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Cell Cycle
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Ibrutinib inhibits BTK-driven NF-κB p65 activity to
overcome bortezomib-resistance in multiple myeloma
a
a
b
c
d
Megan Y Murray , Lyubov Zaitseva , Martin J Auger , Jenny I O Craig , David J MacEwan ,
a
ab
Stuart A Rushworth & Kristian M Bowles
a
Department of Molecular Haematology, Norwich Medical School, University of East Anglia,
Norwich Research Park, Norwich, United Kingdom.
b
Department of Haematology, Norfolk and Norwich University Hospitals NHS Foundation
Trust, Colney Lane, Norwich, United Kingdom.
c
Click for updates
Department of Haematology, Addenbrooke's Hospital, Cambridge Institute for Medical
Research, Wellcome Trust/MRC Building, Cambridge, United Kingdom.
d
Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine,
University of Liverpool, Ashton Street, Liverpool, United Kingdom.
Accepted author version posted online: 07 Jan 2015.
To cite this article: Megan Y Murray, Lyubov Zaitseva, Martin J Auger, Jenny I O Craig, David J MacEwan, Stuart A Rushworth
& Kristian M Bowles (2015): Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomib-resistance in multiple
myeloma, Cell Cycle
To link to this article: http://dx.doi.org/10.1080/15384101.2014.998067
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Combining ibrutinib and bortezomib to treat bortezomib-resistant MM
Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomibresistance in multiple myeloma
cr
MacEwan4, Stuart A Rushworth1* and Kristian M Bowles1, 2*
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Megan Y Murray1, Lyubov Zaitseva1, Martin J Auger2, Jenny I O Craig3, David J
us
Anglia, Norwich Research Park, Norwich, United Kingdom.
an
2.Department of Haematology, Norfolk and Norwich University Hospitals NHS
Foundation Trust, Colney Lane, Norwich, United Kingdom.
M
3.Department of Haematology, Addenbrooke's Hospital, Cambridge Institute for
ed
Medical Research, Wellcome Trust/MRC Building, Cambridge, United Kingdom.
4.Department of Molecular and Clinical Pharmacology, Institute of Translational
pt
Medicine, University of Liverpool, Ashton Street, Liverpool, United Kingdom.
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* SAR and KMB contributed equally
Keywords
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1.Department of Molecular Haematology, Norwich Medical School, University of East
multiple myeloma, bortezomib, ibrutinib, NF-κB, drug-resistance, BTK
Abbreviations
MM – multiple myeloma,
PI – proteasome inhibitor,
1
NF-κB – nuclear factor-kappa B,
BMSC – bone marrow stromal cells,
BTK – Bruton’s tyrosine kinase.
ip
t
Corresponding Author: Kristian Bowles, Email: [email protected]
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Conflicts of interest: The authors declare no competing financial interests.
us
Multiple Myeloma (MM) is a haematologic malignancy characterised by the
an
accumulation of clonal plasma cells in the bone marrow. Over the last 10-15 years
the introduction of the proteasome-inhibitor bortezomib has improved MM prognosis,
M
however relapse due to bortezomib-resistance is inevitable and the disease, at
present, remains incurable. To model bortezomib-resistant MM we generated
ed
bortezomib-resistant MM cell lines (n=4) and utilised primary malignant plasma cells
from patients relapsing after bortezomib treatment (n=6). We identified enhanced
pt
Bruton’s tyrosine kinase (BTK) activity in bortezomib-resistant MM cells and found
ce
that inhibition of BTK, either pharmacologically with ibrutinib (0.5 μM) or via lenti-viral
miRNA-targeted BTK interference, re-sensitised previously bortezomib-resistant MM
cells to further bortezomib therapy at a physiologically relevant concentration (5 nM).
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Abstract
Further analysis of pro-survival signalling revealed a role for the NF-κB p65 subunit
in MM bortezomib-resistance, thus a combination of BTK and NF-κB p65 inhibition,
either pharmacologically or via further lenti-viral miRNA NF-κB p65 interference, also
restored sensitivity to bortezomib, significantly reducing cell viability (37.5±6.9 %,
ANOVA
p≤0.001).
Accordingly,
we
propose
2
the clinical
evaluation
of
a
bortezomib/ibrutinib combination therapy, including in patients resistant to single-
cr
us
an
M
ed
pt
ce
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agent bortezomib.
3
Introduction
Multiple Myeloma (MM) is characterised by the accumulation of clonal plasma cells
in the bone marrow. The American Cancer Society estimates that there will be
approximately 24,000 new cases diagnosed and 11,000 deaths from MM in 2014 in
ip
t
the US1 Clinical manifestations of the disease occur as a consequence of the tumour
bulk; including anaemia and fatigue, immune paresis leading to infection, renal
cr
failure, and osteolytic bone breakdown by activated osteoclasts, resulting in painful
us
therefore, by ‘de-bulking’ the MM tumour mass, thus reducing the associated
an
symptoms and subsequently maintaining remission.
