling and
resulted in even higher protection against BTZ when compared to overexpression of Bcl-
xL alone (Figures 8 and 9). Notably, the cooperative effect of Bcl-xL and Bax was more
noticeable in Z-138 cells, likely due to the existing strong protective effect of Bcl-xL alone on
BTZ-induced apoptosis in Jeko-1 cells. A recent study showed that co-targeting of Bcl-xL
and Bax by ABT-263 (navitoclax), an orally bioavailable inhibitor of Bcl-xL, Bcl-2, and Bcl-w,
and BTSA1.2, an orally bioavailable Bax activator, synergistically increased cytotoxicity in
many hematologic and solid tumor cell lines, regardless of common genetic alterations,
such as TP53 and RAS mutation, when compared to ABT-263 alone. Altogether, these
findings strengthen the concept that co-targeting Bcl-xL and Bax is an attractive therapeutic
strategy for overcoming cancer drug resistance [26]. Earlier, the idea of co-targeting Mcl-1
and Noxa in MCL cells was proposed by combining CDK inhibitor dinaciclib and fatty acid
synthase (FASN) inhibitors, e.g., orlistat [49]. The cytotoxicity of dinaciclib, which caused
Mcl-1 reduction, can be augmented by forced stabilization of Noxa using FASN inhibitors,
thereby supporting that the balance of pro- and antiapoptotic Bcl-2 family proteins is critical
in determining MCL cell fate.
BTZ resistance in MCL cells is a multifactorial phenomenon involving the CSC subpop-
ulation, plasmacytic differentiation, and cellular metabolism [12,14,32]. Our bioinformatics
analyses revealed that MCL patients with low OGT together with high BCL2L1 and low
BAX have poorer prognosis than those patients with high OGT (Figure 10). As cellular
O-GlcNAcylation, which can be endored by either OGA inhibition or OGT activation, can
enhance BTZ sensitivity in de novo BTZ-resistant cells via tBid [31], it is interesting to know
that expression of OGT, BCL2L1, and BAX cooperatively predicts the clinical outcome in
MCL patients.

4. Materials and Methods

4.1. Reagents
Bortezomib was obtained from Janssen-Cilag (Beerse, Belgium). Hoechst 33342 was
obtained from Molecular Probes (Eugene, OR, USA). PE Annexin V Apoptosis Detection
Kit I was obtained from BD Bioscience (BD Bioscience, San Jose, CA, USA). Antibodies
for Bcl-xL, Bax, and β-actin were obtained from Cell Signaling Technology (Danvers, MA,
USA), while an antibody for O-GlcNAc (RL2) was from Abcam (Cambridge, UK).

4.2. Cell Culture

Human MCL-derived Jeko-1 cells (RRID:CVCL_1865) were obtained from American
Type Culture Collection (ATCC; Manassas, MA, USA), while human MCL-derived Z-
138 cells (RRID:CVCL_B077) were a kind gift from Dr. Siwanon Jirawatnotai (Systems
Pharmacology, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand). STR analysis of the
cells (16 loci) was performed at the Department of Forensic Medicine, Faculty of Medicine
Siriraj Hospital, Mahidol University and the profiles can be found in Supplementary Tables
S1 and S2. Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin at
37 ◦ C and 5% CO2 . Mycoplasma contamination was regularly checked using a commercial
test kit (MycoAlert™ PLUS, Lonza, Cologne, Germany) and any cell lines found positive
were discarded.

4.3. Generation of De Novo BTZ-Resistant Cells

BTZ-resistant Z-138 cells were generated by a stepwise selection method as de-
scribed [14]. Parental MCL-derived Z-138 (Z/Parent) cells were continuously exposed to
increasing concentrations of BTZ to the maximum concentration of 100, 250 and 500 nM.
Resistant cells were examined by Annexin V-PE/7-AAD assay, where double negative
(viable) cells were sorted using BD FACSAria cell sorter (BD Bioscience, San Jose, CA, USA)
Int. J. Mol. Sci. 2022, 23, 14474 17 of 21

and designated as Z/100R, Z/250R, and Z/500R cells, respectively. The response curves of
BTZ on parental cells and resistant cells at different degrees were plotted at 24 h and LC50
was calculated using GraphPad Prism software (La Jolla, CA, USA).

