multi-parametricsingle cell profilingdefinesdistinctdrug … · 2019-07-03 ·...
TRANSCRIPT
© FIMM - Institute for Molecular Medicine Finland www.fimm.fi
Multi-Parametric Single Cell Profiling Defines Distinct DrugResponses in Healthy Hematological Cell Lineages That are
Retained in Corresponding Malignant Cell Types
Muntasir Mamun Majumder, PhDTranslational Research and Personalized Cancer Medicine Lab
Institute for Molecular Medicine Finland (FIMM)University of Helsinki
www.fimm.fi
Hematopoiesis: The Process of Producing Distinct Mature Blood Cells
dd.mm.yyyy 2
Hematopoietic Stem Cells
CD34+CD38+CD34+CD38+
Common Myeloid Progenitor
B lymphocyteCD19+
T lymphocyte
Plasma cellCD138+CD38+
NK cellCD56+
CD3+MonocyteCD14+
GranulocyteCD45+SSC++
Cytotoxic T lymphocyte
CD3+CD8+CD3+CD4+CD3+CD56+
T helper cellNK-T cell
Common Lymphoid Progenitor
CD34+CD38-
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fidd.mm.yyyy
Multipotent Stem Cells
Primitive progenitors
Lineage Committed
Cells
Changes in transcriptome and proteome
Distinct cellular phenotype
Specialized functions
Differentiation
Muntasir Mamun Majumder, PhD
Hematopoiesis: The Process of Producing Distinct Mature Blood Cells
Email: [email protected]
www.fimm.fi
Characterizing Lineage Specific Drug Responses is the key to Implement Precision Therapies
Genotype Specific
Cell Lineage Specific
Non Specific
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Cellular Proportions in Patient Biopsies are Heterogeneous
dd.mm.yyyy 5
8.86% Monocyte0.34% HSC1.90% CPC44.39% THC40.50% CTL4.01% B
3.38% Monocyte
46.99% THC24.60% CTL10.05% B
6.01% NK-T
8.97% NK
11.52% Monocyte28.17% HSC50.20% CPC4.99% THC2.33% CTL0.24% NK-T0.72% NK1.84% B
8.04% Monocyte0.73% HSC1.71% CPC15.15% THC39.30% CTL3.98% NK-T15.39% NK5.46% B10.24% Plasma
Healthy BM (n=2) Healthy PB (n=3)
AML (n=11) MM (n=10)
0.40% CPC1.51% THC1.21% CTL75.87% B
CLL (n=7)
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Multiplexed Flow Cytometry
384 well format71 drugs6 cell subtypes3 healthy samples
96 well format6 drugs10 cell subtypes3 healthy, 3 AML and 10MM samples
Identify cell lineage specific drug responses in healthy and patient samples
Healthy PB and BM
Multiple Myeloma
AML
Others
Overview of the Study
Muntasir Mamun Majumder, PhD
Cell density
Antibody dilutions
Incubation Time
Wash vs no wash
Email: [email protected]
www.fimm.fidd.mm.yyyy
SSC-
H
FSC-H
FSC-
H
FSC-A 7-AAD
Anne
xin
V
CD45
SSC-
H
CD138 CD34 CD14 CD19 CD56 CD3
SSC-
H
SSC-
H
CD38 CD38 CD3 CD4
SSC-
H
SSC-
H
Cells Singlets Live cells CD45+
HSCs and CPCsHealthy and clonal
Plasma cells HSPCs Monocytes B cells
CTL and ThNK and NK-T
CD56+ T cells
Muntasir Mamun Majumder, PhD
Gating strategy for immunophenotyping Hematopoietic Cell Subsets
Email: [email protected]
www.fimm.fi
Distinct Drug Responses are Tied to Cell Lineages
dd.mm.yyyy
HC−
1−M
onoc
yte
HC−
2−M
onoc
yte
HC−
3−M
onoc
yte
HC−
2−TH
C
HC−
2−CT
L
HC−
1−TH
C
HC−
1−CT
L
HC−
2−N
K−T
HC−
3−N
K−T
HC−
3−CT
L
HC−
3−TH
C
HC−
1−B
HC−
1−N
K−T
HC−
1−N
K
HC−
2−N
K
HC−
2−B
HC−
3−N
K
HC−
3−B
Carfilzomib
Trametinib
SCH772984
WEHI−539
Pimasertib
Dasatinib
Refametinib
Cladribine
PF−04691502
Omipalisib
Idelalisib
Navitoclax