The proteasome inhibitor (PI) bortezomib, approved for the treatment of MM by the
M
FDA in 20033, is one such de-bulking agent that has greatly contributed to improved
outcomes observed in MM4 Despite this advance, however, relapse following
ed
bortezomib therapy remains inevitable due to the emergence of bortezomib-resistant
plasma cell sub-clones5 For example; recent ‘whole genome’ sequencing studies of
pt
MM patients confirmed a high level of genetic heterogeneity; occurring both between
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separate patients, and within patient samples before and after therapy6, indicating
the presence of a variety of genetically distinct plasma cell ‘sub-clones’7, 8 Different
sub-clones further indicate the presence of numerous minor tumour initiating cell
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lytic bone destruction and hypercalcaemia2 An effective MM therapeutic succeeds,
populations with complex and divergent evolutionary histories9. The potential
quiescence of these MM sub-clones, coupled with their diversity, can contribute to
enhanced tumorigenicity and an intrinsic resistance to therapy10. Therefore; the
eradication of the majority bortezomib-sensitive sub-clones may ultimately promote
the growth of pre-existing minority bortezomib-resistant sub-clones, limiting the
4
efficacy of single-agent bortezomib. This finding is further supported by the clinical
observation that approximately half of initially bortezomib-sensitive MM patients are
no longer able to respond to bortezomib once relapsed.11 This sub-clonal
bortezomib-resistance has been attributed to a range of mechanisms; including
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t
enhanced growth factor expression12, mutated proteasome subunits13, deregulated
plasma cell maturation markers 14, and nuclear factor-kappa B (NF-κB) pathway
cr
‘addiction’15 [for a more in-depth exploration of this topic see Murray et al. 201416].
us
have a broader than anticipated profile in MM whole genome sequencing data.6
These findings are consistent with our previous studies into the role of NF-κB
in
haematological
malignancies17-19,
suggesting
an
signalling
that
a
greater
understanding of the NF-κB signaling network in bortezomib-resistant MM may be
M
central to achieving therapeutic advances in this disease.
ed
In its primary mode of action bortezomib successfully inhibits ‘inducible’ NF-κB
expression in MM cells, such as the expression stimulated by MM-bone marrow
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stromal cell (BMSC) interaction20, via its function as an inhibitor of the 20s
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proteasome β5 subunit.21 Conversely, however, bortezomib also enhances
‘constitutive’ levels of NF-κB through activation of IKKβ, ultimately leading to NF-κB
nuclear translocation and the transcription of multiple NF-κB-induced genes,
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Furthermore, pro-survival NF-κB signalling pathway members were also found to
including Bruton’s tyrosine kinase (BTK).22 BTK, a non-receptor tyrosine kinase, is
now known to be of key importance to a number of haematological malignancies,
including MM23, chronic lymphocytic leukemia (CLL)24 and acute myeloid leukemia
(AML).25 The potential feedback mechanism between NF-κB and BTK signalling,
whereby BTK also lies upstream of several NF-κB inducible signalling pathways 26, 27,
provides a rationale for investigation of combined NF-κB and BTK inhibition in MM.
5
Previously we and others have shown ex vivo efficacy of BTK inhibition in MM.17, 23
Specifically, we have shown that the irreversible BTK inhibitor ibrutinib can enhance
the action of bortezomib via repression of the NF-κB survival pathway in primary
tissue, e.g., bone marrow-derived MM cells from treatment-naïve patients.17 Despite
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these findings, however, early phase II clinical trial data for ibrutinib monotherapy in
MM have so far proved disappointing28, whilst the study of ibrutinib efficacy in
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patients with relapsed of refractory MM is currently recruiting (ClinicalTrials.gov
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Taken together these data highlight the need to develop novel therapeutic strategies
an
that can overcome bortezomib-resistance16, whilst still de-bulking the tumour and
protecting the patient from related organ and tissue impairment.
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MM clonal development and selection is impacted by the timing, order, and
combinations of therapies received; however, all patients treated with bortezomib
ed
therapy are destined to relapse and become bortezomib-resistant. Here, we utilise in
vitro modelling to demonstrate BTK pro-survival pathway activity in bortezomib-
pt
resistant MM. These data provide justification for further assessment of greater
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patient numbers in the clinic, which will establish whether ibrutinib therapy can be
used to overcome bortezomib-resistance in MM in practice.
Results
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Identifier:NCT01478581).
