Therapeutic effect and mechanism of ibrutinib combined with dexamethasone on multiple myeloma
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ORIGINAL ARTICLES
Hematology Department of The Second Hospital1, Cheeloo College of Medicine, Shandong University; Department
of Hematology of Jining No. 1 People’s Hospital2; Institute of Biotherapy for Hematological Malignancies of Shandong
University3; Shandong University-Karolinska Institute Collaborative Laboratory for Stem Cell Research4; Hematology
Department of Linyi Central Hospital5; Hematology Department of Binzhou Medical University Hospital6; Institute of Medical
Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University7, Jinan, Shandong, China
Therapeutic effect and mechanism of ibrutinib combined with dexametha-
sone on multiple myeloma
SHENGLI LI1,2, LIKUN SUN1,3,4, QIAN ZHOU1,5, SHUO LI1,6, XIAOLI LIU1,3,4, JUAN XIAO1,3,4, YAQI XU1,3,4, FANG WANG7, YANG JIANG1,3,4,*,
CHENGYUN ZHENG1,3,4
Received November 14, 2020, accepted December 2020
*Correspondence author: Yang Jiang, Hematology Department, the Second Hospital of Shandong University, 247th
of Beiyuan Rd., Jinan, Shandong, China
yangjiang@email.sdu.edu.cn
Pharmazie 76: 92-96 (2021) doi: 10.1691/ph.2021.0917
Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase and has proven to be an effective agent for
B-cell-mediated hematological malignancies, including multiple myeloma (MM). Several clinical trials of ibrutinib
treatment combined with dexamethasone (DXMS) for relapsed MM have demonstrated high response rates,
however, the mechanism still remains unclear. In this study, we explored the therapeutic effect and mechanism
of ibrutinib combined with DXMS on MM in vitro and vivo. The apoptosis of MM cell lines and mononuclear cells
from MM patients’ bone marrow induced by ibrutinib combined with DXMS was detected by flow cytometry and
the expression of apoptosis-related proteins were detected by Western blot. A mice MM model was established
to verify the therapeutic effect of ibrutinib combined with DXMS on MM. We found that ibrutinib combined
with DXMS increased the apoptosis of MM cell lines through the PI3K/PARP pathway, significantly reduced
CD38 expression in MM cells from patients in vitro, and reduced tumor size and increased the survival time in
mice model. This study provides a theoretical basis for the treatment of relapsed refractory MM with ibrutinib
combined with DXMS, and a potential therapeutic target for MM clinical treatment.
1. Introduction Corticosteroids are standard initial treatments in multiple diseases,
Multiple myeloma (MM), a B-cell hematologic malignancy with many of these showing rapid efficacy. Dexamethasone
characterized by abnormal infiltration of terminally differentiated (DXMS), an important corticosteroid, has been reported to provide
plasma cells in bone marrow, is the second most common hemato- an initial rapid response in several days to weeks (Stasi et al. 1995).
logical malignancy after non-Hodgkin’s lymphoma (Anderson and However, corticosteroid-induced complications have limited its
Carrasco 2011; Siegel et al. 2018). Although numerous therapeutic efficacy as well as long-term and high-dose utility. Consequently,
options have improved outcomes, relapse is frequent, and MM more and more studies have combined other drugs with DXMS to
remains incurable with a 5-year survival rate of 40%. Therefore, improve efficacy and achieve long-term applications (David et al.
novel treatments producing optimal outcomes are urgently needed 2014; Bussel et al. 2014). In patients with relapsed/refractory MM,
(Dimopoulos et al. 2012). ibrutinib combined with DXMS has demonstrated encouraging
Bruton’s tyrosine kinase (BTK) is a B-cell receptor (BCR) activity (Richardson et al. 2018); however, the mechanism of action
signaling kinase expressed by various hematopoietic cells, of this combination is unclear. In the present study, we explored
including B-cell lymphomas and leukemias. Recent studies have the therapeutic effect and mechanism of ibrutinib combined with
shown that BTK is overexpressed on MM cells and implicated DXMS on MM in vitro and in vivo.
in their growth and survival (Liu et al. 2014). Moreover, BTK
expression is correlated with poor prognosis and overexpression 2. Investigations and results
may contribute to the development of drug resistance in MM
cells (Yang et al. 2015). Ibrutinib, an irreversible BTK inhibitor 2.1. Ibrutinib/DXMS complex induced MM cell apoptosis
with excellent pharmacodynamics, is approved for the treatment To confirm the killing effect of ibrutinib combined with DXMS on
of various B-cell malignancies in both the United States and the MM cells, RPMI-8226 and U266 cells were treated with ibrutinib
European Union. Clinical trials of ibrutinib treatment for relapsed with or without DXMS. The apoptosis of cells was evaluated by
mantle cell lymphoma, non-Hodgkin’s lymphoma, and chronic Annexin V/PI using flow cytometry. As shown in Fig. 1, ibrutinib
lymphocytic leukemia have achieved high response rates. Ibrutinib induced RPMI-8226 apoptosis (Fig. 1a and 1b) and U266 apop-
has been found to inhibit tumor growth and improve MM-induced tosis (Fig. 1c and 1d) in a dose-dependent manner (in comparison
osteolysis in a murine model (Tai et al. 2012), and to be cytotoxic with the control group). Although, DXMS did not induce apoptosis
in malignant plasma cells of MM patients in vitro and to synergize in either cell line, the combination of ibrutinib and DXMS signifi-
with bortezomib and lenalidomide (Rushworth et al. 2013). There- cantly increased (compared with ibrutinib alone) RPMI-8226
fore, ibrutinib shows potential as a novel therapeutic approach for apoptosis (Fig. 1a and 1b) and U266 apoptosis (Fig. 1c and 1d).
