Comprehensive proteomic analysis of Ibrutinib mediated...
1
As a core facility, the CUMP offers services in proteomic research, to initiate contact please e-mail Dr. Sebastian Wiese or visit our homepage. [email protected] The enhanced activation of the B cell receptor (BCR) signalling cascade is a crucial contribution in the pathogenesis, progression and/or maintenance of B cell leuke- mia, such as chronic lymphocytic leukemia (CLL). Recently, the irreversible Bru- ton’s tyrosine kinase (BTK) inhibitor Ibrutinib has seen a remarkable success as a first or second-line treatment of patients with various types of B cell malignancies. Despite these successes, the underlying changes, such as modulation of phos- phorylation events on the BTK target PLCγ2 (1-Phosphatidylinositol-4,5-bisphos- phate phosphodiesterase gamma-2) as well as subsequent events in the BCR cascade like the modulation of various post-translational modifications (PTMs) and/or protein levels, induced by Ibrutinib, are poorly understood. Comprehensive proteomic analysis of Ibrutinib mediated changes on proteins and PTMs in malignant human B cells • By targeting the Bruton’s tyrosin kinase and thus disrupting the B cell receptor signalling cascade, Ibrutinib has proven to be an highly efficient drug in the treatment of B cell malignancies. • Detailed studies in a reconstituted system indicate a high number of phos- phorylation events on the PLCγ2 and the dependency on these for enzy- matic activity within the B cell receptor pathway. • Using global phosphoproteomics a comprehensive picture of effects in- duced by this potent drug has been created. • Ibrutinib changes both protein and phosphorylation levels, altering cellular structures and translation events and thus reducing cell survival. Introduction Contact This work was in part funded by the DFG through SFB1074 ”Experimental models and clinical translation in leukemia” (INST 40/514-1) and by strategic investments from the medical faculty, Ulm University. Funding Poster Figure 1: Signaling cascade of the B cell receptor pathway and targets of novel anti-leukemic drugs. Due to its importance in the development of leukemic disease, proteins of the BCR signalosome are targets of novel first and secondary treatments. An array of novel components has entered the clinic in the past years, Ibrutinib being one of the most promising among them. Overview Conclusion/Outlook • Ibrutinib inactivates the BTK and thus prevents phosphorylation of the PLCγ2, subsequently affecting critical pathways in malign B cells and thus dramatically reducing the survival of these cells, causing the success of this high potential drug. • Global proteomic analyses indicate an involvement of various other PTMs as an indirect effect of Ibrutinib treatment. This necessitates characterisa- tin of these modifications by mass spectrometric means. • Drugs targeting other components of the BCR pathway are in clinical use. Their effects on B cell proteomes will be characterized in order to find po- tential prospects for combined treatments. • Despite its novelty, patients showing mutations leading to Ibrutinib resi- stance have been observed. The effects of these mutations will be sub- jects of further research efforts. Reinhild Rösler 1 , Sascha Endres 1,2 , Jennifer Haas 2 , Martin Wist 2 , Claudia Walliser 2 , Heike Wiese 2 , Peter Gierschik 1,2 , Sebastian Wiese 1 1 Core Unit Mass Spectrometry and Proteomics, Medical faculty, Ulm University, 2 Institute of Pharmacology and Toxicology, Ulm University For the analyses of PLCγ2 phosphorylation events, COS-7 cells were transfected as indicated with either empty vector (pcDNA3.1) or vectors encoding PLCγ2, wild-type BTK (BTK-WT) or a constitutive active form of BTK (BTK E41K ). Twen- ty-four hours after transfection, the cells were incubated for 18 h with myo-[2-3H] inositol, and the enzymatic activity was then measured by means of the inositol phosphate formation. Using SILAC-conditions, Jeko-1 cells, human B cells derived from mantle cell lymphoma, were cultivated and exposed to 300 nM or 500 nM Ibrutinib over a course of three