Generation and characterisation of bortezomib-resistant MM cells
To determine the importance of the BTK pro-survival pathway in bortezomibresistance we first generated bortezomib-resistant cells in vitro. We cultured MMderived B lymphocytic cell lines (H929, LP-1, RPMI-8226, and U266) in the
6
continuous presence of bortezomib, increasing bortezomib concentration in stepwise
increments up to 10 nM. Corresponding bortezomib-naïve MM cell lines were used
as a control. Bortezomib-resistance in the cell lines was confirmed by a significant
increase in IC50 log[bortezomib] (Figure 1 A) and enhanced cell viability following
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exposure to physiologically relevant29 levels of bortezomib [5 nM (Figure 1 B)] when
compared to control ‘bortezomib-naïve’ MM cells. In addition, we explored relative
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chymotrypsin-like proteasome activity in bortezomib-naïve and bortezomib-resistant
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bortezomib-resistant MM cells (Figure 1 C), further confirming their inability to
an
respond to bortezomib’s proteasome-inhibitor function.
We also analysed primary human treatment-naïve MM patient samples (primary
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naïve; n=5) and primary human MM samples from patients that had initially
responded to bortezomib but subsequently relapsed (primary relapsed; n=6). All
ed
primary naïve patient samples analysed (n=5/5) showed significantly reduced cell
viability in response to bortezomib in vitro, whilst the majority (n=4/6) of primary
pt
relapsed samples showed no significant reduction in viability. The remaining (n=2/6)
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primary relapsed samples showed significantly decreased cell viability in response to
bortezomib, in-keeping with the clinical observation that approximately half of
patients retreated with bortezomib will respond again 30, 31 (Figure 1 D).30,31
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MM cell lines and observed significantly higher levels of proteasome activity in
Basal BTK activity is enhanced in bortezomib-resistant MM cell lines and is resistant
to inhibition with bortezomib
We have previously shown that the BTK pathway is active in MM.17 Furthermore,
others have shown that bortezomib can reduce the expression of BTK mRNA and
protein via an NF-κB p65-dependent mechanism.22 We therefore examined whether
7
BTK expression and activity in bortezomib-resistant MM cell lines reflects a
mechanistic change in the BTK pro-survival signalling pathway in response to
bortezomib treatment. We first examined basal levels of BTK activity in bortezomibnaïve and bortezomib-resistant U266 MM cells by immunocytochemistry. Utilising
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antibodies against total BTK (BTK) and Y223-phosphorylated BTK (pBTK) we
observed higher levels of pBTK in bortezomib-resistant cells [n ≥ 80] compared to
(Figure
2
A;
left
panel).
Further
quantitative
analysis
of
the
us
immunocytochemistry confirmed significantly enhanced basal pBTK levels in
bortezomib-resistant MM cell lines (Figure 2 A; right panel [n≥20]). We next
an
examined what effect this enhanced level of basal BTK activity has on bortezomibresistant MM cell response to further bortezomib exposure. Following bortezomib
M
treatment (4 h), we observed a significant decrease in relative BTK mRNA in the
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bortezomib-naïve but not bortezomib-resistant MM cell lines (Figure 2 B), suggesting
BTK expression is not inhibited by bortezomib treatment in the bortezomib resistant
pt
cells.
ce
To further investigate this difference in BTK mRNA expression and activity in
bortezomib-resistant MM cells we examined activity of the BTK promoter, utilising a
chemi-luminescent
BTK
promoter-luciferase
reporter
construct,
specifically
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cells
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bortezomib-naïve cells, suggesting a greater level of BTK activity in the resistant
containing two NF-κB transcription factor binding sites [κB1 and κB2]; pGL4.BTK
(Figure 2 C). Following 24 h bortezomib treatment bortezomib-naïve U266 MM cells
transfected with pGL4.BTK showed significantly reduced BTK promoter activity
(Figure 2 D). Conversely, there was no change in BTK promoter activity when
bortezomib-resistant U266 MM cells were transfected with pGL4.BTK, and the level
of BTK promoter activity in these cells remained significantly higher than in
8
bortezomib-naïve cells exposed to bortezomib. These results demonstrate that the
ability of bortezomib to repress BTK promoter activity is lost in bortezomib-resistant
MM cells.
Pharmacological BTK inhibition with ibrutinib restores sensitivity to bortezomib in
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bortezomib-resistant MM cells
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As BTK is emerging as an important regulator of downstream survival pathways in
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bortezomib-resistant MM cells (Figure 2), we investigated the functional effect of
combined pharmacological BTK inhibition and bortezomib treatment in bortezomib-
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naïve and bortezomib-resistant MM cell lines and primary human MM samples.
Ibrutinib ‘pulse’ treatment [0.5 μM/1 h] alone significantly reduced cell viability in
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bortezomib-resistant MM cells lines after 48 h (Figure 3 A). When combined with
bortezomib exposure [5 nM/24 h], the ibrutinib ‘pulse’ also significantly reduced cell
ed
viability in bortezomib-resistant U266 MM cells below levels observed in bortezomibresistant cells receiving bortezomib alone, and bortezomib-naïve cells receiving the
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same combination therapy (Figure 3 B). Importantly, ibrutinib ‘pulse’ treatment
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followed by bortezomib exposure also dramatically reduced cell viability in primary
relapsed MM samples [n=6], likewise below levels observed in primary relapsed
samples receiving bortezomib alone, and primary naïve MM samples [n=5] also
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MM17, 23, and further to our observations of enhanced BTK activity and expression in
receiving the combination therapy (Figure 3 C).