MM, targeting MM cells and the bone marrow microenvironment These data indicate that ibrutinib-induced apoptosis in MM cell
(Tai and Anderson 2012). lines was significantly elevated by the addition of DXMS.
92 Pharmazie 76 (2021)ORIGINAL ARTICLES
2.2. Ibrutinib/DXMS complex reduced the percentage of
CD38+ cells and increased the apoptosis of CD38+ cells in
bone marrow MNCs from MM patients
Fig. 2: Combining ibrutinib and dexamethasone (DXMS) decreased the percentage
Fig. 1: Synergistic effects of ibrutinib and dexamethasone (DXMS) on the induction
of CD38+ cells and increased the apoptosis of CD38+ cells in mononucle-
of cell apoptosis in MM cell lines. (A) The proportion of apoptotic RPMI-
ar cells (MNCs) from the bone marrow of patients with multiple myeloma
8226 cells was assessed by Annexin V-PI staining and flow cytometry after
(MM). (A) The percentage of CD38+ cells in MNCs was detected by flow
treatment with ibrutinib and/or DXMS. (B) The apoptotic RPMI-8226 cell
cytometry after treatment with ibrutinib and/or DXMS. (B) The proportion
proportion in different groups was statistically analyzed by t test. (C) The
of CD38+ cells in different groups was statistically analyzed by t test. (C) The
proportion of apoptotic U266 cells was assessed by Annexin V-PI staining
percentage of CD38+ cell apoptosis was assessed by Annexin V staining and
and flow cytometry after treatment with ibrutinib and/or DXMS. (D) The
flow cytometry after treatment with ibrutinib and/or DXMS. (D) The apop-
apoptotic U266 cell proportion in different groups was statistically analyzed
totic proportion of CD38+ cells in different groups was statistically analyzed
by t test. All experiments were performed three times independently. An-
by t test. The results shown are representative of three independent experi-
nexin V and PI-positive cells were considered as apoptotic cells. *PORIGINAL ARTICLES
smaller than in the control group. Moreover, tumor size in the ibru- lines (Sharma and Lichtenstein 2018). The mechanism underlying
tinib combined with DXMS group was considerably lower than DXMS-induced apoptosis is the transactivation of proapoptotic
that in the ibrutinib or DXMS monotherapy group, demonstrating genes resulting from DXMS binding to its glucocorticoids receptor
the synergistic anti-myeloma effect of DXMS. In addition, ibru- (GR) (Sharma and Lichtenstein 2018). However, MM cell lines
tinib combined with DXMS markedly prolonged the survival of such as RPMI-8226 and U266 were resistant to DXMS-induced
MM mice (Fig. 4b). These results confirm that ibrutinib combined apoptosis (Sharma and Lichtenstein 2018; Salem et al. 2013).
with DXMS demonstrates synergistic anti-tumor activity in vivo. Consistent with these previous studies, our results showed that
DXMS induced slight apoptosis in primary cells but not in RPMI-
8226 or U266 cell lines. DXMS-resistant MM cells have been
demonstrated to overexpress BTK (Chauhan et al. 2002) or PI3K
(Yang et al. 2008), while DXMS resistance in RPMI-8226 or U266
cells may be mediated by activation of BTK (Bose et al. 2014) or
PI3K (Jiang et al. 2018). In the present study, our results show that
RPMI-8226 and U266 cells highly express BTK or PI3K. DXMS
combined with ibrutinib have been shown to display synergistic
anti-tumor effects in CLL and Burkitt lymphoma (Manzoni et al.
2016; Chu et al. 2019). In myeloma, combined use of ibrutinib
and DXMS has achieved good effects in clinical trials, although
the mechanism underlying the combined action of ibrutinib and
DXMS on MM cell apoptosis is not clear. Here, we demonstrate
that DXMS potentiated the apoptotic role of ibrutinib in MM cell
Fig. 4: Treatment of multiple myeloma (MM) mouse model with ibrutinib and lines and bone marrow CD38+ MNCs from MM patients. Surpris-
dexamethasone (DXMS) combination. (A) Tumor diameters were measured ingly, the ibrutinib with DXMS combination not only suppressed
and tumor volume in mm3 was calculated in four groups. n=5, *PORIGINAL ARTICLES
of MM mice in vivo. The synergistic effect of the ibrutinib/ 4.6. Statistical analyses
DXMS combination in inhibiting BTK and inducing apoptosis All statistical analyses and survival curve analysis were performed using the
in MM cells may be mediated by inhibition of the expression of GraphPad Prism 6 software (La Jolla, CA, USA). Unpaired Student’s t tests were
used to compare differences between two groups. Data are shown as mean±SEM. A p
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