Bortezomib
Dexamethasone
Midostaurin
Venetoclax
Clofarabine
0 10 20 30 40DSS
Monocyte T Cell Subsets B and NK Cells
Proteasome Inhibitor
Nucleoside Analog
Glucocorticoid
Multi-kinase inhibitor
Apoptotic Modulator
SRC inhibitor
MEK/ERK inhibitor
PI3K/mTOR inhibitor
Color Key
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi7/1/19 9
Bortezomib
10000nM1000nM100nM10nM1nM
0
20
40
60
80
100
% S
urvi
val HSC
CPCMonocyte
Cytotoxic T cells
B-cellsPlasma cells
NK-Cells
Granulocytes
NK-T cells
Helper T cells
Concentration
Clofarabine
10000nM1000nM100nM10nM1nM
0
20
40
60
80
100
% S
urvi
val HSC
CPCMonocyte
Cytotoxic T cells
B-cellsPlasma cells
NK-Cells
Granulocytes
NK-T cells
Helper T cells
Distinct Drug Responses are Tied to Cell Lineages
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fidd.mm.yyyy 10
Dexamethasone
10000nM1000nM100nM10nM1nM
0
20
40
60
80
100100150200250
% S
urvi
val
HSCCPCMonocyte
Cytotoxic T cells
B-cellsPlasma cells
NK-Cells
Granulocytes
NK-T cells
Helper T cells
Concentration
Distinct Drug Responses are Tied to Cell Lineages
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
B Cells are Inherently Sensitive to Midostaurin
dd.mm.yyyy 11
Midostaurin
10000nM1000nM100nM10nM1nM
0
20
40
60
80
100%
Sur
viva
l HSCCPCMonocyteCytotoxic T cells
B-cellsHelper T cells
Concentration
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
B Cells are Inherently Sensitive to Midostaurin
dd.mm.yyyy 12Muntasir Mamun Majumder, PhD
0 nM
1nM
10 nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100%
Sur
viva
lCPC (CD34+CD38+)
HC-BM (n=2)AML-FLT3-WT (n=3)AML-FLT3-ITD (n=5)
Email: [email protected]
www.fimm.fi
B Cells are Inherently Sensitive to Midostaurin
dd.mm.yyyy 13
0 nM
1nM
10 nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100%
Sur
viva
lB (CD45+CD19+)
AML (FLT3-WT, n=3)HC-BM (n=2)
AML (FLT3-ITD, n= 5)CLL (n=7)
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Venetoclax Displays Dose Dependent Lineage Selectivity toward Lymphocytes
dd.mm.yyyy 14
Venetoclax
10000nM1000nM100nM10nM1nM
0
20
40
60
80
100%
Sur
viva
l HSCCPCMonocyte
Cytotoxic T cells
B-cellsPlasma cells
NK-Cells
Granulocytes
NK-T cells
Helper T cells
Concentration
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Venetoclax Displays Dose Dependent Lineage Selectivity toward Lymphocytes
dd.mm.yyyy 15Muntasir Mamun Majumder, PhD Email: [email protected]
CD45
CD19
SSC
-HSS
C-H
1nM
1nM 10nM
10nM 100nM
100nM 1000nM
1000nM
10 000nM
10 000nM
Venetoclax
32% 7% 5% 3% 2%
86% 72% 61% 39% 32%
0 nM 1n
M10
nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100
% S
urvi
val
CD19+
0 nM 1n
M10
nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100
% S
urvi
val
CD56+
0 nM 1n
M10
nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100
% S
urvi
val
CD3+CD4+
0 nM 1n
M10
nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100%
Sur
viva
l
CD3+CD4-
0 nM 1n
M10
nM
100 n
M
1000
nM
1000
0 nM
0
20
40
60
80
100
% S
urvi
val
CD3+CD56+
Multiple Myeloma (10)AML (3)
Healthy (3)
www.fimm.fi
Basal Signaling Active in Healthy Cell-of-origin is Retained in Leukemic Cells
dd.mm.yyyy 16
Monocytes pDCs B-cellsGranulocytes
pPLCg1pErk
pNFkBp4E-BP1
pCREBpSTAT3pSTAT1
pAktpS6
01.