BTK inhibition via lenti-viral miRNA targeting also restores sensitivity to bortezomib
in bortezomib-resistant MM cells
Although originally developed as an inhibitor of BTK, ibrutinib has more recently
been shown to have ‘off-target’ effects, inhibiting multiple members of the TEC
9
kinase family, including interleukin-2-inducible T-cell kinase (ITK)32 Therefore, to
ensure our results were not due to potential off-target inhibitor activity, we evaluated
bortezomib-resistant and bortezomib-naïve U266 MM cell line response to lenti-viralmediated BTK repression. We generated artificial and exogenous miRNA sequences
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specifically targeting the BTK transcript [miRBTK] and utilised lenti-viral infection to
achieve semi-stable BTK mRNA knockdown [as described previously25] in both
and
bortezomib-resistant
U266
MM cells
(Figure
4 A).
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bortezomib-naïve
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nM/24 h) confirmed that bortezomib-resistant U266 MM cells experienced
significantly reduced cell viability compared to bortezomib-naïve cells also infected
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with miRBTK and exposed to bortezomib, and compared to bortezomib-resistant
cells infected with a non-targeting control miRNA [miRNEG] (Figure 4 B). This
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strongly suggests that the results observed following ibrutinib and bortezomib
ed
combination therapy in bortezomib-resistant cells are due to ibrutinib-driven
irreversible BTK inhibition, rather than any off target ibrutinib effects.
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Bortezomib-resistance is driven by enhanced NF-κB p65 activity and can be
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reversed by BTK inhibition
We have previously shown that BTK inhibition reduces NF-κB p65 activity in
bortezomib-naïve MM cells and primary naïve patient samples.17 To determine the
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Furthermore, the introduction of miRBTK in conjunction with bortezomib treatment (5
role of NF-κB p65 in the regulation of BTK activity in bortezomib-resistant MM cells
we initially examined basal levels of sub-cellular NF-κB p65 protein distribution in
bortezomib-naïve and bortezomib-resistant U266 MM cells by immunocytochemistry.
Utilising an antibody against the NF-κB p65 subunit we observed higher levels p65 in
the nucleus of bortezomib-resistant cells compared to bortezomib-naïve cells [n≥20],
10
suggesting a greater level of NF-κB p65 transcription factor binding, and therefore
NF-κB signalling in the bortezomib-resistant cells (Figure 5 A; inset). Further analysis
of nuclear vs. cytoplasmic NF-κB p65 revealed higher basal levels of nuclear
localisation in bortezomib-resistant compared to bortezomib-naïve U266 MM cells;
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both by quantitative analysis (Figure 5 A) and western immunoblot (Figure 5 B).
Additionally, analysis of sub-cellular NF-κB p65 localisation in response to ibrutinib
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‘pulse’ treatment combined with bortezomib exposure [5 nM/4 h] revealed significant
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naïve U266 MM cells compared to those treated with bortezomib alone (Figure 5 C).
Importantly, bortezomib-resistant cells showed no significant reduction in NF-κB p65
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nuclear localisation in response to bortezomib treatment alone compared to
activity in these cells.
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bortezomib-naïve cells, potentially suggesting a level of constitutive NF-κB p65
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To confirm the functional relevance of enhanced basal nuclear NF-κB p65
localisation in bortezomib-resistant MM cells, and to confirm that any response to
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bortezomib observed was specifically due to its ability to repress inducible NF-κB
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p65 activity, we generated artificial and exogenous miRNA sequences specifically
targeting NF-κB p65 (miRp65) and utilised lenti-viral infection to achieve semi-stable
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repression of NF-κB p65 nuclear localisation in bortezomib-resistant and bortezomib-
p65 mRNA knockdown in bortezomib-naïve and bortezomib-resistant U266 MM cells
(Figure 5 D). Introduction of miRp65, in combination with ibrutinib ‘pulse’ treatment,
significantly reduced cell viability in bortezomib-resistant U266 MM cells compared to
bortezomib-naïve cells exposed to the same treatment, and compared to
bortezomib-resistant cells infected with a non-targeting control miRNEG or ibrutinib
‘pulse’ treatment alone (Figure 5 E).