4.4. Hoechst 33342 Apoptosis Assay

Cells were incubated with 10 µg/mL Hoechst 33342 for 30 min and analyzed for
apoptosis by scoring of cells having condensed (brighter than non-apoptotic) and/or
fragmented nuclei by fluorescence microscopy (Eclipse Ti-U with NiS-Elements, Nikon,
Tokyo, Japan). Faded nuclei, an indication of karyolysis, were sometimes observed. The
apoptotic index was calculated as the percentage of cells with apoptotic nuclei over the
total number of cells.

4.5. Annexin V/7-AAD Assay

Cells were harvested, washed, and stained with Annexin V-PE in binding buffer
and 7-AAD for 15 min at room temperature. Samples were analyzed by BD FACSCanto
flow cytometer (BD Bioscience) using a 488-nm excitation beam and 575-nm and 670-nm
band-pass filters with CellQuest software. Subsequent analysis was performed by Flowjo
(v10.4.1) software.

4.6. RNA Isolation and qPCR

Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) ac-
cording to the manufacturer’s protocols, and cDNA was prepared using SuperScript III
First-Strand Synthesis System and oligo (dT) primers (Invitrogen). qPCR analysis was
carried out on a CFX384 Real-Time PCR (BioRad, Hercules, CA, USA) using a SYBR Green
PCR Master Mix (Applied Biosystems, Waltham, MA). Initial enzyme activation was per-
formed at 95 ◦ C for 10 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s and
primer annealing/extension at 60 ◦ C for 1 min. Relative expression of each gene was
normalized against the housekeeping GAPDH gene product. Heatmap and cluster analysis
were performed using MultiExperiment Viewer (MeV 4.9.0) software.

4.7. BioCarta Pathway Analysis

Lists of significantly changed genes comparing parental Z/Parent and BTZ-resistant
Z/500R cells (p < 0.05) were input into DAVID (v6.8) Bioinformatics Resources, a com-
prehensive set of functional annotation tools. BioCarta pathways were then analyzed
and visualized.

4.8. Overexpression Plasmid and Retroviral Transduction

Retroviral plasmid carrying human Bcl-xL transgene (3541 pMIG Bcl-XL) and GFP
selection marker was a kind gift from Prof. Stanley Korsmeyer (Addgene #8790) [50].
Retrovirus production was performed using Platinum-A packaging cell lines (Cell Bio-
labs, San Diego, CA). Cells were incubated with Bcl-xL viral particles in the presence of
hexadimethrine bromide (HBr) for 48 h and the transduced cells with GFP (FITC)-positive
cells were enriched by BD FACSAria cell sorter (BD Bioscience) and analyzed prior to use
by Western blotting.

4.9. CRISRP/Cas9-Mediated BAX Knockdown

The all-in-one lentiviral plasmid carrying Streptococcus pyogenes Cas9 (SpCas9), puromycin
resistance gene, and sgRNA sequence specific to human BAX was a kind gift from Prof.
Stephen Tait (Addgene #129580) [51]. The plasmid was transfected into HEK293T pack-
aging cells in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G
envelope plasmids (Addgene #8454 and 8455) [52] to produce lentiviral particles. The
particles were transduced into parental cells or Bcl-xL-overexpressed cells in the presence
of 8 µg/mL HBr for 48 h and were treated with 1 µg/mL puromycin for 1 month for the
Int. J. Mol. Sci. 2022, 23, 14474 18 of 21

selection of stably transfected cells. Depletion of Bax was confirmed by Western blotting
prior to the experiments.

4.10. Western Blot Analysis

Cells were incubated in lysis buffer (Cell Signaling Technology) and protease inhibitor
mixtures (Roche Molecular Biochemicals, Indianapolis, IN, USA) at 4 ◦ C for 30 min. The
protein concentration was determined using bicinchoninic acid (BCA) assay (Pierce Biotech-
nology, Rockford, IL, USA). Proteins (30–50 µg) were separated by 12% SDS-PAGE and
transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in
TBST for 1 h at room temperature. Subsequently, membranes were incubated with appro-
priate primary antibodies overnight at 4 ◦ C and incubated with secondary antibodies for
1 h at room temperature. Immunoblots were detected by enhanced the chemiluminescence
system and visualized on an ImageQuantTM digital imager (GE Healthcare, Pittsburgh,
PA, USA).

4.11. Gene Microarray Dataset and Survival Analysis

The published dataset Gene Expression Omnibus (GEO) accession number GSE10793,
which was obtained from tumor biopsies from 71 untreated MCL patients, was used to cal-
culate overall survival associated with BCL2A1, BAX and other related genes [28]. Genechip
measurements were performed using NCI/Staudt human 15K v13 arrays. Kaplan-Meier
survival plot of MCL patients was generated using GraphPad Prism software according
to the level of gene expression with the cutoff value for high expression at upper quartile
(Q3). Expression values below Q3 were defined as low expression.