days. Samples were collected after 6, 24, 48 and 72 hours and subjected to analyses using SDS-PAGE based proteomics, samples collected after 72 hours were also subjected to SCX/TiO2-based phosphoproteomics. All samples were analyzed on an Orbitrap-Velos Pro (Thermo Scientific, Bremen, Germany) online coupled to an RSLCnano (Thermo Scientific, Dreieich, Germa- ny) using Multi-Stage Activation. Data analysis was performed using MaxQuant (MPI Martinsried, München). Methods Results ZYX CDK2 SMARCA5 VIM SNX5 HIVEP2 UBR4 NME1 RPL35 RPL29 RPL14 IRF2BPL RPL24 NCBP1 PLRG1 YARS SRRT U2AF1 RPL15 MARS SNRPD1 HNRNPK NCL SRRM2 RAB7A PUF60 CORO1C LPXN RSF1 HMGA1 RANGAP1 MCM5 NCAPD2 RANBP3 RCC1 SMARCAD1 BUB3 ACTBL2 ZNF207 SMARCA4 LCP1 TWF2 PBRM1 YEATS2 MICALL1 DPYSL2 RAB1B ARFGEF2 AXIN2 MIF ATP6V1A TMOD3 DBN1 DMXL2 MTPN MOB1A PPM1G USP36 UBXN7 NPM1 TPR YBX1 RPS6KB1 NUP153 NUP214 NPM3 ARPC1B GAB1 INPP5D TUBG1 ARPC5 PTPN6 DOK3 SPTBN1 RPL3 EIF5B VARS RPL4 EEF1A2 DIDO1 MAP1B MAP1A SON Legend 6h 1d 2d 3d Ibrutinib- Treatment Phoshosite Figure 4: Interaction network showing effects induced by Ibrutinib treatment. Interaction network of proteins showing either significantly altered protein levels or phosphorylation level changes and having at least binary in- teractions as retrieved from the String Database (www.string-DB.org) are shown. As shown in Figure 3, proteins involved in translation (green square) are affected by Ibrutinib. In addition, proteins known to have important roles in B cell malignancies, such as NPM1 (red square), are altered in either protein or phosphorylation levels. Figure 5: GO-enrichment analysis of proteins and phosphosites regulated upon Ibrutinib treatment. A) Biological process (above) and Cellular compartment (below) GO-terms significantly enriched among the pro- teins differentially expressed upon treatment. B) GO-terms enriched among proteins showing altered phosphoryla- tion levels under Ibrutinib influence. Ibrutinib alters transcription, translation and cellular organization dramatically. A) B) Y242 S443 Y858 Y1125 Y1217 T1219 -6 -4 -2 0 2 -6 -2.03 -0.827 -0.29 -1.78 -5.05 -6 -1.51 -1.67 -2.69 -6.69 -4.2 pcDNA PLCγ2 wt PLCγ2 9Y->F 0 3000 6000 9000 12000 Activity ctrl BTK-WT BTK E41K PLCγ2 + BTK E41K + Ibru/PLCγ2 + BTK E41K PLCγ2 + BTK E41K K430R /PLCγ2 + BTK E41K log 2 ratio Y13 Y100 S677 (Y733) lit PH EF X nSH2 cSH2 SH3 sPHc Y C2 522-634 641-742 774-826 841-913 15-133 160-309 312-457 sPH n 471-514 930-1044 1059-1167 S847 T857 Y858 S789 Y818 S821 Y573 Y611 Y482 Y330 T332 S341 Y345 Y371 S443 S239 Y242 Y936 Y997 S1110 Y1125 Y1197 Y1217 S1236 Y1245 Y1264 S9 S751 Y753 S756 Y759 S763 S828 T829 S2/T3/T4? Figure 2: In vitro phosphorylation analysis of the PLCγ2/BTK system. A) Domain structure of the PLCγ2-pro- tein and phosphorylation sites identified using LC/MS 2 -analyses. Phosphorylation sites highlighted (green, ne- wly identified; red, previously known) were subsequently mutated to render the PLCγ2 inactive. B) Activity of the PCLγ2 in the presence of BTK or BTK E41K , a constitutively active form. Only mutation of several phosphorylation sites inactivates PLCγ2, indicating a tightly controlled enzymatic function. C) Quantitative phosphorylation ana- lyses using SILAC indicates a reduction of phosphorylation levels in PLCγ2 in the presence of Ibrutinib. GTP RAC BCR Igα Igβ LYN SYK BLNK PLCγ2 PIP3 CD19 RAS RAF IKK InsP 3 Ca 2+ DAG ERK JNK p38 AKT GSK3 NF-κB ? NFAT Ibrutinib CB292 Idelalisib ABT-199 Enzastaurin BTK PI3Kδ PKC Bcl-2 p85 p110 Transcripion leading to cell fate decision cell. CytC Apoptosis macromolecular complex subunit organization single-organism organelle organization chromatin remodeling actin filament organization organelle organization 0 10 20 count in data set count in data set 0.00 0.01 0.02 0.03 0.04 0.05 false discovery rate false discovery rate cytoskeleton cytoskeletal part intracellular organelle part focal adhesion cell junction 0 5 10 15 20 25 30 35 40 count in data set 0.0 2.0x10 -4 4.0x10 -4 6.0x10 -4 8.0x10 -4 false discovery rate translation peptide metabolic process protein targeting to membrane SRP-dependent