11
Discussion
The proteasome inhibitor bortezomib is licensed to treat newly diagnosed and
relapsed MM in the clinic. Single agent bortezomib has a response rate of
approximately 30% but, when used in combination with chemotherapy and/or
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corticosteroids, response rates range from approximately 60% to over 90%
depending on the regimen.16 Despite this relative success, however, clinical relapse
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following bortezomib therapy presently remains inevitable and resistance to further
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driving the selection and emergence of drug-resistant clones.7,
8
An improved
an
understanding of the mechanisms underlying bortezomib-resistance is, therefore,
vital for the progressive development of novel pharmacologic strategies to overcome
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the clinical phenomenon of bortezomib-resistance. In this study we have explored
bortezomib-resistance in MM in vitro, utilising bortezomib-resistant MM cell lines
ed
generated in the laboratory, and primary patient samples from both treatment naïve
patients and patients that have relapsed following bortezomib therapy. Despite the
pt
limited sample size, this data provides an important foundation for future in vivo
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assessment of ibrutinib therapy in bortezomib-relapsed MM in a larger number of
patients in the clinic.
We and others have previously demonstrated activity of the BTK survival pathway in
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bortezomib treatment is common30, 31, not only as a consequence of, but also further
treatment-naïve MM cells.17, 33 Furthermore, bortezomib has been reported to inhibit
total BTK expression in a treatment-naive B lymphocyte cell line.22 Here, utilising in
vitro models of post-bortezomib relapsed MM, we describe a role for BTK activity in
bortezomib-resistance, demonstrating higher basal levels of active pBTK protein in
bortezomib-resistant MM cell lines and a failure of these cells to respond to
12
bortezomib in terms of BTK mRNA and BTK promoter activity repression. In
particular, the higher basal BTK activity and altered BTK promoter activity in
bortezomib-resistant cells would be consistent with process of clonal selection,
driven by constant exposure to bortezomib.8 This identifies BTK as a candidate
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therapeutic target in bortezomib-resistant MM. In addition, this may also be relevant
to bortezomib-naïve MM, in which potentially bortezomib-resistant sub-clones as yet
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form a minority population within of the tumour ‘bulk’.
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shown promising clinical activity and a favourable side-effect profile in a range of B
an
cell malignancies.36-39 Ibrutinib has also been found to reduce BTK expression in
MM22 and enhance the cytotoxicity of bortezomib in bortezomib-naïve primary
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malignant plasma cells and MM cell lines.17 By inhibiting BTK with ibrutinib ‘pulse’
treatment we were also able to restore a measurable cytotoxic response to
ed
bortezomib in bortezomib-resistant MM cell lines and primary relapsed MM cells.
Importantly, although ibrutinib is known to target other kinases, we were able to
pt
reproduce these results with highly specific artificial BTK-targeting microRNA,
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miRBTK, introduced by lenti-virus. This suggests that it is BTK inhibition which is
restoring bortezomib activity in bortezomib-resistant MM cells and not an off target
effect of ibrutinib.
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The oral BTK inhibitor ibrutinib, currently licenced for use in CLL and MCL34, 35, has
Multiple mechanisms of action of bortezomib have been described20, 16, including the
inhibition of BTK expression driven via repression of inducible NF-κB transcription
factor translocation to the nucleus.17, 22, 40 We found enhanced nuclear localisation of
NF-kB p65 in the bortezomib-resistant cells. This is in-keeping with previous reports
that bortezomib-resistance in MM is conveyed through the NF-κB signalling network.
13
For example; constitutive expression of NF-κB is frequently seen in bortezomib
refractory primary patient samples.41 Furthermore, the acquisition of bortezomibresistance can follow up-regulation of heat shock protein (HSP)90 and HSP27,
which, in their action as ubiquitin chaperones, facilitate the activation of NF-κB in
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MM.42, 43 Active pBTK is also known to be essential for NF-κB activation and B cell
survival.44 Thus, there exists a positive auto-regulatory feedback loop that stimulates
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transcription of BTK via two functionally competent NF-κB p65 sites in the BTK
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higher basal nuclear NF-κB p65 in bortezomib-resistant MM cells, which, in turn, is
associated with a greater reliance on pBTK for survival. These observations suggest
an
that BTK inhibition by ibrutinib may be useful in the clinical treatment of bortezomib-
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resistant MM.
Taken together, these results show that bortezomib-resistance observed in the clinic
ed
occurs, at least in part, because bortezomib-resistant MM cells lose the capacity to
be influenced by the inhibition of BTK-driven NF-κB p65, and the consequent NF-κB
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p65-driven auto-regulation of BTK. Accordingly, introduction of ibrutinib to the
treatment regimens of bortezomib-resistant MM patients may lead to enhanced cell
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death of the bortezomib-resistant sub-clones through inhibition of this BTK/NF-κB
p65 signalling axis. Here we provide a biologic and molecular rationale for the clinical
evaluation of bortezomib and ibrutinib combination therapy in post-bortezomib
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promoter.22 Here we show that bortezomib-resistance in MM is associated with
relapsed MM, including patients that have previously been refractory to bortezomib.
14
Materials and Methods
Materials
All reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO), unless stated
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otherwise. All MM/B-lymphoblast cell lines were obtained from the European
Collection of Cell Cultures (ECCC).