4.12. Statistical Analysis

The data represent means ± SD from three or more independent experiments as
indicated. Statistical analysis between two groups was performed by two-sided Student’s
t-test at a significance level of p < 0.05.

5. Conclusions
In conclusion, we provide substantial evidence that the Bcl-2 family proteins Bcl-xL
and Bax are key mediators in determining BTZ sensitivity in MCL cells. Overexpression
of Bcl-xL worked cooperatively with depletion of Bax to protect MCL cells against BTZ-
induced apoptosis, causing acquired BTZ resistance. Co-targeting Bcl-xL and Bax, e.g., by
using Bcl-xL inhibitor and Bax activator is, therefore, a promising strategy for overcoming
resistance to BTZ and/or other chemotherapeutic drugs in MCL cells, in which underlying
mechanisms involved the imbalance or aberrant of Bcl-2 family proteins, to improve clinical
outcome and achieve long-term control of disease. It is worth noting that several selective
drugs designed to inhibit antiapoptotic Bcl-2 family proteins, termed BH3 mimetics, have
been developed. Our findings support recent efforts in small molecule drug discovery that
Bax also serves as a promising target and that direct Bax activators could be a new class of
drug candidates for cancer therapy.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms232214474/s1.
Author Contributions: Conceptualization, S.L. and S.I.; methodology, S.L. and P.C.; validation, S.L.,
M.J. and J.Y.; formal analysis, S.L., M.J. and J.Y.; investigation, M.J., J.Y. and J.P.; resources, S.L. and
S.I.; data curation, S.L. and M.J.; writing—original draft preparation, S.L. and J.Y.; writing—review
and editing, S.L.; visualization, S.L.; supervision, P.C. and S.I.; funding acquisition, S.L. and S.I. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Research Council of Thailand (NRCT) and Mahidol
University (N42A650372, to S.L.), Specific League Funds (to S.L.) and Postdoctoral Fellowship Award
(to J.Y.) from Mahidol University, and Siriraj Foundation for Stem Cell Research (D003276).
Institutional Review Board Statement: Not applicable.
Int. J. Mol. Sci. 2022, 23, 14474 19 of 21

Informed Consent Statement: Not applicable.

