Multi-ParametricSingle Cell ProfilingDefinesDistinctDrug … · 2019-07-03 · Multi-ParametricSingle Cell ProfilingDefinesDistinctDrug Responsesin HealthyHematologicalCell LineagesThatare

© 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


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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)


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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


Cell density

Antibody dilutions

Incubation Time

Wash vs no wash


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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


Gating strategy for immunophenotyping Hematopoietic Cell Subsets


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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


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


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



www.fimm.fidd.mm.yyyy 10

Dexamethasone


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



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B Cells are Inherently Sensitive to Midostaurin

dd.mm.yyyy 11

Midostaurin


0

20

40

60

80

100%

Sur

viva

l HSCCPCMonocyteCytotoxic T cells

B-cellsHelper T cells

Concentration


www.fimm.fi


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)


www.fimm.fi


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)


www.fimm.fi

Venetoclax Displays Dose Dependent Lineage Selectivity toward Lymphocytes

dd.mm.yyyy 14

Venetoclax


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


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)

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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


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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+

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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


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


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


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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!!


Multi-ParametricSingle Cell ProfilingDefinesDistinctDrug … · 2019-07-03 · Multi-ParametricSingle Cell ProfilingDefinesDistinctDrug Responsesin HealthyHematologicalCell LineagesThatare

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