503.
01
Med
ian
(Arc
sinh
)
CD4 T-cells CD8 T-cells CD25 T-cells NK-cells Blasts
pPLCg1pErk
pNFkBp4E-BP1
pCREBpSTAT3pSTAT1
pAktpS6
Monocytes pDCs B-cellsGranulocytes CD4 T-cells CD8 T-cells CD25 T-cells NK-cells CD34+ CD38+
Leukemia
Healthy
BMPB
AMLB-ALL
C
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Basal Signaling Active in Healthy Cell-of-origin is Retained in Leukemic Cells
dd.mm.yyyy 17Muntasir Mamun Majumder, PhD Email: [email protected]
0.0
1.0
2.0
3.0
Med
ian
(arc
sinh
)
Healthy Leukemia
BM
PB
AML
B-ALL
0.0
1.0
2.0
3.0
Med
ian
(arc
sinh
)
p4E-
BP1
pPLC
γ1
0.0
1.0
2.0
3.0
Med
ian
(arc
sinh
)
0.0
1.0
2.0
3.0
Med
ian
(arc
sinh
)
#
**** ** ***
*
#
******
Gra
nulo
cyte
s
B-ce
lls
NK
cells
pDCs
Mon
ocyt
es
CD4
T-ce
lls
CD8
T-ce
lls
CD25
T-c
ells
Blas
ts
###*
ns
ns
*
*ns
**
ns
**ns
ns nsns
*
ns ns
nsns
Gra
nulo
cyte
s
B-ce
lls
NK
cells
pDCs
Mon
ocyt
es
CD4
T-ce
lls
CD8
T-ce
lls
CD25
T-c
ells
CD34
/CD
38+
Gra
nulo
cyte
s
B-ce
lls
NK
cells
pDCs
Mon
ocyt
es
CD4
T-ce
lls
CD8
T-ce
lls
CD25
T-c
ells
Blas
ts
Gra
nulo
cyte
s
B-ce
lls
NK
cells
pDCs
Mon
ocyt
es
CD4
T-ce
lls
CD8
T-ce
lls
CD25
T-c
ells
CD34
/CD
38+
www.fimm.fi
STAT3 Phosphorylation Status Correlates withResponse to Venetoclax in Hematopoietic Cells
dd.mm.yyyy 18CD14
+/Monocy
tes
CD4 T ce
lls
CD8 T ce
lls
NK cells
B cells
01020304050
Dru
g Se
nsitiv
ity S
core
CD14+/M
onoc
ytes
CD4 T ce
lls
CD8 T ce
lls
NK cells
B cells
0
2
4
6
8
Med
ian
(arc
sinh
)
Venetoclax
10000nM1000nM100nM10nM1nM0
20
40
60
80
100
% S
urvi
val HSC
CPCMonocyte
Cytotoxic T cells
B-cellsPlasma cells
NK-Cells
Granulocytes
NK-T cells
Helper T cells
Concentration
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Proteome Profile of Hematopoietic Cell Subsets
Presentation name or name of the guestdd.mm.yyyy 19
CD14+
CD19+CD3+
CD14+
CD19+CD3+
CD14+
CD19+CD3+
20
25
30
35
40
Abun
danc
e (L
FQ in
tens
ity)
***
**
******
* **
CAT S100A8 S100A9242628303234
IDH
1G
CA
BASP
1PL
IN3
MAR
CKS
RN
H1
BLVR
BM
THFD
1AL
DH
2AT
P6V1
B2TS
POC
TSB
LAP3
CES
1IT
GAM
ATP6
V1A
TYM
PN
AGK
CYB
BD
PYSL
2M
VPC