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MM cell lines (H929, LP-1, RPMI-8228, and U266) were cultured in a humidified
atmosphere at 37°C and 5% (v/v) CO2 in RPMI-1640 media +L-Glutamine (Gibco
an
Life Technologies) supplemented with 10 % (v/v) foetal bovine serum ([FBS];
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Biosera). Bortezomib-resistant cells were cultured as above but supplemented with
20 % (v/v) FBS. Bortezomib-resistance was selected for by dose escalation of a
ed
once-weekly bortezomib treatment, through 0.5, 1, 2.5 and 5 nM, until cells could
tolerate 10 nM bortezomib (10-15 weeks; confirmed by luminescent cell viability
pt
assay). Each concentration was maintained for approximately 2-3 weeks to allow for
proliferation of bortezomib-resistant sub-clones. Build-up of cell debris due to the
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high level of cell death was removed by centrifugation at 300g for 5 minutes before
gently removing the supernatant and re-suspending the pellet.
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Cell culture
Primary MM cells were obtained under local ethical approval (LREC ref.
07/H0310/146) and were isolated from the bone marrow aspirates of MM patients, as
described previously.17 Primary MM cells from bortezomib-relapsed patients were
not exposed to further bortezomib treatment until experimentation.
Ibrutinib ‘pulse’ treatment
15
To mimic in vivo pharmacokinetics of rapid adsorption and elimination of the
irreversible BTK inhibitor ibrutinib45, we employed a method of ‘pulse’ exposure to
physiologically relevant levels of ibrutinib in vitro, as described previously.46 Briefly,
cells were exposed to 500 nM ibrutinib for 1 h before washing, followed by any
ip
t
additional drug treatment or assay in fresh media.
cr
Luminescent cell viability assay and chymotrypsin-like proteasome activity assay
manufacturer’s
instructions.
Proteasome
us
the CellTiter-Glo® Luminescent Cell Viability Assay (Promega), as per the
activity
was
measured
using
the
an
Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay (Promega), as per the
M
manufacturer’s instructions. Luminescence was measured with the FLUOstar
Omega Microplate Reader (BMGLabtech) and calculated as relative to untreated
ed
control samples.
pt
Quantification of protein activity and subcellular localisation by immunocytochemistry
Relative activity of phosphorylated versus total BTK (pBTK vs. BTK), and subcellular
ce
distribution of nuclear vs. cytoplasmic NF-κB p65 (nuc. vs. cyt. p65) were analysed
by immunocytochemistry. Briefly, 2 x 104 U266 MM cells were fixed onto microscope
slides using the CytoSpin 4 Cytocentrifuge system (Thermo Fisher Scientific) and 4
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1x105 MM cells were treated as indicated. Relative cell viability was assayed using
% (w/v) paraformaldehyde (PFA). Fixed cells were incubated with rabbit anti-human
primary mAbs against pBTK (Tyr223) and goat anti-human primary mAbs against
total BTK, or goat anti-human primary mAbs against NF-κB p65 (Cell Signalling
Technology). Cells were then incubated with Alexa Fluor® goat anti-rabbit and rabbit
anti-goat IgG (H+L) secondary antibodies (Molecular Probes Life Technologies), or
16
rabbit anti-goat IgG alone, respectively. Nuclei were localised by 4',6-diamidino-2phenylindole (DAPI) staining. After mounting, cells were viewed using the Axio
Imager.D2 microscope and Axiovision software (Carl Zeiss Microscopy).
Image analyses and quantification of pBTK vs. BTK, and nuc. vs. cyt. p65 staining
ip
t
intensity was performed using ImageJ 1.46 software (National Institute of Health)
and plugins, as described previously.25 Briefly, a threshold of staining intensity was
cr
us
n≥20 [NF-κB] individual cells). Staining intensity (pixles 2) was converted to a
an
percentage as follows: pBTK/total BTK (%), or, nuclear/[cytoplasmic + nuclear NF-κB
Western Immunoblotting
M
p65] (%).
ed
Protein fractions of equal total protein concentration were extracted from U266 MM
cells using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher
pt
Scientific) according to the manufacturer’s instructions. SDS/PAGE and western
ce
immunoblot analyses were performed as described previously47. ECL detection
using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and
subsequent quantification were performed with the ChemiDoc-It2 Imaging System
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applied and the area above the threshold (pixles2) was measured (n≥80 [BTK] and
(UVP).
RNA extraction and quantitative real time-PCR
Total RNA was extracted from 1 x 106 U266 MM cells using Total RNA Lysis Solution
(Applied Biosystems-Life Technologies) according to the manufacturer’s instructions.