cotranslational protein targeting to membrane viral life cycle 0 5 10 15 count in data set 0.0 5.0x10 -5 1.0x10 -4 1.5x10 -4 2.0x10 -4 false discovery rate extracellular exosome extracellular region part cytosolic large ribosomal subunit membrane-bounded vesicle intracellular non-membrane -bounded organelle 0 5 10 15 20 25 30 35 count in data set 0.0 1.0x10 -6 2.0x10 -6 3.0x10 -6 4.0x10 -6 5.0x10 -6 false discovery rate -5 -4 -3 -2 -1 0 1 2 3 4 5 0 2 4 6 8 10 12 -5 -4 -3 -2 -1 0 1 2 3 4 5 0 2 4 6 8 10 12 SLC1A4 RBM28 PTPN6 PLEK NFKB2 IRF5 IGHMBP2 HVCN1 NDOR1 CD48 CD40 BCAP31 TJP2 MAP3K1 ZNF428 CHD6 ATXN7L3B PDLIM1 RANBP1 TSPYL2 BCL7C DIDO1 BAG6 DDX21 MED1 OTUD5 NCOR2 ZNF687 INCENP REPS1 -log 2 p-value log 2 Ibrutinib/DMSO -log 2 p-value log 2 Ibrutinib/DMSO A) B) Figure 3: Effects of 3d Ibrutinib treatment on the global levels of A) proteins and B) phosphorylations in human B cells. Using SILAC based quantitative phosphoproteomics, 6410 proteins and 4447 phosphosites were quantified. Significantly regulated proteins and phosphorylations indicate the influence of Ibrutinib upon treatment. B) C) A) If you are interested in our work feel free to follow the QR-code to a digital copy of this and other posters illustrating our current research activities.
Transcript of Comprehensive proteomic analysis of Ibrutinib mediated...
As a core facility, the CUMP offers services in proteomic research, to initiate
contact please e-mail Dr. Sebastian Wiese or visit our homepage.
The enhanced activation of the B cell receptor (BCR) signalling cascade is a crucial contribution in the pathogenesis, progression and/or maintenance of B cell leuke-mia, such as chronic lymphocytic leukemia (CLL). Recently, the irreversible Bru-ton’s tyrosine kinase (BTK) inhibitor Ibrutinib has seen a remarkable success as a first or second-line treatment of patients with various types of B cell malignancies. Despite these successes, the underlying changes, such as modulation of phos-phorylation events on the BTK target PLCγ2 (1-Phosphatidylinositol-4,5-bisphos-phate phosphodiesterase gamma-2) as well as subsequent events in the BCR cascade like the modulation of various post-translational modifications (PTMs) and/or protein levels, induced by Ibrutinib, are poorly understood.
Comprehensive proteomic analysis of Ibrutinib mediated changes on proteins and PTMs in malignant human B cells
• By targeting the Bruton’s tyrosin kinase and thus disrupting the B cell receptor signalling cascade, Ibrutinib has proven to be an highly efficient drug in the treatment of B cell malignancies.
• Detailed studies in a reconstituted system indicate a high number of phos-phorylation events on the PLCγ2 and the dependency on these for enzy-matic activity within the B cell receptor pathway.
• Using global phosphoproteomics a comprehensive picture of effects in-
duced by this potent drug has been created.
• Ibrutinib changes both protein and phosphorylation levels, altering cellular structures and translation events and thus reducing cell survival.
Introduction
ContactThis work was in part funded by the DFG through SFB1074 ”Experimental models and
clinical translation in leukemia” (INST 40/514-1) and by strategic investments from the
medical faculty, Ulm University.
FundingPosterFigure 1: Signaling cascade of the B cell receptor pathway and targets of novel anti-leukemic drugs. Due to its importance in the development of leukemic disease, proteins of the BCR signalosome are targets of novel first and secondary treatments. An array of novel components has entered the clinic in the past years, Ibrutinib being one of the most promising among them.
Overview Conclusion/Outlook• Ibrutinib inactivates the BTK and thus prevents phosphorylation of the
PLCγ2, subsequently affecting critical pathways in malign B cells and thus dramatically reducing the survival of these cells, causing the success of this high potential drug.
• Global proteomic analyses indicate an involvement of various other PTMs as an indirect effect of Ibrutinib treatment. This necessitates characterisa-tin of these modifications by mass spectrometric means.