Data Availability Statement: The data used to support the findings of this study are included within
the article.
Acknowledgments: We would like to acknowledge Sirinart Buasumrit for administrative support.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Jain, P.; Wang, M. Mantle cell lymphoma: 2019 update on the diagnosis, pathogenesis, prognostication, and management. Am. J.
Hematol 2019, 94, 710–725. [CrossRef] [PubMed]
2. Teras, L.R.; DeSantis, C.E.; Cerhan, J.R.; Morton, L.M.; Jemal, A.; Flowers, C.R. 2016 US lymphoid malignancy statistics by World
Health Organization subtypes. CA Cancer J. Clin. 2016, 66, 443–459. [CrossRef] [PubMed]
3. Vose, J.M. Mantle cell lymphoma: 2017 update on diagnosis, risk-stratification, and clinical management. Am. J. Hematol. 2017,
92, 806–813. [CrossRef] [PubMed]
4. Kane, R.C.; Dagher, R.; Farrell, A.; Ko, C.W.; Sridhara, R.; Justice, R.; Pazdur, R. Bortezomib for the treatment of mantle cell
lymphoma. Clin. Cancer Res. 2007, 13, 5291–5294. [CrossRef]
5. Goy, A.; Bernstein, S.H.; Kahl, B.S.; Djulbegovic, B.; Robertson, M.J.; De Vos, S.; Epner, E.; Krishnan, A.; Leonard, J.P.; Lonial, S.;
et al. Bortezomib in patients with relapsed or refractory mantle cell lymphoma: Updated time-to-event analyses of the multicenter
phase 2 PINNACLE study. Ann. Oncol. 2009, 20, 520–525. [CrossRef]
6. O’Connor, O.A.; Moskowitz, C.; Portlock, C.; Hamlin, P.; Straus, D.; Dumitrescu, O.; Sarasohn, D.; Gonen, M.; Butos, J.; Neylon,
E.; et al. Patients with chemotherapy-refractory mantle cell lymphoma experience high response rates and identical progression-
free survivals compared with patients with relapsed disease following treatment with single agent bortezomib: Results of a
multicentre Phase 2 clinical trial. Br. J. Haematol. 2009, 145, 34–39.
7. Perez-Galan, P.; Dreyling, M.; Wiestner, A. Mantle cell lymphoma: Biology, pathogenesis, and the molecular basis of treatment in
the genomic era. Blood 2011, 117, 26–38. [CrossRef]
8. Diefenbach, C.S.; O’Connor, O.A. Mantle cell lymphoma in relapse: The role of emerging new drugs. Curr. Opin. Oncol. 2010,
22, 419–423. [CrossRef]
9. Gottesman, M.M.; Fojo, T.; Bates, S.E. Multidrug resistance in cancer: Role of ATP-dependent transporters. Nat. Rev. Cancer 2002,
2, 48–58. [CrossRef]
10. McFadyen, M.C.E.; Melvin, W.T.; Murray, G.I. Cytochrome P450 enzymes: Novel options for cancer therapeutics. Mol. Cancer
Ther. 2004, 3, 363–371. [CrossRef]
11. Pommier, Y.; Sorder, O.; Antony, S.; Hayward, R.L.; Kohn, K.W. Apoptosis defects and chemotherapy resistance: Molecular
interaction maps and networks. Oncogene 2004, 23, 2934–2949. [CrossRef] [PubMed]
12. Pérez-Galán, P.; Mora-Jensen, H.; Weniger, M.A.; Shaffer III, A.L.; Rizzatti, E.G.; Chapman, C.M.; Mo, C.C.; Stennett, L.S.; Rader,
C.; Liu, P.; et al. Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation. Blood 2011,
117, 542–552. [CrossRef] [PubMed]
13. Kim, A.; Seong, K.M.; Kang, H.J.; Park, S.; Lee, S.S. Inhibition of Lyn is a promising treatment for mantle cell lymphoma with
bortezomib resistance. Oncotarget 2015, 6, 38225–38238. [CrossRef]
14. Luanpitpong, S.; Janan, M.; Thumanu, K.; Poohadsuan, J.; Rodboon, N.; Klaihmon, P.; Issaragrisil, S. Deciphering the elevated lipid
via CD36 in mantle cell lymphoma with bortezomib resistance using synchrotron-based Fourier transform infrared spectroscopy
of single cells. Cancers 2019, 11, 576. [CrossRef]
15. Shah, M.A.; Schwartz, G.K. Cell cycle-mediated drug resistance: An emerging concept in cancer therapy. Clin. Cancer Res. 2001,
7, 2168–2181.
16. Dai, B.; Grau, M.; Juilland, M.; Klener, P.; Höring, E.; Molinsky, J.; Schimmack, G.; Aukema, S.M.; Hoster, E.; Vogt, N.; et al. B-cell
receptor-driven MALT1 activity regulates MYC signaling in mantle cell lymphoma. Blood 2017, 129, 333–346. [CrossRef]
17. Bisikirska, B.C.; Adam, S.J.; Alvarez, M.J.; Rajbhandari, P.; Cox, R.; Lefebvre, C.; Wang, K.; Rieckhof, G.E.; Felsher, D.W.; Califano,
A. STK38 is a critical upstream regulator of MYC’s oncogenic activity in human B-cell lymphoma. Oncogene 2013, 32, 5283–5291.
[CrossRef]
18. Vishwamitra, D.; Shi, P.; Wilson, D.; Manshouri, R.; Vega, F.; Schlette, E.J.; Amin, H.M. Expression and effects of inhibition of type
I insulin-like growth factor receptor tyrosine kinasein mantle cell lymphoma. Haematologica 2011, 96, 871–880. [CrossRef]
19. Auer, R. Discovery of Hippo in MCL. Blood 2010, 116, 861–862. [CrossRef]
20. Mathur, R.; Sehgal, L.; Braun, F.K.; Berkova, Z.; Romaguerra, J.; Wang, M.; Rodriguez, M.A.; Fayad, L.; Neelapu, S.S.; Samaniego,
F. Targeting Wnt pathway in mantle cell lymphoma-initiating cells. J. Hematol. Oncol. 2015, 8, 63. [CrossRef]
21. Dennis, G.J.; Sherman, B.T.; Hosack, D.A.; Yang, J.; Gao, W.; Lane, H.C.; Lempicki, R.A. DAVID: Database for annotation,
visualization, and integrated discovery. Genome Biol. 2003, 4, R60. [CrossRef]
22. Imamichi, T.; Yang, J.; Huang, D.W.; Sherman, B.; Lempicki, R.A. Interleukin-27 induces interferon-inducible genes: Analysis of
gene expression profiles using Affymetrix microarray and DAVID. Methods Mol. Biol. 2012, 820, 25–53.
Int. J. Mol. Sci. 2022, 23, 14474 20 of 21