Reverse transcription of total RNA was performed using the GeneAmp® Gold RNA
17
PCR Core Kit (Applied Biosystems-Life Technologies). Resultant cDNA was
analysed by quantitative real-time PCR (qRT-PCR) using LightCycler® 480 SYBR
Green I Master mix (Roche) and qRT-PCR primers for GAPDH [forward 5′ACCAGCCTCAAGATCATCAGC-3′ and reverse 5′-TGCTAAGCAGTTGGTGGTGCBTK
[forward
5′-CACACAGGTGAACTCCAGAAAG-3’
AGAGATACTGCCCATCGATCCAGA-3’]
and
reverse
NF-κB
reverse
5’-
p65
[forward
5′-
5′-GGATGCGCTGACTGATAGC-3′]
cr
ACCGCTGCATCCACAGTT-3′
and
and
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t
3′],
us
(Roche), as previously described.18 Gene expression was analysed using the
against GAPDH expression.
M
Lenti-virus construction and infection
an
comparative cycle threshold algorithm (ΔΔCT); mRNA expression was standardised
ed
Lenti-virus containing artificial exogenous microRNA (miRNA) sequences targeting
human NF-κB p65 (miR-p65 [5′-TACGTTTCTCCTCAATCCGGT-3′]), BTK (miR-BTK
pt
[5′-TTCACTGGACTCTTCACCTCT-3′]) or a control/scrambled sequence (miR-NEG),
and an EmGFP–pre-miRNA encoding fragment, were constructed and produced as
ce
described previously.25 Briefly, artificial miRNA targets were identified and designed
to interact with the relevant exon using Block-iT RNAi designer software (Invitrogen).
This ensures high specificity of miR-p65 and miR-BTK, with no 3’UTR interaction,
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(Invitrogen-Life Technologies), on the LightCycler® 480 Real-Time PCR system
unlike endogenous miRNAs.
5 x 105 U266 MM cells were infected and transduced with each lenti-virus (MOI: 15),
in serum-free medium with 8 µg/mL Polybrene™. Following infection and before
experimentation, transduced cells were analysed for target knock-down and
18
efficiency by qRT-PCR for BTK and NF-κB p65, and flow cytometry to detect GFP
using the Accuri-C6 flow cytometer (BD Biosciences).
Generation and transfection of the wild-type human BTK promoter-luciferase reporter
ip
t
construct
To generate the wild-type human BTK promoter-luciferase reporter construct
genomic
DNA
via
PCR
TATCTCGAGGAAGAAAAGAGCCTGGGCA-3′]
with
and
specific
forward
reverse
us
from
[5′[5′-
ATAAGATCTGTCTTTTTTTCTTCTCAGCAGCA-3′] primers. The amplified fragment
an
was cloned into the Xho I/Bgl II site of the Promoterless Firefly Luciferase
M
‘pGL4.11[luc2P]‘ Vector, according to the manufacturers’ instructions (Promega).
U266 MM cells were co-transfected with a total of 1 μg DNA, composed of the
ed
pGL4.BTK promoter-luciferase reporter construct and a pRL-TK Renilla Luciferase
Control Reporter Vector (Promega) using FuGENE® HD Transfection Reagent
pt
(Promega) and incubated for 24 h at 37°C and 5% (v/v) CO2 before any further
ce
treatment.
Chemi-luminescent luciferase reporter assay
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amplified
cr
(pGL4.BTK), the BTK promoter region, including two innate κB binding sites, was
Firefly and Renilla promoter-luciferase reporter activity was analysed and quantified
sequentially in a single sample with the Dual-Luciferase® Reporter Assay System
(Promega), according to the manufacturer’s instructions, and the EnVision 2103
Multilabel Plate Reader (Perkin Elmer).
19
Financial Support: This work was funded by NIHR Flexibility and Sustainability
Funding, the Big C Appeal, the Association for International Cancer Research, and
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ed
pt
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the Humane Research Trust.
20
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Figure 1. Characterisation of bortezomib-resistant MM cell lines, and primary
relapsed MM patient samples. (A) The IC50 Log[bortezomib] (nM) of bortezomibnaïve and bortezomib-resistant MM cell lines was analysed by luminescent cell
viability assay. (B) Relative cell viability (5 nM/48 h) and (C) chymotrypsin-like
proteasome activity in bortezomib-naïve and bortezomib-resistant MM cell lines in
response to (5 nM/4 h) bortezomib. (D) Relative cell viability of primary naïve and
28
primary relapsed MM patient samples in response to bortezomib (5 nM/24 h).
Statistical significance between treatments was calculated by Student’s t test; *
indicates p ≤ 0.05. Statistical significance between cohorts was calculated by
cr
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ed
pt
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ANOVA; # indicates p ≤ 0.05.
29
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an
M
ed
resistant to inhibition with bortezomib. (A) Representative immunocytochemistry
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pt
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Figure 2. Enhanced BTK activity in bortezomib-resistant MM cell lines is
of basal levels of total and phosphorylated BTK (pBTK) in bortezomib-naïve and
bortezomib-resistant U266 MM cells with DAPI nuclear staining [left panel] and
subsequent quantification of basal pBTK/total BTK (%) staining intensity [right panel].
