The Other Stem Cell in Multiple Myeloma: CD34+ Mobilization,

Exhaustion, and SPMs

Suzanne Lentzsch MD, PhD

Columbia University

Complications of IMiD treatment?

• Most dose-limiting adverse effect of IMiDs is neutropenia (grades 3 or 4: 41.2%)1

• 14.7% of patients develop grade 3 or 4 thrombocytopenia1

• Up to 43% of patients fail to mobilize hematopoietic progenitors for autologous stem cell transplant after lenalidomide2

Effects of IMiDs on hematopoietic stem cells?

Why do IMiDs inhibit CD34+ mobilization ?

1. Weber DM, et al. 2007;357:2133-42. 2. Mazumdar A, et al. Leukemia. 2008;22:1280-81.

Effect of IMiDs on myeloid progenitors?

Pluripotent stem cell

Myeloid progenitors Lymphoid progenitors

Granulocyte progenitors

Granulocyte-macrophage progenitors

Erythropoietic /megakaryocytic

progenitors

NeutrophilsEosinophilsBasophils

MonocytesMacrophages

Lymphocytes/plasma cells

Osteoclastdevelopment? Erythrocytes ?Megakaryocytes

Platelets

Anderson G, et al. Blood. 2006;107:3098-105. Koh KR, et al. Blood. 2005;105:3833-40. Li S, et al. Blood. 2011; 117:5157-65. Pal R, et al. Blood. 2010;115:605-14.

IMiDs induce CFU-G development

Koh KR, et al. Blood. 2005;105:3833-40. Pal R, et al. Blood. 2010;115:605-14.

Nu

mb

er o

f co

lon

ies

BFU-E

Erythroidcolonies

DMSO

10 µM lenalidomide

100 µM lenalidomide

*

0

200

100

160

180

140

120

80

60

40

20

CFU-G CFU-GM

Myeloid colonies

CFU-GEMM

Lenalidomide

*

Nu

mb

er o

f co

lon

ies

BFU-E

Erythroidcolonies

DMSO

10 µM pomalidomide

100 µM pomalidomide

*

0

200

100

160

180

140

120

80

60

40

20

CFU-G CFU-GM

Myeloid colonies

CFU-GEMM

Pomalidomide

*

Cell cycle analysis

G0/G1: 73.3%S: 21.3%G2/M: 5.4%

G0/G1: 50.8%S: 47.2%G2/M: 2.0%

IMiDs induce proliferation and expansion of hematopoietic progenitors with induction

of early myeloid markers

Flow cytometry analysis of CD34+ cells treated +/– IMiDs® for 14 days

POM

DMSO

Lenalidomide induces myeloid maturation arrest with concomitant peripheral neutropenia

Pretreatment WBC nadir

Bone marrow aspirate obtained pretreatment and at the time of WBC nadir (Wright Giemsa,100 x magnification)

Pal R, et al. Blood. 2010;115:605-14.

Pretreatment Cytopenia nadir

M:E ratio Cellularity M:E ratio Cellularity

1 50 2 60

1.2 70 40 50

1 80 2.5 75

0.5 100 1 80

5 50 2 25

3 40 7.5 70

Mean 2.0 65 9.2 60

Below median 1.1 60 2.3 65

SD 1.7 22.6 15.3 20.2

Hypothesis

• PU.1–/– granulocytes do not undergo complete maturation1

• Heterozygous deletion of the PU.1 locus associated with development of human AML2

• IMiD-dependent downregulation of PU.1 induces maturation arrest of granulocytes resulting in peripheral blood neutropenia

1. Dakic A, et al. J Exp Med. 2005;201:1487-502.2. Bonadies N, et al. Blood. 2010;115:331-4. AML = acute myeloid leukaemia.

IMiDs downregulate PU.1 protein in hematopoietic progenitors

DMSO Lenalidomide

0.1% 10 μM 100 μM

Day 6

DMSO Lenalidomide

0.1% 10 μM 100 μM

Day 8

PU.1 PU.1

DMSO Pomalidomide

0.1% 10 μM 100 μM

Day 6

DMSO Pomalidomide

0.1% 10 μM 100 μM

Day 8

PU.1 PU.1

β-Actin β-Actin

β-Actin β-Actin

CD34+ cells cultured with SCF, IL-3, IL-6, and +/–IMiDs® or 0.1% DMSO (control).Cell lysates were subjected to western blotting to determine PU.1 protein expression

Pal R, et al. Blood. 2010;115:605-14.