23. Shamas-Din, A.; Kale, J.; Leber, B.; Andrews, D.W. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb. Perspect. Biol.
2013, 5, a008714. [CrossRef] [PubMed]
24. Kale, J.; Osterlund, E.J.; Andrews, D.W. BCL-2 family proteins: Changing partners in the dance towards death. Cell Death Differ.
2018, 25, 65–80. [CrossRef] [PubMed]
25. Peña-Blanco, A.; García-Sáez, A.J. Bax, Bak and beyond–mitochondrial performance in apoptosis. FEBS J. 2018, 285, 416–431.
[CrossRef] [PubMed]
26. Lopez, A.; Reyna, D.E.; Gitego, N.; Kopp, F.; Zhou, H.; Miranda-Roman, M.A.; Nordstrøm, L.U.; Narayanagari, S.R.; Chi, P.; Vilar,
E.; et al. Co-targeting of BAX and BCL-XL proteins broadly overcomes resistance to apoptosis in cancer. Nat. Commun. 2022, 13,
1199. [CrossRef] [PubMed]
27. Fernandez-Luna, J.L. Regulation of pro-apoptotic BH3-only proteins and its contribution to cancer progression and chemoresis-
tance. Cell Signal. 2008, 20, 1921–1926. [CrossRef] [PubMed]
28. Blenk, S.; Engelmann, J.C.; Pinkert, S.; Weniger, M.; Schultz, J.; Rosenwald, A.; Müller-Hermelink, H.K.; Müller, T.; Dandekar,
T. Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by
explorative CGH analysis. BMC Cancer 2008, 8, 106. [CrossRef]
29. Bond, M.R.; Hanover, J.A. A little sugar goes a long way: The cell biology of O-GlcNAc. J. Cell Biol. 2015, 208, 869–880. [CrossRef]
30. Jóźwiak, P.; Forma, E.; Bryś, M.; Krześlak, A. O-GlcNAcylation and metabolic reprogramming in cancer. Front. Endocrinol. 2014, 5, 145.
31. Luanpitpong, S.; Chanthra, N.; Janan, M.; Poohadsuan, J.; Samart, P.; U-Pratya, Y.; Rojanasakul, Y.; Issaragrisil, S. Inhibition of
O-GlcNAcase sensitizes apoptosis and reverses bortezomib resistance in mantle cell lymphoma through modification of truncated
Bid. Mol. Cancer Ther. 2018, 17, 484–496. [CrossRef] [PubMed]
32. Luanpitpong, S.; Poohadsuan, J.; Samart, P.; Kiratipaiboon, C.; Rojanasakul, Y.; Issaragrisil, S. Reactive oxygen species mediate
cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma. Biochim. Biophys. Acta
Mol. Basis Dis. 2018, 1864, 3739–3753. [CrossRef] [PubMed]
33. Gonzalez-Santamarta, M.; Quinet, G.; Reyes-Garau, D.; Sola, B.; Roué, G.; Rodriguez, M.S. Resistance to the proteasome inhibitors:
Lessons from multiple myeloma and mantle cell lymphoma. Adv. Exp. Med. Biol. 2020, 1233, 153–174. [PubMed]
34. Luanpitpong, S.; Chanvorachote, P. Nitric oxide and aggressive behavior of lung cancer cells. Anticancer Res. 2015, 35, 4585–4592.
35. Medan, D.; Luanpitpong, S.; Azad, N.; Wang, L.; Jiang, B.H.; Davis, M.E.; Barnett, J.B.; Guo, L.; Rojanasakul, Y. Multifunctional role
of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells. PLoS ONE 2012, 7, e37045. [CrossRef]
36. Delbridge, A.R.; Strasser, A. The BCL-2 protein family, BH3-mimetics and cancer therapy. Cell Death Differ. 2015, 22, 1071–1080.
[CrossRef]
37. Maji, S.; Panda, S.; Samal, S.K.; Shriwas, O.; Rath, R.; Pellecchia, M.; Emdad, L.; Das, S.K.; Fisher, P.B.; Dash, R. Bcl-2 antiapoptotic
family proteins and chemoresistance in cancer. Adv. Cancer Res. 2018, 137, 37–75.
38. Kapoor, I.; Bodo, J.; Hill, B.T.; His, E.D.; Almasan, A. Targeting BCL-2 in B-cell malignancies and overcoming therapeutic