APN
2R
HO
GW
ARS
ACAD
VLG
PX1
PYC
ARD
LGAL
S1TP
P1IT
GB2
FABP
3SP
TBN
1SP
TAN
1LM
NA
SEPT
9R
PL27
RPL
13A
RPS
20R
PL7A
ARH
GEF
1O
GD
HSH
3KBP
1D
LST
NAP
1L4
RPL
38AL
DH
9A1
ALYR
EFPA
RP1
IL16
EVL
ANP3
2AH
P1BP
3SE
RPI
NB9
SEPT
7SE
PT6
GIM
AP4
DC
DLM
NB2
KRT2
S100A9
S100A8
LYZ
AHNAK
PLEC
CAPG
MNDA
CAT
MPO
GSN
PRTN3
HC-2-CD14HC-1-CD14HC-1-CD19HC-1-CD3HC-2-CD19HC-2-CD3
LFQ intensity
Hea
lthy
LYZ
S100A8
S100A9
LMN
AG
IMAP
4C
NN
2EV
LIL
16SE
PT9
MN
DA
CA1
ALD
H2
CTS
BIT
GAM
ATP6
V1A
TYM
PN
AGK
IDH
1D
PYSL
2G
PX1
ATP6
V1B2
WAR
SC
YBB
GC
AM
ARC
KSBA
SP1
MM-4-CD3MM-1-CD3MM-3-CD3MM-2-CD3MM-1-CD19MM-4-CD14MM-2-CD14MM-1-CD14MM-4-CD19MM-3-CD14
242628303234
LFQ intensity
Mye
lom
a
www.fimm.fidd.mm.yyyy 20
Cell-of-Origin Tied Response Recapitulates Their Drug Response In Malignant State
Venetoclax
CD34CD14
MDS
CMMLCML
AML(D)
AML (R)
CD19 CLLALL
B-PLL
CD3
T-PLL
T-ALL
CD138
MM (D)
MM (R)
0
10
20
30
40
DS
S
Myeloid Lineage Lymphoid Lineage
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Conclusions
dd.mm.yyyy 21
ü Drug responses in hematopoietic cells are often lineage specific
ü Basal signaling activation was comparable between healthy and leukemic cell types
ü Drug responses tied to the cell of origin are predictive of the responses observed in the malignant counterpart
Muntasir Mamun Majumder, PhD Email: [email protected]
www.fimm.fi
Acknowledgements
Translational Research and Personalized Medicine Lab, FIMM
Aino Maija Leppä
Alun ParsonsAlina Malyutina
Caroline A. HeckmanHUCH/HruHProf. Kimmo Porkka
Pekka AnttilaMika KontroJuha LievonenScience for Life Laboratory, Karolinska Institute, SwedenProf. Olli Kallioniemi
Kuopio University HospitalRaija Silvennoinen
University of Bergen
Monica Hellesøy
Prof. Bjørn T. Gjertsen
Mater Misericordiae University Hospital, IrelandDespina BazouProf. Peter O’Gorman
National University of IrelandPaul Dowling
Biotech Research & Innovation CentreProf. Krister Wennerberg
Hematology Research UnitEmma AnderssonProf. Satu Mustjoki
The Patients!!
Muntasir Mamun Majumder, PhD Email: [email protected]