PU.1 is downregulated by IMiDs in myeloid progenitors of MM patient

PU.1 (black)/ myeloperoxidase (red) double labelling immunohistochemistry on bone marrow biopsies from patients treated with lenalidomide

Pretreatment During treatment

Pat 1: Lenalidomide

Pat 2: Lenalidomide

Pat 3: Control

Pal R, et al. Blood. 2010;115:605-14.

Summary

Pal R, et al. Blood. 2010;115:605-14.

Maturation block with accumulation of immature granulocytes

Treatment with IMiDs

PU.1

Cathepsin GNeutrophil elastase

Neutrophil Neutropenia

Myeloblast

N. promyelocyte

Effect of IMiDs on megakaryopoiesis?

Pluripotent stem cell

Myeloid progenitors Lymphoid progenitors

Granulocyte progenitors

Granulocyte-macrophage progenitors

Megakaryocytic progenitors

Neutrophils

MonocytesMacrophages

Lymphocytes/plasma cells

Osteoclastdevelopment

Erythrocytes ?Megakaryocytesplatelets

Anderson G, et al. Blood. 2006;107:3098-105; Koh KR, et al. Blood. 2005;105:3833-40. Li S, et al. Blood. 2011. March 9 [Epub ahead of print]. Pal R, et al. Blood. 2010;115:605-14.

Erythropoietic progenitors

PU. 1

CD34+ cells cultured with POM or 0.1% DMSO in MegaCult-C assay and stained with CD41 (GPIIb/IIIa)Ab

IMiDs significantly induce formation of megakaryocytic colony (CFU-Mk)

*p < 0.05 **p < 0.001

Anti-CD41 ab (GPIIb/IIIa)

CFU-Mk small CFU-Mk med/big CFU-Mk mixed

CF

U-M

k/3

10

4 C

D34

+ c

ells

0

20

60

120

40

80

100DMSO

POM

**

**

*

IMiDs inhibit apoptosis and induce expansion of megakaryocyte progenitors

CD34+ cells cultured with TPO +/–IMiDs®. A. Flow cytometry (propidium iodide) for apoptosis and cell cycle; B. Proliferation profiles of CD34+ cells expanded in serum-free liquid culture

A B

58% 22% 6%

7 days

S-G2M

GOG1

Apop

LENDMSO POM0%

100%

20%

60%

80%

40%

68% 24% 8%

14 days

LENDMSO POM0%

100%

20%

60%

80%

40%

S-G2M

GOG1

Apop

Via

ble

ce

lls

(x

10

5 c

ell

s/m

L)

0

*

0

30

10

20

25

5

15

2 4 6 8 10 12 14

* *

**

****

**

DMSOLEN

POM

* p < 0.05** p < 0.001

Days

IMiDs induce the expansion of early CD34+ cells and development of CD33+/CD41+ hybrids

CD34+ cells treated for 14 days with POM +/- TPO or vehicle

Maintaining and expanding CD34+ cells for up to 4 month in the presence of POM

010

110

210

310

100

101

102

103

CD45 APC-Cy7

Nucleated singlets

D: 86.57%

E: 6.64%

CD

34 A

PC

-A70

0

010

110

210

310

100

101

102

103

CD33 PC5

CD

11

b A

PC

[E]

Q3:0.01% Q4:8.14%

Q1:0.37% Q2:91.48%

010

110

210

310

100

101

102

103

CD41 PC7

CD

11

b A

PC

[E]

R3:0.13% R4:6.80%

R1:1.47% R2:91.60%

010

110

210

310

100

101

102

103

CD61 PE

CD

11

b A

PC

[E]

S3:0.06% S4:6.46%

S1:1.39% S2:92.09%

010

110

210

310

100

101

102

103

CD33 PC5

CD

11

b A

PC

[D]