resistance. Cell Death Dis. 2020, 11, 941. [CrossRef]
39. Thus, Y.J.; Eldering, E.; Kater, A.P.; Spaargaren, M. Tipping the balance: Toward rational combination therapies to overcome
venetoclax resistance in mantle cell lymphoma. Leukemia 2022, 36, 2165–2176. [CrossRef]
40. Tessoulin, B.; Papin, A.; Gomez-Bougie, P.; Bellanger, C.; Amiot, M.; Pellat-Deceunynck, C.; Chiron, D. BCL2-family dysregulation
in B-cell malignancies: From gene expression regulation to a targeted therapy biomarker. Front. Oncol. 2019, 8, 645. [CrossRef]
41. Xerri, L.; Parc, P.; Brousset, P.; Schlaifer, D.; Hassoun, J.; Reed, J.C.; Krajewski, S.; Birnbaum, D. Predominant expression of the
long isoform of Bcl-x (Bcl-xL) in human lymphomas. Br. J. Haematol. 1996, 92, 900–906. [CrossRef] [PubMed]
42. Morales-Martínez, M.; Vega, M.I. Roles and regulation of BCL-xL in hematological malignancies. Int. J. Mol. Sci. 2022, 23, 2193.
[CrossRef] [PubMed]
43. Hagenbuchner, J.; Ausserlechner, M.J.; Porto, V.; David, R.; Meister, B.; Bodner, M.; Villunger, A.; Geiger, K.; Obexer, P. The
anti-apoptotic protein BCL2L1/Bcl-xL is neutralized by pro-apoptotic PMAIP1/Noxa in neuroblastoma, thereby determining
bortezomib sensitivity independent of prosurvival MCL1 expression. J. Biol. Chem. 2010, 285, 6904–6912. [CrossRef]
44. Minn, A.J.; Rudin, C.M.; Boise, L.H.; Thompson, C.B. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood
1995, 86, 1903–1910. [CrossRef] [PubMed]
45. Haselager, M.; Thijssen, R.; West, C.; Young, L.; Van Kampen, R.; Willmore, E.; Mackay, S.; Kater, A.; Eldering, E. Regulation of
Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL. Cell Death Differ. 2021, 28, 1658–1668.
[CrossRef]
46. Liu, Z.; Ding, Y.; Ye, N.; Wild, C.; Chen, H.; Zhou, J. Direct activation of Bax protein for cancer therapy. Med. Res. Rev. 2016,
36, 313–341. [CrossRef] [PubMed]
47. Fresquet, V.; Rieger, M.; Carolis, C.; García-Barchino, M.J.; Martinez-Climent, J.A. Acquired mutations in BCL2 family proteins
conferring resistance to the BH3 mimetic ABT-199 in lymphoma. Blood 2014, 123, 4111–4119. [CrossRef]
48. Reed, J.C. Balancing cell life and death: Bax, apoptosis, and breast cancer. J. Clin. Investig. 1996, 97, 2403–2404. [CrossRef]
49. Höring, E.; Montraveta, A.; Heine, S.; Kleih, M.; Schaaf, L.; Vöhringer, M.C.; Esteve-Arenys, A.; Roué, G.; Colomer, D.; Campo,
E.; et al. Dual targeting of MCL1 and NOXA as effective strategy for treatment of mantle cell lymphoma. Br. J. Haematol. 2017,
177, 557–561. [CrossRef]
50. Cheng, E.H.; Wei, M.C.; Weiler, S.; Flavell, R.A.; Mak, T.W.; Lindsten, T.; Korsmeyer, S.J. BCL-2, BCL-X(L) sequester BH3
domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 2001, 8, 705–711. [CrossRef]
Int. J. Mol. Sci. 2022, 23, 14474 21 of 21

51. Lopez, J.; Bessou, M.; Riley, J.S.; Giampazolias, E.; Todt, F.; Rochegue, T.; Oberst, A.; Green, D.R.; Edlich, F.; Ichim, G.; et al.
Mito-priming as a method to engineer Bcl-2 addiction. Nat. Commun. 2016, 7, 10538. [CrossRef] [PubMed]
52. Stewart, S.A.; Dykxhoorn, D.M.; Palliser, D.; Mizuno, H.; Yu, E.Y.; An, D.S.; Sabatini, D.M.; Chen, I.S.; Hahn, W.C.; Sharp, P.A.;
et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 2003, 9, 493–501. [CrossRef] [PubMed]