Box and whisker indicates the mean ± SEM (n ≥ 80). (B) qRT-PCR analysis of BTK
mRNA following bortezomib (5 nM/4 h) exposure in bortezomib-naïve and
30
bortezomib-resistant U266 MM cells relative to GAPDH. Values indicate the mean ±
SEM from 3 independent experiments. (C) Representation of wild type pGL4.BTK
promoter-luciferase vector [including κB binding sites] transfected into bortezomibnaïve and bortezomib-resistant U266 MM cells. (D) Luciferase activity of pGL4.BTK
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promoter-luciferase vectors in bortezomib-naïve and bortezomib-resistant U266 MM
cells following bortezomib (5 nM/24 h) exposure, normalised by co-transfection with
cr
pRL-TK Renilla Luciferase Reporter Vectors. Values indicate the mean ± SEM from 3
ce
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ed
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calculated by ANOVA; # indicates p ≤ 0.01.
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by Student’s t test; * indicates p ≤ 0.05. Statistical significance between cohorts was
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independent experiments. Statistical significance between treatments was calculated
31
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cr
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an
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ed
pt
naïve and bortezomib-resistant MM cells. (A) Relative cell viability of bortezomib-
ce
naïve and bortezomib-resistant MM cell lines 48 h post-ibrutinib ‘pulse’ treatment. (B)
Relative cell viability of bortezomib-naïve and bortezomib-resistant MM cells postibrutinib ‘pulse’ treatment in combination with bortezomib (5 nM/24 h). (C) Relative
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Figure 3. BTK inhibition enhances sensitivity to bortezomib in bortezomib-
cell viability of primary naïve and primary relapsed MM patient samples post in vitro
ibrutinib ‘pulse’ treatment in combination with bortezomib (5 nM/24 h). Statistical
significance between treatments was calculated by Student’s t test; * indicates p ≤
0.05. Statistical significance between cohorts was calculated by ANOVA; # indicates
p ≤ 0.01.
32
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cr
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bortezomib in bortezomib-naïve and bortezomib-resistant MM cells. (A) qRT-
an
PCR analysis of basal BTK mRNA expression in bortezomib-naïve and bortezomibresistant MM U266 cells infected with lenti-viral miRNA constructs targeting BTK
M
(miRBTK) transcription relative to GAPDH. (B) Relative cell viability of bortezomibnaïve and bortezomib-resistant MM U266 cells infected with lenti-viral miRBTK
ed
before and after bortezomib treatment (5 nM/24 h). Values indicate the mean ± SEM
from 3 independent experiments. Statistical significance between treatments was
pt
calculated by Student’s t test; * indicates p ≤ 0.05. Statistical significance between
ce
cohorts was calculated by ANOVA; # indicates p ≤ 0.01.
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Figure 4. BTK inhibition via lenti-viral miRNA targeting enhances sensitivity to
33
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Figure 5. Bortezomib-resistance is driven by enhanced NF-κB p65 activity and
can be reversed by BTK inhibition. (A) Quantification of staining intensity of
nuclear p65/total NF-κB p65 (%) in bortezomib-naïve and bortezomib-resistant U266
34
MM cells. Box and whisker indicates the mean ± SEM (n ≥ 20). Statistical
significance between cohorts was calculated by ANOVA; # indicates p ≤ 0.01. Inset
shows example immunocytochemistry for NF-κB p65. Dotted line indicates nucleus
as determined by DAPI staining. (B) Western immunoblot of bortezomib-naïve [N]
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and bortezomib-resistant [R] U266 nuclear [Nuc.] and cytoplasmic [Cyt.] cell
fractions. GAPDH and Histone 3 [H3] show equal loading for cyt. and nuc. fractions,
cr
respectively. (C) Quantification of staining intensity of nuclear NF-κB p65/total NF-κB
us
whisker indicates the mean ± SEM (n ≥ 20). Statistical significance between cohorts
was calculated by ANOVA; # indicates p ≤ 0.05. (D) qRT-PCR analysis of basal NF-
an
κB p65 mRNA expression in bortezomib-naïve and bortezomib-resistant MM U266
cells infected with lenti-viral miRNA constructs targeting NF-κB p65 (miRp65)
M
transcription relative to GAPDH. (E) Relative cell viability of bortezomib-naïve and
ed
bortezomib-resistant MM U266 cells infected with lenti-viral miRp65 24 h before and
after ibrutinib ‘pulse’ treatment. Values indicate the mean ± SEM from 3 independent
pt
experiments. Statistical significance between treatments was calculated by Student’s
t test; * indicates p ≤ 0.05. Statistical significance between cohorts was calculated by
ce
ANOVA; # indicates p ≤ 0.01.
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p65 (%) in bortezomib-naïve and bortezomib-resistant U266 MM cells. Box and
35