N3:0.46% N4:17.80%

N1:0.96% N2:80.77%

010

110

210

310

100

101

102

103

CD41 PC7

CD

11

b A

PC

[D]

O3:0.99% O4:15.81%

O1:22.82% O2:60.39%

010

110

210

310

100

101

102

103

CD61 PE

CD

11

b A

PC

[D]

P3:1.17% P4:17.09%

P1:23.87% P2:57.86%

CD45+34+ are the hybrids because they are CD33+11b+41+61+

CD45+34- are more differentiated

CD34+ cells cultured for 4 month in the presence of POM

IMiDs induce expansion of immature megakaryocytes (CD 41a+/CD42b–)

Flow cytometry analysis showing the percentage of immature megakaryocytic cells (CD41a+/CD42b–) and more mature megakaryocytic cells (CD41a+/CD42b+) of CD34 cells treated with TPO+/– IMiDs®

DMSO LEN POM

7 d

10 d

14 d

CD42b-FITC

CD

41a-

PE

16%

65%

32%

66%50%

51%

23%

51%

23%

IMiDs inhibit the maturation of megakaryocytes

CD34+ cells grown in serum-free liquid culture with 10ng/mL TPO

Number of nuclei and centromeric enumeration probes (CEP 6) signals for each megakaryocyte (CD61 FITC stained-green) were recorded. FISH signals were only counted from the green FITC-stained cells.

DMSO POM

Data 2

0

2

4

6100

110

120

130

140DMSOPOM

Total nuclei numbers Mean ploidy numbers

Data 1

2N 4N ≥8N0

10

20

30

40

50DMSOPOM

% o

f M

egak

aryo

cyte

s

IMiDs inhibit the polyploidization of megakaryocytes

IMiDs downregulate GATA-1 expression in megakaryocytic progenitors

Overexpression of GATA1 rescues hematopoietic progenitors from Effects of IMiDs

IMiDs inhibit the process of megakaryopoiesis through suppression of GATA-1

IMiDs

Po

lyp

loid

izat

ion

Term

inal

dif

fere

nti

atio

n

Cyclin-D1

p21

FOG

p45-NF-E2

GATA-1

Cell division

HSC

HSC

CFU-Mix

CFU-Meg

Meg-precursor

Megakaryocyte

Commitment

Proliferation

Maturation

Proplatelet producing megakaryocyte

Conclusion

• IMiDs induce expansion of hematopoietic progenitors with concomitant inhibition of maturation

• IMiDs induce myeloid development• Inhibition of maturation via down regulation of critical

transcription factors such as PU.1 and GATA1• Significance of the results are not completely clear but

mutations of PU.1 and GATA1 have been described in hematologic malignancies

• Treatment break for maintenance suggested to allow maturation of hematopoietic progenitors

• Up to 43% of patients fail to mobilize haematopoietic progenitors after prolonged lenalidomide treatment2

• Often mobilization failure can be overcome by Plerixafor – a CXCR4 antagonist

• Role of SDF-1a/ CXCR4 in Len induced mobilization failure?

Why do IMiDs inhibit CD34+ mobilization ?

2. Mazumdar A, et al. Leukemia. 2008;22:1280-81.

LEN up regulates CXCR4 on the CD34+ cell surface

LEN upregulates CXCR4 on the CD34+ cell surface

LEN enhances the SDF-1α driven CD34+ cell migration

LEN inhibits SDF-1α induced CXCR4 internalization

Conclusion

• Len induces localization of CXCR4 to the cell membrane

• Len inhibits internalization of CXCR4 resulting in increased migration

• Inhibition of internalization of CXCR4 results in increased binding of CD34+ cells to the bone marrow niche

• Blocking the CXCR4 receptor with Plerixafor releases CD34+ cells from the increased binding to the bone marrow niche

Acknowledgments

University of Pittsburgh,Pittsburgh

Ailing Liu, PhDRekha Pal, PhDAlbert Donnenberg, PhDVera Donnenberg, PhDDonna B. Stolz, PhDSusanne Gollin, PhD

Columbia University,New York

Shirong Li, PhDJing FuMarkus Y. Mapara, MD

University of Texas, Dallas

Sara A. Monaghan, MD