• Drugs targeting other components of the BCR pathway are in clinical use.Their effects on B cell proteomes will be characterized in order to find po-tential prospects for combined treatments.
• Despite its novelty, patients showing mutations leading to Ibrutinib resi-stance have been observed. The effects of these mutations will be sub-jects of further research efforts.
Reinhild Rösler1, Sascha Endres1,2, Jennifer Haas2, Martin Wist2, Claudia Walliser2, Heike Wiese2, Peter Gierschik1,2, Sebastian Wiese1
1Core Unit Mass Spectrometry and Proteomics, Medical faculty, Ulm University, 2Institute of Pharmacology and Toxicology, Ulm University
For the analyses of PLCγ2 phosphorylation events, COS-7 cells were transfected as indicated with either empty vector (pcDNA3.1) or vectors encoding PLCγ2, wild-type BTK (BTK-WT) or a constitutive active form of BTK (BTKE41K). Twen-ty-four hours after transfection, the cells were incubated for 18 h with myo-[2-3H]inositol, and the enzymatic activity was then measured by means of the inositol phosphate formation.Using SILAC-conditions, Jeko-1 cells, human B cells derived from mantle cell lymphoma, were cultivated and exposed to 300 nM or 500 nM Ibrutinib over a course of three days. Samples were collected after 6, 24, 48 and 72 hours and subjected to analyses using SDS-PAGE based proteomics, samples collected after 72 hours were also subjected to SCX/TiO2-based phosphoproteomics. All samples were analyzed on an Orbitrap-Velos Pro (Thermo Scientific, Bremen, Germany) online coupled to an RSLCnano (Thermo Scientific, Dreieich, Germa-ny) using Multi-Stage Activation. Data analysis was performed using MaxQuant (MPI Martinsried, München).
Methods
Results
ZYX
CDK2
SMARCA5
VIM
SNX5
HIVEP2
UBR4
NME1
RPL35RPL29
RPL14
IRF2BPL
RPL24
NCBP1
PLRG1
YARS
SRRT
U2AF1
RPL15
MARS
SNRPD1
HNRNPKNCL
SRRM2
RAB7APUF60
CORO1C
LPXN
RSF1
HMGA1
RANGAP1
MCM5
NCAPD2
RANBP3RCC1
SMARCAD1
BUB3
ACTBL2
ZNF207
SMARCA4LCP1
TWF2
PBRM1YEATS2
MICALL1
DPYSL2
RAB1B
ARFGEF2
AXIN2
MIF
ATP6V1A
TMOD3
DBN1
DMXL2
MTPN
MOB1A
PPM1G
USP36
UBXN7
NPM1
TPR
YBX1RPS6KB1
NUP153NUP214
NPM3
ARPC1B
GAB1
INPP5D
TUBG1
ARPC5
PTPN6
DOK3
SPTBN1
RPL3EIF5B
VARS
RPL4
EEF1A2
DIDO1
MAP1B
MAP1ASON
Legend
6h 1d 2d 3d
Ibrutinib-Treatment
Phosho
site
Figure 4: Interaction network showing effects induced by Ibrutinib treatment. Interaction network of proteins showing either significantly altered protein levels or phosphorylation level changes and having at least binary in-teractions as retrieved from the String Database (www.string-DB.org) are shown. As shown in Figure 3, proteins involved in translation (green square) are affected by Ibrutinib. In addition, proteins known to have important roles in B cell malignancies, such as NPM1 (red square), are altered in either protein or phosphorylation levels.
Figure 5: GO-enrichment analysis of proteins and phosphosites regulated upon Ibrutinib treatment. A) Biological process (above) and Cellular compartment (below) GO-terms significantly enriched among the pro-teins differentially expressed upon treatment. B) GO-terms enriched among proteins showing altered phosphoryla-tion levels under Ibrutinib influence. Ibrutinib alters transcription, translation and cellular organization dramatically.
A) B)
Y242 S443 Y858 Y1125 Y1217 T1219
-6
-4
-2
0
2
-6
-2.03
-0.827-0.29
-1.78
-5.05-6
-1.51
-1.67-2.69
-6.69
-4.2
pcDNA
PLCγ2
wt
PLCγ2
9Y->F
0
3000
6000
9000
12000
Act
ivity
ctrlBTK-WTBTKE41K
PLCγ2 + BTKE41K + Ibru/PLCγ2 + BTKE41K
PLCγ2 + BTKE41K K430R/PLCγ2 + BTKE41Klo
g 2ra
tio
Y13Y100
S677(Y733)lit
PH EF X nSH2 cSH2 SH3 sPHc Y C2
522-634 641-742 774-826 841-91315-133 160-309 312-457
sPHn
471-514 930-1044 1059-1167
S847T857Y858
S789Y818S821
Y573Y611Y482
Y330T332S341Y345Y371S443
S239Y242
Y936Y997
S1110Y1125
Y1197Y1217S1236Y1245Y1264
S9S751Y753S756Y759S763
S828T829
S2/T3/T4?
Figure 2: In vitro phosphorylation analysis of the PLCγ2/BTK system. A) Domain structure of the PLCγ2-pro-tein and phosphorylation sites identified using LC/MS2-analyses. Phosphorylation sites highlighted (green, ne-wly identified; red, previously known) were subsequently mutated to render the PLCγ2 inactive. B) Activity of the PCLγ2 in the presence of BTK or BTKE41K, a constitutively active form. Only mutation of several phosphorylation sites inactivates PLCγ2, indicating a tightly controlled enzymatic function. C) Quantitative phosphorylation ana-lyses using SILAC indicates a reduction of phosphorylation levels in PLCγ2 in the presence of Ibrutinib.
GTPRAC
BCR
Igα Igβ
LYN
SYKBLNK PLCγ2
PIP3
CD19
RAS
RAF
IKK
InsP3
Ca2+
DAG
ERK JNK p38
AKT
GSK3
NF-κB ? NFAT
IbrutinibCB292
Idelalisib
ABT-199
Enzastaurin
BTK
PI3Kδ
PKC Bcl-2
p85
p110
Transcripion leading to cell fate decision
cell.CytC
Apoptosis
macrom
olecu
larco
mplex
subu
nitorg
aniza
tion
single
-orga
nism
organ
elle org
aniza
tion
chrom
atin rem
odeli
ng
actin
filamen
t
organ
izatio
n
organ
elle org
aniza
tion
0
10
20
count in data set
coun
tin
data
set
0.00
0.01
0.02
0.03
0.04
0.05false discovery rate
fals
edi
scov
ery
rate
cytos
kelet
on
cytos
kelet
alpa
rt
intrac
ellula
r organ
elle pa
rt
focal
adhe
sion
cell j
uncti
on0
5
10
15
20
25
30
35
40
45
coun
tin
data
set
0.0
2.0x10-4
4.0x10-4
6.0x10-4
8.0x10-4
1.0x10-3
fals
edi
scov
ery
rate
trans
lation
pepti
de
metabo
licpro
cess
protei
n targe
ting
tomem
brane
SRP-depe
nden
t cotr
ansla
tiona
l
protei
n targe
ting to
membra
ne
viral
lifecy
cle0
5
10
15
coun
tin
data
set
0.0
5.0x10-5
1.0x10-4
1.5x10-4
2.0x10-4
fals
edi
scov
ery
rate
extra
cellu
larex
osom
e
extra
cellu
larreg
ionpa
rt
cytos
olic lar
ge
ribos
omal
subu
nit
membra
ne-bo
unde
d
vesic
le
intrac
ellula
r non-m
embra
ne
-boun
ded org
anell
e
0
5
10
15
20
25
30
35
coun
tin
data
set
0.0
1.0x10-6
2.0x10-6
3.0x10-6
4.0x10-6
5.0x10-6
fals
edi
scov
ery
rate
-5 -4 -3 -2 -1 0 1 2 3 4 50
2
4
6
8
10
12
-5 -4 -3 -2 -1 0 1 2 3 4 50
2
4
6
8
10
12
SLC1A4RBM28PTPN6
PLEK
NFKB2
IRF5
IGHMBP2
HVCN1NDOR1CD48
CD40
BCAP31TJP2MAP3K1
ZNF428
CHD6
ATXN7L3B
PDLIM1
RANBP1
TSPYL2
BCL7CDIDO1
BAG6DDX21
MED1OTUD5
NCOR2ZNF687
INCENP
REPS1
-log 2
p-va
lue
log2 Ibrutinib/DMSO
-log 2
p-va
lue
log2 Ibrutinib/DMSO
A) B)
Figure 3: Effects of 3d Ibrutinib treatment on the global levels of A) proteins and B) phosphorylations in human B cells. Using SILAC based quantitative phosphoproteomics, 6410 proteins and 4447 phosphosites were quantified. Significantly regulated proteins and phosphorylations indicate the influence of Ibrutinib upon treatment.
B) C)
A)
If you are interested in our work feel free to follow the QR-code to a digital
copy of this and other posters illustrating our current research activities.
